Arthritis & Rheumatism

Volume 56, Issue 4, Pages 1037-1370 (April 2007)

ACR Presidential Address Mentors and heroes: The foundation and future of rheumatology (p 1037-1043) Mary K. Crow Published Online: 28 Mar 2007 DOI: 10.1002/art.22503

Editorials The role of varus and valgus alignment in knee osteoarthritis (p 1044-1047) Leena Sharma Published Online: 28 Mar 2007 DOI: 10.1002/art.22514

Estrogens and lupus: Bubbling cauldron or another overrated Witches' Brew? (p 1048-1050) Michael D. Lockshin, Jill P. Buyon Published Online: 28 Mar 2007 DOI: 10.1002/art.22631

Review Psoriatic arthritis: Current concepts on pathogenesis-oriented therapeutic options (p 1051-1066) Anthony M. Turkiewicz, Larry W. Moreland Published Online: 28 Mar 2007 DOI: 10.1002/art.22489

Research Articles Decoy receptor 3 expressed in rheumatoid synovial fibroblasts protects the cells against fas-induced apoptosis (p 1067-1075) Shinya Hayashi, Yasushi Miura, Takayuki Nishiyama, Makoto Mitani, Koji Tateishi, Yoshitada Sakai, Akira Hashiramoto, Masahiro Kurosaka, Shunichi Shiozawa, Minoru Doita Published Online: 28 Mar 2007 DOI: 10.1002/art.22494

Up-regulation of stromal cell-derived factor 1 (CXCL12) production in rheumatoid synovial fibroblasts through interactions with T lymphocytes: Role of interleukin-17 and CD40L-CD40 interaction (p 1076-1086) Kyoung-Woon Kim, Mi-La Cho, Hae-Rim Kim, Ji-Hyeon Ju, Mi-Kyung Park, Hye-Jwa Oh, Joon-Seok Kim, Sung- Hwan Park, Sang-Heon Lee, Ho-Youn Kim Published Online: 28 Mar 2007 DOI: 10.1002/art.22439

Histone deacetylase/acetylase activity in total synovial tissue derived from rheumatoid arthritis and osteoarthritis patients (p 1087-1093) Lars C. Huber, Matthias Brock, Hossein Hemmatazad, Olivier T. Giger, Falk Moritz, Michelle Trenkmann, Jörg H. W. Distler, Renate E. Gay, Christoph Kolling, Holger Moch, Beat A. Michel, Steffen Gay, Oliver Distler, Astrid Jüngel Published Online: 28 Mar 2007 DOI: 10.1002/art.22512

Analysis of vascular gene expression in arthritic synovium by laser-mediated microdissection (p 1094- 1105) Atsushi Hashimoto, Ingo H. Tarner, Rainer M. Bohle, Andreas Gaumann, Mirko Manetti, Oliver Distler, Jürgen Steinmeyer, Ann-Kristin Ulfgren, Andreas Schulz, Steffen Gay, Ulf Müller-Ladner, Elena Neumann Published Online: 28 Mar 2007 DOI: 10.1002/art.22450

Light up-regulated on B lymphocytes and monocytes in rheumatoid arthritis mediates cellular adhesion and metalloproteinase production by synoviocytes (p 1106-1117) Young Mo Kang, So Young Kim, Jin Hee Kang, Seung Woo Han, Eon Jeong Nam, Hee Soo Kyung, Jae Yong Park, In San Kim Published Online: 28 Mar 2007 DOI: 10.1002/art.22493

Bone erosions and bone marrow edema as defined by magnetic resonance imaging reflect true bone marrow inflammation in rheumatoid arthritis (p 1118-1124) Esther Jimenez-Boj, Iris Nöbauer-Huhmann, Beatrice Hanslik-Schnabel, Ronald Dorotka, Axel-Hugo Wanivenhaus, Franz Kainberger, Siegfried Trattnig, Roland Axmann, Wayne Tsuji, Sonja Hermann, Josef Smolen, Georg Schett Published Online: 28 Mar 2007 DOI: 10.1002/art.22496

Risk of serious bacterial infections among rheumatoid arthritis patients exposed to tumor necrosis factor antagonists (p 1125-1133) Jeffrey R. Curtis, Nivedita Patkar, Aiyuan Xie, Carolyn Martin, Jeroan J. Allison, Michael Saag, Deborah Shatin, Kenneth G. Saag Published Online: 28 Mar 2007 DOI: 10.1002/art.22504

Aberrant expression of BAFF by B lymphocytes infiltrating the salivary glands of patients with primary Sjögren's syndrome (p 1134-1144) Capucine Daridon, Valérie Devauchelle, Pascal Hutin, Rozenn Le Berre, Christine Martins-Carvalho, Boutahar Bendaoud, Maryvonne Dueymes, Alain Saraux, Pierre Youinou, Jacques-Olivier Pers Published Online: 28 Mar 2007 DOI: 10.1002/art.22458

Interferon- regulates susceptibility to collagen-induced arthritis through suppression of interleukin-17 (p 1145-1151) Cong-Qiu Chu, David Swart, Dina Alcorn, Joel Tocker, Keith B. Elkon Published Online: 28 Mar 2007 DOI: 10.1002/art.22453

Blockade of the interleukin-21/interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis (p 1152-1163) Deborah A. Young, Martin Hegen, Hak Ling Margery Ma, Matthew J. Whitters, Leo M. Albert, Leslie Lowe, Mayra Senices, Paul W. Wu, Barbara Sibley, Yelena Leathurby, Tom P. Brown, Cheryl Nickerson-Nutter, James C. Keith Jr., Mary Collins Published Online: 28 Mar 2007 DOI: 10.1002/art.22452

A tumor necrosis factor receptor loop peptide mimic inhibits bone destruction to the same extent as antitumor necrosis factor monoclonal antibody in murine collagen-induced arthritis (p 1164-1174) Hiroaki Saito, Takefumi Kojima, Mariko Takahashi, William C. Horne, Roland Baron, Teruo Amagasa, Keiichi Ohya, Kazuhiro Aoki Published Online: 28 Mar 2007 DOI: 10.1002/art.22495

Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen- induced arthritis (p 1175-1186) Andrea Augello, Roberta Tasso, Simone Maria Negrini, Ranieri Cancedda, Giuseppina Pennesi Published Online: 28 Mar 2007 DOI: 10.1002/art.22511

Selective therapeutic control of C5a and the terminal complement complex by anti-C5 single-chain Fv in an experimental model of antigen-induced arthritis in rats (p 1187-1197) Fabio Fischetti, Paolo Durigutto, Paolo Macor, Roberto Marzari, Renzo Carretta, Francesco Tedesco Published Online: 28 Mar 2007 DOI: 10.1002/art.22492

A randomized crossover trial of a wedged insole for treatment of knee osteoarthritis (p 1198-1203) Kristin Baker, Joyce Goggins, Hui Xie, Karen Szumowski, Michael LaValley, David J. Hunter, David T. Felson Published Online: 28 Mar 2007 DOI: 10.1002/art.22516

Association between valgus and varus alignment and the development and progression of radiographic osteoarthritis of the knee (p 1204-1211) G. M. Brouwer, A. W. Van Tol, A. P. Bergink, J. N. Belo, R. M. D. Bernsen, M. Reijman, H. A. P. Pols, S. M. A. Bierma-Zeinstra Published Online: 28 Mar 2007 DOI: 10.1002/art.22515

Knee alignment does not predict incident osteoarthritis: The Framingham osteoarthritis study (p 1212- 1218) David J. Hunter, Jingbo Niu, David T. Felson, William F. Harvey, K. Douglas Gross, Paula McCree, Piran Aliabadi, Burton Sack, Yuqing Zhang Published Online: 28 Mar 2007 DOI: 10.1002/art.22508

Examining the role of CD1d and natural killer T cells in the development of nephritis in a genetically susceptible lupus model (p 1219-1233) Jun-Qi Yang, Xiangshu Wen, Hongzhu Liu, Gbolahan Folayan, Xin Dong, Min Zhou, Luc Van Kaer, Ram Raj Singh Published Online: 28 Mar 2007 DOI: 10.1002/art.22490

Structural insertion/deletion variation in IRF5 is associated with a risk haplotype and defines the precise IRF5 isoforms expressed in systemic lupus erythematosus (p 1234-1241) Sergey V. Kozyrev, Susanna Lewén, Prasad M. V. Linga Reddy, Bernardo Pons-Estel, Argentine Collaborative Group, Torsten Witte, German Collaborative Group, Peter Junker, Helle Laustrup, Carmen Gutiérrez, Ana Suárez, Maria Francisca González-Escribano, Javier Mart ´n, Spanish Collaborative Group, Marta E. Alarcón-Riquelme Published Online: 28 Mar 2007 DOI: 10.1002/art.22497

Interleukin-6 and chemokines in the neuropsychiatric manifestations of systemic lupus erythematosus (p 1242-1250) H. Fragoso-Loyo, Y. Richaud-Patin, A. Orozco-Narváez, L. Dávila-Maldonado, Y. Atisha-Fregoso, L. Llorente, J. Sánchez-Guerrero Published Online: 28 Mar 2007 DOI: 10.1002/art.22451

Reproductive and menopausal factors and risk of systemic lupus erythematosus in women (p 1251-1262) Karen H. Costenbader, Diane Feskanich, Meir J. Stampfer, Elizabeth W. Karlson Published Online: 28 Mar 2007 DOI: 10.1002/art.22510

Histopathologic and clinical outcome of rituximab treatment in patients with cyclophosphamide-resistant proliferative lupus nephritis (p 1263-1272) Iva Gunnarsson, Birgitta Sundelin, Thorunn Jónsdóttir, Stefan H. Jacobson, Elisabet Welin Henriksson, Ronald F. van Vollenhoven Published Online: 28 Mar 2007 DOI: 10.1002/art.22505

The clinical continuum of cryopyrinopathies: Novel CIAS1 mutations in North American patients and a new cryopyrin model (p 1273-1285) Ivona Aksentijevich, Christopher D. Putnam, Elaine F. Remmers, James L. Mueller, Julie Le, Richard D. Kolodner, Zachary Moak, Michael Chuang, Frances Austin, Raphaela Goldbach-Mansky, Hal M. Hoffman, Daniel L. Kastner Published Online: 28 Mar 2007 DOI: 10.1002/art.22491

Positive association of SLC26A2 gene polymorphisms with susceptibility to systemic-onset juvenile idiopathic arthritis (p 1286-1291) Rebecca Lamb, Wendy Thomson, British Society of Paediatric and Adolescent Rheumatology, Emma M. Ogilvie, Rachelle Donn Published Online: 28 Mar 2007 DOI: 10.1002/art.22444

Interstitial pneumonitis in Blau syndrome with documented mutation in CARD15 (p 1292-1294) Mara L. Becker, Tammy M. Martin, Trudy M. Doyle, Carlos D. Rosé Published Online: 28 Mar 2007 DOI: 10.1002/art.22509

Clinical and immunogenetic features of patients with autoantibodies to asparaginyl-transfer RNA synthetase (p 1295-1303) Michito Hirakata, Akira Suwa, Tetsuya Takada, Shinji Sato, Sonoko Nagai, Ekkehard Genth, Yeong W. Song, Tsuneyo Mimori, Ira N. Targoff Published Online: 28 Mar 2007 DOI: 10.1002/art.22506

A new murine model to define the critical pathologic and therapeutic mediators of polymyositis (p 1304- 1314) Takahiko Sugihara, Chiyoko Sekine, Takashi Nakae, Kuniko Kohyama, Masayoshi Harigai, Yoichiro Iwakura, Yoh Matsumoto, Nobuyuki Miyasaka, Hitoshi Kohsaka Published Online: 29 Mar 2007 DOI: 10.1002/art.22521

Role of matrix metalloproteinases, proinflammatory cytokines, and oxidative stress-derived molecules in hepatitis C virus-associated mixed cryoglobulinemia vasculitis neuropathy (p 1315-1324) David Saadoun, Ivan Bieche, François-Jérome Authier, Ingrid Laurendeau, Florence Jambou, Jean Charles Piette, Michel Vidaud, Thierry Maisonobe, Patrice Cacoub Published Online: 28 Mar 2007 DOI: 10.1002/art.22456

High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory lyme arthritis (p 1325-1335) Junghee J. Shin, Lisa J. Glickstein, Allen C. Steere Published Online: 28 Mar 2007 DOI: 10.1002/art.22441

Gabapentin in the treatment of fibromyalgia: A randomized, double-blind, placebo-controlled, multicenter trial (p 1336-1344) Lesley M. Arnold, Don L. Goldenberg, Sharon B. Stanford, Justine K. Lalonde, H. S. Sandhu, Paul E. Keck Jr., Jeffrey A. Welge, Fred Bishop, Kevin E. Stanford, Evelyn V. Hess, James I. Hudson Published Online: 28 Mar 2007 DOI: 10.1002/art.22457

Arthritic pain is processed in brain areas concerned with emotions and fear (p 1345-1354) B. Kulkarni, D. E. Bentley, R. Elliott, P. J. Julyan, E. Boger, A. Watson, Y. Boyle, W. El-Deredy, A. K. P. Jones Published Online: 28 Mar 2007 DOI: 10.1002/art.22460

Risk factors for more severe regional musculoskeletal symptoms: A two-year prospective study of a general working population (p 1355-1364) Johan H. Andersen, Jens P. Haahr, Poul Frost Published Online: 28 Mar 2007 DOI: 10.1002/art.22513

Concise Communication Liver X receptor is a therapeutic target in collagen-induced arthritis (p 1365-1367) Subba R. Chintalacharuvu, George E. Sandusky, Thomas P. Burris, Glenna C. Burmer, Sunil Nagpal Published Online: 28 Mar 2007 DOI: 10.1002/art.22528

Letters What is juvenile psoriatic arthritis? Comment on the article by Stoll et al (p 1368) Alberto Martini Published Online: 28 Mar 2007 DOI: 10.1002/art.22519

The irreversible component of the disability index of the health assessment questionnaire: Comment on the article by Aletaha et al (p 1368-1369) James F. Fries Published Online: 28 Mar 2007 DOI: 10.1002/art.22517

Reply (p 1369-1370) Daniel Aletaha, Josef S. Smolen, Michael M. Ward Published Online: 28 Mar 2007 DOI: 10.1002/art.22518

Erratum Erratum (p 1370) Published Online: 28 Mar 2007 DOI: 10.1002/art.22648

ACR Announcements ACR Announcements (p A12) Published Online: 28 Mar 2007 DOI: 10.1002/art.22649

ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1037–1043 DOI 10.1002/art.22503 © 2007, American College of Rheumatology Arthritis & Rheumatism An Ofﬁcial Journal of the American College of Rheumatology www.arthritisrheum.org and www.interscience.wiley.com ACR PRESIDENTIAL ADDRESS

Mentors and Heroes

The Foundation and Future of Rheumatology

Mary K. Crow

I have had a wonderful and stimulating year fully pushed to restore funding to the Centers for serving as your President, and while I am relieved to be Disease Control for lupus registries. The ACR staff and passing the responsibility of guiding the American Col- volunteers battle every day for rheumatologists and our lege of Rheumatology on to our next President in a few patients. And at the end of the day, they are what days, this evening I want to report several of my obser- matters—our colleagues and our patients. My message vations and convey my optimism regarding the future of today is that rheumatology will continue to thrive as the rheumatology. most interesting and rewarding specialty if we can Rheumatology has always attracted physicians succeed in developing a next generation of leaders as who enjoy solving problems and dealing with the most committed as those we have had—that it is all about the fascinating and complex patients in all of medicine. At people. least in part because the patients are so challenging, rheumatologists generally love their work and love their patients. Rheumatologists have filled many of the lead- Personal mentors and giants of rheumatology ership positions in American medical education, and I will begin by acknowledging my personal men- through basic and clinical research have made significant tors and role models, Henry Kunkel and Charles Chris- contributions to understanding disease. And rheumatol- tian (Figure 1). Henry and Chuck have changed our ogy can take credit for introducing the life-changing understanding of two of the most significant rheumatic biologic therapies to our patients and to medicine. The diseases, rheumatoid arthritis (RA) and systemic lupus day-to-day work of the ACR can easily obscure that big erythematosus (SLE), based on their detailed studies of picture, the place that rheumatology holds in medicine patients (1–5). Lupus autoantibodies, the components of as a specialty leading the way in innovative patient care immune complexes, and rheumatoid factor were all and in academic medicine and research. characterized in a meticulous fashion, and in the classic During this year, ACR has fought hard to push papers of Estes and Christian describing the clinical for reform of the sustainable growth rate formula that is manifestations of lupus (5) and of Koffler, Schur, and threatening physician reimbursement. We have argued Kunkel documenting deposition of antibodies in lupus for the value of bone density testing. We have success- kidneys (3), their studies of serum factors were interpreted in the context of the disease manifestations they Presented at the 70th Annual Scientific Meeting of the saw in their patients. If you go back to the 1960s, it is American College of Rheumatology, November 11, 2006. difficult to find a volume of the Journal of Experimental Mary K. Crow, MD: Hospital for Special Surgery, New York, New York; President, American College of Rheumatology, 2005–2006. Medicine that does not include a paper by Henry or Address correspondence and reprint requests to Mary K. Chuck that advanced our concepts of the pathogenesis Crow, MD, Hospital for Special Surgery, 535 East 70th Street, New of rheumatic disease. While their contributions to sci- York, NY 10021. E-mail: [email protected]. Submitted for publication January 2, 2007; accepted January ence and rheumatology are huge, what stays with me is 3, 2007. a sense of their total commitment and engagement in

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impressive contributions to rheumatology through their work in the ACR. It is always dangerous to select out some individuals to specifically mention, as clearly many are involved in the work of the College, in fact, more than 300 physician and health professional volunteers. But I am talking about special individuals, and Walter Barr, who is completing a 3-year term as Chair of ACR’s Committee on Rheumatology Training and Workforce Issues, and Sherine Gabriel, Chair of ACR’s Committee on Quality Measures, have led initiatives that will have a lasting impact on our field. When Walter took on the chairmanship of his committee, he laid out a 3-year plan Figure 1. Personal mentors and giants of rheumatology. to assure that we would have a strong and highly competent workforce. Among his many accomplish- ments, he guided David Daikh and other volunteers to solving the mysteries of rheumatic diseases and mentor- develop a comprehensive ACR Core Curriculum Out- ing others to do the same. line for rheumatology fellowship training programs. This The discoveries made by Christian and Kunkel document will set the standard for fellowship training have been followed by significant advances by others and has been recognized as a model for other subspe- based on a similar approach, characterization of the cialties. mediators responsible for disease. Confirmation of tu- A second accomplishment this year was comple- mor necrosis factor (TNF) as a rational therapeutic tion of a survey commissioned by the ACR to under- target in RA has been followed by designation of stand the factors that will affect the future supply of and interferon-␣ (IFN␣), interleukin-6, and B lymphocyte demand for rheumatologists (Paul F. Hogan and Ellen stimulator, or the cells that make them, as candidate Bouchery, The Lewin Group, for the American College targets in lupus. of Rheumatology. Rheumatologist Survey Overview: While rheumatologists continue to unravel the Final Report, June 2006). A task force led by Chad Deal mechanisms of rheumatic diseases, we must recognize is preparing a manuscript that will describe the findings. that partnerships with basic scientists from other fields, As we might have predicted, the demand for rheuma- including those in industry, will lead us most expedi- tologists is expected to exceed supply over the next 20 tiously toward improved therapies. I have never met years, based on aging of the population and increasing Anthony Cerami, Jan Vilcek, or David Goeddel, but prevalence of musculoskeletal disorders. While the frus- these outstanding individuals must be recognized for a trations related to inappropriate reimbursement for level of commitment and focus that complements the services were reported in the survey, the overall level of achievements of rheumatology’s best scientists. Tony satisfaction of rheumatologists with their practice is Cerami studied the biologic properties of what he called high, as it should be based on the opportunities to have cachectin, and we now call TNF (6). Jan Vilcek not only an impact on our patients. The survey will guide the essentially wrote the book on interferon, but also helped ACR as it develops strategies to respond to our changing to develop the anti-TNF monoclonal antibody that be- health care environment and improve the efficiency of came infliximab (7,8). David Goeddel did the molecular medical practice. biology groundwork that resulted in the cloning of both Sherine Gabriel is another terrific leader and role TNF and IFN␣ (9,10). Future breakthroughs in rheu- model who has led the ACR in its efforts to respond to matology will increasingly depend on partnerships that the emerging quality trend in health care. The need to bring together dedicated individuals with distinct exper- align accountability with improvement in quality of care tise from both within and outside of rheumatology. and patient outcomes was the focus of a highly cited 2001 report by the Institute of Medicine (11). This and other studies have led to the expectation that our Guiding rheumatology to a leadership position in the medical system should promote the application of changing health care environment evidence-based medicine and information technology. I want to acknowledge some current role models Some insurance companies are already recognizing phy- who have worked tirelessly for all of us and have made sicians for their use of defined quality indicators, and we ACR PRESIDENTIAL ADDRESS 1039

Table 1. ACR initiatives in the changing health care environment Challenges to practice of rheumatology Accelerating cost of medical care and challenges to reimbursement Pressure to incorporate information technology into rheumatology practice Pressure to incorporate process management into rheumatology practice Goal of improving disease control by quantifying disease activity and managing care based on measurable data Advances in therapeutics requiring expert patient management ACR quality of care initiatives ACR Committee on Quality Measures—addresses need for common guidelines, standards of care, and tools to improve outcomes in rheumatic disease patients ACR-sponsored Rheumatology Quality Stakeholders’ Summit, August 2006—common goal of quality of care for rheumatic disease patients ACR Quality Leadership Council—coordinates quality initiatives across ACR committees and prioritizes communication of quality of care efforts to ACR members

predict that the need for increased documentation of Leading a new initiative to fund innovative research medical care will only increase in the coming years. I want to tell you about two more ACR leaders While none of us would argue with the goal of who had the vision to identify an essential issue, inves- improving patient outcomes, it is sometimes difficult to tigate the problem, recommend a response, and see their connect the dots between collection of data and out- work take shape as a major initiative. About 3 years ago, comes. Recognizing the significance of this new era of we had come through a period of unprecedented growth accountability for the practice of rheumatology, recent ACR leaders put in place a new committee, the Com- in the level of research funding from the National mittee on Quality Measures, to serve our members Institutes of Health, with the total NIH budget having through development of the measures and tools that doubled over a relatively short period. But Jane Salmon, rheumatologists will need to meet these challenges Chair of the ACR Committee on Research, and Mike (Table 1). It is this committee that Sherine has led. Holers, the prior committee chair, saw that in spite of In addition to developing disease criteria and this generous funding across a spectrum of research treatment guidelines that will be the gold standard for areas and the dramatic impact of the new biologic patient care, Sherine and her committee organized a therapies, we still did not understand RA with sufficient meeting this past August, the Rheumatology Quality specificity to achieve a state of remission. In the case of Stakeholders’ Summit, bringing together representatives SLE we had had no new therapies in 40 years, and in from organizations expert in developing quality indica- scleroderma the situation remained grim. tors for medical practice, as well as from government Jane appointed Mike to head a Research Fund- agencies, the insurance and pharmaceutical industries, ing Task Force that was charged with identifying the and others. These participants recognized the leadership most effective approach to achieve research funding that taken by the ACR to develop evidence-based measures would ultimately impact the development of new thera- that will help physicians improve the care of rheumatic pies for rheumatic diseases. The task force’s conclusion disease patients. If rheumatologists are asked to rede- was that disease-focused research funding and partner- sign the way they practice medicine—documenting their ship structures described the research funding model actions, measuring outcomes, and implementing new that had been most successful in other specialty areas. information technology—ACR is determined to help The bold recommendation from this group was that our members respond to this changing environment. ACR’s Research and Education Foundation (REF) Walter and Sherine are examples of rheumatolo- should build on its recent successes in supporting train- gists who somehow find time to make significant contri- ees and rheumatologists in the early stage of their butions to rheumatology while they carry on with their careers and extend its support to significant research day jobs—seeing patients, running training programs, projects through a disease-targeted research initiative. leading departments. These are the people who are Jim O’Dell, President of the ACR REF, has willing to commit whatever it takes to make our specialty described the results of this recommendation, the Within thrive. Our Reach campaign to support RA research, the first 1040 CROW

New activism in advocacy Three years ago ACR developed its most recent strategic plan and identified the quality of the professional life of our members as its highest priority. Again, it is all about people. We will need to fight every day to achieve that goal in view of the pressures imposed on us by the accelerating cost of health care in our country. But I can tell you that we have never been more active in advocating at the national level for policies that will permit rheumatologists to do what they love while being Figure 2. Increasing number of adult and pediatric rheumatology reimbursed for the work they do. And we will continue trainees, 2001–2006. (Adult data from Journal of the American Medical to develop the infrastructure to facilitate communication Association, September of each year. Pediatric data from American Academy of Pediatrics.) between rheumatologists and the ACR through the Regional Advisory Council of the Committee on Rheu- matologic Care. It is unlikely that we will ever arrive at a day when we have no battles to fight and no problems disease area that will be targeted by the REF (12). to manage. But I am confident that rheumatology will Funding of the first set of grants is anticipated next continue to thrive, developing the most novel therapies, summer. This program is even more essential to the treating the most fascinating and complex patients, and future of rheumatology now that our federal budget is enjoying the high level of satisfaction that the workforce holding NIH support at its current level, which in reality survey identified. translates into an absolute decrease in research funds for current grant holders and a significant decrease in Our greatest challenge: recruiting and retaining our funding of new grants. I want to emphasize that support academic leaders for research funding not only benefits researchers. It is the work of investigators both inside and outside the While I am an optimist, I do have a concern. I rheumatology community that leads to new insights into have described my personal role models and my heroes the diseases that we treat and to discoveries that im- among our current colleagues, but to assure that we have prove patient care and outcomes. Sponsorship and sup- a next generation of role models and heroes, we need a port of the RA Initiative by the REF and ACR represent talented pipeline of fellowship candidates, as well as an investment in the future of rheumatology and in the strong and supportive academic rheumatology programs future of our patients. to guide and mentor those trainees. In February of 1998,

Table 2. Challenges faced in academic rheumatology Facts of life for academic rheumatologists* Academic salaries are ϳ30% lower than private practice compensation Only 28% of academic rheumatologists are tenured, while another 16% are eligible for tenure Half of academic faculty members require at least 7 years to achieve independent status Challenges for rheumatology divisions Increasing demands to meet departmental financial goals Decreased NIH and uncertain Medicare funding means decreased division funds No hard money for basic and clinical researchers Limited salary lines for clinician educators and program directors Uneven distribution of resources among institutions Challenges for junior faculty Decreased NIH funding means decreased training grants and career development awards Limited departmental funds for junior faculty support Burdensome student loans Modest junior faculty salaries Limited job security * Data derived from Paul F. Hogan and Ellen Bouchery, The Lewin Group, for the American College of Rheumatology. Rheumatologist Survey Overview: Final Report, June 2006. ACR PRESIDENTIAL ADDRESS 1041

an ACR Blue Ribbon Committee on the Future of Table 3. Strategies to increase the strength of academic Academic Rheumatology, led by Dr. Eng Tan, issued a rheumatology report describing great concern regarding the survival of Encourage partnerships among NIH, foundations, industry, and academic rheumatology. Well, rheumatology is in a very private philanthropy to increase research funding Develop collaborations between academic rheumatology and different and stronger place now. Through the contribu- industry investigators—rather than industry recruiting the best of tions of supporters of the REF, including our industry academic rheumatologists partners, we have had impressive success in turning Promote cohesion among rheumatologists—clinicians, researchers, educators around our previous deficit in recruitment of qualified Focus on people Ͼ infrastructure applicants to fellowship programs (Figure 2). We have Communicate more than met the initial REF target for adult rheumatology fellows, and although the total number is still small, the number of pediatric rheumatology trainees has more than doubled since 2001. So we are doing very have been among the most outstanding triple-threat well in stimulating entry into fellowship programs. physicians who have not only led rheumatology divisions But the concern relates to our academic faculty. but have often gone on to lead departments of medicine Throughout the history of our specialty, rheumatologists or medical schools as deans. But our workforce survey raises some flags of concern, and the data from the survey resonate with my own sense of worry (Table 2). The survey confirmed what we know—that academic salaries are lower than those of rheumatologists in practice. While most of those who choose a career in academics readily accept that differential, more difficult to live with are the insecurities related to the challenges in achieving a tenured position and NIH-funded research grants. Many of us know of leaders and outstanding scientists who have left their rheumatology divisions, and recently a startling number have gone to work in the pharmaceutical or biotech industry. I cannot help but feel that on balance rheumatology has suffered a significant loss from these career changes. We should not blame industry. Why wouldn’t they make every effort to recruit the most talented and creative rheumatologists? And it is difficult to question the decisions of our colleagues when they are presented with offers that are hard to refuse. A career in industry can be a fine and valid choice. The problem is that to some degree industry’s gain is rheumatology’s loss. Yes, these individuals might contribute to development of new therapies. But we need them even more as role models and mentors for our trainees and to lead the educational and research programs at our academic centers. Why do our valued colleagues so readily slip away? To understand what is happening to our leaders we need to look at our academic centers, their priorities, Figure 3. Challenges in academic rheumatology. Top, Share of aca- their financial structure, and their sources of financial demic rheumatologists’ salary required to be covered by grant support. support. While some institutions are thriving, many are Bottom, Reasons given by those who have left academic rheumatology not, and uncertain Medicare funding and reduced NIH programs in the past 5 years. (Data derived from Paul F. Hogan and Ellen Bouchery, The Lewin Group, for the American College of support put pressure on division chiefs and on those Rheumatology. Rheumatologist Survey Overview: Final Report, June rheumatologists trying to succeed as clinician educators 2006.) or researchers. Our academic medical centers have 1042 CROW

essential responsibilities to the public and our health ACR staff. Together, they do the work that will grow and care system, and in most respects they meet those sustain rheumatology as the most rewarding medical responsibilities. They do an excellent job of caring for specialty. The runner-up as most important lesson of the patients and educating medical students and residents, year is that it is not possible to put too much effort into and they are the center of the medical research enter- communicating effectively with our members. A con- prise. stant refrain this year has been: “We need to communi- But I am concerned that an equally important cate this more effectively to our members.” I am thrilled responsibility, mentoring and recruiting the next gener- to alert you to a new ACR publication, The Rheumatol- ation of role models and leaders, may become difficult if ogist, that is edited by another extraordinary ACR rheumatology faculty cannot be retained. New junior volunteer and leader, David Pisetsky. We are very proud faculty, who want nothing more than to make a career as of the job that David has done to craft a compelling and educators or investigators, struggle to deal with financial informative publication that will bring you the latest burdens. Those are barely manageable, but together news in rheumatology patient care, research, and edu- with the insecurity inherent in our current academic cation, as well as updates on the policy and advocacy structure, it is difficult to maintain the commitment to an academic career. The workforce survey commissioned issues that have become increasingly important to us in by ACR showed that 10% of non-academic rheumatolo- our professional lives and in the ACR. I hope you enjoy gists had left academics within the past 5 years. Their The Rheumatologist and consider contributing your ideas reasons for leaving included a lack of support, difficulty for future articles. funding their research, and a desire for higher pay I will end with some special thanks to two more (Figure 3). It should be noted that nearly half of those personal mentors. Both show a characteristic that tends surveyed who are in academic medicine are required to to be a feature of rheumatologists, dedication, but in support 75% of their salary through grants. In an each case this dedication is taken to the limit. Steve environment in which achieving NIH funding is difficult, Paget is as dedicated to his patients as a physician can it is not surprising that an attractive job offer might possibly be, and he never fails to be supportive of me and prove irresistible. all of his colleagues. Peter Lipsky shows absolute devo- We need to develop strategies that will help our tion to mastering an understanding of the complex academic institutions improve recruitment and retention diseases that rheumatologists study and treat. No one of our rheumatology faculty (Table 3). While we can knows the research literature better or more thought- hope that NIH funding will improve in the next few fully applies it to advancing therapies for patients. Both years, in the meantime research programs can be sus- Steve and Peter have sometimes served as behind-the- tained if they are pursued through partnerships with scenes mentors, and I value their support. industry and foundations, in addition to the NIH. Can I want to thank my colleagues on the ACR we encourage industry to work with our talented scien- Executive Committee and the ACR staff. We all truly tists and clinicians collaboratively, rather than recruiting work as a team, a necessary approach in view of the them away from our academic programs? We need to diversity of issues that ACR manages. Mark Andrejeski strengthen communication among those within our med- has been the ultimate role model and mentor through ical centers to optimize research and education re- the past 10 years of my work with ACR. He has taught sources and facilitate the success of our faculty. A me the value of organizational structures that work; promising new NIH program that funds clinical and clearly ACR does work well, with much of the credit translational science awards aims to do just that. And going to Mark. Finally, I recognize David and Will, my most of all, the academic medical centers need to recognize the value of people. Many of our centers have sons, who have tolerated my work schedule and sup- put significant resources into building beautiful new ported my own devotion to rheumatology, science, and facilities. As nice as these are, if we do not dedicate medicine. sufficient resources for people, these buildings will not suffice to assure a strong future for rheumatology. REFERENCES A new and comprehensive ACR publication 1. Fudenberg HH, Kunkel HG. Specificity of the reaction between The most important lesson of my year as Presi- rheumatoid factors and gamma globulin. J Exp Med 1961;114: dent is the value of the volunteers and the outstanding 257–78. ACR PRESIDENTIAL ADDRESS 1043

2. Tan EM, Kunkel HG. Characteristics of a soluble nuclear antigen 8. Knight DM, Trinh H, Le J, Siegel S, Shealy D, McDonough M, precipitating with sera of patients with systemic lupus erythema- et al. Construction and initial characterization of a mouse- tosus. J Immunol 1966;96:464–71. human chimeric anti-TNF antibody. Mol Immunol 1993;30: 3. Koffler D, Schur PH, Kunkel HG. Immunological studies concern- 1443–53. ing the nephritis of systemic lupus erythematosus. J Exp Med 9. Goeddel DV, Yelverton E, Ullrich A, Heyneker HL, Miorzzari G, 1967;126:607–24. Holmes W, et al. Human leukocyte interferon produced by E. coli 4. Abruzzo JL, Christian CL. The induction of a rheumatoid factor- is biologically active. Nature 1980;287:411–6. like substance in rabbits. J Exp Med 1961;114:791–806. 10. Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, 5. Estes D, Christian CL. The natural history of systemic lupus Palladino MA, et al. Human tumour necrosis factor: precursor erythematosus by prospective analysis. Medicine (Baltimore) structure, expression and homology to lymphotoxin. Nature 1984; 1971;50:85–95. 312:724–9. 6. Beutler B, Greenwald D, Hulmes JD, Chang M, Pan YC, Mathi- 11. Committee on Quality of Health Care in America, Institute of son J, et al. Identity of tumour necrosis factor and the macro- Medicine. Crossing the quality chasm: a new health care system for phage-secreted factor cachectin. Nature 1985;316:552–4. the 21st century. Washington: The Institute; 2001. 7. Vilcek J. Fifty years of interferon research: aiming at a moving 12. O’Dell JR. A cure for RA—within our reach. From the College target. Immunity 2006;25:343–8. 2006;2:1–7. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1044–1047 DOI 10.1002/art.22514 © 2007, American College of Rheumatology

EDITORIAL

The Role of Varus and Valgus Alignment in Knee Osteoarthritis

Leena Sharma

In this issue of Arthritis & Rheumatism, Brouwer (versus the absence) of a varus/valgus deformity by and colleagues present the first report that knee mal- inspection on physical examination did not differ be- alignment increases the risk of development of knee tween patients in whom OA progressed and those in osteoarthritis (OA) (1). In the bigger picture, this is also whom OA did not progress (5). The results reported by the first report of a local mechanical factor increasing Brouwer et al are consistent not only with most of these the risk of incident OA at the knee. Brouwer et al natural history studies but also with numerous studies additionally confirm a previously reported relationship suggesting that malalignment worsens the outcome of between malalignment and progression of knee OA. surgery (e.g., arthroplasty, osteotomy, meniscectomy, These are important findings. OA is the most and meniscal debridement) in OA knees. common arthritis in humans, and knee OA is a major The relationship between malalignment and pro- cause of chronic disability. There are no treatments that gression of knee OA was perhaps not as strong in the change the natural course of OA, and knee OA that current study as in previous natural history studies in advances to end-stage disease is the leading indication which the alignment angle was measured. As noted by for total joint replacement surgery. Without disease- Brouwer et al, this difference may relate to method- altering treatment, prevention strategies become para- ologic differences between studies or to a lower mean mount, and identification of risk factors supports the body mass index (BMI) in this sample than in the development of prevention strategies. In a nutshell, not previously described cohorts. The mean BMI of the full only do the findings of the Brouwer study advance sample was 26 kg/m2; the BMI of the subgroup in whom understanding of the role of a pivotal factor in the OA progressed (the group analogous to those in previ- evolution of knee OA, they also support further devel- ous studies) may have been higher than that of the opment of novel strategies to improve tibiofemoral load sample as a whole but was likely to have been lower than distribution in malaligned knees. that in US progression cohorts. In their present study in knees with OA at Another reason may relate to the OA disease baseline, Brouwer et al confirm a relationship between stage at study baseline. In the population-derived sub- malalignment at baseline and progression, i.e., worsen- cohort of patients with diseased knees in the study by ing, of established OA. This is consistent with the results Brouwer et al, knees with mild disease (Kellgren/ of 2 previous studies in which alignment was measured Lawrence [K/L] grade 2) (6) predominated; the small on full-limb radiographs (2,3). Earlier studies had also proportion of knees with K/L grade 3 precluded mean- examined the malalignment effect on progression of ingful analysis, and those knees were excluded. Previous OA, albeit without measuring the alignment angle. Schouten et al (4) reported that, in patients with knee studies of OA progression, which recruited participants OA at study baseline, a recollection of “bow-legs or from both community and clinical sources, included in knock-knees in childhood” was associated with an in- the analyses a larger proportion of moderately diseased creased risk of OA progression during the followup knees (2,3). As reported by Cerejo et al (7), malalign- period. Other investigators observed that the presence ment has a more deleterious effect in more damaged (and therefore more vulnerable) knees, although at some point a ceiling effect emerges. Leena Sharma, MD: Feinberg School of Medicine, North- western University, Chicago, Illinois. Other possible reasons for the relatively weaker Address correspondence and reprint requests to Leena risk of progression observed in the current study include Sharma, MD, Arthritis Division, Northwestern University, Ward the use of conventional, extended-knee radiography and Building, 3-315, 303 East Chicago Avenue, Chicago, IL 60611. Submitted for publication December 4, 2006; accepted in a global rather than a compartment-specific approach to revised form January 16, 2007. assessing OA progression. As noted by Brouwer and

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colleagues, they detected a relationship between knee significant (1). Similarly, in the cohort of patients in malalignment and OA progression in spite of these whom OA progression occurred, the results (not strati- approaches; thus, their results may underestimate the fied by BMI) were significant for varus alignment only, true relationship. not for valgus alignment. From the perspective of bio- The study by Brouwer and colleagues is the first mechanics, the difference between the impact of varus to examine the link between knee malalignment and alignment and that of valgus alignment is not surprising. incident knee OA. It is also the first study in which the As noted above, during gait in the neutrally aligned effect of malalignment on incident OA and the effect of knee, load is disproportionately transmitted to the me- malalignment on progressive OA were examined simul- dial tibiofemoral compartment (10,11). Varus malalign- taneously. If the effect on incidence differed from the ment further increases the total load passing medially effect on progression when these outcomes were exam- during gait (13). Although valgus malalignment is asso- ined in separate studies, it would remain possible that ciated with an increase in lateral compartment peak the difference was linked to methodologic differences pressures (14), the medial compartment often continues between studies (8). According to the results reported by to bear more load than the lateral compartment until Cerejo et al (7), it is not surprising that the effect of more severe valgus malalignment is present (15,16). malalignment on incident OA is weaker than that on OA Varus/valgus alignment is important for reasons progression, given the healthier condition of the joint in in addition to these direct effects at the knee. First, the former condition compared with the latter condition. malalignment very likely puts stress not only on articular What is the mechanism of the malalignment hyaline cartilage but also on other joint tissue, e.g., effect? In theory, any shift from neutral alignment of the menisci, subchondral bone, and ligaments, which may hip, knee, and ankle affects load distribution at the knee contribute to the development and progression of OA. (9). A line drawn from the middle of the femoral head to Second, malalignment may participate in a vicious circle, the middle of the ankle represents the load-bearing axis. with knee OA worsening (e.g., from malalignment to In a knee with varus alignment, this line passes medial to worsening of OA to worse malalignment). The issue of the knee, and a moment arm is created that increases cause and effect plagues the question of what role is forces across the medial tibiofemoral compartment (9). played by local mechanical factors in general, and mal- In a knee with valgus alignment, the load-bearing axis alignment in particular, in the development and progres- line passes lateral to the knee, and the moment arm sion of OA. Malalignment predating knee OA may be created increases forces across the lateral tibiofemoral attributable to genetic, developmental, or traumatic compartment. Due to a stance phase knee adduction factors. Prior to the study by Brouwer et al (1), the major moment, even during normal gait in healthy knees, more evidence suggesting that preexisting malalignment may load passes through the medial tibiofemoral compart- contribute to the initial development of OA came from ment than through the lateral compartment (10,11). animal models and studies of complicated traumatic Varus alignment is a key determinant of the magnitude fracture in humans (9); there was little to suggest what of this adduction moment. The magnitude of the peak its role might be in primary idiopathic knee OA. The adduction moment predicted knee OA progression (12); results reported by Brouwer et al imply that, prior to the an increase in the adduction moment may lie in the onset of radiographic OA (using accepted and widely causal pathway between varus alignment and progres- used definitions of what constitutes OA), malalignment sion of knee OA. increases the risk of development of OA. Of note, the The concept of dynamic varus/valgus alignment prevalence of malalignment in knees without OA at during gait is not identical to the knee adduction mo- baseline was not low; using a definition of neutral ment during gait, although it is likely that they would be alignment as being 182° to 184°, 25% of 2,290 knees had highly correlated. Varus/valgus alignment under dy- varus alignment, and 36% had valgus alignment. namic conditions might have an even stronger relation- Malalignment resulting from knee OA may be ship (compared with alignment measured on a static attributable to loss of cartilage and bone height. The full-limb radiograph) with the development and progres- progression of OA observed in this and other studies sion of knee OA. implies that whatever the original cause(s) of the mal- In their separate examinations of varus and val- alignment, malalignment assessed at baseline increases gus malalignment, Brouwer et al observed a borderline the risk of knee OA progression in the period fol- effect of valgus malalignment on the risk of incident OA, lowing the baseline evaluation. In other words, in a while varus malalignment had a larger effect, which was specific knee, malalignment may postdate the onset of 1046 SHARMA

OA and still contribute to progression. From a bio- good adjudication process. Dichotomous outcome defi- mechanical perspective, the most likely possibility is that nitions were wisely applied, given the need to rely on the relationship between malalignment and knee OA conventional radiography. Accepted definitions for what development/progression is bidirectional. constitutes knee OA incidence and progression were Third, alignment may modify the effect of other used. Data for both knees were used, applying statistical risk factors. There is biomechanical and some epidemi- methods that account for the correlation between knees ologic evidence (17–20) of this for at least 2 factors: in an individual person. quadriceps strength and BMI. Local factors that alter The study has some limitations, particularly the load distribution (malalignment is a prototypic example) possibility that patients who returned for the followup influence how well the joint copes with muscle forces. evaluation were healthier than the original population as Woo et al (21) compared this to a hammer (the muscle) a whole. Perhaps related to this, the rates of incident driving a nail (the joint), while a hand (the local envi- knee OA and OA progression were relatively low for a ronment) holds the nail in place. The stabilizing hand study of this duration. The authors discuss this issue allows larger forces from the hammer. In other words, a clearly and explicitly and acknowledge the possibility healthy environment contributes to safe muscle force that patients with greater pain and/or disability may have distribution over the menisci, articular cartilage, and been more likely to miss followup visits. This subgroup other tissues. Alternatively, with malalignment, muscle may have had more advanced and perhaps even end- forces may increase stress on localized areas of these stage knee OA. The loss of patients with more advanced tissues. Similarly, Marks et al (22) theorized that local disease at baseline would not have affected the pre- joint abnormalities could render muscle forces patho- sented results, because analyses of incident OA dealt genic. These concepts have contributed to an increasing with knees with K/L grade 0 or 1 at baseline, and awareness that a generic strengthening program will not analyses of progression dealt with knees with K/L grade serve all knee subsets, and to an emerging emphasis on 2 at baseline. muscle training rather than brute strengthening. The authors note that their results may have been How alignment and BMI or body weight interact stronger if full-limb radiographs had been acquired and in any effect on OA progression is a subject of much if hip and ankle landmarks had been incorporated into interest. As illustrated by Brouwer and associates, there the measurement of alignment, instead of measurement is more than one way to think about this. Previous of the femorotibial angle from the knee radiograph. reports suggest that alignment modifies the effect of Actually, other studies suggest that the correlation be- BMI on OA knees (19,20). Brouwer et al examined the tween the 2 approaches is high (with correlation coeffi- alignment effect on the risk of incident knee OA in cients ranging from 0.75 to 0.98), measuring the femo- patients stratified according to BMI, as follows: healthy rotibial angle from knee radiographs acquired using a weight, overweight, and obese. Notably, in their report, variety of approaches (23–26). The calculated offset/ the valgus malalignment effect became significant only correction factors for men and women were similar in in the obese stratum. The varus effect was detectable in the 2 studies reporting this (24,25). After correction, the the overweight group and was perhaps even stronger in sensitivity, specificity, and area under the receiver oper- the obese group, although the size of some of these ating curve were high for femorotibial angle identifica- groups was fairly small. The smaller sample size of the tion of both a varus and valgus hip–knee–ankle angle cohort of patients in which OA progressed precluded (26). Finally, the severity of varus alignment worsened stratified examination of the effect of alignment on comparably with each alignment measure as the medial progression of knee OA. lesion (cartilage, bone, and meniscus) score on magnetic The study by Brouwer et al has several strengths. resonance imaging worsened, and, laterally, as the lesion Because the sample was population-based, the goal of score worsened, comparably worse valgus malalignment looking at the effect of alignment on incident and was seen with either approach to the assessment of progressive knee OA was accomplished within the same alignment (26). These results suggest that the offset- source population and by applying the same methods to corrected femorotibial angle assessment from the knee the subcohorts at risk for disease development and radiograph is an acceptable alternative to full-limb radi- disease progression. The definitions of neutral, varus, ography and should be considered in research and and valgus alignment were appropriate, and offset cor- clinical settings. A sex-specific offset correction would be rection of the measured femorotibial angle was per- ideal. However, it seems unlikely that this would have formed. The study relied on 2 radiograph readers and a altered the results of the current study. EDITORIAL 1047

In summary, the report by Brouwer et al is 10. Andriacchi TP. Dynamics of knee malalignment. Orthop Clin important, as the first report of a relationship between North Am 1994;25:395–403. 11. Morrison JB. The mechanics of the knee joint in relation to malalignment and incident idiopathic knee OA, as the normal walking. J Biomech 1970;3:51–61. first report of a relationship between a local mechanical 12. Miyazaki T, Wada M, Kawahara H, Sato M, Baba H, Shimada S. factor and incident idiopathic knee OA, and as a con- Dynamic load at baseline can predict radiographic disease progression in medial compartment knee osteoarthritis. Ann Rheum firmation that malalignment influences knee OA pro- Dis 2002;61:617–22. gression, especially in patients who are overweight. 13. Hsu RW, Himeno S, Coventry MB, Chao EY. Normal axial There will be opportunities to confirm these results in alignment of the lower extremity and load-bearing distribution at the knee. Clin Orthop 1990;255:215–27. the ongoing Multicenter Osteoarthritis Study and the 14. Bruns J, Volkmer M, Luessenhop S. Pressure distribution at the Osteoarthritis Initiative. Future studies should explore knee joint: influence of varus and valgus deviation without and what portion of primary knee OA is attributable to with ligament dissection. Arch Orthop Trauma Surg 1993;133: 12–9. malalignment and what portion is attributable to the 15. Johnson F, Leitl S, Waugh W. The distribution of load across the combination of malalignment and increased BMI. Strat- knee: a comparison of static and dynamic measurements. J Bone egies to improve load distribution in malaligned knees, Joint Surg Br 1980;62:346–9. 16. Harrington IJ. Static and dynamic loading patterns in knee joints some of which are fairly simple and inexpensive, should with deformities. J Bone Joint Surg Am 1983;65:247–59. be further developed. 17. Sharma L, Dunlop DD, Cahue S, Song J, Hayes KW. Quadriceps strength and osteoarthritis progression in malaligned and lax knees. Ann Intern Med 2003;138:613–9. REFERENCES 18. Sharma L, Dunlop DD, Hayes K. Is a strong quadriceps muscle bad for a patient with knee osteoarthritis? [letter]. Ann Intern 1. Brouwer GM, van Tol AW, Bergink AP, Belo JN, Bernsen RM, Med 2004;140:150. Reijman M, et al. Association between valgus and varus alignment 19. Sharma L, Lou C, Cahue S, Dunlop DD. The mechanism of the and the development and progression of radiographic osteoarthri- effect of obesity in knee osteoarthritis: the mediating role of tis of the knee. Arthritis Rheum 2007;1204–11. malalignment. Arthritis Rheum 2000;43:568–75. 2. Sharma L, Song J, Felson DT, Cahue S, Shamiyeh E, Dunlop DD. 20. Felson DT, Goggins J, Niu J, Zhang Y, Hunter DJ. The effect of The role of knee alignment in disease progression and functional body weight on progression of knee osteoarthritis is dependent on decline in knee osteoarthritis. JAMA 2001;286:188–95. alignment. Arthritis Rheum 2004;50:3904–9. 3. Felson DT, McLaughlin S, Goggins J, LaValley MP, Gale ME, 21. Woo SL, Fenwick JA, Kanamori A, Gil JE, Chan Saw SS, Vogrin Totterman S, et al. Bone marrow edema and its relation to TM. Biomechanical consideration of joint function. In: Moskowitz progression of knee osteoarthritis. Ann Intern Med 2003;139(5 Pt RW, Howell DS, Altman RD, Buckwalter JA, Goldberg VM, 1):330–6. editors. Osteoarthritis: diagnosis and medical/surgical manage- 4. Schouten JS, van den Ouweland FA, Valkenburg HA. A 12 year ment. Philadelphia: WB Saunders; 2001. p. 145–69. follow up study in the general population on prognostic factors of 22. Marks R, Percy JS, Semple J, Kumar S. Quadriceps femoris cartilage loss in osteoarthritis of the knee. Ann Rheum Dis activation changes in genu varum: a possible biomechanical factor 1992;51:932–7. in the pathogenesis of osteoarthrosis. J Theor Biol 1994;170:283–9. 5. Dougados M, Gueguen A, Nguyen M, Thiesce A, Listrat V, Jacob 23. Jokio PJ, Lindholm TS, Vankka E. Comparison between radio- L, et al. Longitudinal radiologic evaluation of osteoarthritis of the logic lower extremity angles of femur and tibia and articular knee. J Rheumatol 1992;19:378–84. surface pressure measurements in gonarthrosis treated by high 6. Kellgren JH, Lawrence JS. Radiological assessment of osteo- tibial osteotomy. Acta Orthop Belg 1984;50:802–14. arthrosis. Ann Rheum Dis 1957;16:494–502. 24. Kraus VB, Vail TP, Worrell T, McDaniel G. A comparative 7. Cerejo R, Dunlop DD, Cahue S, Channin D, Song J, Sharma L. assessment of alignment angle of the knee by radiographic and The influence of alignment on risk of knee osteoarthritis progres- physical examination methods. Arthritis Rheum 2005;52:1730–5. sion according to baseline stage of disease. Arthritis Rheum 25. Hinman RS, May RL, Crossley KM. Is there an alternative to the 2002;46:2632–6. full-leg radiograph for determining knee joint alignment in osteo- 8. Felson DT, Nevitt MC. Epidemiologic studies for osteoarthritis: arthritis? Arthritis Rheum 2006;55:306–13. new versus conventional study design approaches [review]. Rheum 26. Issa SN, Dunlop D, Chang A, Song J, Prasad PV, Guermazi A, et Dis Clin North Am 2004;30:783–97. al. Full-limb and knee radiography assessments of varus-valgus 9. Tetsworth K, Paley D. Malalignment and degenerative arthro- alignment and their relationship to osteoarthritis disease features pathy. Orthop Clin North Am 1994;25:367–77. by magnetic resonance imaging. Arthritis Rheum. In press. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1048–1050 DOI 10.1002/art.22631 © 2007, American College of Rheumatology

EDITORIAL

Estrogens and Lupus: Bubbling Cauldron or Another Overrated Witches’ Brew?

Michael D. Lockshin1 and Jill P. Buyon2

The 9:1 female:male ratio in systemic lupus ery- Non–estrogen-related aspects of sex that may thematosus (SLE) (in addition to diseases such as Sjo¨- inform an understanding of high female:male ratios are gren’s syndrome, Hashimoto thyroiditis, Graves’ disease, just beginning to be studied, including genomic hetero- and primary biliary cirrhosis) is a startling, unexplained geneity within women because of the presence of 2 clinical fact that, once understood, will likely lead to X-chromosomes or between men and women because of profound new insights into both the biology of these the Y-chromosome, imprinting, X-inactivation, epige- illnesses and the biology of sex and may also lead to new netic modification, microchimerism and other aspects of interventions. High sex ratios attract the attention of pregnancy, chronobiologic effects of menstrual cycling, epidemiologists for easily understandable reasons, epi- sex differences in metabolism and pharmacokinetics, demiology being the study of population discrepancies in possible microbiologic exposures due to anatomic differ- disease. However, placing excessive blame for the initi- ences, and sociobiologic differences (3,4). Notwithstand- ation or exacerbation of SLE on the use of exogenous ing the widely studied, but largely in vitro, effects of female hormones or on the timing of physiologic hor- estrogen on immune response, sex differences encom- monal events is a concern. Even less understandable, pass more than the differential effects of estrogen and except perhaps if one is devoted to the notion of an testosterone. These topics have been reviewed else- excessive influence of estrogen, is the insistence that the where (1,5). secondary effects of estrogen explain the sex ratio In this issue of Arthritis & Rheumatism, Cos- differences. tenbader and colleagues, taking the epidemiologists’ Many other kinds of diseases, for instance, can- approach, focus on surrogates of estrogen burden (his- cers of the lung or head and neck, have strikingly torical data on age at first menses, use of oral contra- unequal sex ratios, but these are unequivocally attribut- ceptives and estrogen replacement, number of pregnan- able to occupational or personal habit exposures and not cies, duration of breastfeeding, and age at menopause) to hormone or other physiologic measures. Sex discrep- to argue that estrogen is important in the pathophysio- ancies in infections (tuberculosis, venereal diseases) and logic development of SLE (6). The strength of their inflammatory diseases (pneumoconiosis, atherosclero- study is that it uses prospective data from a huge database sis) have also been established, more so by epidemio- of illness that was self-reported by well-informed par- logic than by clinical or basic science research. Even ticipants, comprising professional nurses from the some autoimmune diseases have sex ratios clearly attrib- Nurses’ Health Study. The Nurses’ Health Study is a utable to exposure to exogenous agents rather than to brilliantly conceived enterprise that, like the Framing- hormones per se; these diseases include drug-induced lupus, toxic oil syndrome, polyvinyl chloride–associated ham Study, has and will continue to alter the paradigms scleroderma, eosinophilia-myalgia syndrome, and possi- by which physicians function. The weaknesses of the bly even primary biliary cirrhosis (1,2). study by Costenbader et al are that the illnesses were based on self-diagnosis (although supported by detailed 1Michael D. Lockshin, MD: Hospital for Special Surgery, questionnaire and chart review) in the absence of veri- Barbara Volcker Center for Women and Rheumatic Diseases, and fication studies of control subjects who did not self- Weill Medical College of Cornell University Medical Center, New diagnose SLE. York, New York; 2Jill P. Buyon, MD: Hospital for Joint Diseases, New York University Medical Center, New York, New York. This issue regarding what are the false-positive Address correspondence and reprint requests to Michael D. and false-negative rates of SLE diagnosis in this cohort Lockshin, MD, Hospital for Special Surgery, 535 East 70th Street, New is highlighted, as acknowledged by Costenbader et al, by York, NY 10021. E-mail: [email protected]. Submitted for publication December 8, 2006; accepted in the fact that of the 3,563 of 14,607 women who screened revised form January 4, 2007. positive for SLE by initial questionnaire and who were

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then diagnosed from the medical records as having 3 of not be clinically meaningful. The mean age at meno- the American College of Rheumatology criteria for SLE pause in women who developed SLE was 51.6 years and meeting the diagnosis according to “reviewer con- compared with 52.7 years for women who did not sensus” (a curious choice for epidemiologists, to eschew develop SLE, a difference that is statistically significant, the well-accepted 4 criteria requirement for diagnosis), although one can question the biologic relevance of a only 262 women (7% of those who screened positive by 1.1-year difference, especially since menopause cannot questionnaire and only 1.8% of all women who initially be precisely timed in most women. Furthermore, if the stated that they had been diagnosed as having SLE) age at menopause did in fact show the “burden hypothe- were actually accepted as having incident disease. In sis of estrogen,” then why would menopause not have other words, the false-positive rate of the questionnaire occurred later in the women destined to develop SLE? was 93%, and among all of the women who stated they On closer inspection, indeed, several of the epidemio- had SLE, the false-positive rate was 98%. These num- logic findings seem to cloud rather than clarify the bers raise serious questions about the use of the term matter. The authors found that the risk of developing SLE and about how it is diagnosed in the community. SLE was the highest in women with a short duration of Furthermore, the authors offer no indication of screen- oral contraceptive use, and that the effects of oral ing the false-negative population, that is, women who contraceptive use were long lasting. Women who under- said they did not have SLE but whose charts, if reviewed, went natural menopause before age 47 years had twice might have revealed some findings that would have fit the risk of incident SLE. Is timing the critical link? Or the criteria used for diagnosis, especially those who were does this information weaken the link? diagnosed as having other connective tissue disease. With regard to estrogen burden, the authors cite Another important concern is that, as Costen- references for the self-fulfilling (because only women bader et al also acknowledge, the target group of the experience pregnancy) prophecy that pregnancy, the study, almost exclusively white women well into their physiologic state in which estrogens reach levels higher, professional years, excluded those patients most likely to in order of magnitude, than in any other state, exacer- develop SLE (inner-city black women ranging in age bates both the signs and the symptoms of SLE. Although from teenage up to age 30 years). Moreover, other one could argue that a simultaneous rise in glucocorti- groups who were excluded were women already diag- coids might counterbalance the evils of estrogen, most nosed as having SLE before entry into the study, and investigative groups report little to no difference in because of the sociology of the profession and the era disease activity in pregnant versus nonpregnant patients, during which the study was initiated, most black and no recent study has presented a strong argument to women—those groups of highest interest. Indeed, support the notion that pregnancy worsens SLE. 121,700 women were at least age 30 years when enrolled, The effect of exogenous estrogen on existing and all of the 238,308 women were at least age 25 years. lupus has been considered a litmus test of the influence This issue is important not only because of the higher of sex hormones on disease activity. Flying in the face of rates of lupus flare in black women under age 25 years, strongly held ide´es rec¸ues, large studies from the US and but also because young black women experience puberty Mexico have shown that oral contraceptives do not earlier and have very different estrogen exposures than increase the risk of either mild/moderate or severe SLE do older professional white women. activity (7–9). The American studies included the full These concerns aside, the study by Costenbader ethnic spectrum of women, and the results are likely to and colleagues is the best available study of this type that be more generalizable than those from the Nurses’ Health can be done. It shows a doubled risk of developing SLE Study. Although patients in these intervention studies did in middle-age professional white women who possess have inactive disease or stable disease activity at the time of certain surrogates for estrogen exposure, such as enrollment, there was no restriction with regard to past younger age at first menses (by recall), menstrual irreg- severity of disease. In the oral contraceptive study from the ularity (by recall; whether oral contraceptives were US, more than one-third of the patients had a history of prescribed because of this menstrual irregularity is not lupus nephritis (7). stated), hysterectomy-oophorectomy (the risk for this Costenbader et al conclude that estrogen has an group is, however, based on only 35 women), and important influence on the development of incident younger age at onset of menopause. SLE. We acknowledge the population effect but argue This study illustrates the truism that large de- that the very low relative risks, 1.7–2.3, in subsets of the nominators yield statistically important results that may nurse participants in the study are insufficient to explain 1050 LOCKSHIN AND BUYON

a 9:1 sex ratio. In fact, since a doubling of risk is a 3. Invernizzi P, Miozzo M, Selmi C, Persani L, Battezzati PM, Zuin relatively small increase, the data may indicate that M, et al. X chromosome monosomy: a common mechanism for autoimmune diseases. J Immunol 2005;175:575–8. estrogen is not a critical determinant of the sex ratio in 4. Ballestar E, Esteller M, Richardson BC. The epigenetic face of SLE. Rather, epidemiologic studies of exposure to ex- systemic lupus erythematosus. J Immunol 2006;176:7143–7. ternal agents and biologic studies of non–hormone- 5. Lockshin MD, on behalf of the Mary Kirkland Center Research related aspects of sex are more likely to account for the Consortium. Biology of the sex and age distribution of systemic lupus erythematosus. Arthritis Rheum. In press. sex ratio. High sex ratios still remain an extraordinarily 6. Costenbader KH, Feskanich D, Stampfer MJ, Karlson EW. Re- interesting, unexplained clinical fact that is likely to lead productive and menopausal factors and risk of systemic lupus to a new understanding of the biology—or sociology—of erythematosus in women. Arthritis Rheum 2007;56:1251–62. 7. Petri M, Kim MY, Kalunian KC, Grossman J, Hahn BH, autoimmune disease. Sammaritano LR, et al, for the OC-SELENA Trial. Combined oral contraceptives in women with systemic lupus erythematosus. REFERENCES N Engl J Med 2005;353:2550–8. 8. Buyon JP, Petri MA, Kim MY, Kalunian KC, Grossman J, Hahn 1. Wiseman T, Pardue ML. Exploring the biological contributions to BH, et al. The effect of combined estrogen and progesterone human health: does sex matter? In: Institute of Medicine Report. hormone replacement therapy on disease activity in systemic lupus Washington: National Academy Press; 2001. erythematosus: a randomized trial. Ann Intern Med 2005;142(12 2. Gershwin ME. Environment and the induction of autoimmunity: Pt 1):953–62. the case of primary biliary cirrhosis [abstract]. Autoimmunity 9. Sanchez-Guerrero J, Uribe AG, Jimenez-Santana L, Mestanza- reviews: abstracts from the 5th International Congress on Auto- Peralta M, Lara-Reyes P, Seuc AH, et al. A trial of contraceptive immunity in Sorrento, Italy, November 29–December 3, 2006. methods in women with systemic lupus erythematosus. N Engl URL: www.elsevier.com/locate/autrev. J Med 2005;353:2539–49. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1051–1066 DOI 10.1002/art.22489 © 2007, American College of Rheumatology

REVIEW

Psoriatic Arthritis

Current Concepts on Pathogenesis-Oriented Therapeutic Options

Anthony M. Turkiewicz and Larry W. Moreland

Introduction conventional therapies and to anti–tumor necrosis factor (anti-TNF) agents that are currently approved for the Psoriatic arthritis (PsA) is a chronic inflamma- treatment of PsA. A number of novel agents beyond tory arthropathy of the peripheral joints, spine, and TNF blockade are under investigation for PsA, under- entheses, associated with psoriasis and characterized by diverse phenotypic subtypes and a variable clinical scoring the diverse mechanisms likely to be at play in course. Much progress has been made in identifying the these heterogeneous phenotypic subtypes of PsA. distinctive characteristics of this disease since Alibert first described the association between psoriasis and Classification and diagnostic strategies arthritis in 1818 in “Lepre squammeuse,” his discourse Defining PsA—a work in progress. In any discus- on skin diseases (1). Recent insights into the immunopathogenic mechanisms of PsA have revealed disease sion on PsA, acknowledging the difficulties that underlie characteristics in the synovium, vascular structures, en- the classification and diagnosis of this heterogeneous theses, and bone of PsA patients that are similar to, and disease is important. In clinical practice and in interven- distinct from, those of rheumatoid arthritis (RA) as well tion studies, as well as in designing and executing proper as other forms of spondylarthritis (SpA), including prospective epidemiologic investigations which are ankylosing spondylitis (AS), reactive arthritis, and en- sorely needed for this disease, there is a clear unmet teropathic arthritis. need for a valid, accepted, and easy-to-use set of classi- Such investigations, along with advances in fication criteria for PsA. biotechnology and the development of a number of It is accepted that PsA, as a member of the SpA targeted biologic response modifiers (BRMs) with dem- family, is characterized by infrequent seropositivity for onstrated effectiveness for both skin and joint manifes- rheumatoid factor (RF) and anti–cyclic citrullinated tations, have led to substantial progress in the treatment peptide (anti-CCP), as well as an association with HLA– of PsA and a renewed interest in the mechanistic B27 alleles, particularly in those patients with axial processes behind this complex disease. As with RA and involvement. Additional clinical features can include SpA, a subset of patients with PsA fail to respond to enthesitis, dactylitis, iritis, peripheral arthritis (both oligoarticular asymmetric and polyarticular symmetric), spondylitis, and a variable clinical course. It is this Anthony M. Turkiewicz, MD, Larry W. Moreland, MD: University of Alabama at Birmingham. heterogeneity in clinical presentation and course that Dr. Turkiewicz has received consulting and speaking fees (less makes PsA a particularly challenging disease to diagnose than $10,000) from Abbott and honoraria (less than $10,000 each) and classify relative to other forms of SpA and inflam- from Abbott, Amgen, and Wyeth. Dr. Moreland has received consulting and speaking fees and/or honoraria (less than $10,000 each) from matory arthropathies. Indeed, the variety of classifica- Abbott, Amgen, Centocor, Genentech, and Wyeth and from Bristol- tion criteria proposed over the past 30 years by numer- Myers Squibb (more than $10,000). ous investigators (2–10) highlights this point. To date, Address correspondence and reprint requests to Anthony M. Turkiewicz, MD, University of Alabama at Birmingham, Department the seminal work of Moll and Wright more than 30 years of Medicine, Division of Clinical Immunology and Rheumatology, ago, in which they described their experiences with a 1530 3rd Avenue South, FOT 848, Birmingham, AL 35294-3408. large case series of patients with PsA (4), still remains E-mail: [email protected]. Submitted for publication April 12, 2006; accepted in revised the key reference by which PsA is divided into its 5 form December 21, 2006. clinical subtypes (Table 1).

1051 1052 TURKIEWICZ AND MORELAND

Table 1. Moll and Wright clinical subtypes for psoriatic arthritis* important discussion on the need to identify accurate, Polyarticular, symmetric arthritis (RA-like) agreed-upon, and clinically applicable classification cri- Oligoarticular (Ͻ5 joints), asymmetric arthritis teria, since numerous therapeutic agents for PsA cur- Distal interphalangeal joint predominant Spondylitis predominant rently being studied (and many on the horizon) require Arthritis mutilans rigorous clinical trials which rely upon standardized and * To meet the Moll and Wright 1973 classification criteria for psoriatic validated enrollment criteria. arthritis (4), a patient with psoriasis and inflammatory arthritis who is seronegative for rheumatoid arthritis (RA) must present with 1 of the above 5 clinical subtypes. Criteria specificity is 98% and sensitivity is 91%. Pathophysiologic mechanisms Interplay of genes, environment, and immuno- CASPAR contributions. As a result of the inter- logic factors. Although the etiology of PsA remains national collaborative efforts of the CASPAR (ClASsi- unknown, recent research has elucidated the changes fication criteria for Psoriatic ARthritis) Study Group, a occurring in the skin, synovium, enthesium, and bone of new set of PsA classification criteria were recently patients with PsA, illuminating features of this disease published, based on a large prospective study from 30 both similar to, and distinct from, other forms of SpA rheumatology clinics in 13 countries (11). In the CAS- and inflammatory arthritides such as RA. Such basic, PAR study, patient-derived data collected on 588 con- translational, and clinical investigations, along with consecutive clinic patients with PsA and 536 control subjects tinued advances in biotechnology, have provided a num- with other inflammatory arthritides (including RA, AS, ber of attractive and logical targets for the treatment of and connective tissue disorders), who were matched for both individual and collective features of the disease. approximate disease duration, were used to develop the The interplay between genetic, environmental, and im- new classification criteria (Table 2). munologic factors has become more apparent and pro- Compared with the other previously published vides important clues that will enable the further inves- PsA criteria, the CASPAR schematic was found to have tigation into arenas of potential therapies. better specificity but less sensitivity than the criteria that Genetic factors. Studies have documented that performed best of the existing classification methods to more than 40% of patients with PsA have a first-degree date (6,11). Of note, the CASPAR criteria were devel- family member with either psoriasis or PsA (5,12). oped using data from patients with established PsA Moreover, an increased frequency of psoriasis and PsA ϳ (mean disease duration 12.5 years), and therefore the has been observed in monozygotic and dizygotic twins utility of these criteria for patients presenting at an early (13). Additional investigations have proposed several stage of disease is unclear; however, this limitation of genetic susceptibility loci, with the strongest effect re- identifying and classifying patients with early rheumatic siding within the major histocompatibility complex disease is clearly not limited to PsA. The work by the (MHC) (14). PsA population studies have shown an CASPAR Study Group provides a timely and critically increased frequency of HLA–B13, B17, B27, B38, B39, DR4, DR7, and Cw6 (12,15). Genes at the HLA region Table 2. CASPAR criteria for psoriatic arthritis* may be relevant to disease expression in PsA, as suggested by studies showing that HLA–B27 has been Inflammatory articular disease (joint, spine, or entheseal) plus the following: associated with radiographic sacroiliitis and axial in- Psoriasis: current (2), history of (1), family history of (1)† volvement (16), while HLA–B22 was shown to be pro- Nail dystrophy (1) ϳ Negative rheumatoid factor (1) tective of damage in a cohort of 300 patients with PsA Dactylitis: Current (1), history of (1)‡ who were followed up prospectively (17). Karason et al Radiographs (hand or foot) with juxtaarticular new bone (18) performed a genotype analysis of 100 patients with formation (1)§ PsA from 39 families, which revealed a paternal trans- * To meet the ClASsification criteria for Psoriatic ARthritis (CASPAR) mission associated with chromosome 16q. Linkage to 2006 classification criteria for psoriatic arthritis, a patient must have inflammatory articular disease and Ն3 points from the remaining this region has also been observed in other SpA subsets, categories; the assigned scores are in parentheses. Criteria specificity is including AS and Crohn’s disease (19–21). Furthermore, 98.7% and sensitivity is 91.4%. a recent study further characterized the association of † Patient-reported history in a first- or second-degree relative. ‡ As recorded by a rheumatologist. susceptibility genes in PsA and Crohn’s disease, an § Excludes osteophyte formation. association independent of both psoriasis and undiffer- THERAPEUTIC OPTIONS FOR PsA 1053

entiated inflammatory arthritis (22) and suggesting that circulating antibodies (e.g., RF and anti-CCP) (28). T common inflammatory pathways underlie both of these cell–derived cytokines, including interleukin-1␤ (IL-1␤), diseases. IL-2, IL-10, IFN␥, and TNF␣, dominate the psoriatic Other genes within the MHC region have been synovium and skin (31). explored. MHC class I chain–related A (MICA) A9 and There is an association between human immuno- Cw6 have been touted as the strongest genetic suscepti- deficiency virus (HIV) infection and severe PsA (32–35), bility factors in PsA (16). Previous results have also providing some suggestion of immunologic mechanisms ␣ suggested that TNF promoter polymorphisms or a at play. HIV preferentially depletes T helper (CD4ϩ) ␣ gene in linkage disequilibrium with TNF predispose cells; diseases dependent on these cells, such as RA and the patient to psoriasis and PsA (23). In patients treated systemic lupus erythematosus, typically improve in the ␥ ␥ with interferon- (IFN ), which is a potent inducer of presence of HIV. Thus, an interaction of class I HLA class II MHC expression, PsA can develop (24), a molecules and CD8ϩ T cells may be fundamental to the finding that is suggestive of the molecular involvement pathogenesis of PsA (36,37). In further support of this of class II HLA. In contrast to patients with RA, the notion, a predominance of CD8ϩ T lymphocytes has prevalence of the HLA–DRB1 shared epitope (SE) is been noted in both the synovial fluid and entheses of not increased in patients with PsA; however, when present in PsA, the SE is associated with more erosive patients with PsA (38). Interestingly, this CD4:CD8 ratio disease compared with that in patients lacking the observed in PsA synovial fluid is reversed in RA synovial SE (19). fluid. ␣ Environmental factors. Trauma and infection have Proinflammatory cytokines, including TNF , ac- been implicated as causative agents in PsA. Psoriatic tivate endothelial cells, leading to expression of a variety lesions arising at areas of trauma (Koebner phenome- of adhesion molecules, including vascular cell adhesion non) have been described in a significant percentage of molecule 1, intercellular adhesion molecule 1 (ICAM- patients with psoriasis (25), and Langevitz et al de- 1), and E-selectin, resulting in lymphocyte migration to scribed PsA following trauma to a joint in 24% of sites of inflammation (23,28,39). TNF␣ also plays a role patients in an observational cohort (26). Both bacterial in cartilage degradation via increased production of and viral pathogens have been suggested as etiologic matrix metalloproteinases (MMPs), which are thought agents in PsA. Evidence of a possible link between to mediate cartilage erosion (40) and up-regulation of gram-positive infection and PsA was shown in a study by angiogenic factors such as vascular endothelial growth Vasey and colleagues in which sera from PsA patients factor (VEGF) and transforming growth factor ␤ showed higher levels of antibody to streptococcal infec- (TGF␤), providing the distinctive tortuous vessels tion (27). Indirect observation of enhanced humoral and noted in psoriatic skin and synovium (23,28,41). cellular immunity to gram-positive bacteria typically In addition, TNF␣ mediates a number of biologic found in psoriatic plaques supports the role of bacterial processes that can result in joint damage, including antigens in the pathogenesis of psoriasis and PsA (14). inhibition of bone formation, inhibition of proteoglycan Immunologic factors. Although studies of the synthesis, and stimulation of bone resorption (14). In- genetic and environmental aspects of PsA have provided terestingly, in a study of 20 PsA patients treated with key insights into potential pathophysiologic mechanisms, infliximab monotherapy, serum levels of TNF␣ mea- they are not, at this time, a primary target for developing sured during the initial (Ͻ12 weeks) phases of infliximab therapies. In contrast, a number of investigations have elucidated the immunologically mediated processes at treatment did not correlate with the prompt resolution play in both the skin and joints of PsA patients, and of the cutaneous and synovial symptoms observed. How- these findings serve as a foundation ripe for potential ever, there were notable decreases in serum levels of therapeutic strategies. Both the skin and the joint in PsA IL-6, VEGF, E-selectin, and MMP-2 after the initial possess a prominent lymphocytic infiltrate, comprising infusions of infliximab (42). Such observations of tem- activated T cells localized to dermal papillae in skin and poral cytokine behavior in response to anti-TNF␣ ther- to the sublining stroma in the joint as well as the apy provide further insight into which specific mediators inflamed enthesis (28–30). An abundance of B lympho- may contribute more fully to specific disease character- cytes forming primitive germinal centers is also present, istics and, consequently, provide the foundation for the the significance of which is not clear, because neither development of biomarkers to allow a tailored individ- psoriasis nor PsA is associated with high levels of ualized therapeutic regimen. 1054 TURKIEWICZ AND MORELAND

Distinctions from RA, including clinical correlates. When examining the immunohistologic characteristics of involved structures (synovium, bone, skin, vascular beds) in PsA compared with those observed in RA, a number of important distinctions can be made. PsA synovial lining cells exhibit less hyperplasia with fewer monocyte/macrophages than those in RA, whereas PsA synovium is more vascular than RA synovium and has a characteristic tortuosity of the vascular beds (28). Sim- ilary, and likely related, VEGF, along with TGF␤,is more highly expressed in PsA synovium as compared with RA synovium (43). Although the levels of TNF␣ are not significantly different between PsA (joint and skin lesions) and RA, IL-1␤ levels are significantly higher in PsA (31). Compared with healthy controls, the serum levels of osteoclast precursors (OCPs) are higher in PsA patients, particularly those with radiographically evident erosive changes, and the numbers of OCPs are reduced following anti-TNF therapy (44). A number of clinical characteristics differentiat- ing PsA from RA have also been observed. Although erosive disease can be observed in both diseases, new bone formation and the enthesopathy commonly noted in PsA are rarely seen in RA. Asymmetry and a tendency Figure 1. Five general pathogenesis-oriented therapeutic principles toward oligoarticular involvement are seen in PsA com- targeting the key pathogenic mechanisms of psoriasis and psoriatic pared with RA. Unlike RA, PsA is not associated with arthritis. The first involves inhibition of T cell activation through vasculitis or its clinical correlates. PsA affects men and inhibition of molecules involved in the formation of the immunologic women almost equally (13), compared with an ϳ3:1 synapse (A). The second principle is depletion of pathogenic T cells through targeting molecules expressed by activated T cells, such as female:male ratio observed in seropositive RA. As interleukin-2 (IL-2) receptor or CD4 (B). The third approach involves noted, seropositivity for RF and anti-CCP is not char- inhibition of leukocyte recruitment to the inflamed skin, such as acteristic of PsA, although recent investigations indi- inhibition of adhesion molecules (C). The fourth principle is inhibition cated that anti-CCP antibodies can be found in a small of key inflammatory cytokines, tumor necrosis factor ␣ (TNF␣) being proportion of PsA patients (5–8%) and, when present, the most prominent example (D). The final approach is inducing an immune deviation to shift the cytokine milieu dominated by Th1 cells are associated with polyarticular involvement and a to a milieu weighted with Th2 cells, as has been demonstrated with propensity for erosive arthritis (45–47). IL-10 and IL-4 (E). TCR ϭ T cell receptor; rIL-10 ϭ recombinant IL-10. Adapted, with permission, from Schon MP, Boehncke WH. Psoriasis. N Engl J Med 2005;352:1899–912. Overview of therapeutic options General considerations. With the advent of targeted BRMs and their successful application in a num- oriented therapeutic principles include the following: ber of chronic inflammatory disorders, a surge of inter- inhibition of T cell activation via a number of costimu- est in the pathophysiologic mechanisms of autoimmune latory blockades; depletion of pathogenic T cells diseases has developed. Elucidating the role of key through targeting molecules expressed by activated T cytokines, including TNF␣, IL-1, and IL-6, involved in cells; inhibition of leukocyte recruitment; and induction RA, the most common of the inflammatory rheumatic of an immune system deviation shift from Th1 to Th2. disorders, has led to the development of a number of These strategies have led to the development of a BRMs approved or in late-phase clinical trials for the number of targeted therapies that are currently being treatment of RA as well as PsA. Beyond inhibition of a investigated in both diseases (Figure 1). single proinflammatory cytokine, other therapeutic tar- Although RA and PsA share a number of patho- gets in PsA are logically based on an improved under- physiologic mechanisms (which may explain the shared standing of disease pathogenesis. Such mechanistically success of some of the BRMs in both disorders), the THERAPEUTIC OPTIONS FOR PsA 1055

unique clinical and pathologic characteristics of PsA, as ration, and desquamation along with the extent of previously discussed, necessitate individual investiga- lesions per site) (57). These measures have performed tions of each BRM in the treatment of PsA, as opposed reliably in clinical trials and are considered to possess to simple empiric application of RA therapies in PsA adequate discriminant capability when comparing active patients. Key investigations into the pathophysiologic treatment with placebo (58,59). Efforts are under way to mechanisms of PsA and the comparative analyses be- develop PsA-specific instruments, including assessment tween PsA and RA have shed light on the misconception tools for enthesitis, axial disease, and dactylitis, to more that PsA is the mild-to-moderate cousin of RA with a accurately evaluate the functional and disease outcomes rash. Recent studies have suggested that the clinical in PsA. severity of PsA may be greater than initially observed. An evidence-based approach to customized man- Peripheral joint involvement in PsA is often progressive, agement. The efforts headed by the Group for Research despite treatment with conventional disease-modifying and Assessment of Psoriasis and Psoriatic Arthritis antirheumatic drugs (DMARDs) (48,49), with ϳ20% of (GRAPPA) to provide a comprehensive and phenotype- PsA patients developing a severe destructive and de- specific (peripheral arthritis, spondylitis, skin and nail forming arthritis (50). The functional disability and disease, enthesopathy, dactylitis) approach to PsA man- reduced quality of life observed in PsA are comparable agement, based on the best available evidence, have with those documented in RA (51,52). recently been published (60–68). While a complete RA therapies such as methotrexate (MTX) may discussion of the findings of the GRAPPA is beyond the have not only differing efficacy in PsA, but also different scope of this review, their work sought to provide toxicity patterns (53–56). The challenge with treatment evidence-based recommendations obtained from the of PsA comes with the diversity of the disease pheno- published literature, with grading according to the types as well as the degrees of severity of individual strength of the evidence along with expert consensus disease manifestations in both the skin and joints. Cer- opinion for those areas in which scientific evidence was tain patients may have extensive skin involvement with lacking. A summary of their treatment recommenda- little joint involvement, whereas the converse may be the tions and ratings of the quality of evidence for these case; other patients with seemingly benign mono- or guidelines are outlined in Figure 2. There are a number oligoarticular joint involvement may require aggressive of guidelines that still rely on expert consensus because management to avoid long-term disability. Matching the of the lack of published information and/or accepted disease presentation, its severity, and its likelihood of outcome measures specific to PsA (particularly dactylitis progressive damage with the appropriate therapeutic and PsA axial disease involvement). However, these choices is essential. The cost-to-benefit ratio, as well as recommendations provide the groundwork for formaliz- the associated toxicities with each therapy, must be ing a customized management approach to PsA. In considered. addition, they provide a challenge for future PsA inter- In assessing the efficacy and outcomes of thera- vention trials to include specific and validated outcomes peutic interventions in PsA, it is important to note that for enthesitis, dactylitis, and axial disease in PsA, which, the majority of the measures currently used were devel- at the current time, are lacking. oped and validated in patients with either RA or pso- Conventional drugs. Nonsteroidal antiinflamma- riasis; these have, for the most part, not been validated tory drugs (NSAIDs), both selective and nonselective, in PsA. Such measures include the following: the Amer- are often used as first-line therapy for the arthritic ican College of Rheumatology (ACR) response criteria component of PsA, typically in patients with mild-to- (ACR20, ACR50, and ACR70), the Psoriatic Arthritis moderate symptoms and without evidence of progressive Response Criteria (PsARC) (a composite index that joint damage. NSAIDs have the ability to reduce inflam- calls for improvement of at least 2 of the following mation and improve pain and joint mobility, although elements: tender or swollen joint count by Ն30%, phy- gastrointestinal side effects have been a limiting factor in sician’s or patient’s assessment of global improvement some PsA patients (69). Oral and intralesional cortico- by at least 1 point on a 5-point Likert scale, one of which steroids have been associated with rebound worsening of must be joint assessment, and no worsening of any psoriasis upon withdrawal of the drug (70), and are element), and a composite psoriasis score, the Psoriasis therefore to be used with caution. Area and Severity Index (PASI) (a composite skin score DMARDs, including MTX, sulfasalazine, le- of 4 sites [head, upper extremities, trunk, and lower flunomide, and cyclosporine, are used in PsA patients extremities] that assesses the extent of erythema, indu- whose disease remains unresponsive to NSAID therapy 1056 TURKIEWICZ AND MORELAND

Figure 2. Group for Research and Assessment of Psoriasis and Psoriatic Arthritis treatment guidelines for psoriatic arthritis (PsA), including quality indices for each recommendation. Individual clinical manifestations in PsA warrant tailored guidelines for specific manifestations. Recommendation quality indices (levels, grades) are denoted in brackets next to each therapeutic agent, and correspond to the guidelines issued by the Agency for Health Care Policy Research as outlined. NSAID ϭ nonsteroidal antiinflammatory drug; IA ϭ intraarticular; DMARDs ϭ disease-modifying antirheumatic drugs; MTX ϭ methotrexate; CsA ϭ cyclosporin A; SSZ ϭ sulfasalazine; Lef ϭ leflunomide; anti-TNF ϭ anti–tumor necrosis factor; PUVA ϭ psoralen plus ultraviolet light A; UVB ϭ ultraviolet light B; PT ϭ physical therapy. Adapted, with permission, from ref. 60. or who exhibit progressive disease. MTX is often used as intake, with repeat biopsies performed as indicated by the primary DMARD in PsA because of its proven the results of liver enzyme tests (75). efficacy in treating both the skin and joint involvement Sulfasalazine has been proven effective for treat- in the disease (71,72); however, the toxic effects of ing peripheral arthritis in PsA but has no proven effect MTX, including an apparent yet unexplained increased on axial disease or skin lesions (76). Leflunomide was proclivity for hepatotoxicity in psoriasis patients versus demonstrated to have statistically significant efficacy for RA patients (73), have elicited concerns regarding its both skin and joint involvement, compared with placebo, long-term use. A discord still exists among the derma- in a recent study of 190 PsA patients (59% of tology and rheumatology communities regarding pub- leflunomide-treated patients versus 30% of placebo- lished guidelines for MTX monitoring (53); the Ameri- treated patients achieved a PsARC response) (77). can College of Dermatology recommends pretreatment Treatment with leflunomide was generally well toler- liver biopsy and repeat biopsies after a 1.5 gram accu- ated, with the most common side effects being diarrhea mulated dose has been achieved in psoriasis patients and increased transaminase levels (77). Cyclosporine, (74), whereas the ACR guidelines (developed for RA) while effective for both skin and joint manifestations, is recommend pretreatment liver biopsies only for patients less commonly used because of its toxic effects, most with persistently elevated liver enzymes, a history of notably hypertension and nephrotoxicity (78,79). chronic hepatitis B or C infection, or significant alcohol Other DMARDs, including hydroxychloroquine, THERAPEUTIC OPTIONS FOR PsA 1057

have been used in PsA, although use of hydroxychloro- data from a phase III trial (96). In the phase II trial, 47% quine has been reported to exacerbate skin disease of patients were on stable doses of MTX and had (80,81). In a case–control study by Gladman and col- longstanding disease (a mean 20-year history of psoriasis leagues in which 32 PsA patients were treated with and 15-year history of inflammatory arthritis). At the chloroquine, 75% of the chloroquine-treated patients end of the 3-month placebo-controlled phase, the etan- had a Ͼ30% reduction in active joint inflammation over ercept group showed significant improvement in all a 6-month period, compared with 58% of control sub- outcome measures compared with the placebo group, jects. Six of the control subjects experienced a flare of with the primary end point (the PsARC response) their skin disease, and 6 of the chloroquine-treated achieved by 87% of the patients receiving etanercept patients had a psoriasis flare but no exfoliative derma- versus 23% of the placebo group. Of note, there was no titis (82). Gold salts have also been reported to improve significant difference in the response (joints or skin) in arthritis in PsA patients, but their use is limited due to patients receiving background MTX versus those who toxicity. A discrepancy between formulations and their were not taking background MTX, a finding that has respective efficacies in PsA has been observed; for also been noted in the majority of PsA trials with other example, in a study of 82 patients with PsA, only anti-TNF agents. intramuscular gold compounds, and not the oral formu- In the large phase III multicenter trial that lations, resulted in a statistically significant improvement followed, significant improvements in skin lesions, qual- in outcomes compared with the placebo group (83). Of ity of life, and function were noted in the etanercept note, none of these conventional DMARDs are ap- group (Table 3). In addition, inhibition of disease pro- proved by the US Food and Drug Administration (FDA) gression, as measured radiographically, was demon- for the treatment of PsA. strated. In the 48-week open-label radiographic followup period, there was no worsening of the total Sharp score for radiographic joint damage in the active treatment Targeted strategies for manipulating the mechanisms group; moreover, the original placebo group, once of disease switched over to etanercept, showed no further disease Biologic response modifiers. Currently available progression. As before, background use of MTX (42% TNF␣ antagonists. As a central mediator in a number of of patients in this trial) did not affect the outcomes. inflammatory arthropathies, including RA and PsA, Interestingly, there appeared to be a lag in skin response TNF␣ emerged as an attractive target for biologic when compared with that in the joints; the full extent of therapies in the 1990s. The 3 currently available TNF␣ joint improvement occurred sooner than the full skin inhibitors, etanercept (a soluble TNF receptor [p75]- response, and improvement in the skin was still occur- IgG1 fusion protein), infliximab (a chimeric monoclonal ring well into the open-label aspect of the phase II trial antibody specific for soluble and membrane-bound (92,96,97). TNF␣), and adalimumab (a fully human anti-TNF Infliximab. Infliximab, subsequent to its successes monoclonal antibody), have since gained US FDA ap- in improving skin and joint disease in open-label studies proval for the treatment of RA and PsA, along with a of PsA (98,99), was studied in 2 major placebo- number of other indications. Etanercept (84,85) and controlled trials using a similar study design. In these infliximab (86–88) were the first agents to show impres- trials, the initial Infliximab Multinational Psoriatic Ar- sive results in reducing the signs and symptoms of thritis Controlled Trial (IMPACT) (93) and the fol- established RA, inhibiting the progression of structural lowup trial IMPACT II (94), background MTX was damage (86,89) and substantially improving the func- allowed, but not required, so that ϳ50% of patients who tional status and quality of life of patients (90,91). were enrolled in both studies received concomitant Following the proven successes in RA, these agents were MTX. The IMPACT study allowed continuation of then studied in PsA, with demonstrated dramatic im- DMARDs other than MTX (total of 64% receiving a provements in all facets of the disease, including the background DMARD, 46% of whom were taking skin, joints, and entheses (92–95). Table 3 provides a MTX), while IMPACT II limited DMARD use to MTX. summary of the key clinical investigations of anti-TNF In the IMPACT study, enthesitis and dactylitis therapy in PsA. measures were formally assessed for the first time; these Etanercept. Etanercept gained US FDA approval improved significantly among infliximab users. No dif- for the treatment of PsA in 2002, following the comple- ference in radiographic outcomes was noted between the tion of a published phase II trial (92) and publication of treatment groups over 1 year, although the short dura- 1058

Table 3. Key clinical trials of anti–tumor necrosis factor agents for psoriatic arthritis (PsA)* % of patients meeting response criteria in Study No. of Study type/ active treatment/ P, active vs. reference Agent patients duration Outcome measure placebo group placebo Comments 92 Etanercept 25 mg 60 RDBPC/12 weeks PsARC 87/23 All Ͻ0.0001 HAQ significantly improved with twice weekly ACR20 73/13 etanercept; no significant difference (in skin and joints) in MTX vs. non-MTX users

96 Etanercept 25 mg 205 RDBPC/12 weeks ACR20 59/15 Ͻ0.001 Inhibition of radiographic progression (TSS) twice weekly PsARC 72/31 Ͻ0.0001 at 48 weeks in etanercept group versus PASI50 47/18 Յ0.001 worsening in placebo group; no difference PASI75 23/3 Յ0.001 between groups in PsA-specific radiographic changes (osteolysis, periostitis)

93 Infliximab 5 mg/kg 104 RDBPC/16 weeks; ACR20 (week 16) 65/10 Ͻ0.0001 All patients crossed over to active drug at on weeks 0, 2, 6, BSC/through 50 ACR50 (week 16) 46/0 Ͻ0.001 week 17; no progression of joint damage then every 8 weeks† ACR70 (week 16) 29/0 Ͻ0.001 observed in either the original infliximab- weeks PsARC (week 16) 75/21 Ͻ0.001 treated or the placebo-treated patients at ACR20 (week 50) 68/69 Ͻ0.0001 week 50; enthesitis and dactylitis ACR50 (week 50) 42/53 Ͻ0.001 significantly improved in infliximab group ACR70 (week 50) 34/39 Ͻ0.001 PsARC (week 50) 76/74 Ͻ0.001

94 Infliximab 5 mg/kg 200 RDBPC/14 weeks ACR20 58/11 All Ͻ0.001 Similar results noted up to week 24; on weeks 0, 2, 6, ACR50 36/3 significant PASI response observed in then every 8 ACR70 15/1 both ACR20 responders and ACR20 weeks PsARC 77/27 nonresponders PASI50 82/9 PASI75 64/2 PASI90 41/0

95 Adalimumab 40 313 RDBPC/24 weeks ACR20 57/15 All Ͻ0.001 Inhibition of radiographic progression mg every other ACR50 39/6 (mTSS) in adalimumab group at week 24 week ACR70 23/1 and maintained at week 48; significant PsARC 60/23 improvements in quality of life and PASI50 75/12 disability measures in adalimumab group MORELAND AND TURKIEWICZ PASI75 59/1 PASI90 42/0 PASI100 29/0

103 Onercept 50 mg 126 DBPC/12 weeks PsARC 86/45 Ͻ0.001 Results listed are those observed in the 100 and 100 mg ACR20 67/31 0.001 mg arm; placebo responses were higher subcutaneously than in etanercept PsA trials 3 times/week

* The initial outcome measure listed for each study was the primary outcome assessed. RDBPC ϭ randomized, double-blind, placebo-controlled; PsARC ϭ Psoriatic Arthritis Response Criteria; ACR20 ϭ American College of Rheumatology 20% response criteria; HAQ ϭ Health Assessment Questionnaire; MTX ϭ methotrexate; PASI50 ϭ Psoriasis 50% Area and Severity Index; TSS ϭ total Sharp score; BSC ϭ blinded single-crossover; mTSS ϭ modified TSS. † Values through week 50 are patients who were crossed over from placebo to infliximab/patients remaining on infliximab. THERAPEUTIC OPTIONS FOR PsA 1059

tion of the placebo treatment (14 weeks) was considered observed more frequently in the onercept group. In a to be a likely contributor to this lack of difference. Of phase III psoriasis trial of onercept, 2 patients developed note, the calculated annual radiographic progression sepsis, one of whom subsequently died (104). Two phase rate was reduced in both treatment arms, suggesting that III clinical trials of onercept have been discontinued infliximab treatment, even after a 14-week delay in (105). A number of other anti-TNF␣ agents are under initiating treatment, inhibits radiographic progression in investigation in PsA, including a human monoclonal PsA (100). antibody for subcutaneous administration (golimumab In IMPACT II, an analysis of skin response was [CNTO 148]), a PEGylated Fab fragment of a monoclo- carried out in ACR20 responders as compared with nal antibody (certolizumab pegol [CDP870]), and oral ACR20 nonresponders, with results showing that the compounds possessing TNF␣-inhibitory action (106). median improvement in the PASI was 87% in ACR20 TNF␣ antagonists in PsA. In general, the reported responders versus 74% in nonresponders, supporting the PsA trials revealed no new significant adverse events or notion that skin response can be achieved with inflix- safety issues with anti-TNF therapy other than those imab in the absence of significant joint response. Signif- reported in RA trials. Regarding skin-specific issues, no icantly less radiographic progression was noted at 24 increases in the frequency of Koebner phenomenon or weeks in the infliximab-treated group compared with the cellulitis were observed in PsA patients. placebo group. As with the other anti-TNF␣ agents, Of note, several case series have been published there was no difference between the treatment groups describing what appears to be a paradoxical adverse when PsA-specific radiographic features, including gross reaction following treatment with the 3 anti-TNF ther- osteolysis and pencil-in-cup deformities, were examined, apies, in that all 3 therapies induced psoriasiform skin and this was considered to possibly be due to the lesions (typically palmoplantar pustulosis) in previously chronicity and fixed nature of such lesions (101). Once unaffected individuals (the majority of whom had RA) again, treatment with background MTX did not have an without a personal or family history of psoriasis (107– impact on efficacy in either of the IMPACT studies. 112). While the underlying pathophysiologic mechanism Adalimumab. Adalimumab, the most recently for these observations is still unknown, hypotheses in- approved anti-TNF agent for PsA, was initially observed clude infectious (bacterial) and autoimmune (activation as an effective treatment for PsA in an open trial (n ϭ of autoreactive T cells or altered immunity induced by 15) (102). In the Adalimumab Effectiveness in PsA Trial anti-TNF activity in predisposed individuals) etiologies. (ADEPT) of 313 patients with PsA, 57% of the patients Because arthritis in some of these patients continued to treated with adalimumab versus 15% of the placebo improve despite the cutaneous eruptions, these observa- group achieved the primary end point (the ACR20 tions again highlight the need for more complete defi- response). Significant improvements in skin disease nition of the mechanisms of disease in the skin and joint. along with significant inhibition of radiographic progres- Although there have been no trials directly com- sion of disease and improvement in quality of life and paring TNF antagonists in PsA, switching from one functional indices were also observed in the anti-TNF agent to another due to inefficacy, loss of adalimumab-treated patients during the initial 24-week efficacy, or side effects may be a reasonable option, as ADEPT study, with continued response through 48 described in a small number of patients with PsA and weeks (95) (Table 3). The safety profile of adalimumab those with SpA (113). Similar experiences with switching in patients with PsA appears to be consistent with the anti-TNF therapies in the management of RA have been safety profile observed in clinical trials of adalimumab reported (114,115). in RA. Modulators of T lymphocyte function. Psoriasis has Other TNF␣ antagonists. Onercept is a recombi- been described as a T lymphocyte–driven disease (116); nant, fully human type I TNF receptor (p55). A phase II therefore, utilizing BRMs that interfere with T cell placebo-controlled trial of 126 patients with PsA was activation and/or trafficking of T cells is a logical ap- recently completed (103), involving treatment with 2 proach for targeted therapies in psoriasis and PsA subcutaneous doses (50 mg and 100 mg) of onercept (Figures 1 and 3). T cell activation is a 2-step process. administered 3 times per week; the 100 mg dose of The first signal involves the recognition of an MHC– onercept was more effective than the 50 mg dose, with antigen complex presented on an antigen-presenting cell both doses generally well tolerated. Side effects were (APC) by the complementary T cell receptor. The similar between the patients receiving onercept and the second signal consists of a variety of receptor/ placebo group. Of note, injection site reactions were counterreceptor couplings, which can produce stimula- 1060 TURKIEWICZ AND MORELAND

inhibition of T cell activation and depletion of T cells via interactions through the Fc portion of the molecule (118,119). Alefacept is administered as a weekly intramuscular injection, alternating 12 weeks on and 12 weeks off to allow for recovery of CD4 cells, the levels of which are monitored during treatment (120,121). Following a small, successful open-label trial of alefacept in patients with PsA (122), a randomized, placebo-controlled phase II study involving 185 patients with PsA who were taking MTX was performed, with results showing significant improvements in both skin and joint outcomes in patients receiving alefacept 15 mg intramuscularly in combination with MTX versus those receiving placebo plus MTX (117). At week 24, 54% of the patients in the alefacept plus MTX group achieved an ACR20 response, compared with 23% of patients in the placebo plus MTX group (P Ͻ 0.001). Improvements in the ACR20 response continued after the active treatment phase. No serious infections were reported and no Figure 3. Costimulatory blockade strategies. T cell activation requires malignancies were diagnosed in the alefacept plus MTX 2 signals. The first signal (signal 1) consists of recognition of a major ϩ histocompatibility complex (MHC)–antigen complex presented on an arm. The predictable reductions in CD4 T cell counts antigen-presenting cell (APC) by the complementary T cell receptor in patients treated with the alefacept plus MTX combi- (TCR). A number of costimulatory pathways serve as second signals nation were consistent with those reported in the phase (signal 2) through which full T cell activation occurs. Interfering with III clinical trials of alefacept monotherapy in patients these second signal costimulatory pathways serves as the mechanism of with psoriasis, with similarities in the overall safety action for a number of biologic agents used in psoriasis and psoriatic arthritis. Alefacept is a dimeric human fusion protein that binds to profile observed between the psoriasis and PsA trials CD2 on T cells and blocks its interaction with leukocyte function– (117,120,121). associated antigen 3 (LFA-3) on APCs, in addition to binding to the Efalizumab. Efalizumab, approved in the US as Fc␥III region of natural killer lymphocytes, resulting in apoptosis of T a weekly subcutaneous injection for treatment of cells expressing CD2. Efalizumab is a recombinant humanized mono- moderate-to-severe plaque psoriasis (123), is a human- clonal IgG1 antibody to the CD11a subunit of the LFA-1 on T cells that prevents adhesion of LFA-1 to intercellular adhesion molecule 1 ized monoclonal IgG antibody that binds to the CD11a (ICAM-1) on APCs. Abatacept is a recombinant CTLA-4Ig fusion subunit of LFA-1 (an adhesion molecule expressed on T protein that selectively modulates costimulation via interruption of the cells), thereby inhibiting LFA-1 binding to ICAM-1 on CD28:CD80/86 pathway. the APC and resulting in inhibition of T cell activation. It also interferes with migration of cells from the circu- tory responses (costimulatory pathways). Examples of such couplings include leukocyte function–associated Table 4. Status of development of biologic response modifiers in antigen 3 (LFA-3) and CD2, LFA-1 and ICAM-1, and psoriasis and psoriatic arthritis* CD80/CD86 and CD28/cytotoxic T lymphocyte– Status in psoriatic associated 4 (CTLA-4). Many of these pathways have Agent Target Status in psoriasis arthritis been targeted by specific therapeutic agents that are Infliximab TNF␣ FDA-approved FDA-approved currently being investigated in clinical trials for use in Etanercept TNF␣ FDA-approved FDA-approved ␣ psoriasis and PsA (Table 4). Adalimumab TNF Phase II and III FDA-approved trials completed Alefacept. Alefacept, approved in the US in 2003 Alefacept CD2 FDA-approved Phase II trial for the treatment of psoriasis and currently being inves- completed tigated in phase II clinical trials for its use in PsA (117), Efalizumab CD11a (LFA-1) FDA-approved Phase II trial completed is a fully human LFA-3/IgG1 fusion protein that binds to Abatacept CD80/CD86 Phase II trial No trials CD2 receptor on T cells to block the interaction between completed completed LFA-3 on APCs and CD2 on T cells. This blockade of * TNF␣ ϭ tumor necrosis factor ␣; FDA ϭ US Food and Drug the LFA-3/CD2 costimulatory pathway results in the Administration; LFA-1 ϭ leukocyte function–associated antigen 1. THERAPEUTIC OPTIONS FOR PsA 1061

lation to sites of inflammation (106). A phase II study in designed to suppress IL-15 are in development (anti– 117 PsA patients failed to show significant improve- IL-15 [AMG714, HuMax-IL15], IL-15:Fc mutant [CRB- ments in the ACR20 response at 12 weeks; 28% of the 15], soluble IL-15 receptor ␣) (131). Arthroscopic eval- patients receiving the active drug achieved an ACR20 uations of synovial membrane from PsA patients, response versus 19% in the placebo arm (P ϭ 0.2717) obtained prior to and following a 3-month course of (124). Given the short duration of this trial, it is un- MTX, demonstrated that expression of IL-15 messenger known if more robust improvements with efalizumab RNA and protein was modified but not abrogated by would have been evident with a longer trial (125). MTX, providing the groundwork to consider a combi- Abatacept. Abatacept, recently approved in the nation of IL-15 antagonist with MTX as a plausible US for treatment of RA, is a recombinant CTLA-4Ig therapeutic strategy for PsA (131). A pilot trial of fusion protein that selectively modulates costimulation anti–IL-15 has shown efficacy in PsA (132). via interruption of the CD28:CD80/86 pathway (126). It On the opposite end of the spectrum, cytokines is administered as a monthly intravenous infusion. The that possess antiinflammatory effects, such as IL-10 and efficacy of abatacept has been demonstrated in a phase IL-11, could be attractive agents for the treatment of II psoriasis trial (127), although it has yet to be studied PsA. IL-10 promotes Th2-biased cytokine secretion and formally in PsA. inhibits IFN␥ (133). It appears that recombinant IL-10 is Other potential targets. Do we need more targeted successful in attenuating skin disease but not joint therapies? Although the introduction of anti-TNF␣ ther- inflammation, as indicated in a controlled study of PsA apies has provided a much-needed option in the arma- patients (134). Recombinant human IL-11, which has mentarium of PsA therapies, approximately one-third of demonstrated antiinflammatory activity, has been tested patients with moderate-to-severe PsA have insufficient in a small number of patients with psoriasis, resulting in responses to these therapies (14). The successes and some improvement in skin scores (135). failures of the anti-TNF␣ agents and costimulatory Additional strategies of T cell manipulation. blockers in the management of psoriasis and PsA have HuOKT3␥1 (ala-ala), a non–FcR-binding monoclonal sparked considerable interest in exploring other avenues antibody to the T cell receptor complex component for targeting the diverse immune responses considered CD3, anergizes type 1 lymphocytes and serves as a to be at play. targeted therapeutic option for type 1 lymphocyte– More cytokines, both pro- and antiinflammatory, to mediated diseases such as PsA. In a pilot study of 7 consider. Examples of other potential therapeutic targets patients with active PsA whose condition failed to in PsA include a variety of antagonistic approaches respond to at least one remittive agent, improvements in (neutralizing monoclonal antibody, soluble forms of skin and joint outcomes were reported (136). A human- receptor antagonists, and cytokine:Fc mutant fusion ized anti-CD80 monoclonal antibody (IDEC-114, galix- proteins) to interfere with the actions of so-called proin- imab) has been studied in a phase I/II trial of 35 patients flammatory cytokines, including IL-1, IL-6, IL-8, IL-12, with plaque psoriasis; improvements in skin scores and IL-15, and IL-18. Anakinra, the IL-1 receptor antagonist good tolerability were observed (137). approved for RA, has been investigated for use in PsA, Targeting the vasculature and mediators of bone although significant efficacy has not been observed dysregulation. The findings of elevated angiogenic factors (128). Plans are under way to test IL-1 Trap, a weekly in PsA suggest that reducing synovial vascularity may be subcutaneous IL-1 antagonist, in PsA (97). A monoclo- an alternative mechanistic target in this disease. Anti- nal antibody to the IL-6 receptor (tocilizumab) is cur- TNF therapy has been shown to produce significant rently being investigated in a number of phase III reductions in the circulating concentrations of VEGF in clinical trials in RA and would be a logical therapy to RA (138,139) and PsA (140). Blockade of neovascular- test in PsA. ization utilizing a number of angiogenesis inhibitor As a key cytokine in Th1 immune responses, strategies (VEGF antibody, small-molecule inhibitors of IL-12 is also a potential target in psoriasis and PsA; a tyrosine kinases) has been effective in patients with solid number of anti–IL-12 therapies are under development tumors (141,142), and such strategies continue to be for the treatment of psoriasis. IL-15, a pleiotropic cyto- investigated. Angiogenesis inhibitor strategies could be kine with proinflammatory effects in a number of considered for the treatment of PsA to target the highly chronic inflammatory diseases, has been demonstrated vascular PsA synovium, likely in combination with other to have increased levels in the skin and synovium of therapies such as anti-TNF, particularly in those patients psoriasis patients (129,130), and a number of agents with a suboptimal response to anti-TNF; i.e., target the 1062 TURKIEWICZ AND MORELAND

inflammatory and vascular components, possibly with- the quest to join the ranks of approved PsA therapies, out increasing infection risk. they will have a large burden of proof to achieve Pioglitazone, a member of the thiazolidinedione recognition as a truly successful treatment for PsA. In family of insulin-sensitizing drugs developed for the developing future therapeutic strategies for PsA, one treatment of type II diabetes mellitus, is an agonist for must consider the need for efficacy in both skin and joint the peroxisome proliferator–activated receptor ␥ responses, an acceptable safety and tolerability profile, (PPAR␥). Beyond its role in adipocyte differentiation, the ability to inhibit disease progression as is currently PPAR␥ has been considered a potential target for the measured radiographically, and the capacity to improve treatment of inflammatory rheumatic conditions, given quality of life and functional status, all within the context observations that it leads to marked inhibition of angio- of achieving favorable cost-effective results. genesis and down-regulation of proinflammatory cytokines (53). In an uncontrolled study of 10 patients with PsA treated with PPAR␥ for 12 weeks, 60% of patients Conclusions were considered PsARC responders (the primary end With its uniquely diverse pathophysiologic and point) and 50% reached an ACR20 response. Three clinical features and the ability to progress into one of patients were withdrawn from the study due to treat- the most destructive arthritides known, PsA remains a ment inefficacy and adverse events, with edema in the challenging disease deserving of the attention it has been lower extremities and weight gain as the most common receiving in recent years. A surge of critically important adverse events (143). observations regarding the immunopathogenetic mech- Strategies targeting abnormalities in bone re- anisms of PsA, including the finding that the disease is modeling characteristic of PsA could also be considered. paradoxically both similar to, and distinct from, other As described by Ritchlin and colleagues, a bidirectional inflammatory arthropathies, has heightened the aware- model for the extensive erosive changes observed in ness that novel biologic agents used in RA cannot simply patients with PsA may exist, whereby circulating OCPs be empirically applied to PsA. Rather, these agents and migrate to the inflamed synovium and are induced to others soon to come have the burden of showing efficacy ␬ become osteoclasts by the NF- B ligand (RANKL) that in a number of immunologic, clinical, and functional is expressed by synoviocytes; in addition, OCPs also outcomes specific to PsA. Measures of these outcomes traverse the subchondral bone endothelium, where they are sorely needed to standardize the expected response are exposed to TNF␣-induced RANKL on osteoblasts in this disease and to determine the natural history of and stromal cells. The result is osteoclastogenesis at the PsA. synovium and in subchondral bone (44). An inhibitor of Although there are clear indications that the RANKL, denosumab, which is currently being investi- same pathogenetic processes occurring in the skin are gated in postmenopausal osteoporosis and RA, could be likely occurring in the joints and vice versa, the clinical considered a viable target in PsA among those patients experience with therapies that preferentially improve with erosive disease that is not completely controlled the condition in either the skin or the joints indicates with their current DMARD. that more factors are at play. As in RA, but perhaps even MMPs, regulated by IL-1 and TNF␣, and tissue more so in PsA, there exists a clear unmet need for inhibitors of MMPs (TIMPs) may also be attractive targeted therapies for affected patients, who are in- targets for PsA therapy. MMP-3 (an activator of other flicted with 2 incurable diseases for which true disease- MMPs [144]), along with MMP-1, has been demon- modifying agents are needed. Although the early data strated in synovial tissue and skin samples from PsA seem to support the potential of biologic agents to patients (145). In addition, patients with polyarticular achieve significant benefits, particularly with anti-TNF PsA were observed to have increased serum levels of therapies, we are still in the infancy of the biologic era in MMP-1 and TIMP-1 compared with controls (146). PsA. Further studies are needed to confirm the long- Once again, since these strategies utilize targets “down- term safety and efficacy of currently available therapeutic stream” from currently available targets (TNF␣, IL-1, strategies and those on the horizon. IL-6), they could serve as vital adjuncts to the PsA regimen, with the potential to have a favorable toxicity profile in combination with DMARD and/or biologic REFERENCES therapies. 1. Alibert J. Precis theorique et pratique sur les maladies de la peau. As novel agents are discovered and entered into Paris: Caille et Ravier; 1818. THERAPEUTIC OPTIONS FOR PsA 1063

2. Helliwell PS, Taylor WJ. Classification and diagnostic criteria for riasis and psoriatic arthritis. Ann Rheum Dis 2005;64 Suppl psoriatic arthritis. Ann Rheum Dis 2005;64 Suppl 2:ii3–8. 2:ii26–9. 3. Gladman DD. Psoriatic arthritis. Baillieres Clin Rheumatol 24. O’Connell PG, Gerber LH, Digiovanna JJ, Peck GL. Arthritis in 1995;9:319–29. patients with psoriasis treated with ␥-interferon. J Rheumatol 4. Moll JM, Wright V. Psoriatic arthritis. Semin Arthritis Rheum 1992;19:80–2. 1973;3:55–78. 25. Melski JW, Bernhard JD, Stern RS. The Koebner (isomorphic) 5. Gladman DD, Shuckett R, Russell ML, Thorne JC, Schachter response in psoriasis: associations with early age at onset and RK. Psoriatic arthritis (PSA): an analysis of 220 patients. Q multiple previous therapies. Arch Dermatol 1983;119:655–9. J Med 1987;62:127–41. 26. Langevitz P, Buskila D, Gladman DD. Psoriatic arthritis precip- 6. Vasey F, Espinoza LR. Psoriatic arthropathy. In: Calin A, editor. itated by physical trauma. J Rheumatol 1990;17:695–7. Spondyloarthropathies. Orlando (FL): Grune & Stratton; 1984. 27. Vasey FB, Deitz C, Fenske NA, Germain BF, Espinoza LR. p. 151–85. Possible involvement of group A streptococci in the pathogenesis 7. Dougados M, van der Linden S, Juhlin R, Huitfeldt B, Amor B, of psoriatic arthritis. J Rheumatol 1982;9:719–22. Calin A, et al. The European Spondylarthropathy Study Group 28. Veale D, Yanni G, Rogers S, Barnes L, Bresnihan B, FitzGerald preliminary criteria for the classification of spondylarthropathy. O. Reduced synovial membrane macrophage numbers, ELAM-1 Arthritis Rheum 1991;34:1218–27. expression, and lining layer hyperplasia in psoriatic arthritis as 8. McGonagle D, Conaghan PG, Emery P. Psoriatic arthritis: a compared with rheumatoid arthritis. Arthritis Rheum 1993;36: unified concept twenty years on. Arthritis Rheum 1999;42: 893–900. 1080–6. 29. Veale DJ, Barnes L, Rogers S, FitzGerald O. Immunohistochem- 9. Fournie B, Crognier L, Arnaud C, Zabraniecki L, Lascaux- ical markers for arthritis in psoriasis. Ann Rheum Dis 1994;53: Lefebvre V, Marc V, et al. Proposed classification criteria of 450–4. psoriatic arthritis: a preliminary study in 260 patients. Rev Rhum 30. Laloux L, Voisin MC, Allain J, Martin N, Kerboull L, Chevalier Engl Ed 1999;66:446–56. X, et al. Immunohistological study of entheses in spondyloar- 10. Bennett RM. Psoriatic arthritis. In: McCarty DJ, editor. Arthritis thropathies: comparison in rheumatoid arthritis and osteoarthri- and allied conditions: a textbook of rheumatology. Philadelphia: tis. Ann Rheum Dis 2001;60:316–21. Lea & Febiger; 1979. p. 645. 31. Ritchlin C, Haas-Smith SA, Hicks D, Cappuccio J, Osterland CK, 11. Taylor W, Gladman D, Helliwell P, Marchesoni A, Mease P, Looney RJ. Patterns of cytokine production in psoriatic syno- Mielants H, and the CASPAR Study Group. Classification vium. J Rheumatol 1998;25:1544–52. criteria for psoriatic arthritis: development of new criteria from a 32. Berman A, Espinoza LR, Diaz JD, Aguilar JL, Rolando T, Vasey large international study. Arthritis Rheum 2006;54:2665–73. FB, et al. Rheumatic manifestations of human immunodeficiency 12. Gladman DD, Anhorn KA, Schachter RK, Mervart H. HLA virus infection. Am J Med 1988;85:59–64. antigens in psoriatic arthritis. J Rheumatol 1986;13:586–92. 33. Espinoza LR, Jara LJ, Espinoza CG, Silveira LH, Martinez- 13. Moll JM, Wright V. Familial occurrence of psoriatic arthritis. Osuna P, Seleznick M. There is an association between human Ann Rheum Dis 1973;32:181–201. immunodeficiency virus infection and spondyloarthropathies. 14. Cassell S, Kavanaugh A. Psoriatic arthritis: pathogenesis and Rheum Dis Clin North Am 1992;18:257–66. novel immunomodulatory approaches to treatment. J Immune 34. Espinoza LR, Berman A, Vasey FB, Cahalin C, Nelson R, Based Ther Vaccines 2005;3:6. Germain BF. Psoriatic arthritis and acquired immunodeficiency 15. Sakkas LI, Loqueman N, Bird H, Vaughan RW, Welsh KI, syndrome. Arthritis Rheum 1988;31:1034–40. Panayi GS. HLA class II and T cell receptor gene polymorphisms 35. Duvic M, Johnson TM, Rapini RP, Freese T, Brewton G, Rios A. in psoriatic arthritis and psoriasis. J Rheumatol 1990;17:1487–90. Acquired immunodeficiency syndrome-associated psoriasis and 16. Gonzalez S, Martinez-Borra J, Lopez-Vazquez A, Garcia-Fer- Reiter’s syndrome. Arch Dermatol 1987;123:1622–32. nandez S, Torre-Alonso JC, Lopez-Larrea C. MICA rather than 36. Winchester R, Brancato L, Itescu S, Skovron ML, Solomon G. MICB, TNFA, or HLA-DRB1 is associated with susceptibility to Implications from the occurrence of Reiter’s syndrome and psoriatic arthritis. J Rheumatol 2002;29:973–8. related disorders in association with advanced HIV infection. 17. Gladman DD, Farewell VT, Kopciuk KA, Cook RJ. HLA Scand J Rheumatol Suppl 1988;74:89–93. markers and progression in psoriatic arthritis. J Rheumatol 37. Brancato L, Itescu S, Skovron ML, Solomon G, Winchester R. 1998;25:730–3. Aspects of the spectrum, prevalence and disease susceptibility 18. Karason A, Gudjonsson JE, Upmanyu R, Antonsdottir A, Hauks- determinants of Reiter’s syndrome and related disorders associ- son VB, Runasdottir EH, Jonsson HH, et al. A susceptibility gene ated with HIV infection. Rheumatol Int 1989;9:137–41. for psoriatic arthritis maps to chromosome 16q: evidence for 38. Costello P, Bresnihan B, O’Farrelly C, FitzGerald O. Predomi- imprinting. Am J Hum Genet 2003;72:125–31. nance of CD8ϩ T lymphocytes in psoriatic arthritis. J Rheumatol 19. Korendowych E, Dixey J, Cox B, Jones S, McHugh N. The 1999;26:1117–24. influence of the HLA-DRB1 rheumatoid arthritis shared epitope 39. Veale D, Rogers S, Fitzgerald O. Immunolocalization of adhe- on the clinical characteristics and radiological outcome of psori- sion molecules in psoriatic arthritis, psoriatic and normal skin. atic arthritis. J Rheumatol 2003;30:96–101. Br J Dermatol 1995;132:32–8. 20. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos 40. Fraser A, Fearon U, Billinghurst RC, Ionescu M, Reece R, R, et al. A frameshift mutation in NOD2 associated with suscep- Barwick T, et al. Turnover of type II collagen and aggrecan in tibility to Crohn’s disease. Nature 2001;411:603–6. cartilage matrix at the onset of inflammatory arthritis in humans: 21. Laval SH, Timms A, Edwards S, Bradbury L, Brophy S, Milicic A, relationship to mediators of systemic and local inflammation. et al. Whole-genome screening in ankylosing spondylitis: evi- Arthritis Rheum 2003;48:3085–95. dence of non-MHC genetic-susceptibility loci. Am J Hum Genet 41. Fearon U, Reece R, Smith J, Emery P, Veale DJ. Synovial 2001;68:918–26. cytokine and growth factor regulation of MMPs/TIMPs: implica- 22. Ho P, Bruce IN, Silman A, Symmons D, Newman B, Young H, et tions for erosions and angiogenesis in early rheumatoid and al. Evidence for common genetic control in pathways of inflam- psoriatic arthritis patients. Ann N Y Acad Sci 1999;878:619–21. mation for Crohn’s disease and psoriatic arthritis. Arthritis 42. Mastroianni A, Minutilli E, Mussi A, Bordignon V, Trento E, Rheum 2005;52:3596–602. D’Agosto G, et al. Cytokine profiles during infliximab mono- 23. Veale DJ, Ritchlin C, FitzGerald O. Immunopathology of pso- therapy in psoriatic arthritis. Br J Dermatol 2005;153:531–6. 1064 TURKIEWICZ AND MORELAND

43. Fearon U, Griosios K, Fraser A, Reece R, Emery P, Jones PF, et 63. Soriano ER, McHugh NJ. Therapies for peripheral joint disease al. Angiopoietins, growth factors, and vascular morphology in in psoriatic arthritis: a systematic review. J Rheumatol 2006;33: early arthritis. J Rheumatol 2003;30:260–8. 1422–30. 44. Ritchlin CT, Haas-Smith SA, Li P, Hicks DG, Schwarz EM. 64. Ritchlin CT. Therapies for psoriatic enthesopathy: a systematic Mechanisms of TNF-␣- and RANKL-mediated osteoclastogen- review. J Rheumatol 2006;33:1435–8. esis and bone resorption in psoriatic arthritis. J Clin Invest 65. Nash P. Therapies for axial disease in psoriatic arthritis: a 2003;111:821–31. systematic review. J Rheumatol 2006;33:1431–4. 45. Bogliolo L, Alpini C, Caporali R, Scire CA, Moratti R, Mon- 66. Helliwell PS. Therapies for dactylitis in psoriatic arthritis: a tecucco C. Antibodies to cyclic citrullinated peptides in psoriatic systematic review. J Rheumatol 2006;33:1439–41. arthritis. J Rheumatol 2005;32:511–5. 67. Cassell S, Kavanaugh AF. Therapies for psoriatic nail disease: a 46. Vander Cruyssen B, Hoffman IE, Zmierczak H, Van den Berghe systematic review. J Rheumatol 2006;33:1452–6. M, Kruithof E, De Rycke L, et al. Anti-citrullinated peptide 68. Boehncke WH, Prinz J, Gottlieb AB. Biologic therapies for antibodies may occur in patients with psoriatic arthritis. Ann psoriasis: a systematic review. J Rheumatol 2006;33:1447–51. Rheum Dis 2005;64:1145–9. 69. Nash P, Clegg DO. Psoriatic arthritis therapy: NSAIDs and 47. Korendowych E, Owen P, Ravindran J, Carmichael C, McHugh traditional DMARDs. Ann Rheum Dis 2005;64 Suppl 2:ii74–7. N. The clinical and genetic associations of anti-cyclic citrullinated 70. Witman PM. Topical therapies for localized psoriasis. Mayo Clin peptide antibodies in psoriatic arthritis. Rheumatology (Oxford) Proc 2001;76:943–9. 2005;44:1056–60. 71. Black RL, O’Brien WM, Vanscott EJ, Auerbach R, Eisen AZ, 48. McHugh NJ, Balachrishnan C, Jones SM. Progression of peri- Bunim JJ. Methotrexate therapy in psoriatic arthritis; double- pheral joint disease in psoriatic arthritis: a 5-yr prospective study. blind study on 21 patients. JAMA 1964;189:743–7. Rheumatology (Oxford) 2003;42:778–83. 72. Willkens RF, Williams HJ, Ward JR, Egger MJ, Reading JC, 49. Gladman DD, Stafford-Brady F, Chang CH, Lewandowski K, Clements PJ, et al. Randomized, double-blind, placebo con- Russell ML. Longitudinal study of clinical and radiological trolled trial of low-dose pulse methotrexate in psoriatic arthritis. progression in psoriatic arthritis. J Rheumatol 1990;17:809–12. Arthritis Rheum 1984;27:376–81. 50. Gladman DD. Natural history of psoriatic arthritis. Baillieres 73. Whiting-O’Keefe QE, Fye KH, Sack KD. Methotrexate and Clin Rheumatol 1994;8:379–94. histologic hepatic abnormalities: a meta-analysis. Am J Med 51. Sokoll KB, Helliwell PS. Comparison of disability and quality of 1991;90:711–6. life in rheumatoid and psoriatic arthritis. J Rheumatol 2001;28: 74. Roenigk HH Jr, Auerbach R, Maibach HI, Weinstein GD. 1842–6. Methotrexate in psoriasis: revised guidelines. J Am Acad Der- 52. Husted JA, Gladman DD, Farewell VT, Cook RJ. Health-related matol 1988;19(1 Pt 1):145–56. quality of life of patients with psoriatic arthritis: a comparison 75. Kremer JM, Alarcon GS, Lightfoot RW Jr, Willkens RF, Furst with patients with rheumatoid arthritis. Arthritis Rheum 2001;45: DE, Williams HJ, et al. Methotrexate for rheumatoid arthritis: 151–8. suggested guidelines for monitoring liver toxicity. Arthritis 53. Ruderman EM. Treatment advances in psoriatic arthritis. Curr Rheum 1994;37:316–28. Rheumatol Rep 2005;7:313–8. 76. Clegg DO, Reda DJ, Abdellatif M. Comparison of sulfasalazine 54. Heydendael VM, Spuls PI, Bossuyt PM, Bos JD, de Rie MA. and placebo for the treatment of axial and peripheral articular Analysis of risk factors in psoriatic patients with methotrexate- manifestations of the seronegative spondylarthropathies: a De- induced increases in transaminase levels. Arch Dermatol 2004; partment of Veterans Affairs cooperative study. Arthritis Rheum 140:1289–90. 1999;42:2325–9. 55. Ujfalussy I, Koo E, Sesztak M, Gergely P. Termination of 77. Kaltwasser JP, Nash P, Gladman D, Rosen CF, Behrens F, Jones disease-modifying antirheumatic drugs in rheumatoid arthritis P, et al. Efficacy and safety of leflunomide in the treatment of and in psoriatic arthritis: a comparative study of 270 cases. Z psoriatic arthritis and psoriasis: a multinational, double-blind, Rheumatol 2003;62:155–60. randomized, placebo-controlled clinical trial. Arthritis Rheum 56. Furst DE. The rational use of methotrexate in rheumatoid 2004;50:1939–50. arthritis and other rheumatic diseases. Br J Rheumatol 1997;36: 78. Spadaro A, Taccari E, Mohtadi B, Riccieri V, Sensi F, Zoppini A. 1196–204. Life-table analysis of cyclosporin A treatment in psoriatic arthri- 57. Clegg DO, Reda DJ, Mejias E, Cannon GW, Weisman MH, tis: comparison with other disease-modifying antirheumatic Taylor T, et al. Comparison of sulfasalazine and placebo in the drugs. Clin Exp Rheumatol 1997;15:609–14. treatment of psoriatic arthritis: a Department of Veterans Affairs 79. Ellis CN, Fradin MS, Messana JM, Brown MD, Siegel MT, cooperative study. Arthritis Rheum 1996;39:2013–20. Hartley AH, et al. Cyclosporine for plaque-type psoriasis: results 58. Gladman DD, Helliwell P, Mease PJ, Nash P, Ritchlin C, Taylor of a multidose, double-blind trial. N Engl J Med 1991;324: W. Assessment of patients with psoriatic arthritis: a review of 277–84. currently available measures [review]. Arthritis Rheum 2004;50: 80. Luzar MJ. Hydroxychloroquine in psoriatic arthropathy: exacer- 24–35. bations of psoriatic skin lesions. J Rheumatol 1982;9:462–4. 59. Mease PJ, Antoni CE, Gladman DD, Taylor WJ. Psoriatic 81. Vine JE, Hymes SR, Warner NB, Cohen PR. Pustular psoriasis arthritis assessment tools in clinical trials. Ann Rheum Dis induced by hydroxychloroquine: a case report and review of the 2005;64 Suppl 2:ii49–54. literature. J Dermatol 1996;23:357–61. 60. Kavanaugh AF, Ritchlin CT, and the GRAPPA Treatment 82. Gladman DD, Blake R, Brubacher B, Farewell VT. Chloroquine Guideline Committee. Systematic review of treatments for pso- therapy in psoriatic arthritis. J Rheumatol 1992;19:1724–6. riatic arthritis: an evidence based approach and basis for treat- 83. Palit J, Hill J, Capell HA, Carey J, Daunt SO, Cawley MI, et al. ment guidelines. J Rheumatol 2006;33:1417–21. A multicentre double-blind comparison of auranofin, intramus- 61. Mease PJ, Kivitz AJ, Burch FX, Siegel EL, Cohen SB, Ory P, et cular gold thiomalate and placebo in patients with psoriatic al. Continued inhibition of radiographic progression in patients arthritis. Br J Rheumatol 1990;29:280–3. with psoriatic arthritis following 2 years of treatment with etan- 84. Moreland LW, Baumgartner SW, Schiff MH, Tindall EA, ercept. J Rheumatol 2006;33:712–21. Fleischmann RM, Weaver AL, et al. Treatment of rheumatoid 62. Strober BE, Siu K, Menon K. Conventional systemic agents for arthritis with a recombinant human tumor necrosis factor recep- psoriasis: a systematic review. J Rheumatol 2006;33:1442–6. tor (p75)-Fc fusion protein. N Engl J Med 1997;337:141–7. THERAPEUTIC OPTIONS FOR PsA 1065

85. Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleisch- Controlled Trial (IMPACT): results of radiographic analyses mann RM, Fox RI, et al. A trial of etanercept, a recombinant after 1 year. Ann Rheum Dis 2006;65:1038–43. tumor necrosis factor receptor:Fc fusion protein, in patients with 101. Van der Heijde D, Gladman DD, Kavanaugh A, Antoni CE, rheumatoid arthritis receiving methotrexate. N Engl J Med Guzzo C, Krueger GG, et al. Infliximab inhibits progression of 1999;340:253–9. radiographic damage in patients with active psoriatic arthritis: 54 86. Maini R, St.Clair EW, Breedveld F, Furst D, Kalden J, Weisman week results from IMPACT 2 [abstract]. Arthritis Rheum M, et al, for the ATTRACT Study Group. Infliximab (chimeric 2005;52 Suppl 9:S281. anti-tumour necrosis factor ␣ monoclonal antibody) versus pla- 102. Ritchlin C, Anandarajaha A, Totterman S, Shao T, Kemshetti S, cebo in rheumatoid arthritis patients receiving concomitant Badger W, et al. Preliminary data from a study of adalimumab in methotrexate: a randomised phase III trial. Lancet 1999;354: the treatment of psoriatic arthritis [abstract]. Ann Rheum Dis 1932–9. 2004;63 Suppl 1:403. 87. Maini RN, Breedveld FC, Kalden JR, Smolen JS, Davis D, 103. Nikas SN, Drosos AA. Onercept. Curr Opin Investig Drugs Macfarlane JD, et al. Therapeutic efficacy of multiple intrave- 2003;4:1369–76. nous infusions of anti–tumor necrosis factor ␣ monoclonal anti- 104. Kincaid L. Psoriasis: TNF-␣ inhibitors and beyond. Drug Discov body combined with low-dose weekly methotrexate in rheuma- Today 2005;10:884–6. toid arthritis. Arthritis Rheum 1998;41:1552–63. 105. Gladman DD. Traditional and newer therapeutic options for 88. Lipsky PE, van der Heijde DM, St.Clair EW, Furst DE, Breed- psoriatic arthritis: an evidence-based review. Drugs 2005;65: veld FC, Kalden JR, et al, for the Anti–Tumor Necrosis Factor 1223–38. Trial in Rheumatoid Arthritis with Concomitant Therapy Study 106. Mease P. Current treatment for psoriatic arthritis and other Group. Infliximab and methotrexate in the treatment of rheuma- spondyloarthritides. In: Kavanaugh A, editor. Rheum Dis Clin toid arthritis. N Engl J Med 2000;343:1594–602. North Am - Updates; 2006. p. 4–13. 89. Genovese MC, Bathon JM, Martin RW, Fleischmann RM, 107. Dereure O, Guillot B, Jorgensen C, Cohen JD, Combes B, Tesser JR, Schiff MH, et al. Etanercept versus methotrexate in Guilhou JJ. Psoriatic lesions induced by antitumour necrosis patients with early rheumatoid arthritis: two-year radiographic factor-␣ treatment: two cases. Br J Dermatol 2004;151:506–7. and clinical outcomes. Arthritis Rheum 2002;46:1443–50. 108. Sari I, Akar S, Birlik M, Sis B, Onen F, Akkoc N. Anti-tumor 90. Kavanaugh A, Cohen S, Cush JJ. The evolving use of tumor necrosis factor-␣-induced psoriasis. J Rheumatol 2006;33: necrosis factor inhibitors in rheumatoid arthritis. J Rheumatol 1411–4. 2004;31:1881–4. 109. Sfikakis PP, Iliopoulos A, Elezoglou A, Kittas C, Stratigos A. 91. Moreland LW, Cohen SB, Baumgartner SW, Tindall EA, Bulpitt Psoriasis induced by anti–tumor necrosis factor therapy: a para- K, Martin R, et al. Long-term safety and efficacy of etanercept in doxical adverse reaction. Arthritis Rheum 2005;52:2513–8. patients with rheumatoid arthritis. J Rheumatol 2001;28: 110. Kary S, Worm M, Audring H, Huscher D, Renelt M, Sorensen H, 1238–44. et al. New onset or exacerbation of psoriatic skin lesions in 92. Mease PJ, Goffe BS, Metz J, VanderStoep A, Finck B, Burge DJ. patients with definite rheumatoid arthritis receiving tumour Etanercept in the treatment of psoriatic arthritis and psoriasis: a necrosis factor ␣ antagonists. Ann Rheum Dis 2006;65:405–7. randomised trial. Lancet 2000;356:385–90. 111. Beuthien W, Mellinghoff HU, von Kempis J. Skin reaction to 93. Antoni CE, Kavanaugh A, Kirkham B, Tutuncu Z, Burmester adalimumab. Arthritis Rheum 2004;50:1690–2. GR, Schneider U, et al. Sustained benefits of infliximab therapy 112. Cohen JD, Bournerias I, Buffard V, Paufler A, Chevalier X, for dermatologic and articular manifestations of psoriatic arthri- Bagot M, et al. Psoriasis induced by tumor necrosis factor-␣ tis: results from the Infliximab Multinational Psoriatic Arthritis antagonist therapy: a case series. J Rheumatol 2006;33:1–6. Controlled Trial (IMPACT). Arthritis Rheum 2005;52:1227–36. 113. Delaunay C, Farrenq V, Marini-Portugal A, Cohen JD, Chevalier 94. Antoni C, Krueger GG, de Vlam K, Birbara C, Beutler A, Guzzo X, Claudepierre P. Infliximab to etanercept switch in patients C, et al. Infliximab improves signs and symptoms of psoriatic with spondyloarthropathies and psoriatic arthritis: preliminary arthritis: results of the IMPACT 2 trial. Ann Rheum Dis 2005; data. J Rheumatol 2005;32:2183–5. 64:1150–7. 114. Hansen KE, Cush JJ, Patel S, Genovese MC, Schiff M. The 95. Mease PJ, Gladman DD, Ritchlin CT, Ruderman EM, Steinfeld efficacy of switching from etanercept to infliximab in patients SD, Choy EH, et al. Adalimumab for the treatment of patients with rheumatoid arthritis [abstract]. Arthritis Rheum 2002;46 with moderately to severely active psoriatic arthritis: results of a Suppl 9:S538. double-blind, randomized, placebo-controlled trial. Arthritis 115. Haraoui B, Keystone EC, Thorne JC, Pope JE, Chen I, Asare Rheum 2005;52:3279–89. CG, et al. Clinical outcomes of patients with rheumatoid arthritis 96. Mease PJ, Kivitz AJ, Burch FX, Siegel EL, Cohen SB, Ory P, et after switching from infliximab to etanercept. J Rheumatol al. Etanercept treatment of psoriatic arthritis: safety, efficacy, 2004;31:2356–9. and effect on disease progression. Arthritis Rheum 2004;50: 116. Krueger JG. The immunologic basis for the treatment of psoriasis 2264–72. with new biologic agents. J Am Acad Dermatol 2002;46:1–23. 97. Mease PJ. Psoriatic arthritis therapy advances. Curr Opin Rheu- 117. Mease PJ, Gladman DD, Keystone EC, on behalf of the Alefa- matol 2005;17:426–32. cept in Psoriatic Arthritis Study Group. Alefacept in combination 98. Ogilvie AL, Antoni C, Dechant C, Manger B, Kalden JR, Schuler with methotrexate for the treatment of psoriatic arthritis: results G, et al. Treatment of psoriatic arthritis with antitumour necrosis of a randomized, double-blind, placebo-controlled study. Arthri- factor-␣ antibody clears skin lesions of psoriasis resistant to tis Rheum 2006;54:1638–45. treatment with methotrexate. Br J Dermatol 2001;144:587–9. 118. Braun J, Sieper J. Role of novel biological therapies in psoriatic 99. Van den Bosch F, Kruithof E, Baeten D, De Keyser F, Mielants arthritis: effects on joints and skin. BioDrugs 2003;17:187–99. H, Veys EM. Effects of a loading dose regimen of three infusions 119. Weinberg JM. An overview of infliximab, etanercept, efalizumab, of chimeric monoclonal antibody to tumour necrosis factor ␣ and alefacept as biologic therapy for psoriasis. Clin Ther 2003; (infliximab) in spondyloarthropathy: an open pilot study. Ann 25:2487–505. Rheum Dis 2000;59:428–33. 120. Krueger GG, Papp KA, Stough DB, Loven KH, Gulliver WP, 100. Kavanaugh A, Antoni CE, Gladman D, Wassenberg S, Zhou B, Ellis CN. A randomized, double-blind, placebo-controlled phase Beutler A, et al. The Infliximab Multinational Psoriatic Arthritis III study evaluating efficacy and tolerability of 2 courses of 1066 TURKIEWICZ AND MORELAND

alefacept in patients with chronic plaque psoriasis. J Am Acad 134. McInnes IB, Illei GG, Danning CL, Yarboro CH, Crane M, Dermatol 2002;47:821–33. Kuroiwa T, et al. IL-10 improves skin disease and modulates 121. Lebwohl M, Christophers E, Langley R, Ortonne JP, Roberts J, endothelial activation and leukocyte effector function in patients Griffiths CE. An international, randomized, double-blind, place- with psoriatic arthritis. J Immunol 2001;167:4075–82. bo-controlled phase 3 trial of intramuscular alefacept in patients 135. Trepicchio WL, Ozawa M, Walters IB, Kikuchi T, Gilleaudeau P, with chronic plaque psoriasis. Arch Dermatol 2003;139:719–27. Bliss JL, et al. Interleukin-11 therapy selectively downregulates 122. Kraan MC, van Kuijk AW, Dinant HJ, Goedkoop AY, Smeets type I cytokine proinflammatory pathways in psoriasis lesions. TJ, de Rie MA, et al. Alefacept treatment in psoriatic arthritis: J Clin Invest 1999;104:1527–37. reduction of the effector T cell population in peripheral blood 136. Utset TO, Auger JA, Peace D, Zivin RA, Xu D, Jolliffe L, et al. and synovial tissue is associated with improvement of clinical Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II signs of arthritis. Arthritis Rheum 2002;46:2776–84. clinical trial. J Rheumatol 2002;29:1907–13. 123. Gottlieb AB, Krueger JG, Wittkowski K, Dedrick R, Walicke PA, 137. Gottlieb AB, Kang S, Linden KG, Lebwohl M, Menter A, Garovoy M. Psoriasis as a model for T-cell-mediated disease: Abdulghani AA, et al. Evaluation of safety and clinical activity of immunobiologic and clinical effects of treatment with multiple multiple doses of the anti-CD80 monoclonal antibody, galiximab, doses of efalizumab, an anti-CD11a antibody. Arch Dermatol in patients with moderate to severe plaque psoriasis. Clin Immu- 2002;138:591–600. nol 2004;111:28–37. 124. Papp KA, Mease P, Garovoy M. Efalizumab in patients with 138. Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M, psoriatic arthritis: results of a phase II randomized double-blind Maini RN. Modulation of angiogenic vascular endothelial growth placebo controlled study. In: The International Psoriasis Sympo- factor by tumor necrosis factor ␣ and interleukin-1 in rheumatoid sium; Toronto, Ontario; June 10-14, 2004. arthritis. Arthritis Rheum 1998;41:1258–65. 125. Mease PJ, Antoni CE. Psoriatic arthritis treatment: biological response modifiers. Ann Rheum Dis 2005;64 Suppl 2:ii78–82. 139. Paleolog EM, Miotla JM. Angiogenesis in arthritis: role in 126. Teng GG, Turkiewicz AM, Moreland LW. Abatacept: a costimu- disease pathogenesis and as a potential therapeutic target. An- latory inhibitor for treatment of rheumatoid arthritis. Expert giogenesis 1998;2:295–307. Opin Biol Ther 2005;5:1245–54. 140. Veale DJ, Markham T, Fearon U, Rogers S, Golden-Mason L, 127. Abrams JR, Lebwohl MG, Guzzo CA, Jegasothy BV, Goldfarb Bresnihan B, et al. Therapy in psoriasis and psoriatic arthritis: ␣ MT, Goffe BS, et al. CTLA4Ig-mediated blockade of T-cell anti-TNF clinical and angiogenic responses [abstract]. Arthritis costimulation in patients with psoriasis vulgaris. J Clin Invest Rheum 2003;48 Suppl 9:S168. 1999;103:1243–52. 141. Margolin K, Gordon MS, Holmgren E, Gaudreault J, Novotny 128. Gibbs A, Gogarty M, Veale D, Bresnihan B, FitzGerald O. W, Fyfe G, et al. Phase Ib trial of intravenous recombinant Efficacy of anakinra (Kineret) in psoriatic arthritis, a clinical and humanized monoclonal antibody to vascular endothelial growth immunohistological study. Ann Rheum Dis 2006;65 Suppl factor in combination with chemotherapy in patients with ad- 2:216–7. vanced cancer: pharmacologic and long-term safety data. J Clin 129. Ong PY, Hamid QA, Travers JB, Strickland I, Al Kerithy M, Oncol 2001;19:851–6. Boguniewicz M, et al. Decreased IL-15 may contribute to ele- 142. Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res vated IgE and acute inflammation in atopic dermatitis. J Immu- 2002;4 Suppl 3:S81–90. nol 2002;168:505–10. 143. Bongartz T, Coras B, Vogt T, Scholmerich J, Muller-Ladner U. 130. Ruckert R, Asadullah K, Seifert M, Budagian VM, Arnold R, Treatment of active psoriatic arthritis with the PPAR␥ ligand Trombotto C, et al. Inhibition of keratinocyte apoptosis by IL-15: pioglitazone: an open-label pilot study. Rheumatology (Oxford) a new parameter in the pathogenesis of psoriasis? J Immunol 2005;44:126–9. 2000;165:2240–50. 144. Ribbens C, Martin y Porras M, Franchimont N, Kaiser MJ, 131. McInnes IB. Cytokine targeting in psoriasis and psoriatic arthri- Jaspar JM, Damas P, et al. Increased matrix metalloproteinase-3 tis: beyond TNF␣. Ernst Schering Res Found Workshop 2006; serum levels in rheumatic diseases: relationship with synovitis and 56:29–44. steroid treatment. Ann Rheum Dis 2002;61:161–6. 132. McInnes IB, Gracie JA. Interleukin-15: a new cytokine target for 145. Fraser A, Fearon U, Reece R, Emery P, Veale DJ. Matrix the treatment of inflammatory diseases. Curr Opin Pharmacol metalloproteinase 9, apoptosis, and vascular morphology in early 2004;4:392–7. arthritis. Arthritis Rheum 2001;44:2024–8. 133. Wang P, Wu P, Siegel MI, Egan RW, Billah MM. Interleukin 146. Myers A, Lakey R, Cawston TE, Kay LJ, Walker DJ. Serum (IL)-10 inhibits nuclear factor ␬B (NF ␬B) activation in human MMP-1 and TIMP-1 levels are increased in patients with psori- monocytes: IL-10 and IL-4 suppress cytokine synthesis by differ- atic arthritis and their siblings. Rheumatology (Oxford) 2004;43: ent mechanisms. J Biol Chem 1995;270:9558–63. 272–6. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1067–1075 DOI 10.1002/art.22494 © 2007, American College of Rheumatology

Decoy Receptor 3 Expressed in Rheumatoid Synovial Fibroblasts Protects the Cells Against Fas-Induced Apoptosis

Shinya Hayashi, Yasushi Miura, Takayuki Nishiyama, Makoto Mitani, Koji Tateishi, Yoshitada Sakai, Akira Hashiramoto, Masahiro Kurosaka, Shunichi Shiozawa, and Minoru Doita

Objective. Decoy receptor 3 (DcR3), a newly iden- ptosis in FLS. Down-regulation of DcR3 in FLS by tified member of the tumor necrosis factor receptor siRNA increased Fas-induced apoptosis. TNF␣ in- (TNFR) superfamily, is a soluble receptor that binds to creased DcR3 expression and inhibited Fas-induced members of the TNF family, including FasL, LIGHT, apoptosis in RA FLS, but not in OA FLS. and TNF-like molecule 1A. DcR3 is mostly expressed in Conclusion. DcR3 expressed in RA FLS is in- tumor cells, and it competitively inhibits binding of TNF creased by TNF␣ and protects the cells against Fas- to TNFRs. The present study was undertaken to inves- induced apoptosis. These findings indicate that DcR3 tigate DcR3 expression in fibroblast-like synoviocytes may be a possible therapeutic target in RA. (FLS) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the effects of DcR3 Rheumatoid arthritis (RA) is an inflammatory on Fas-induced apoptosis in RA FLS. joint disease characterized by hyperplasia of synovial Methods. Expression of DcR3 in FLS was mea- tissue and formation of pannus, which grows invasively sured by reverse transcriptase–polymerase chain reac- into the cartilage, causing cartilage and bone destruction (RT-PCR) and Western blotting. FLS were incu- tion. Analyses of hyperplastic synovial tissue from pa- bated with DcR3–Fc chimera protein or transfected with tients with RA have revealed features of transformed DcR3 small interfering RNA (siRNA) using the lipofec- long-living cells, such as the presence of somatic mutation method, before induction of apoptosis. Apoptosis tions, expression of oncogenes, and resistance to apo- induced by Fas in FLS was detected with TUNEL ptosis (1–4). Resistance to apoptosis further contributes staining and Western blotting of caspase 8 and to synovial hyperplasia and is closely linked to the poly(ADP-ribose) polymerase. Finally, FLS were incu- invasive phenotype of synovial fibroblasts (5,6). bated with TNF␣ prior to Fas-induced apoptosis, ex- Decoy receptor 3 (DcR3)/TR6/M68 is a recently pression of DcR3 was analyzed by quantitative RT-PCR, identified member of the tumor necrosis factor receptor and apoptosis was measured. (TNFR) superfamily (7). DcR3 lacks the transmem- Results. DcR3 was expressed in both RA FLS and brane domain of conventional TNFRs and is believed to OA FLS. DcR3–Fc protein inhibited Fas-induced apo- be a secreted protein (7). It is overexpressed mainly in tumor cells, including lung and colon cancers (7), glio- Supported in part by the Japan Society for the Promotion of mas, gastrointestinal tract tumors (8), and virus- Science (grant-in-aid for scientific research 17591572). associated leukemia (9). In addition, DcR3 is expressed Shinya Hayashi, MD, Yasushi Miura, MD, PhD, Takayuki Nishiyama, MD, PhD, Makoto Mitani, MD, PhD, Koji Tateishi, MD, in some normal tissue, including the colon, stomach, Yoshitada Sakai, MD, PhD, Akira Hashiramoto, MD, PhD, Masahiro spleen, lymph node, spinal cord, pancreas, and lung Kurosaka, MD, PhD, Shunichi Shiozawa, MD, PhD, Minoru Doita, (7,8); however, it is not expressed in human fibroblast MD, PhD: Kobe University School of Medicine, Kobe, Japan. Address correspondence and reprint requests to Yasushi NIH3T3 cells (10), and its expression in RA fibroblast- Miura, MD, PhD, Division of Orthopedic Sciences, Faculty of Health like synoviocytes (FLS) has not been investigated pre- Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, viously. Suma, Kobe 654-0142, Japan. E-mail: [email protected]. Submitted for publication January 30, 2006; accepted in Overexpression of DcR3 in tumors might be revised form December 21, 2006. beneficial in terms of protecting against the cytotoxic

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and regulatory effects of FasL (7), LIGHT (11), and TNF-like molecule 1A (12). Both rheumatoid synovial cells and tumor cells are resistant to apoptosis, and rheumatoid synovial cells proliferate aggressively in the same manner as tumors. Hence, we speculated that DcR3 might contribute to the pathogenesis of RA by down-regulating apoptosis in RA FLS. In this study, we investigated the expression of DcR3 in RA FLS and its effects on Fas-induced apoptosis. Our results suggest that DcR3 might be a therapeutic target in RA. MATERIALS AND METHODS Isolation and culture of synovial fibroblasts. FLS from 19 patients with RA fulfilling the criteria of the American College of Rheumatology (formerly, the American Rheuma- tism Association) (13) were obtained during total knee replacement surgery, in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. FLS from 14 patients with osteoarthritis (OA) were obtained in a similar manner. Tissue specimens were minced and digested in Dul- becco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) containing 0.2% collagenase (Sigma, St. Louis, MO) for 2 hours at 37°C. Dissociated cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD) and 100 units/ml of Figure 1. a, Reverse transcriptase–polymerase chain reaction (RT- penicillin/streptomycin. After overnight culture, nonadherent PCR) analysis of the expression of decoy receptor 3 (DcR3) mRNA in cells were removed, and adherent cells were further incubated fibroblast-like synoviocytes (FLS). FLS from each rheumatoid arthritis in fresh medium. All experiments were conducted using cells (RA) patient and each osteoarthritis (OA) patient expressed DcR3 from passages 3–4 (14). mRNA. Numbers below the panels are the expression ratios of DcR3 Reverse transcriptase–polymerase chain reaction (RT- mRNA (quantified by real-time PCR) versus RA FLS sample 1. PCR) analysis. RA FLS and OA FLS were cultured in 6-well Values were normalized to GAPDH expression. b, Western blot plates with various stimulants, and RNA was extracted with a analysis of the expression of DcR3 protein in the cytoplasm of RA FLS QIAshredder and RNeasy Mini kit according to the protocol and OA FLS. FLS from each RA patient and each OA patient of the manufacturer (Qiagen, Hilden, Germany). One micro- expressed DcR3 protein. c, Western blot analysis of the expression of gram of total RNA was reverse-transcribed to first-strand DcR3 protein in the supernatant of RA FLS and OA FLS. FLS from complementary DNA with 1.25 ␮M oligo(dT) primer in 40 ␮l each RA patient and each OA patient secreted DcR3 protein in the PCR buffer II containing 2.5 mM MgC12, 0.5 mM dNTP supernatant. Each lane in a–c represents 1 patient. mixture, 0.5 units RNase inhibitor, and 1.25 units Moloney murine leukemia virus RT (PerkinElmer, Foster City, CA), for

60 minutes at 42°C. The PCR buffer contained 1.5 mM MgC12, the supernatants were concentrated 20 times using Centriplus 0.2 mM dNTP mixture, 0.5 ␮M sense and antisense primer, 1.5 (Millipore, Bedford, MA). The concentrated supernatants units AmpliTaq Gold DNA polymerase, and 1 ␮lofRT were incubated for 1 hour with protein G–agarose beads and reaction mixture in 20 ␮l PCR buffer II. Thermal cycling centrifuged prior to incubation with antibody to eliminate conditions for DcR3 consisted of 40 cycles of denaturation at proteins nonspecifically binding to protein G. The superna- 93°C for 30 seconds, annealing at 58°C for 60 seconds, and tants were then incubated overnight with rabbit anti-human extension at 72°C for 45 seconds. The primers used were DcR3 polyclonal antibody (Santa Cruz Biotechnology, Santa 5Ј-TCAATGTGCCAGGCTCTTC-3Ј (sense) and 5Ј- Cruz, CA). The beads were then washed 3 times with hypo- GCCTCTTGATGGAGATGTCC-3Ј (antisense). tonic lysis buffer, and 3ϫ electrophoresis sample buffer was Cell fractionation and immunoprecipitation. For iso- added (Bio-Rad, Hercules, CA). lation of cytoplasmic proteins, adherent cells were washed 3 Western blotting. Cytoplasmic proteins and concen- times with phosphate buffered saline (PBS) and lysed in trated supernatants were quantified with protein assay reagent hypotonic lysis buffer (25 mM Tris, 1% Nonidet P40, 150 mM (Bio-Rad) by the Bradford method, and diluted to equal NaCl, 1.5 mM EGTA) supplemented with protease and phos- concentrations with hypotonic buffer. Each sample was mixed phatase inhibitor mix (Roche Diagnostics, Basel, Switzerland) with 3ϫ electrophoresis sample buffer, electrophoresed on on ice for 20 minutes. The lysates were centrifuged for 20 7.5–15% polyacrylamide gradient gels (Biocraft, Tokyo, Ja- minutes at 15,000 revolutions per minute to remove cellular pan), and transblotted electrically onto the blotting membrane debris, and the supernatants were collected. For detection of (Amersham Biosciences, Arlington Heights, IL). Expression of DcR3 in the supernatant, FLS were maintained in Opti-MEM DcR3 protein was detected using rabbit anti-human DcR3 (Invitrogen, Carlsbad, CA) without serum for 48 hours, and polyclonal antibody (R&D Systems, Minneapolis, MN) and DcR3 INHIBITION OF FAS-INDUCED APOPTOSIS IN RA FLS 1069

Figure 2. Effects of DcR3 small interfering RNA (siRNA) on DcR3 expression in FLS. a and b, DcR3 mRNA levels in RA FLS (a) and OA FLS (b), analyzed by real-time PCR. Values were normalized to GAPDH expression and are the mean and SD of 5 individual samples per group. DcR3 mRNA expression in RA FLS and OA FLS was significantly down-regulated at 0.5, 1, 2, 6, and 12 hours after transfection with DcR3 siRNA, compared with that observed after transfection with control siRNA (P Ͻ 0.01). c and d, DcR3 protein expression in RA FLS (c) and OA FLS (d), determined by Western blot analysis. DcR3 protein expression in RA FLS and OA FLS was inhibited by transfection with DcR3 siRNA, compared with that observed after transfection with control siRNA. Numbers in boxes below the panels are the ratios of DcR3 expression obtained with DcR3 siRNA treatment to that obtained with control siRNA treatment. Results shown are representative of 5 independent experiments. See Figure 1 for other definitions. horseradish peroxidase (HRP)–conjugated goat anti-rabbit diluted in 1 ml of Opti-MEM. The diluted mixture was applied IgG antibody (Amersham Biosciences), and visualized using to 3 ϫ 105 FLS rinsed with Opti-MEM. After incubation for 6 ECL Plus reagent (Amersham Biosciences) and a Chemilu- hours, the mixture was replaced with DMEM supplemented mino analyzer LAS-3000 mini (Fuji, Tokyo, Japan). Fas- with 10% FBS and incubated for 0.5, 1, 2, 6, or 12 hours before induced apoptotic signals were confirmed by detection of RNA extraction for real-time RT-PCR. full-length and cleaved caspase 8 (15) and poly(ADP-ribose) Quantification of DcR3 messenger RNA (mRNA) ex- polymerase (PARP) (16). Expression of full-length and pression. The relative levels of mRNA encoding DcR3 in FLS cleaved caspase 8 was detected using mouse anti-human were compared by TaqMan real-time PCR using an ABI Prism caspase 8 monoclonal antibody (Cell Signaling Technology, 7700 sequence detection system (Applied Biosystems, Foster Beverly, MA) and conjugated sheep anti-mouse IgG antibody City, CA). Predesigned primers and probes for human DcR3 (Amersham Biosciences). Expression of full-length and and human GAPDH as control were obtained from Applied cleaved PARP was detected using rabbit anti-human PARP Biosystems. polyclonal antibody (Cell Signaling Technology) and HRP- Fas-induced apoptosis in FLS pretreated with cyclo- conjugated goat anti-rabbit IgG antibody. Protein expression heximide. FLS were incubated with 100 ␮g/ml cycloheximide was determined by semiquantification of digitally captured (Wako, Osaka, Japan) for 12 hours, followed by incubation images using the public-domain NIH Image program (http:// with 100 ng/ml recombinant human FasL (rHuFasL; R&D rsb.info.nih.gov/nih-image/). Values were normalized to Systems) for an additional 12 hours. ␣-tublin expression. Pretreatment of FLS with DcR3–Fc protein before Transfection of DcR3 small interfering RNA (siRNA) apoptosis induction. FLS were pretreated with 0.1 ng/ml, 1 into FLS. DcR3 siRNA and nonspecific siRNA control (both ng/ml, or 10 ng/ml recombinant human DcR3–Fc chimera from Dharmacon, Chicago, IL) were transfected into FLS protein (DcR3–Fc; R&D Systems) for 24 hours in DMEM using Lipofectamine Plus reagent according to the protocol of supplemented with 10% FBS, and cells were washed 3 times the manufacturer (Invitrogen), with minor modifications. with PBS. Before induction of apoptosis, FLS were cultured Briefly, 100 nM siRNA was formulated with liposomes and for 24 hours in Opti-MEM. 1070 HAYASHI ET AL

Figure 3. Effects of cycloheximide (CHX), recombinant human FasL (hFasL), and DcR3–Fc chimera protein on Fas-induced apoptosis. a and b, TUNEL-positive apoptotic cells were not increased when RA FLS (a)orOAFLS (b) were incubated with 100 ␮g/ml CHX alone for 24 hours or with 100 ng/ml hFasL alone for 12 hours. However, TUNEL-positive apoptotic cells were significantly increased when FLS were incubated with 100 ␮g/ml CHX for 12 hours followed by incubation with 100 ng/ml hFasL for an additional 12 hours. Furthermore, TUNEL-positive apoptotic cells induced by treatment with CHX plus FasL were significantly and dose-dependently decreased by treatment with DcR3–Fc protein. In each experiment, at least 300 cells were counted by a blinded observer. Values are the mean and SD percent TUNEL-positive cells in FLS from 5 individual patients per group. c, Western blot analysis of the induction of caspase 8 and poly(ADP-ribose) polymerase (PARP) cleavage in RA FLS. Treatment with the combination of CHX and hFasL induced cleavage of caspase 8 and PARP; this cleavage was inhibited by treatment with 100 ng/ml DcR3–Fc protein. Numbers in boxes below the panels are the ratios of expression of cleaved caspase 8 and PARP obtained with 100 ng/ml DcR3–Fc treatment to those obtained with culture media only. Results shown are representative of 5 independent experiments. See Figure 1 for other definitions.

Treatment of FLS with TNF␣ protein. FLS were expressed in each individual RA FLS and OA FLS treated with 1 ng/ml or 10 ng/ml rHuTNF␣ (R&D Systems) for sample (Figure 1a). Western blotting confirmed that 24 hours in DMEM supplemented with 10% FBS. DcR3 protein was expressed in the cytoplasm of RA TUNEL staining. FLS (2 ϫ 105) were cultured in 8-well chamber slides (Nunc, Roskilde, Denmark). After ad- FLS and OA FLS (Figure 1b) and revealed secretion of dition of various stimulants, the cultured FLS were fixed for 10 DcR3 protein in the RA FLS and OA FLS culture minutes with 4% neutral buffered formalin, and apoptotic cells supernatants (Figure 1c). were determined using a TUNEL assay kit, according to the DcR3 siRNA–induced reduction of DcR3 expres- protocol of the manufacturer (Wako). sion in FLS. Time-course experiments showed that the Statistical analysis. Data are expressed as the mean Ϯ SD. For normally distributed data, Student’s 2-tailed t-test was expression of DcR3 mRNA in RA FLS and OA FLS was used for comparisons between groups. P values less than 0.05 significantly decreased as early as 0.5 hours after trans- were considered significant. fection with DcR3 siRNA, and remained decreased for at least 12 hours (Figures 2a and b). Western blotting RESULTS experiments confirmed that the expression of DcR3 DcR3 mRNA expression and protein production protein in the cytoplasm was also decreased 6 hours after in FLS, and secretion in the culture supernatant. RT- transfection with DcR3 siRNA. The expression of DcR3 PCR and real-time PCR revealed that DcR3 mRNA was protein was inhibited by DcR3 siRNA to 5% of that DcR3 INHIBITION OF FAS-INDUCED APOPTOSIS IN RA FLS 1071

Figure 4. Effects of DcR3 small interfering RNA (siRNA) on Fas-induced apoptosis. a and b, TUNEL-positive cells were significantly increased in cycloheximide plus recombinant human FasL–treated RA FLS (a) and OA FLS (b) when DcR3 siRNA was introduced. Values are the mean and SD percent TUNEL-positive cells in FLS from 5 individual patients per group. c and d, Western blot analysis of the induction of caspase 8 and poly(ADP-ribose) polymerase (PARP) cleavage in RA FLS (c) and OA FLS (d). Caspase 8 and PARP cleavage in both RA FLS and OA FLS was increased when DcR3 siRNA was introduced. Numbers in boxes below the panels are the ratios of expression of cleaved caspase 8 and PARP obtained with DcR3 siRNA treatment to those obtained with control siRNA treatment. Results shown are representative of 5 independent experiments. See Figure 1 for other definitions.

observed with control siRNA in RA FLS (Figure 2c), manner, when FLS were preincubated with DcR3–Fc and to 4% of that observed with control siRNA in OA (Figures 3a and b). Cleavage of caspase 8 and PARP was FLS (Figure 2d). also inhibited in RA FLS (Figure 3c). The cleavage of Failure of rHuFasL alone or cycloheximide alone caspase 8 was inhibited to 6% of control and the to induce apoptosis in FLS. Compared with findings in cleavage of PARP was completely inhibited by pretreat- untreated FLS, the number of Fas-induced TUNEL- ment with 10 ng/ml DcR3–Fc. positive apoptotic cells was not significantly increased Down-regulation of endogenous DcR3 by siRNA when RA FLS or OA FLS were treated with rHuFasL increases Fas/cycloheximide-induced apoptosis in FLS. alone or cycloheximide alone. However, cycloheximide Compared with control, the number of TUNEL-positive sensitized FLS to Fas-induced apoptosis (Figures 3a and apoptotic cells induced by rHuFasL and cycloheximide b). Cleavage of caspase 8 and PARP was not increased was significantly increased in RA FLS (Figure 4a) and when RA FLS were treated with rHuFasL alone or OA FLS (Figure 4b) when DcR3 siRNA was introduced. cycloheximide alone. However, cleavage of caspase 8 Cleavage of caspase 8 and PARP was also increased and PARP was increased when RA FLS were treated (4.7- and 6.0-fold, respectively, in RA FLS and 4.4- and with both rHuFasL and cycloheximide (Figure 3c). 8.5-fold, respectively, in OA FLS) when DcR3 siRNA Inhibition of Fas/cycloheximide-induced apopto- was introduced (Figures 4c and d). sis in FLS by DcR3–Fc protein. TUNEL-positive apo- Induction of DcR3 expression by rHuTNF␣ in ptotic cells induced by rHuFasL and cycloheximide in RA FLS, but not in OA FLS. Real-time PCR showed FLS were significantly decreased, in a dose-dependent that the expression of DcR3 mRNA was significantly 1072 HAYASHI ET AL

Figure 5. Effects of tumor necrosis factor ␣ (TNF␣) on DcR3 expression in FLS. a and b, DcR3 mRNA levels in RA FLS (a) and OA FLS (b), analyzed by real-time PCR. Values are the mean and SD of 5 individual samples per group. DcR3 mRNA expression in RA FLS was significantly and dose-dependently increased by 24-hour treatment with recombinant human TNF␣ (hTNF␣), whereas expression of DcR3 mRNA in OA FLS was not increased by treatment with TNF␣. c and d, DcR3 protein expression in TNF␣-treated RA FLS (c) and OA FLS (d), determined by Western blot analysis. DcR3 protein expression was increased in RA FLS, but not in OA FLS, after 24-hour incubation with TNF␣. Numbers in boxes below the panels are the ratios of expression of DcR3 obtained with TNF␣ treatment to that obtained with culture media only. Results shown are representative of 5 independent experiments. See Figure 1 for other definitions. increased when RA FLS were treated for 24 hours with tion with 1 or 10 ng/ml hTNF␣ (Figure 6a), and cleavage Ն1 ng/ml of rHuTNF␣ (Figure 5a). However, the ex- of caspase 8 and PARP in OA FLS was not affected by pression of DcR3 mRNA was not increased when OA preincubation with 10 ng/ml rHuTNF␣ (Figure 6c). FLS were treated with rHuTNF␣, even at 10 ng/ml (Figure 5b). Western blotting experiments confirmed DISCUSSION that expression of DcR3 protein followed the same pattern as DcR3 mRNA expression (Figures 5c and d). In many cancers, a mechanism develops by which Inhibition of Fas/cycloheximide-induced apopto- cancer cells escape the immune surveillance of the hosts sis by rHuTNF␣ in RA FLS, but not in OA FLS. (17) and are protected against Fas-induced growth inhi- TUNEL-positive apoptotic cells induced by rHuFasL bition signals in spite of the expression of Fas (1). and cycloheximide in RA FLS were significantly de- Recent studies have provided evidence that DcR3 ex- creased, in a dose-dependent manner, when RA FLS pressed in cancer cells might play an important role in were preincubated with 1 ng/ml or 10 ng/ml rHuTNF␣ this mechanism. For example, Tsuji et al demonstrated (Figure 6a). Cleavage of caspase 8 and PARP in RA that DcR3 is highly expressed in many pancreatic can- FLS was also reduced by preincubation with 10 ng/ml cers and that endogenous DcR3 blocks the growth rHuTNF␣ (Figure 6b). However, TUNEL-positive apo- inhibition signals mediated by FasL (18). ptotic cells induced by rHuFasL and cycloheximide in Lymphocytes, macrophages, and plasma cells in- OA FLS were not significantly decreased by preincuba- filtrate the synovial tissue in RA (19). Both synoviocytes DcR3 INHIBITION OF FAS-INDUCED APOPTOSIS IN RA FLS 1073

FLS to apoptosis in the absence of Fas (22). Further- more, Fas-induced apoptosis has been found to vary in different FLS lines (22). Therefore, our results in which 100 ng/ml FasL plus 100 ␮g/ml cycloheximide induced apoptosis in ϳ60% of RA FLS are consistent with previous reports (22,23). In the present study, we demonstrated that DcR3 is expressed in FLS and that DcR3–Fc inhibits Fas- induced apoptosis in FLS. We further showed that the reduction of DcR3 in FLS by siRNA was associated with increased Fas-induced apoptosis. These results strongly suggest that DcR3 actually protects rheumatoid synovial cells against death via Fas-induced apoptosis. Although several antiapoptotic molecules, including Bcl-2 (24), sentrin 1 (25), very late activation antigen 5 (26), and FLIP (27), have been identified in inflamed rheumatoid synovial fibroblasts, it remains unclear as to why RA FLS are so resistant to apoptosis induction in vivo (28). Kim et al demonstrated that human intestinal epithelial cell lines selectively increase DcR3 release in response to lipopolysaccharide, and human dermal microvascular endothelial cells increase DcR3 release in response to proinflammatory cytokines such as TNF␣ and interleukin-1␤ (IL-1␤). Moreover, increased expression of DcR3 in appendix epithelia from Figure 6. Effects of tumor necrosis factor ␣ (TNF␣) on Fas-induced patients with acute appendicitis was demonstrated (29). apoptosis. a, TUNEL-positive cells were significantly decreased in RA We examined the expression of DcR3 mRNA in FLS compared with OA FLS when the FLS were preincubated with 1 RA and OA FLS by quantitative real-time PCR and ␣ ␣ ng/ml or 10 ng/ml recombinant human TNF (hTNF ) for 24 hours found that this expression was not significantly different before apoptosis was induced with cycloheximide (CHX) plus recombinant human FasL (hFasL). Values are the mean and SD of 5 between RA FLS and OA FLS when the cells were not individual samples per group (same samples as in Figures 5a and b). b stimulated. In contrast, DcR3 expression in RA FLS, but and c, Western blot analysis of caspase 8 and poly(ADP-ribose) not in OA FLS, was highly induced by TNF␣. Our polymerase (PARP) cleavage in RA FLS (b) and OA FLS (c). Caspase results therefore suggest that DcR3 release may be 8 and PARP cleavage in RA FLS was inhibited by 24-hour incubation ␣ ␣ selectively increased in RA FLS in response to proin- with 10 ng/ml TNF , but TNF treatment did not inhibit cleavage in ␣ OA FLS. Numbers in boxes below the panels are the ratios of flammatory cytokines including TNF . We further dem- expression of cleaved caspase 8 and PARP obtained with 10 ng/ml onstrated that preincubation with TNF␣ induces DcR3 TNF␣ treatment to that obtained with culture media only. Results expression and inhibits Fas/cycloheximide-induced apo- shown are representative of 5 independent experiments (using the ptosis in RA FLS but not OA FLS. same samples as in Figures 5c and d). See Figure 1 for other Rosengren et al measured TNF␣ in the superna- definitions. tant of cultured RA FLS and found the concentration to be 0.13 pg/ml (30). However, Ribbens and colleagues and lymphocytes in rheumatoid synovium express func- demonstrated that the concentration of TNF␣ in rheu- tional Fas antigen, and these cells are sensitive to a matoid synovial fluid was Ͼ100 pg/ml (31). Therefore, Fas-induced apoptosis in vitro (20,21). Despite this, in the concentration of TNF␣ in cultured supernatant may both RA and OA, DNA strand breaks have been not be enough to sensitize RA FLS to increase DcR3 observed mainly in synovial lining macrophages, but not expression, resulting in a lack of significant difference in FLS (19). Fas apoptosis signaling plays a role in defec- DcR3 expression between cultured RA FLS and OA tive down-modulation of the hyperimmune response FLS in vitro. Findings of several previous studies have observed in human autoimmune diseases such as RA also suggested that high concentrations of TNF␣ and (6). Cycloheximide sensitizes FLS to Fas-mediated apo- IL-1␤ can be detected in the serum of patients with RA, ptosis; however, cycloheximide alone does not sensitize and that the expression of IL-1␤, IL-6, IL-8, and TNF␣ 1074 HAYASHI ET AL

in serum is significantly different between RA and OA Statistical analysis. Hayashi, Miura, Sakai. (32). Responses of FLS to TNF␣ in terms of the Sample preparation. Nishiyama, Kurosaka, Doita. production of osteoprotegerin and proinflammatory angiopoietin 1 (33,34) also differ significantly in RA and REFERENCES OA, similar to our findings with DcR3. 1. Chou CT, Yang JS, Lee MR. Apoptosis in rheumatoid arthritis— Inhibition of Fas/cycloheximide-induced apopto- expression of Fas, Fas-L, p53, and Bcl-2 in rheumatoid synovial sis by TNF␣ was so potent that other antiapoptotic tissues. J Pathol 2001;193:110–6. molecules in addition to DcR3 might be simultaneously 2. Tak PP, Zvaifler NJ, Green DR, Firestein GS. Rheumatoid ␣ ␣ ␬ arthritis and p53: how oxidative stress might alter the course of induced by TNF . Indeed, TNF also stimulates NF- B inflammatory diseases. Immunol Today 2000;21:78–82. activation and protects cells against apoptosis through 3. Yamanishi Y, Boyle DL, Rosengren S, Green DR, Zvaifler NJ, expression of TNFR-associated factor in RA FLS (35). Firestein GS. Regional analysis of p53 mutations in rheumatoid arthritis synovium. Proc Natl Acad SciUSA2002;99:10025–30. However, DcR3 seems to play a substantial antiapop- 4. Shiozawa S, Shiozawa K, Fujita T. Morphologic observations in totic role in RA FLS, as a decoy receptor against Fas. the early phase of the cartilage–pannus junction: light and electron Furthermore, TNF␣ receptors are expressed in the microscopic studies of active cellular pannus. Arthritis Rheum 1983;26:472–8. synovium, and expression levels are higher in RA FLS 5. Baier A, Meineckel A, Gay S, Pap T. Apoptosis in rheumatoid than in OA FLS (36). Our results indicate that DcR3 arthritis. Curr Opin Rheumatol 2003;15:274–9. expression, under the control of proinflammatory 6. Mountz JD, Zhang HG. Regulation of apoptosis of synovial ␣ fibroblasts. Curr Dir Autoimmun 2001;3:216–39. TNF , is increased in the synovial tissue of patients with 7. Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd RA and that DcR3 in synovial tissue protects against P, et al. Genomic amplification of a decoy receptor for Fas ligand apoptosis induced by Fas. in lung and colon cancer. Nature 1998;396:699–703. 8. Bai C, Connolly B, Metzker ML, Hilliard CA, Liu X, Sandig V, et Recently, Hsu et al demonstrated that the Th2 al. Overexpression of M68/DcR3 in human gastrointestinal tract cell–biased phenotype in DcR3-transgenic mice could tumors independent of gene amplification and its location in a be attributed to decreased IL-2 secretion by T cells, four-gene cluster. Proc Natl Acad SciUSA2000;97:1230–5. ϩ 9. Ohshima K, Haraoka S, Sugihara M, Suzumiya J, Kawasaki C, resulting in the suppression of IL-2–dependent CD4 T Kanda M, et al. Amplification and expression of a decoy receptor cell proliferation (37). Although RA is considered a Th1 for fas ligand (DcR3) in virus (EBV or HTLV-I) associated cell–biased disease and a Th1 cell–biased phenotype is lymphomas. Cancer Lett 2000;160:89–97. 10. Chen J, Zhang L, Kim S. Quantification and detection of DcR3, a required for induction of the disease, after disease onset decoy receptor in TNFR family. J Immunol Methods 2004;285: the cytokine balance shifts to a Th2 bias (38,39). There- 63–70. fore, DcR3 may contribute to the pathogenesis of RA by 11. Yu KY, Kwon B, Ni J, Zhai Y, Ebner R, Kwon BS. A newly identified member of tumor necrosis factor receptor superfamily affecting not only rheumatoid synoviocytes, but also T (TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem 1999; lymphocytes. 274:13733–6. We suggest that DcR3 expressed in RA FLS is 12. Migone TS, Zhang J, Luo X, Zhuang L, Chen C, Hu B, et al. TL1A increased by TNF␣, making it one of the pathologic is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity 2002;16:479–92. factors that induces hyperplasia of rheumatoid syno- 13. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, vium. Thus, strategies aimed at down-regulation of Cooper NS, et al. The American Rheumatism Association 1987 DcR3 in FLS warrant further investigation as a possible revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. therapeutic approach in RA. 14. Mitani M, Miura Y, Saura R, Kitagawa A, Fukuyama T, Hash- iramoto A, et al. Estrogen specifically stimulates expression and production of osteoprotegerin from rheumatoid synovial fibro- ACKNOWLEDGMENTS blasts. Int J Mol Med 2005;15:827–32. 15. Santiago B, Galindo M, Palao G, Pablos JL. Intracellular regula- We thank Kyoko Tanaka, Minako Nagata, and Masako tion of Fas-induced apoptosis in human fibroblasts by extracellular Sakaguchi for technical assistance, and Janina Tubby for factors and cycloheximide. J Immunol 2004;172:560–6. English rewriting. 16. Itoh K, Hase H, Kojima H, Saotome K, Nhishioka K, Kobata T. Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts. Rheumatology (Oxford) 2004;43: AUTHOR CONTRIBUTIONS 277–85. Dr. Miura had full access to all of the data in the study and 17. Strand S, Galle PR. Immune evasion by tumours: involvement of takes responsibility for the integrity of the data and the accuracy of the the CD95 (APO-1/Fas) system and its clinical implications. Mol data analysis. Med Today 1998;4:63–8. Study design. Hayashi, Miura. 18. Tsuji S, Hosotani R, Yonehara S, Masui T, Tulachan SS, Nakajima Acquisition of data. Hayashi, Miura, Mitani, Tateish, Hashiramoto. S, et al. Endogenous decoy receptor 3 blocks the growth inhibition Analysis and interpretation of data. Hayashi, Miura. signals mediated by Fas ligand in human pancreatic adenocarci- Manuscript preparation. Hayashi, Miura, Shiozawa. noma. Int J Cancer 2003;106:17–25. 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19. Firestein GS. Evolving concepts of rheumatoid arthritis (review). decoy receptor 3 in acutely inflamed intestinal epithelia. Clin Nature 2003;423:356–61. Immunol 2005;115:286–94. 20. Nakajima T, Aono H, Hasunuma T, Yamamoto K, Shirai T, 30. Rosengren S, Firestein GS, Boyle DL. Measurement of inflamma- Hirohata K, et al. Apoptosis and functional Fas antigen in tory biomarkers in synovial tissue extracts by enzyme-linked rheumatoid arthritis synoviocytes. Arthritis Rheum 1995;38: immunosorbent assay. Clin Diagn Lab Immunol 2003;10:1002–10. 485–91. 31. Ribbens C, Andre B, Kaye O, Kaiser MJ, Bonnet V, Jaspar JM, et 21. Hoa TT, Hasunuma T, Aono H, Masuko K, Kobata T, Yamamoto al. Synovial fluid matrix metalloproteinase-3 levels are increased in K, et al. Novel mechanisms of selective apoptosis in synovial T inflammatory arthritides whether erosive or not. Rheumatology cells of patients with rheumatoid arthritis. J Rheumatol 1996;23: (Oxford) 2000;12:1357–65. 1332–7. 32. Tetta C, Camussi G, Modena V, Vittorio CD, Baglioni C. Tumour 22. Palao G, Santiago G, Galindo M, Paya M, Ramirez JC, Pablos JL. necrosis factor in serum and synovial fluid of patients with active Down-regulation of FLIP sensitizes rheumatoid synovial fibro- and severe rheumatoid arthritis. Ann Rheum Dis 1990;49:665–7. blasts to Fas-mediated apoptosis (published erratum appears in 33. Kubota A, Hasegawa K, Suguro T, Koshihara Y. Tumor necrosis Arthritis Rheum 2005;52:678). Arthritis Rheum 2004;50:2803–10. factor-␣ promotes the expression of osteoprotegerin in rheuma- 23. Knight MJ, Riffkin CD, Muscat AM, Ashley DM, Hawkins CJ. toid synovial fibroblasts. J Rheumatol 2004;31:426–35. Analysis of FasL and TRAIL induced apoptosis pathways in 34. Gravallese EM, Pettit AR, Lee R, Madore R, Manning C, Tsay A, glioma cells. Oncogene 2001;20:5789–98. et al. Angiopoietin-1 is expressed in the synovium of patients with 24. Matsumoto S, Muller-Ladner U, Gay RE, Nishioka K, Gay S. rheumatoid arthritis and is induced by tumour necrosis factor ␣. Ultrastructural demonstration of apoptosis, Fas and Bcl-2 expres- Ann Rheum Dis 2003;62:100–7. sion of rheumatoid synovial fibroblasts. J Rheumatol 1996;23: 35. Youn J, Kim HY, Park JH, Hwang SH, Lee SY, Cho CS, et al. 1345–52. Regulation of TNF-␣-mediated hyperplasia through TNF recep- 25. Franz JK, Pap T, Hummel KM, Nawrath M, Aicher WK, ␬ Shigeyama Y, et al. Expression of sentrin, a novel antiapoptotic tors, TRAFs, and NF- B in synoviocytes obtained from patients molecule, at sites of synovial invasion in rheumatoid arthritis. with rheumatoid arthritis. Immunol Lett 2002;83:85–93. Arthritis Rheum 2000;43:599–607. 36. Deleuran BW, Chu CQ, Field M, Brennan FM, Mitchell T, 26. Kitagawa A, Miura Y, Saura R, Mitani M, Ishikawa H, Hash- Feldmann M, et al. Localization of tumor necrosis factor receptors iramoto A, et al. Anchorage on fibronectin via VLA-5 (␣5␤1 in the synovial tissue and cartilage–pannus junction in patients integrin) protects rheumatoid synovial cells from Fas-induced with rheumatoid arthritis: implications for local actions of tumor ␣ apoptosis. Ann Rheum Dis 2006;65:721–7. necrosis factor . Arthritis Rheum 1992;35:1170–8. 27. Perlman H, Pagliari LJ, Liu H, Koch AE, Haines GK III, 37. Hsu TL, Wu YY, Chang YC, Yang CY, Lai MZ, Su WB, et al. Pope RM. Rheumatoid arthritis synovial macrophages express the Attenuation of Th1 response in decoy receptor 3 transgenic mice. Fas-associated death domain–like interleukin-1␤–converting J Immunol 2005;175:5135–45. enzyme–inhibitory protein and are refractory to Fas-mediated 38. Doncarli A, Stasiuk LM, Fournier C, Abehsira AO. Conversion in apoptosis. Arthritis Rheum 2001;44:21–30. vivo from an early dominant Th0/Th1 response to a Th2 pheno- 28. Ceponis A, Hietanen J, Tamulaitiene M, Partsch G, Patiala H, type during the development of collagen-induced arthritis. Eur Konttinen YT. A comparative quantitative morphometric study of J Immunol 1997;27:1451–8. cell apoptosis in synovial membranes in psoriatic, reactive and 39. Yoshino S. Effect of a monoclonal antibody against interleukin-4 rheumatoid arthritis. Rheumatology (Oxford) 1999;38:431–40. on collagen-induced arthritis in mice. Br J Pharmacol 1998;123: 29. Kim S, Fotiadu A, Kotoura V. Increased expression of soluble 237–42. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1076–1086 DOI 10.1002/art.22439 © 2007, American College of Rheumatology

Up-Regulation of Stromal Cell–Derived Factor 1 (CXCL12) Production in Rheumatoid Synovial Fibroblasts Through Interactions With T Lymphocytes Role of Interleukin-17 and CD40L–CD40 Interaction

Kyoung-Woon Kim,1 Mi-La Cho,1 Hae-Rim Kim,2 Ji-Hyeon Ju,1 Mi-Kyung Park,1 Hye-Jwa Oh,1 Joon-Seok Kim,1 Sung-Hwan Park,1 Sang-Heon Lee,2 and Ho-Youn Kim1

Objective. Stromal cell–derived factor 1 (SDF-1) activator protein 1 (AP-1). When FLS were cocultured is a potent chemoattractant for memory T cells in with T cells, SDF-1 production was up-regulated, espe- inflamed rheumatoid arthritis (RA) synovium. This cially in the presence of IL-17, but FLS were inhibited by study was undertaken to investigate the effect of neutralizing anti–IL-17 and anti-CD40L antibodies. Ad- interleukin-17 (IL-17) and CD40–CD40L interaction on dition of RA SF to cultured RA FLS significantly SDF-1 production in RA fibroblast-like synoviocytes up-regulated SDF-1 messenger RNA expression, which (FLS). was hampered by pretreatment with anti–IL-17 anti- Methods. Synovial fluid (SF) and serum levels of body. SDF-1 in RA patients were measured by enzyme-linked Conclusion. SDF-1 is overproduced in RA FLS, immunosorbent assay (ELISA). The SDF-1 produced by and IL-17 could up-regulate the expression of SDF-1 in cultured RA FLS was evaluated by real-time polymerase RA FLS via pathways mediated by PI 3-kinase, NF-␬B, chain reaction and ELISA after FLS were treated with and AP-1. Our findings suggest that inhibition of the IL-17 and inhibitors of intracellular signal molecules. interaction between IL-17 from T cells and SDF-1 in The SDF-1 level was also determined after FLS were FLS may provide a new therapeutic approach in RA. cocultured with T cells in the presence and absence of IL-17. Rheumatoid arthritis (RA) is a systemic inflam- Results. Concentrations of SDF-1 in the sera and matory disease characterized by synovial inflammation SF were higher in RA patients than in osteoarthritis and progressive joint destruction. Although the definite patients, although the increase in the serum levels did etiology remains unknown, the complex and delicate not reach statistical significance. The production of networks of various inflammatory cells, cytokines, and SDF-1 in RA FLS was enhanced by IL-17 stimulation. chemokines may play a central role in the pathogenesis This effect of IL-17 was blocked by inhibitors of phos- of RA. RA synovium is characterized by the infiltration phatidylinositol 3-kinase (PI 3-kinase), NF-␬B, and of T cells and monocytes as well as the proliferation of synoviocytes. These cells costimulate each other, resulting in a vicious circle of synovial inflammation. Supported by the Korea Science and Engineering Foundation The predominant T cell subset in the sublining of through the Rheumatism Research Center at Catholic University of ϩ Korea (grant R11-2002-07003-0). RA synovium is CD4 cells. The majority of these 1Kyoung-Woon Kim, MS, Mi-La Cho, PhD, Ji-Hyeon Ju, CD4ϩ T cells are mature memory CD45ROϩ T cells MD, Mi-Kyung Park, MS, Hye-Jwa Oh, MD, Joon-Seok Kim, MD, (1). The migration of T cells, B cells, and monocyte/ Sung-Hwan Park, MD, Ho-Youn Kim, MD: Catholic University of Korea, Seoul, Korea; 2Hae-Rim Kim, MD, Sang-Heon Lee, MD: macrophages into the inflamed synovium is promoted by Konkuk University Hospital, Seoul, Korea. a chemokine, stromal cell–derived factor 1 (SDF-1; Drs. Kyoung-Woon Kim and Cho contributed equally to this CXCL12). SDF-1 is produced by bone marrow stromal work. Address correspondence and reprint requests to Sang-Heon cells, fibroblast-like synoviocytes (FLS), macrophages, Lee, MD, Department of Internal Medicine, Konkuk University and endothelial cells in RA. The concentration of SDF-1 Hospital, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, Korea. is elevated in plasma and synovial fluid (SF) specimens E-mail: [email protected]. Submitted for publication April 18, 2006; accepted in revised from patients with RA (2,3), and overexpression of form December 6, 2006. SDF-1 is also observed in RA synovial tissue (4,5).

1076 IL-17 INDUCES SDF-1 PRODUCTION IN RA FLS 1077

SDF-1 promotes CD4ϩ memory T cell recruit- SDF-1 production. In addition, we attempted to identify ment and accumulation (6) and monocyte migration into which signaling pathways are involved in the reciprocal synovium (7) and induces pseudoemperipolesis (e.g., effect of synovial SDF-1 on T cell migration into the migration into synovium) of both T and B cells through inflamed synovium. a vascular cell adhesion molecule 1–dependent mechanism (8). In addition, SDF-1 can promote joint destruc- PATIENTS AND METHODS tion by inducing angiogenesis through immobilization of Patients. SF and sera were obtained from 20 RA endothelial cells on heparin sulfate molecules and by patients fulfilling the 1987 revised criteria of the American inducing angiogenesis via activation of chondrocytes and College of Rheumatology (formerly, the American Rheuma- osteoclasts through matrix metalloproteinase 3 tism Association) (23). Twenty age- and sex-matched patients (MMP-3) and MMP-9, respectively (5). Inhibition of with osteoarthritis (OA) were studied as controls. Informed consent was obtained from all patients, and the experimental SDF-1 decreases the development of collagen-induced protocol was approved by the Catholic University of Korea arthritis (CIA) (9), and the inhibitor of CXCR4, which is Human Research Ethics Committee. Synovial tissue specimens an SDF-1 receptor, also reduces the incidence and were isolated from 8 additional RA patients with a mean Ϯ clinical severity of CIA (10). SDF-1 potentially plays an SEM age of 63.4 Ϯ 4.6 years (range 38–76 years) who were important role in inflammatory response, neovascular- undergoing total knee replacement surgery. ization, and joint destruction in the pathogenesis of RA. Reagents. Recombinant IL-17, CD40L, macrophage migration inhibitory factor (MIF), and anti–IL-17 neutralizing SDF-1 is generally known to be mainly a stimu- antibody were purchased from R&D Systems (Minneapolis, lating chemokine, and investigators have focused on its MN), recombinant TNF␣ and IL-1␤ from Endogen (Cam- role in the stimulation and induction of other molecules. bridge, MA), and concanavalin A (Con A) from Sigma (St. Therefore, the regulation and induction mechanism of Louis, MO). Pyrrolidine dithiocarbamate (PDTC), curcumin, SDF-1 remains unknown. It has been demonstrated that and parthenolide were obtained from Sigma. LY294002, wort- hypoxia enhances the expression of SDF-1 messenger mannin, PD98059, JNK inhibitor, SB203580, and SP600125 were obtained from Calbiochem (Schwalbach, Germany). RNA (mRNA) in synovial fibroblasts, but cytokines such Isolation of FLS. Synoviocytes were isolated by enzy- as transforming growth factor ␤, interleukin-1␤ (IL-1␤), matic digestion of synovial tissue specimens obtained from and tumor necrosis factor (TNF) do not affect SDF-1 patients with RA and patients with OA undergoing total joint production (11). replacement surgery. The tissue samples were minced into IL-17, a major T cell–derived proinflammatory 2–3-mm pieces and treated for 4 hours with 4 mg/ml of type I collagenase (Worthington, Freehold, NJ) in Dulbecco’s mod- cytokine in RA synovium, stimulates FLS to produce ified Eagle’s medium (DMEM) at 37°C in 5% CO2. Dissoci- inflammatory cytokines and chemokines (12). Previ- ated cells were then centrifuged at 500g, resuspended in ously, we observed reciprocal activation of antigen- DMEM supplemented with 10% fetal calf serum (FCS), 2 mM stimulated T cells and FLS from RA patients, where L-glutamine, 100 units/ml of penicillin, and 100 ␮g/ml of IL-17 production from T cells increased upon coculture streptomycin, and plated in 75-cm2 flasks. After overnight with FLS (13). Synovial tissue and FLS express IL-17 culture, the nonadherent cells were removed, and the adherent cells were cultivated in DMEM supplemented with 20% FCS. receptors (14,15), and FLS have the potential to respond The cultures were kept at 37°C in 5% CO2, and the medium to IL-17 produced by activated T cells. CD40L, a was replaced every 3 days. When the cells approached conflu- member of the TNF superfamily, is a 30–33-kd type II ence, they were passaged after dilution (1:3) with fresh me- transmembrane protein expressed on activated T cells, dium. mast cells, basophils, and eosinophils (16,17). It has been Synoviocytes from passages 4–8 were used in each experiment. The cells were morphologically homogeneous and reported that stimulation with CD40L-expressing cells exhibited the appearance of synovial fibroblasts, with typical or purified recombinant CD40L induces the secretion of bipolar configuration under inverse microscopy. The purity of proinflammatory cytokines such as IL-1, IL-6, IL-8, and cells (1 ϫ 104) was tested by flow cytometric analysis using TNF␣ from monocytes, dendritic cells, epithelial cells, phycoerythrin (PE)–conjugated anti-CD14 (PharMingen, San and fibroblasts, and augments the expression of adhe- Diego, CA) and fluorescein isothiocyanate–conjugated anti- CD3 or anti–Thy-1 (CD90) monoclonal antibodies (PharMin- sion molecules and metalloproteinases (18–22). gen). At passage 4, most cells (Ͼ95%) expressed the surface In this study, we hypothesized that the interaction markers for fibroblasts (CD90ϩ), whereas 3.5% of the cells between the T cell–derived cytokine IL-17 and FLS may were CD14ϩ, and Ͻ1% of the cells were CD3ϩ. -affect the production of SDF-1 by FLS through some CD4؉ T cell isolation by magnetic-activated cell sort distinct pathway. We assessed whether IL-17 (a repre- ing (MACS). Anti-CD4 microbeads were used according to the recommendations of the manufacturer (Miltenyi Biotec, sentative cytokine from activated T cells) and physical Sunnyvale, CA) (24). Peripheral blood mononuclear cells were interplay between FLS and T cells through CD40– resuspended in 80 ␮l FCS staining buffer. Anti-CD4 mi- CD40L interaction have a role in the regulation of crobeads (20 ␮l) were added and incubated for 15 minutes at 1078 KIM ET AL

6–12°C. Saturating amounts of fluorochrome-conjugated anti- standard, ranging from 10 pg/ml to 2,000 pg/ml. A standard bodies were added, and cells were incubated for an additional curve was drawn by plotting OD versus the log of the concen- 10 minutes. Cells were diluted in 2.5 ml 2% FCS staining tration of recombinant cytokine. buffer, pelleted, resuspended in 500 ␮l FCS, and magnetically Expression of SDF-1 mRNA determined by reverse separated, usually on an AutoMACS magnet (Miltenyi Biotec, transcriptase–polymerase chain reaction (RT-PCR). FLS were Bergisch Gladbach, Germany) fitted with a MACS mass incubated with various concentrations of IL-17 in the presence spectrophotometry column. Flow-through and two 1-ml or absence of various signal inhibitors (LY294002, wortman- washes were collected as the negative fraction. Enriched cells nin, SB203580, PD98059, JNK inhibitor, curcumin, SP600125, were collected in two 0.5-ml aliquots from the column after PDTC, and parthenolide). After 12 hours of incubation, removal from the magnet. Alternatively, cells stained with mRNA was extracted using RNAzol B, according to the PE-conjugated anti-CD4 were washed, magnetically labeled recommendations of the manufacturer (Biotecx, Houston, with anti-PE microbeads (20 ␮l added to an 80-␮l cell suspen- TX). Reverse transcription of 2 ␮g total mRNA was carried sion for 15 minutes at 6–12°C), and magnetically separated as out at 42°C using the Superscript reverse transcription system described above. The purity of cells was assessed by flow (Takara, Shiga, Japan). PCR amplification of complementary cytometric analysis of stained cells on a FACSVantage sorter. DNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 Most of the isolated cells (Ͼ97%) exhibited the CD4 T cell units Taq DNA polymerase (Takara), and 0.25 ␮M sense and marker. antisense primers. The reaction took place in 25 ␮lofPCR

Coculture of RA T cells and FLS. The FLS were buffer, consisting of 1.5 mM MgCl2,50mM KCl, and 10 mM seeded in 24-well plates at 5 ϫ 104 cells/well in 1 ml serum-free Tris HCl (pH 8.3). DMEM/insulin–transferrin–selenium A (Life Technologies, The following primers were used for each molecule: Gaithersburg, MD). Subsequently, CD4ϩ T cells (5 ϫ 105 for SDF-1, 5Ј-ATG-AAC-GCC-AAG-GTC-GTG-GTC-3Ј cells/well) were added to the FLS monolayers, and the culture (sense) and 5Ј-TGG-CTG-TTG-TGC-TTA-CTT-GTT-T-3Ј plates were incubated at a ratio of FLS to T cells of 1:10 for 48 (antisense); for GAPDH, 5Ј-CGA-TGC-TGG-GCG-TGA- hours alone or with 10–100 ng/ml of IL-17. The culture GTA-C-3Ј (sense) and 5Ј-CGT-TCA-GCT-CAG-GGA-TGA- supernatants were collected and stored at Ϫ20°C until assayed. CC-3Ј (antisense). Reactions were processed in a DNA ther- All cultures were set up in triplicate. In some experiments, mal cycler (PerkinElmer Cetus, Wellesley, MA) through 25 neutralizing monoclonal antibodies to IL-17, CD40L, cycles of 30 seconds of denaturation at 94°C, 1 minute of interferon-␥ (all from R&D Systems), or isotype-matched annealing at 56°C, followed by 30 seconds of elongation at mouse IgG1 were added to the cocultures for 48 hours. 72°C. PCR products were run on a 1.5% agarose gel and Concentrations of SDF-1 determined by sandwich stained with ethidium bromide. Results were expressed as the enzyme-linked immunosorbent assay (ELISA). Concentra- ratio of SDF-1 product to GAPDH product. tions of SDF-1 in sera and SF were measured by sandwich Expression of SDF-1, IL-17, and CD40L mRNA deter- ELISA, as follows. Antibody to human SDF-1 (4 ␮g/ml; R&D mined by real-time PCR with SYBR Green I. Real-time PCRs Systems) was added to a 96-well plate (Nunc, Roskilde, were performed in 20-␮l final volumes in capillary tubes in a Denmark) and incubated overnight at 4°C. After treatment LightCycler instrument (Roche Diagnostics, Mannheim, Ger- with blocking solution (phosphate buffered saline [PBS] con- many). The following primers were used for each molecule: for taining 1% bovine serum albumin and 0.05% Tween 20) for 2 SDF-1, 5Ј-ATG-AAC-GCC-AAG-GTC-GTG-GTC-3Ј (sense) hours at room temperature, test samples and the standard and 5Ј-TGG-CTG-TTG-TGC-TTA-CTT-GTT-T-3Ј (anti- recombinant SDF-1 (R&D Systems) were added to the 96-well sense); for IL-17, 5Ј-TGG-AGG-CCA-TAG-TGA-AGG-3Ј plate and incubated at room temperature for 2 hours. (sense) and 5Ј-GGC-CAC-ATG-GTG-GAC-AAT-3Ј (anti- Biotinylated SDF-1 polyclonal antibody to human cy- sense); for CD40L, 5Ј-CCA-GGT-GCT-TCG-GTG-TTG-3Ј tokines (400 ng/ml; R&D Systems) was added after washing 4 (sense) and 5Ј-GAC-GTG-AAG-CCA-GTG-CCA-T-3Ј (anti- times with PBS containing Tween 20, and the reactions were sense); for ␤-actin, 5Ј-GGA-CTT-CGA-GCA-AGA-GAT- allowed to proceed for 2 hours at room temperature. After GG-3Ј (sense) and 5Ј-TGT-GTT-GGC-GAT-CAG-GTC- further washing, 2,000-fold diluted streptavidin–alkaline phos- TTT-G-3Ј (antisense). phate (Sigma) was added, and the reactions were again Reaction mixtures contained 2 ␮l of LightCycler Fast- allowed to proceed for 2 hours. Fifty microliters of diluted Start DNA mastermix for SYBR Green I (Roche Diagnostics), ␮ ␮ avidin–peroxidase (1:2,000 in diluent) was added after 4 addi- 0.5 M each primer, 4 mM MgCl2, and 2 l of template DNA. tional washes. After incubation for 2 hours at room tempera- All capillaries were sealed, centrifuged at 500g for 5 seconds, ture, 50 ␮l tetramethylbenzidine substrate solution (Kirke- and then amplified in a LightCycler instrument, with activation gaard & Perry, Guildford, UK) was added to each well and of polymerase (95°C for 10 minutes), followed by 45 cycles of incubated for 20–30 minutes. 10 seconds at 95°C, 10 seconds at 60°C, and 10 seconds at 72°C. Initially, the reaction produced a blue color that was The temperature transition rate was 20°C/second for all steps. monitored by absorbance at 595 nm with a microplate reader Double-stranded PCR product was measured during the 72°C (MRX Revelation; Dynex Technologies, Chantilly, VA). extension step by detection of fluorescence associated with the When the desired intensity was reached (optical density [OD] binding of SYBR Green I to the product. Fluorescence curves Ͻ0.8), sulfuric acid (2.0 moles/liter) was added to each 50-␮l were analyzed with LightCycler software, version 3.0. For well to stop the color-generating reaction. An automated quantification analysis of SDF-1, IL-17, or CD40L mRNA, microplate reader (Vmax; Molecular Devices, Palo Alto, CA) LightCycler was used. set at 450 nm was used to measure OD. The limit of sensitivity Relative expression levels of samples were calculated for SDF-1 was 15.6 pg/ml. Recombinant human cytokines by normalizing SDF-1, IL-17, or CD40L levels to the endog- diluted in the culture medium were used as a calibration enously expressed housekeeping gene (␤-actin). Melting curve IL-17 INDUCES SDF-1 PRODUCTION IN RA FLS 1079

second (hold time) at 95°C. The temperature change rate was 20°C/second, except in the final step, in which it was 0.1°C/ second. The melt peak generated represented the specific amplified product. The crossing point was defined as the maximum of the second derivative from the fluorescence curve. Negative controls, which contained all the elements of the reaction mixture except template DNA, were also included. All samples were processed in duplicate. Cell viability measured by trypan blue dye exclusion. Trypan blue dye exclusion was performed as previously described (25) to evaluate the potential for direct cytotoxic effects of the chemical inhibitors on the cultured cells. Follow- ing 24 hours of incubation, the cells were harvested, and the percentage of cell viability was expressed using the formula 100 ϫ (number of viable cells/number of both viable and dead cells). Statistical analysis. Data are expressed as the mean Ϯ SEM. Statistical analysis was performed using the Mann- Whitney U test for independent samples and Wilcoxon’s signed rank test for related samples. P values less than 0.05 were considered significant.

RESULTS SDF-1 concentrations in RA sera and SF. Serum Figure 1. Concentrations of stromal cell–derived factor 1 (SDF-1) in and SF levels of SDF-1 in 20 RA patients and 20 OA sera and synovial fluid samples from patients with rheumatoid arthritis patients were measured by sandwich ELISA. The 20 (RA) and patients with osteoarthritis (OA). SDF-1 concentrations patients with RA included 3 men and 17 women, with a were determined by enzyme-linked immunosorbent assay. Each circle mean Ϯ SEM age of 50.4 Ϯ 1.5 years (range 23–77 ϭ Ͻ ء represents an individual patient; bars show the mean. P 0.05. years) and a mean Ϯ SEM disease duration of 71.5 Ϯ 8.2 Ϯ analysis was performed immediately after the amplification months (range 3–240 months). The mean SEM eryth- protocol under the following conditions: 0 second (hold time rocyte sedimentation rate was 40.2 Ϯ 3.8 mm/hour, and on reaching temperatures) at 95°C, 15 seconds at 65°C, and 0 the mean Ϯ SEM C-reactive protein level was 2.6 Ϯ 0.4

Figure 2. Quantitation of stromal cell–derived factor 1 (SDF-1) production by fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis. FLS were cultured with 50 ng/ml interleukin-17 (IL-17), 10 ng/ml tumor necrosis factor ␣ (TNF␣), 10 ng/ml IL-1␤, 10 ng/ml macrophage migration inhibitory factor (MIF), 10 ng/ml CD40L, 0.1 ␮g/ml concanavalin A (Con A), or 1 ␮g/ml Con A for 48 hours, and the SDF-1 concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay. Values are the mean and SEM of triplicate .ϭ P Ͻ 0.05 versus untreated cells ء .cultures 1080 KIM ET AL

Figure 3. A, Dose-dependent effects of IL-17 on the expression of SDF-1 mRNA in rheumatoid arthritis (RA) FLS. FLS from RA patients were cultured in the presence of recombinant human IL-17 (1–100 ng/ml) for 48 hours. Total RNA was extracted and analyzed by real-time polymerase chain reaction with SYBR Green I, using specific primers for human SDF-1 cDNA sequences. For an internal control, ␤-actin mRNA was used. Values are the mean and SEM from 1 representative experiment with FLS from 4 RA patients. B, Quantitation of SDF-1 production by FLS from RA patients. FLS were cultured with 10–100 ng/ml of recombinant IL-17 for 48 hours, and the SDF-1 concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay. Values are the mean and SEM .ϭ P Ͻ 0.005 versus untreated cells. See Figure 2 for other definitions ءء .of triplicate cultures mg/dl in the patients with RA. Rheumatoid factor was Regulation of RA FLS production of SDF-1 by positive in 24 of 28 patients (85.7%), at a mean Ϯ SEM various cytokines. To determine the cytokines that cause titer of 110.8 Ϯ 27.7 IU/ml (range 6.7–1,060 IU/ml). SDF-1 overproduction in RA, FLS were stimulated with In RA patients, the mean Ϯ SEM concentration various inflammatory cytokines. RA FLS were isolated of SDF-1 in SF was 1,001.5 Ϯ 103.9 pg/ml. In patients and cultured in the presence of 50 ng/ml IL-17, 10 ng/ml with OA, the concentration was 779.2 Ϯ 30.8 pg/ml. IL-1␤, 10 ng/ml TNF␣, 10 ng/ml CD40L, or 0.1 ␮g/ml Statistical analysis revealed that RA patients had higher Con A, and the concentrations of SDF-1 in the culture concentrations compared with OA patients (P ϭ 0.04). supernatants were determined by ELISA. When FLS The serum concentrations of SDF-1 tended to be were stimulated with exogenous IL-17, IL-1␤, MIF, or higher in RA patients than in OA patients, although CD40L, as well as with Con A, the production of SDF-1 the difference was not statistically significant (749.3 Ϯ increased significantly. However, TNF␣ did not affect 103.6 pg/ml versus 516.4 Ϯ 31.4 pg/ml; P ϭ 0.08) the production of SDF-1 in the FLS culture supernatants (Figure 1). (Figure 2). IL-17 INDUCES SDF-1 PRODUCTION IN RA FLS 1081

Figure 4. A, Effects of protein kinase inhibitors on levels of SDF-1 produced by IL-17 stimulation. Rheumatoid arthritis FLS were pretreated with 20 ␮M LY294002, 100 nM wortmannin, 10 ␮M SB203580, 1 ␮M PD98059, 20 ␮M JNK inhibitor, 10 ␮M curcumin, 1 ␮M SP600125, 100 ␮M pyrrolidine dithiocarbamate (PDTC), or 10 ␮M parthenolide, in combination with 10 ng/ml IL-17. SDF-1 levels in culture supernatants were ء .determined by enzyme-linked immunosorbent assay, as described in Patients and Methods. Values are the mean and SEM of triplicate cultures ϭ P Ͻ 0.05 versus cells treated with IL-17 alone. B, Effects of protein kinase inhibitors on FLS viability. FLS were cultured at a concentration of 5 ϫ 104 cells/well with medium, IL-17, and inhibitors, as described in Patients and Methods. After 24 hours of treatment, cell viability was assessed by trypan blue dye exclusion and expressed as a percentage using the formula 100 ϫ (number of viable cells/number of both viable and dead cells). Percentages Ͼ100 indicate cell growth. Values are the mean and SEM of triplicate cultures. See Figure 2 for other definitions.

Dose-dependent IL-17 enhancement of SDF-1 (inhibitors of NF-␬B activation), 20 ␮M LY294002 and production by RA FLS. To better characterize the effects 100 nM wortmannin (inhibitors of phosphatidylinositol of IL-17 on SDF-1 production by RA FLS, we performed 3-kinase [PI 3-kinase] activation), and 10 ␮M SB203580 dose-response studies on IL-17–induced SDF-1 produc- (an inhibitor of p38 MAPK). Curcumin (10 ␮M) and 1 tion. After FLS was cultured with IL-17, the production of ␮M SP600125 were tested as antagonists of activator SDF-1 in culture supernatants was determined by ELISA, protein 1 (AP-1), and 1 ␮M PD98059 as an inhibitor of and the expression of SDF-1 mRNA by RA FLS was MEK-1/2. evaluated by real-time PCR. As shown in Figure 3, IL-17 RA FLS were preincubated for 1 hour with the treatment increased the production of SDF-1 in FLS inhibitors, and stimulated with 10 ng/ml of IL-17 for 12 culture supernatants and the expression of SDF-1 mRNA hours. The production of SDF-1 in the culture superna- in RA FLS in a dose-dependent manner. tants was determined by ELISA. SDF-1 production was Signal pathways involving IL-17–induced SDF-1 decreased after inhibition of PI 3-kinase, NF-␬B, and production in RA FLS. To determine the signal trans- AP-1 (Figure 4A). In contrast, disruption of p38 MAPK duction pathways mediating the production of SDF-1 by and JNK activities showed no effect on IL-17–induced IL-17, we used 100 ␮M PDTC and 10 ␮M parthenolide SDF-1 production. At the experimental concentrations 1082 KIM ET AL

used, the chemical inhibitors exhibited no cytotoxic effects on FLS (Figure 4B). For determination of the inhibitory effects at the level of transcription, RA FLS were preincubated for 1 hour with LY294002, SP600125, or parthenolide, and cultured in the presence of 10 ng/ml of IL-17 for 12 hours. The expression of SDF-1 mRNA was determined by RT-PCR. SDF-1 mRNA expression was completely blocked when PI 3-kinase, NF-␬B, and AP-1 were inhibited (Figure 5). CD4؉ T cell augmentation of SDF-1 production by RA FLS. To determine the effect of T cells on SDF-1 production by FLS, we isolated and cultured FLS with or without CD4ϩ T cells for 24–48 hours. The SDF-1 concentration in the culture supernatants was determined using ELISA. The production of SDF-1 by CD4ϩ T cells was minimal, whereas RA FLS produced moderate basal levels of SDF-1 (mean Ϯ SEM 273.7 Ϯ 24.8 pg/ml). However, when RA FLS were cocultured with Figure 5. Effects of protein kinase inhibitors and IL-17 on SDF-1 CD4ϩ T cells, SDF-1 production was augmented to mRNA expression in rheumatoid arthritis (RA) FLS. Top, FLS were 492.7 Ϯ 49.6 pg/ml (P ϭ 0.05 versus FLS cells cultured left untreated (lane 1), treated with 10 ng/ml IL-17 (lane 2), or ␮ ␮ without T cells). pretreated with 20 M LY294002 (lane 3), 1 M SP600125 (lane 4), or 10 ␮M parthenolide (lane 5) in the presence of 10 ng/ml IL-17. Total Since increased SDF-1 production was observed RNA was extracted and analyzed by reverse transcriptase–polymerase in the T cell–FLS coculture system, we hypothesized that chain reaction (RT-PCR) using specific primers for human SDF-1 T cell–derived cytokines and/or physical interaction be- cDNA sequences. GAPDH mRNA was used as an internal control. tween T cells and FLS may contribute to the augmenta- Bottom, Quantitation of the RT-PCR results. Values are the mean tion of SDF-1 production by RA FLS. The addition of a from 1 representative experiment with FLS from 3 RA patients. PI3K ϭ phosphatidylinositol 3-kinase; AP-1 ϭ activator protein 1 (see neutralizing anti–IL-17 monoclonal antibody to cocul- Figure 2 for other definitions). tures of FLS and CD4ϩ T cells decreased SDF-1 production significantly (to 361.0 Ϯ 46.3 pg/ml, versus 492.7 Ϯ 49.6 pg/ml; P ϭ 0.04) (Figure 6A). of recombinant IL-17 (Figure 6B), suggesting a direct To further define the nature of interaction be- effect of IL-17 on the production of SDF-1 by RA FLS. tween RA T cells and FLS, we tested the effect of To identify time-dependent effects on the expres- inserting a barrier in the coculture chamber. Disrupting sion of IL-17 and CD40L mRNA in the FLS–T cell direct cell contact by placing the 2 cell types in chambers coculture system, T cells were cultured with or without separated by a barrier reversed the induction effect on FLS for 12–48 hours. IL-17 and CD40L mRNA expres- SDF-1 production, and SDF-1 levels returned almost, sion were augmented at 24–48 hours (Figure 6C). but not completely, to the levels produced by FLS alone. In addition, we tested whether IL-17 in RA SF In addition, pretreatment with anti-CD40L anti- specimens could induce SDF-1 production by RA FLS. body, which blocks the CD40L–CD40 interaction be- Addition of RA SF (with a mean Ϯ SEM IL-17 concen- tween T cells and FLS, also significantly decreased tration of 790 Ϯ 153 pg/ml) mimicked the effect of IL-17 SDF-1 production by RA FLS. Combined treatment treatment on SDF-1 production and mRNA expression. with anti–IL-17 antibody and anti-CD40L antibody To further define the stimulatory effect of RA SF on showed additive effects on the decreased production of SDF-1 production, we added neutralizing monoclonal SDF-1. A similar tendency was observed in OA FLS, but antibodies to IL-17 to cultured FLS incubated with RA the difference was not as evident as with RA FLS SF. The addition of anti–IL-17 antibody (10 ␮g/ml) (Figure 6A). abrogated the elevation of SDF-1 production induced by To investigate the role of exogenous IL-17 in the RA SF, whereas the equivalent concentration of isotype- FLS–T cell coculture system, we added IL-17 in various matched control monoclonal antibody had no effect concentrations to FLS–T cell cocultures. SDF-1 produc- (Figure 6D). These findings support the notion that the tion was augmented in a dose-dependent manner when IL-17 present at elevated levels in RA SF can directly FLS was cocultured with CD4ϩ T cells in the presence stimulate SDF-1 production in RA FLS. IL-17 INDUCES SDF-1 PRODUCTION IN RA FLS 1083

Figure 6. A, SDF-1 production by rheumatoid arthritis (RA) FLS cultured alone, with T cells, and with T cells in the presence of a transwell membrane (pore size 0.45 ␮m) between the 2 cell groups, or in the presence of anti–IL-17 monoclonal antibody, anti-CD40L monoclonal antibody, anti–IL-17 monoclonal antibody and anti-CD40L monoclonal antibody combined, or isotype-matched mouse IgG1 (all at 10 ␮g/ml). SDF-1 concentrations in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Values are the mean and SEM from 3 independent experiments ϭ P Ͻ 0.005 versus coculture of ءء ;ϭ P Ͻ 0.05 versus coculture of FLS and T cells ء .performed using 3 different lines of RA peripheral blood T cells FLS and T cells. B, SDF-1 production by RA FLS cocultured for 48 hours with T cells in the presence of 10–100 ng/ml IL-17. SDF-1 concentrations in ϭ P Ͻ 0.005 versus coculture of FLS and T cells ءء .culture supernatants were determined by ELISA. Values are the mean and SEM of triplicate cultures in the absence of IL-17. C, Time-dependent effects on the expression of IL-17 and CD40L mRNA by T cells cultured for 12–48 hours alone or with FLS from RA patients. Total RNA was extracted and analyzed by real-time polymerase chain reaction (PCR) with SYBR Green I using specific primers for human IL-17 or CD40L cDNA sequences. For an internal control, ␤-actin mRNA was used. Values are the mean and SEM from 1 representative ϭ P Ͻ 0.005 versus coculture of T cells and ءء ;ϭ P Ͻ 0.05 versus coculture of T cells and FLS at 12 hours ء .experiment with T cells from 3 RA patients FLS at 12 hours. D, Stimulatory effect of IL-17 in RA synovial fluid (SF) on SDF-1 production by RA FLS. FLS were incubated for 12–48 hours with RA SF. Neutralizing antibody to IL-17 (aIL-17 Ab; 10 ␮g/ml) or isotype-matched control antibody (10 ␮g/ml) was added to 50 ␮l RA SF. Total RNA was extracted and analyzed by real-time PCR with SYBR Green I using specific primers for human SDF-1 cDNA sequences. For an internal control, ␤-actin mRNA was used. SDF-1 concentrations in culture supernatants were determined by ELISA. Values are the mean and SEM from 3 representative .ϭ P Ͻ 0.005 versus FLS cultured alone. See Figure 2 for other definitions ءء .experiments using triplicate cultures of SF from 4 RA patients 1084 KIM ET AL

DISCUSSION SDF-1, as well as the expression of SDF-1 mRNA, in cultured FLS in a dose-dependent manner. This result SDF-1 is a potent CXC chemokine which binds to implies that there is a reciprocal action between IL-17 its single receptor, CXCR4, and has a unique role in the from T cells and SDF-1 from FLS, in RA pathogenesis. regulation of cell retention or homing (26). Interaction T cells, which migrate into the inflamed synovium with between SDF-1 and CXCR4 plays an important role in the guidance of SDF-1 from FLS, produce IL-17, result- many diseases, such as human immunodeficiency virus ing in the induction of SDF-1 from FLS. SDF-1 over- (27), cancer (28), autoimmune diabetes (29), and RA. production induces the migration of T cells into the Although the important roles of SDF-1 in the pathogen- synovial tissue, resulting in the augmentation and accel- esis of RA have been demonstrated, the regulation eration of the inflammatory response and bone destruc- mechanism of SDF-1 is not fully understood. Unlike tion in RA. other chemokines, SDF-1 is constitutively expressed in We used higher concentrations of IL-17 (1–50 numerous normal tissue types (30). Therefore, it was ng/ml) than the actual level of IL-17 in SF (1–27 ng/ml) thought that SDF-1 was not specifically regulated, and to stimulate RA FLS. Therefore, there was some dis- little attention has been paid to its regulation mecha- crepancy between the SF level of IL-17 and the concen- nism. The few previous studies of SDF-1 regulation that tration of the cytokine used in our experiments. This have been conducted have shown that anti-CD40 stim- discrepancy can be rationalized by the fact that the ulation enhances the production of cultured RA FLS cytokine environment in vivo is complicated and is (6), hypoxia induces SDF-1 expression in RA FLS (11), influenced by other serum factors. and IL-1␤ and TNF␣ decrease the production and expres- Of special interest in the present study was the sion of SDF-1 in dermal and gingival fibroblasts (31). result of experiments in which RA FLS were cocultured In this study, we hypothesized that a specific with CD4ϩ T cells. SDF-1 concentrations are negligible molecule, such as a certain cytokine, is able to regulate in the culture supernatants of CD4ϩ T cells; this means the expression of SDF-1 in RA, and that the production that T cells are not the source of SDF-1 in RA synovium. and action of SDF-1 results from the interplay of T cells SDF-1 production increased spontaneously in cultured and FLS. T cells express CXCR4 (6) and respond to FLS over time, and its production was augmented when SDF-1 produced by FLS. It is possible that a specific FLS were cocultured with CD4ϩ T cells. These findings molecule from T cells activated by SDF-1 may stimulate indicate that there may be some synergistic interactions FLS. We hypothesized that IL-17, a T cell–derived proinflammatory cytokine, might stimulate FLS recipro- between FLS and T cells that affect the production of cally. FLS express IL-17 receptors on their cell surface SDF-1 in RA synovium. (14,15) and have the potential to respond to stimulation Certain molecules or pathways are thought to be by IL-17. IL-17 is mainly produced by CD4ϩ, involved in the reciprocal effects of these 2 types of RA CD45ROϩ memory T cells, and overexpression of IL-17 cells, and in this study we presumed IL-17 was a is observed in the SF and synovial tissue of RA patients potential candidate. For evaluation of the effect of IL-17 (32,33). on the production of SDF-1 in cocultures of FLS with ϩ IL-17 plays a critical role in the inflammatory CD4 T cells, we used anti–IL-17 monoclonal antibody. ϩ process in RA, and recently it has become a subject of When FLS were cultured with CD4 T cells in the special interest in RA pathogenesis (34,35). It stimulates presence of anti–IL-17 antibody, the production of the production and expression of proinflammatory cyto- SDF-1 was inhibited significantly. Similar results were kines from monocyte/macrophages (34,36) and IL-6 and obtained when cells were pretreated with anti-CD40L IL-8 from RA synovial fibroblasts (12,37). It also stim- antibody. Moreover, when FLS were cultured with ulates chemokines, such as CCL20 (38). Furthermore, CD4ϩ T cells in the presence of recombinant human IL-17 contributes to bone erosion and tissue destruction IL-17, the production of SDF-1 was enhanced in a in RA. It induces chondrocytes and synovial fibroblasts dose-dependent manner. to produce prostaglandin E2 (39) and up-regulates nitric On the basis of these findings, it can be proposed oxide production (40). IL-17 induces IL-6, osteoclast that CD4ϩ T cells may play an important role in the differentiation factor, and RANKL production by T cells production of SDF-1 by FLS, through physical interac- and osteoblasts (14). Taken together, these findings tion with FLS and through the T cell–derived cytokine provide evidence that IL-17 regulates the inflammatory IL-17. To simulate the in vivo condition, RA SF was and destructive process in RA. added to cultured RA FLS, and this also significantly We found that IL-17 induced the production of up-regulated the expression of SDF-1 mRNA. This IL-17 INDUCES SDF-1 PRODUCTION IN RA FLS 1085

effect was mediated mainly by the IL-17 present in RA Statistical analysis. Sung-Hwan Park. SF, as confirmed in experiments using anti–IL-17. In early stages of inflammatory cell migration REFERENCES into inflamed synovium, FLS may initiate the migration 1. Hoy MD, O’Donnell JL, Hart DN. Dual CD45RA, CD45RO of immune cells, but as the inflammatory response positive T-lymphocytes within rheumatoid arthritic joints. Pathol- progresses, T cells may maintain and augment their ogy 1993;25:167–73. migration through IL-17–induced FLS production of 2. Mittal GA, Joshi VR, Deshpande A. Stromal cell-derived factor-1 ␣ SDF-1. However, the role of IL-17 in SDF-1 induction in rheumatoid arthritis. Rheumatology (Oxford) 2003;42:915–6. 3. Kanbe K, Takagishi K, Chen Q. Stimulation of matrix metallopro- should be confirmed in studies with genetically defined tease 3 release from human chondrocytes by the interaction of strains of mice and models of experimental synovitis in stromal cell–derived factor 1 and CXC chemokine receptor 4. future investigations. The role of monocytes and B cells Arthritis Rheum 2002;46:130–7. 4. Grassi F, Cristino S, Toneguzzi S, Piacentini A, Facchini A, in the induction of SDF-1 and augmentation of their Lisignoli G. CXCL12 chemokine up-regulates bone resorption and migration is also an attractive candidate for future MMP-9 release by human osteoclasts: CXCL12 levels are in- experiments. creased in synovial and bone tissue of rheumatoid arthritis patients. J Cell Physiol 2004;199:244–51. Although the SDF-1/CXCR4–mediated signaling 5. Pablos JL, Santiago B, Galindo M, Torres C, Brehmer MT, Blanco pathways have been documented widely (41), there are FJ, et al. Synoviocyte-derived CXCL12 is displayed on endothe- no published studies of the mechanism of regulation of lium and induces angiogenesis in rheumatoid arthritis. J Immunol SDF-1 production by specific signaling pathways. There- 2003;170:2147–52. 6. Nanki T, Hayashida K, El-Gabalawy HS, Suson S, Shi K, Girschick fore, we used inhibitors of signal transduction molecules HJ, et al. Stromal cell-derived factor-1-CXC chemokine receptor 4 to evaluate which signaling pathway was primarily in- interactions play a central role in CD4ϩ T cell accumulation in volved in the induction of SDF-1 in RA FLS. We rheumatoid arthritis synovium. J Immunol 2000;165:6590–8. 7. Blades MC, Ingegnoli F, Wheller SK, Manzo A, Wahid S, Panayi demonstrated that IL-17–induced SDF-1 production in GS, et al. Stromal cell–derived factor 1 (CXCL12) induces mono- RA FLS was significantly hampered by inhibitors of cyte migration into human synovium transplanted onto SCID NF-␬B (PDTC and parthenolide), PI 3-kinase mice. Arthritis Rheum 2002;46:824–36. 8. Burger JA, Zvaifler NJ, Tsukada N, Firestein GS, Kipps TJ. (LY294002 and wortmannin), and AP-1 (curcumin and Fibroblast-like synoviocytes support B-cell pseudoemperipolesis SP600125). via a stromal cell-derived factor-1- and CD106 (VCAM-1)-depen- The direct relationship between these molecules dent mechanism. J Clin Invest 2001;107:305–15. needs to be investigated in future studies. Our data 9. De Klerck B, Geboes L, Hatse S, Kelchtermans H, Meyvis Y, Vermeire K, et al. Pro-inflammatory properties of stromal cell- demonstrated that PI 3-kinase/Akt could be the up- derived factor-1 (CXCL12) in collagen-induced arthritis. Arthritis stream arbitrator of IL-17–mediated up-regulation of Res Ther 2005;7:R1208–20. SDF-1 in RA. We postulate that subsequent activation 10. Tamamura H, Fujisawa M, Hiramatsu K, Mizumoto M, Na- ␬ kashima H, Yamamoto N, et al. Identification of a CXCR4 of NF- B and AP-1 may also have contributed to the antagonist, a T140 analog, as an anti-rheumatoid arthritis agent. increased binding of inflammatory transcription factors FEBS Lett 2004;569:99–104. to the promoter of SDF-1 in IL-17–stimulated synovial 11. Hitchon C, Wong K, Ma G, Reed J, Lyttle D, El-Gabalawy H. fibroblasts. Hypoxia-induced production of stromal cell–derived factor 1 (CXCL12) and vascular endothelial growth factor by synovial In summary, SDF-1 is overproduced in RA, and fibroblasts. Arthritis Rheum 2002;46:2587–97. IL-17 could regulate the expression of SDF-1 in RA FLS 12. Hwang SY, Kim JY, Kim KW, Park MK, Moon Y, Kim WU, et al. via pathways mediated by PI 3-kinase, NF-␬B, and AP-1. IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-␬B- and PI3-kinase/Akt-dependent SDF-1 production in RA FLS was enhanced by cocul- pathways. Arthritis Res Ther 2004;6:R120–8. ture with CD4ϩ T cells through IL-17 itself and through 13. Cho ML, Yoon CH, Hwang SY, Park MK, Min SY, Lee SH, et al. CD40–CD40L interaction. These results suggest that Effector function of type II collagen–stimulated T cells from rheumatoid arthritis patients: cross-talk between T cells and inhibition of the interaction between IL-17 in T cells and synovial fibroblasts. Arthritis Rheum 2004;50:776–84. SDF-1 in FLS may provide a new molecular target for 14. Kotake S, Udagawa N, Takahashi N, Matsuzaki K, Itoh K, future treatment of RA. Ishiyama S, et al. IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103:1345–52. AUTHOR CONTRIBUTIONS 15. Hwang SY, Kim HY. Expression of IL-17 homologs and their receptors in the synovial cells of rheumatoid arthritis patients. Mol Dr. Lee had full access to all of the data in the study and takes Cells 2005;19:180–4. responsibility for the integrity of the data and the accuracy of the data 16. Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immu- analysis. nity. Annu Rev Immunol 1998;16:111–35. Study design. Lee, Ho-Youn Kim. 17. Banchereau J, Bazan F, Blanchard D, Briere F, Galizzi JP, van Acquisition of data. Kyoung-Woon Kim, Mi-Kyung Park, Oh. Kooten C, et al. The CD40 antigen and its ligand. Annu Rev Analysis and interpretation of data. Cho, Ju, Joon-Seok Kim. Immunol 1994;12:881–922. Manuscript preparation. Hae-Rim Kim, Cho. 18. Malik N, Greenfield BW, Wahl AF, Kiener PA. Activation of 1086 KIM ET AL

human monocytes through CD40 induces matrix metalloprotein- 30. Shirozu M, Nakano T, Inazawa J, Tashiro K, Tada H, Shinohara T, ases. J Immunol 1996;156:3952–60. et al. Structure and chromosomal localization of the human 19. Yellin MJ, Winikoff S, Fortune SM, Baum D, Crow MK, Leder- stromal cell-derived factor 1 (SDF1) gene. Genomics 1995;28: man S, et al. Ligation of CD40 on fibroblasts induces CD54 495–500. (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 produc- 31. Fedyk ER, Jones D, Critchley HO, Phipps RP, Blieden TM, tion and proliferation. J Leukoc Biol 1995;58:209–16. Springer TA. Expression of stromal-derived factor-1 is decreased 20. Kiener PA, Moran-Davis P, Rankin BM, Wahl AF, Aruffo A, by IL-1 and TNF and in dermal wound healing. J Immunol Hollenbaugh D. Stimulation of CD40 with purified soluble gp39 2001;166:5749–54. induces proinflammatory responses in human monocytes. J Immu- 32. Chabaud M, Durand JM, Buchs N, Fossiez F, Page G, Frappart L, nol 1995;155:4917–25. et al. Human interleukin-17: a T cell–derived proinflammatory 21. Caux C, Massacrier C, Vanbervliet B, Dubois B, van Kooten C, cytokine produced by the rheumatoid synovium. Arthritis Rheum Durand I, et al. Activation of human dendritic cells through CD40 1999;42:963–70. cross-linking. J Exp Med 1994;180:1263–72. 33. Honorati MC, Meliconi R, Pulsatelli L, Cane S, Frizziero L, 22. Sekine C, Yagita H, Miyasaka N, Okumura K. Expression and Facchini A. High in vivo expression of interleukin-17 receptor in function of CD40 in rheumatoid arthritis synovium. J Rheumatol synovial endothelial cells and chondrocytes from arthritis patients. 1998;25:1048–53. Rheumatology (Oxford) 2001;40:522–7. 23. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, 34. Lubberts E, Koenders MI, van den Berg WB. The role of T cell Cooper NS, et al. The American Rheumatism Association 1987 interleukin-17 in conducting destructive arthritis: lessons from revised criteria for the classification of rheumatoid arthritis. animal models. Arthritis Res Ther 2005;7:29–37. Arthritis Rheum 1988;31:315–24. 35. Chabaud M, Lubberts E, Joosten L, van den Berg W, Miossec P. 24. Stanciu LA, Shute J, Holgate ST, Djukanovic R. Production of IL-17 derived from juxta-articular bone and synovium contributes IL-8 and IL-4 by positively and negatively selected CD4ϩ and to joint degradation in rheumatoid arthritis. Arthritis Res 2001;3: CD8ϩ human T cells following a four-step cell separation method 168–77. including magnetic cell sorting (MACS). J Immunol Methods 36. Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He 1996;189:107–15. Y, Zhang M, et al. IL-17 stimulates the production and expression 25. Perry SW, Epstein LG, Gelbard HA. In situ trypan blue staining of of proinflammatory cytokines, IL-␤ and TNF-␣, by human macro- monolayer cell cultures for permanent fixation and mounting. phages. J Immunol 1998;160:3513–21. Biotechniques 1997;22:1020–1, 1024. 37. Yamamura Y, Gupta R, Morita Y, He X, Pai R, Endres J, et al. 26. Murdoch C. CXCR4: chemokine receptor extraordinaire. Immu- Effector function of resting T cells: activation of synovial fibro- nol Rev 2000;177:175–84. blasts. J Immunol 2001;166:2270–5. 27. Mbemba E, Benjouad A, Saffar L, Gattegno L. Glycans and 38. Chabaud M, Page G, Miossec P. Enhancing effect of IL-1, IL-17, proteoglycans are involved in the interactions of human immuno- and TNF-␣ on macrophage inflammatory protein-3␣ production deficiency virus type 1 envelope glycoprotein and of SDF-1␣ with in rheumatoid arthritis: regulation by soluble receptors and Th2 membrane ligands of CD4ϩ CXCR4ϩ cells. Virology 1999;265: cytokines. J Immunol 2001;167:6015–20. 354–64. 39. Stamp LK, Cleland LG, James MJ. Upregulation of synoviocyte 28. Burger JA, Kipps TJ. CXCR4: A key receptor in the cross talk COX-2 through interactions with T lymphocytes: role of interleu- between tumor cells and their microenvironment. Blood 2006;107: kin 17 and tumor necrosis factor-␣. J Rheumatol 2004;31:1246–54. 1761–7. 40. Miljkovic D, Trajkovic V. Inducible nitric oxide synthase activation 29. Dubois-Laforgue D, Hendel H, Caillat-Zucman S, Zagury JF, by interleukin-17. Cytokine Growth Factor Rev 2004;15:21–32. Winkler C, Boitard C, et al. A common stromal cell-derived 41. Kucia M, Jankowski K, Reca R, Wysoczynski M, Bandura L, factor-1 chemokine gene variant is associated with the early onset Allendorf DJ, et al. CXCR4-SDF-1 signalling, locomotion, che- of type 1 diabetes. Diabetes 2001;50:1211–3. motaxis and adhesion. J Mol Histol 2004;35:233–45. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1087–1093 DOI 10.1002/art.22512 © 2007, American College of Rheumatology

Histone Deacetylase/Acetylase Activity in Total Synovial Tissue Derived From Rheumatoid Arthritis and Osteoarthritis Patients

Lars C. Huber,1 Matthias Brock,1 Hossein Hemmatazad,1 Olivier T. Giger,2 Falk Moritz,1 Michelle Trenkmann,1 Jo¨rg H. W. Distler,1 Renate E. Gay,1 Christoph Kolling,3 Holger Moch,2 Beat A. Michel,1 Steffen Gay,1 Oliver Distler,1 and Astrid Ju¨ngel1

Objective. Rheumatoid arthritis (RA) is a chronic 4.2 ␮moles/␮g) synovial tissue samples were signifi- inflammatory disorder of unknown origin. Histone cantly higher. Histone acetylase activity reached similar deacetylase (HDA) activity is considered to play a major levels in RA and OA tissues and in normal tissues. The role in the transcriptional regulation of proinflamma- ratio of HDA activity to histone acetylase activity in RA (tory genes. We undertook this study to investigate the synovial tissue was significantly reduced (12 ؎ 2% .(balance of histone acetylase and HDA activity in syno- compared with that in OA synovial tissue (26 ؎ 3% vial tissue from RA patients compared with that from The activity ratio in normal control samples was arbi- -patients with osteoarthritis (OA) and normal controls. trarily set at 100 ؎ 40%. In addition, the tissue expres Methods. Activity of histone acetylases and HDAs sion of HDA-1 and HDA-2 proteins was clearly lower in was measured in nuclear extracts of total synovial tissue RA samples than in OA samples. samples, which were obtained from RA and OA patients Conclusion. The balance of histone acetylase/ undergoing surgical joint replacement, and compared HDA activities is strongly shifted toward histone hyper- with the activity in synovial tissues from patients with- acetylation in patients with RA. These results offer out arthritis. Tissue expression of HDAs 1 and 2 was novel molecular insights into the pathogenesis of the quantified by Western blotting. In addition, immunohis- disease that might be relevant to the development of tochemistry was performed for HDA-2. future therapeutic approaches. Results. Mean ؎ SEM HDA activity in synovial tissue samples derived from patients with RA was Rheumatoid arthritis (RA) is a chronic polyartic- ␮ ␮ ؎ measured as 1.5 0.3 moles/ g, whereas the activity ular disease that is characterized by inflammation and ؎ ␮ ␮ ؎ levels in OA (3.2 0.7 moles/ g) and normal (7.1 progressive destruction of the articular cartilage. Gene transcription of chemotactic and inflammatory media- Supported by the Swiss National Science Foundation (grant 3200BO-103691) and the European Community’s Sixth Framework tors is regulated, at least in part, by the tight balance Programme for Research and Technological Development (FP6). Dr. between histone acetylation and histone deacetylation Kolling’s work was supported in part by the Georg and Bertha (1–4). Histone acetylases and histone deacetylases Schwyzer-Winiker Foundation. This publication reflects only the authors’ views. The Euro- (HDAs) induce posttranslational modifications of the pean Community is not liable for any use that may be made of the N-terminal tails of the nuclear histone proteins that information herein. impact chromatin structure and gene transcription. The 1Lars C. Huber, MD, Matthias Brock, MSc, Hossein Hem- matazad, MD, Falk Moritz, MD, Michelle Trenkmann, MSc, Jo¨rg chromatin is compactly organized in nucleosomes as an H. W. Distler, MD, Renate E. Gay, MD, Beat A. Michel, MD, Steffen octomeric core unit of 4 histones. Modifications are Gay, MD, Oliver Distler, MD, Astrid Ju¨ngel, PhD: Center of Exper- regulated by 2 groups of enzymes (i.e., histone acetylases imental Rheumatology, University Hospital Zurich, and Zurich Center of Integrative Human Physiology, Zurich, Switzerland; 2Olivier T. and HDAs). In inactivated cells, the dense DNA– Giger, MD, Holger Moch, MD: University Hospital Zurich, Zurich, protein package prevents accessibility of transcription Switzerland; 3Christoph Kolling, MD: Schulthess Clinic, Zurich, Swit- factors and nucleic acid polymerases (5,6). Acetylation zerland. Address correspondence and reprint requests to Lars C. of histones is thought to occur on actively transcribed Huber, MD, Center of Experimental Rheumatology, University Hos- chromatin only (7), thus allowing gene transcription pital Zurich, Gloriastrasse 25, CH-8091 Zurich, Switzerland. E-mail: during cell activation (8,9). [email protected]. Submitted for publication July 12, 2006; accepted in revised Histone acetylases can be separated into 2 cate- form January 4, 2007. gories (i.e., type A and type B) depending on their

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Table 1. Characteristics of the study subjects* RA patients OA patients Normal subjects Age, mean Ϯ SEM (range) years 63 Ϯ 6 (43–79) 73 Ϯ 3 (62–80) 68 Ϯ 10 (49–81) No. of women/no. of men 6/1 5/1 2/3 Disease duration, mean Ϯ SEM 26 Ϯ 5 (10–38) Ͼ10 NA (range) years Origin of synovial tissue Shoulder 2 2 1 Elbow 1 0 0 Finger 2 0 0 Hip 1 1 0 Knee 1 3 1 Sternoclavicular joints 0 0 3 Medication Oral corticosteroid 2 0 1 Methotrexate 2 0 0 Anti-TNF␣ 30 0 * Except where indicated otherwise, values are the number of subjects. RA ϭ rheumatoid arthritis; OA ϭ osteoarthritis; NA ϭ not applicable; anti-TNF␣ ϭ anti–tumor necrosis factor ␣. subcellular localization. Type A histone acetylases are (11). In this context, HDAs have been described as key found in the nucleus. Since histone acetylases acetylate enzymes in the repression of proinflammatory cytokines nucleosomal histones, they are closely linked to the in alveolar macrophages, as seen for example in the transcriptional regulation of gene expression. On the clinical exacerbation of chronic obstructive pulmonary other hand, type B histone acetylases acetylate newly disease (COPD) (12). Increased acetylation of histones, synthesized histones that are free in the cytoplasm. Once moreover, has been associated with the activation of acetylated, these histones are shuttled into the nucleus, NF-␬B and activator protein 1, two major transcription where they may be deacetylated and incorporated into factors involved in the pathogenesis of RA (13–15). chromatin. Some histone acetylases also have coactivat- In the present study, we addressed the activity ing roles for different transcription factors. levels of HDAs and histone acetylases in nuclear ex- The reverse reaction (deacetylation of histone tracts of synovial tissue of RA and osteoarthritis (OA) proteins) is catalyzed by HDAs, which can be divided patients. Novel insights into the imbalanced expression into 3 different classes based on sequence homologies to of epigenetic markers such as histone acetylase/HDA yeast proteins. Class I HDAs (HDAs 1, 2, 3, 8, and 11) provide important information about the molecular fea- are closely related to the yeast transcriptional regulator tures of the rheumatoid synovium. reduced potassium dependency 3. Class II HDAs (HDAs 4, 5, 6, 7, 9, and 10) show homologies to the yeast PATIENTS AND METHODS deacetylase HDA-1. Class II HDAs are similar to the silent information regulator 2 family of NADϩ- Reagents. Recombinant human tumor necrosis factor dependent HDAs. HDAs of class I are expressed in all ␣ (TNF␣) was purchased from R&D Systems (Minneapolis, cell types, whereas the expression of class II HDAs is MN). Trichostatin A was from Sigma (St. Louis, MO). Rabbit anti-human HDA-1 and HDA-2 were from Santa Cruz Bio- more restricted and might show tissue-specific expres- technology (Santa Cruz, CA). Mouse anti-human ␣-tubulin sion patterns (10). was from Sigma. Polyclonal rabbit anti-mouse IgG conjugated The extent of gene transcription is regulated by to horseradish peroxidase (HRP) were from Dako (Zug, the equilibrium of histone acetylation (increasing gene Switzerland), and goat anti-rabbit IgG conjugated to HRP transcription) and histone deacetylation (blocking gene were purchased from Jackson ImmunoResearch (Soham, UK). Mouse anti-human CD68 was from Dako. transcription). Hyperacetylation of histones is achieved Patients. Synovial tissue specimens were obtained through an increase in histone acetylase activity and, from 7 RA patients and 6 OA patients undergoing surgical conversely, through a decrease in HDA activity. In joint replacement at the Clinic of Orthopedic Surgery, Schul- either case, the local unwinding of nucleosomal DNA thess Hospital Zurich. All RA patients fulfilled the 1987 results in a loosened state of DNA, which allows tran- revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (16). An scription factors and RNA polymerase II to bind. overview of the clinical characteristics of the subjects is pro- So far, decreased levels of active HDAs have vided in Table 1. mainly been reported in inflammatory lung diseases To establish HDA and histone acetylase activity as HISTONE DEACETYLASE/ACETYLASE ACTIVITY IN RA AND OA 1089

epigenetic markers, as well as to determine their protein standard (for HDA) or the CoA standard curve (for histone expression, normal synovial tissue was required for quality acetylase) as ⌬OD/␮M. assurance. Synovial tissues (Ͻ5mm3 in size) from patients Immunohistochemistry. Immunohistochemistry was without arthritis undergoing surgical amputation of a limb performed using a standard indirect immunoperoxidase (n ϭ 2) and from sternoclavicular joints removed during method (18). Sections from formalin-fixed, paraffin-embedded autopsies (n ϭ 3) were used as normal controls. All tissue tissues were deparaffinized and pretreated at 80°C for 30 analyses were performed according to the regulations of the minutes in 10 mmoles/liter citrate buffer (pH 6.0) for antigen Ethical Committee Zurich. retrieval. Preparation of nuclear extracts. Total synovial tissue To determine the cell type expressing HDA-2 in specimens (ϳ3–5 mm3 in size) were used for preparation of synovial tissues, double labeling with immunohistochemistry nuclear extracts as described previously (12,17). Briefly, fresh was performed. Mouse anti-human CD68 (1:100) was used for tissue was transferred in hypotonic buffer consisting of 10 mM double staining visualized by nitroblue tetrazolium/BCIP. In HEPES (pH 7.9), 1.5 mM magnesium chloride, 10 mM potas- control experiments, matched mouse IgG isotypes (1:50,000; sium chloride, 10 mM 2-mercaptoethanol, and a mixture of Dako) were used instead of the primary antibodies. To block protease inhibitors (2 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 nonspecific binding, slides were incubated for 1 hour in ␮g/ml pepstatin A [Sigma]) as well as 1 mM phenylmethylsul- blocking solution consisting of 4% nonfat dry milk and 2% fonyl fluoride (PMSF; Calbiochem, Dietikon, Switzerland) and horse serum in Tris buffered saline (TBS) at pH 7.4. Slides homogenized in a tissue lyser (Qiagen, Hilden, Germany) for were then incubated for 1 hour with polyclonal rabbit anti- 2 minutes twice at 20 Hz. The samples were left on ice for 15 human HDA-2 antibodies (200 ␮g/ml diluted 1:500 in phos- minutes before adding Nonidet P40 to a final concentration of phate buffered saline [PBS]). Bound primary antibodies were 0.5%. The samples were centrifuged at 3,000 revolutions per detected using biotinylated goat anti-rabbit IgG in PBS (1 minute for 1 minute to pellet the larger cellular debris. The mg/ml, diluted 1:1,000). Labeling was performed for 20 min- resulting supernatants were centrifuged at 14,000 rpm for 30 utes with an HRP-conjugated streptavidin complex (Bio- seconds at 4°C to obtain the nuclear-rich fraction. The pellet Genex, San Ramon, CA). Antigens were visualized using ␮ was then resuspended in 100 l nuclear buffer (20 mM HEPES aminoethylcarbazole chromogen and H2O2 as substrate. All [pH 7.9], 0.42 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, steps were performed at room temperature. and1mM PMSF, which was added immediately before use) Western blot analysis. For Western blot analysis, and incubated on ice for 15 minutes with additional vortexing whole cell lysates were prepared by lysing confluent cells (1 ϫ every 5 minutes. Finally, the samples were centrifuged at 106)in2ϫ concentrated Laemmli buffer (100 mM Tris HCl 14,000 rpm for 15 minutes at 4°C, the supernatants (nuclear [pH 6.8], 40% glycerol, 10% sodium dodecyl sulfate [SDS], extracts) were transferred to ice-chilled tubes, and the protein 0.7M ␤-mercaptoethanol, and 0.0005% bromphenol blue). concentration of each sample was analyzed (Bradford Bio-Rad Proteins were separated on a 10% SDS–polyacrylamide gel Protein assay kit; Bio-Rad, Hercules, CA) with bovine serum and transferred to nitrocellulose membranes. Membranes albumin (Sigma) used as a standard. were blocked for 1 hour at room temperature in 5% nonfat dry Measurement of histone acetylase activity and HDA milk with 0.05% Tween 20 in TBS (pH 7.4) and were probed activity. Total histone acetylase activity and HDA activity were overnight at 4°C with antibodies against HDA-1 or ␣-tubulin. measured using colorimetric assay kits (BioVision, Mountain After incubation for 30 minutes at room temperature with View, CA) according to the manufacturer’s instructions. In this HRP-conjugated secondary antibodies (HRP-conjugated goat way, acetylation of peptides by active histone acetylases re- anti-rabbit or HRP-conjugated rabbit anti-mouse) in 5% non- leases the histone acetylase cofactor acetyl-coenzyme A fat dry milk with 0.05% Tween 20 in TBS (pH 7.4), bound (acetyl-CoA). Free CoA serves as an essential coenzyme for antibodies were visualized using enhanced chemiluminescence producing NADH. New generated NADH can be measured (Amersham Pharmacia Biotech, Little Chalfont, UK). Evalu- spectrophotometrically upon reacting with a soluble tetrazo- ation of the expression of specific proteins was performed by lium dye. To measure the HDA activity, samples are incubated the Alpha Imager Software system (Alpha Innotech, San with a colorimetric substrate, which includes an acetylated Leandro, CA) via pixel quantification of the electronic image. lysine side chain. Deacetylation of the substrate sensitizes the Statistical analysis. All data are expressed as the mean Ϯ SEM. Statistical analysis was performed using Graph- substrate to react with a chromophore that can be measured Pad Prism software, version 4.03 (GraphPad Software, San spectroscopically. Diego, CA). For analysis between different groups, the Mann- Briefly, 25–100 ␮g of nuclear extracts was prepared in Whitney U test was used. P values less than 0.05 were 40 ␮l water and added to a 96-well plate including blank considered significant. samples and positive controls (for histone acetylase activity, CoA [Sigma]; for HDA activity, HeLa nuclear extract provided with the kit). After addition of assay buffer and enzyme mix, RESULTS the plate was incubated at 37°C for 2 hours. Samples were then read at 500 nm (for histone acetylase activity) or incubated HDA activity and histone acetylase activity in after the addition of a lysine developer (HDA activity kit) for total synovial tissue. Decreased levels of HDA activity an additional 30 minutes at 37°C before reading the plate at have been associated with inflammatory diseases, in 405 nm. Data were analyzed by using Revel software (version G 3.2; Dynex Technologies, West Sussex, UK). Activity was particular with inflammatory lung diseases. In this con- analyzed as the relative optical density (OD) value per ␮gof text, we investigated the levels of active HDA in synovial protein sample and was recalculated using the deacetylated tissues. The HDA activity in synovial tissues from pa- 1090 HUBER ET AL

tients with RA was ϳ2-fold lower than that in synovial tissues from patients with OA or from normal controls. In particular, the mean Ϯ SEM HDA activity levels in RA were determined to be 1.5 Ϯ 0.3 ␮moles/␮g, expressed as ␮moles of deacetylated product from ␮gof total protein. On the other hand, the levels were 3.2 Ϯ 0.7 ␮moles/␮g in OA samples and 7.1 Ϯ 4.2 ␮moles/␮g in normal control samples. As shown in Figure 1A, the difference between the HDA activity in RA synovial tissue versus OA synovial tissue (P ϭ 0.008) as well as between HDA activity in RA synovial tissue versus normal control tissue (P ϭ 0.01) was statistically significant (Figure 1A), whereas no significant difference was observed between OA and normal control samples. The extent of gene transcription is regulated by the tight balance between HDA and histone acetylase activities. Thus, we next tested whether the decreased activity levels of HDA in RA synovial tissue samples were compensated by an adequate decrease in histone acetylase activity. As shown in Figure 1B, we found similar activity levels of histone acetylase in synovial tissues from RA patients (57.0 Ϯ 25.1 ␮moles/␮g) and OA patients (61.6 Ϯ 26.0 ␮moles/␮g) and in those from normal controls (72 Ϯ 39.2 ␮moles/␮g). To detect the resulting activity levels within RA and OA synovial tissues, the ratio of HDA activity to histone acetylase activity was calculated. The HDA activity:histone acetylase activity ratio in normal synovial tissue was arbitrarily set at 100% (100 Ϯ 40%). In OA samples, the ratio decreased to 26 Ϯ 3%. In RA samples, a further decrease in the HDA activity:histone

Figure 1. Histone deacetylase (HDA) activity versus histone acetylase (HAT) activity in synovial tissue. A, HDA activity in synovial tissue from normal controls compared with that in synovial tissue from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). HDA activity was significantly reduced in RA patients (1.5 Ϯ 0.3 ␮moles/␮g) compared with OA patients (3.2 Ϯ 0.7 ␮moles/␮g) and normal controls (7.1 Ϯ 4.2 ␮moles/␮g). Data were calculated as ␮moles of deacetylated product from ␮g of total protein content. B, Histone acetylase activity measured in the same samples as in A. Data were calculated as ␮moles of product from ␮g of total protein content. No significant differences in histone acetylase activity could be detected between OA, RA, and normal samples. C, Ratio of HDA activity to Figure 2. HDA-1 protein expression in synovial tissue. Representa- histone acetylase activity. Activity is strongly shifted toward hyper- tive Western blot showing down-regulation of HDA-1 protein in RA acetylation in RA synovial tissue samples (12 Ϯ 2%) compared with synovial tissue compared with OA synovial tissue. Whole cell lysates OA synovial tissue samples (26 Ϯ 3%) and normal synovial tissue were analyzed and normalized to ␣-tubulin protein. Numbers repre- samples (arbitrarily set at 100 Ϯ 40%). Values are the mean and SEM. sent individual patients. See Figure 1 for definitions. HISTONE DEACETYLASE/ACETYLASE ACTIVITY IN RA AND OA 1091

acetylase activity ratio to 12 Ϯ 2% could be observed. enzymatic counterpart histone acetylase have been The alteration toward acetylation of histones in RA found between all conditions investigated. patients reached statistical significance between OA and When the ratio of both involved molecular play- RA synovial samples (P ϭ 0.009) (Figure 1C), but not ers was calculated, the overall histone status was shifted between normal samples and OA synovial samples. toward histone hyperacetylation in RA. These changes Protein expression of HDA-1 and HDA-2 in reached significance between RA and OA synovial synovial tissue. To analyze whether the activity levels tissue, indicating that chronic inflammatory processes correlate with protein expression, we performed West- are closely linked to reduced activity of HDAs. This ern blotting for HDA-1 and HDA-2. After normalizing tendency is further supported by our observation that protein to ␣-tubulin protein, Western blots were quan- the levels of HDA activity are even higher within the tified by Alpha Imager Software as electronic images. total synovium of healthy individuals. Reduced levels of HDA-1 (51 Ϯ 26%) (Figure 2) and Our data are consistent with other studies that HDA-2 (70 Ϯ 18%) were found in RA synovial tissues showed histone hyperacetylation in chronic inflamma- (n ϭ 8) compared with expression in OA synovial tissues tory lung diseases due to decreased HDA activity with- (n ϭ 6), which was arbitrarily set at 100%. out alterations of histone acetylase activity (12). To date, To determine the morphologic localization of however, this is the first study to analyze HDA activity HDA expression in the synovium, HDA-2 protein was within the synovial tissue. Of interest, several studies analyzed by immunohistochemistry and evaluated by have suggested beneficial effects of HDA inhibitors in analyzing several tissue slides. In normal healthy syno- the treatment of RA and other inflammatory processes vium as well as in OA synovial tissue, most of the cells (20,21). By showing a clear reduction of HDA activity in were strongly positive for nuclear HDA-2 expression the rheumatoid synovium, however, our data strongly (Figures 3a and b). However, in RA synovial tissue (n ϭ challenge the idea of epigenetically modulating molec- 4), the expression of nuclear HDA-2 was strongly re- ular targets by HDA inhibitors for therapeutic purposes duced (Figure 3c). When the tissue slides were double in RA. stained against HDA-2 and CD68, no merging of CD68 With respect to NF-␬B, which is one of the and HDA-2 could be observed (Figures 3e and f). best-characterized proinflammatory transcription fac- Taken together, our results show that the total tors, it is a matter of debate whether HDA inhibitors HDA activity as well as distinct isoforms of HDA lead to induction (22,23) or inhibition (24) of NF-␬B. proteins are down-regulated in RA synovial tissue com- Both scenarios are probably possible, depending on pared with OA synovial tissue. These data suggest a specificity, the proinflammatory mediator investigated, pathophysiologic association between reduced HDA ac- or the cell type (21). tivity and chronic inflammatory processes of the joint. Previous work by Ito et al (12) revealed that the reduction of total HDA activity in inflammatory diseases is mainly due to changes in the expression of class I DISCUSSION HDAs (i.e., HDAs 1, 2, 3, 8, and 11), particularly Enhanced histone acetylation or histone hyper- HDA-2. We therefore focused on the tissue expression acetylation leads to local unwinding of chromatin and is of HDA-1 and HDA-2 proteins, both of which we found generally associated with induction of gene expression to be clearly reduced in RA synovial tissue compared due to increased gene transcription rates. The acetyla- with OA or normal synovial tissue. tion status is regulated by the action of 2 distinct groups Still, it remains rather unclear whether the ob- of enzymes (i.e., histone acetylases and HDAs). Persis- served reduction in HDA activity is “the chicken or the tent alterations in the tight equilibrium between histone egg” in the pathogenesis of RA. We cannot exclude the acetylase and HDA activities have been associated with possibility that the reduced HDA activity reflects an pathologic gene expression patterns and the develop- epiphenomenon of ongoing inflammation. However, Ito ment of chronic diseases, such as COPDs and other et al (23) have shown that HDA-2 suppresses NF-␬B– related inflammatory disorders of the lungs (for review, mediated gene expression. In RA synovial cells, NF-␬B see refs. 11 and 19). is highly activated, leading subsequently to the expres- Our present data show that the levels of total sion of several proinflammatory mediators including HDA activity are strongly decreased in synovial tissue TNF␣, interleukin-6 (IL-6), IL-8, and cyclooxygenase 2, homogenates from patients with RA compared with the as well as matrix-degrading enzymes (for review, see ref. respective activity in those from OA patients and normal 25). In concert with the findings of Ito and coworkers, controls. In addition, no different activity levels of the we hypothesize that class I HDAs appear to act up- 1092 HUBER ET AL

Figure 3. HDA-2 protein expression in synovial tissue. a–c, Representative immunohistochemistry for HDA-2 protein in normal synovial tissue (a), OA synovial tissue (b), and RA synovial tissue (c). d, Isotype control. e and f, Double labeling for CD68 after immunohistochemistry for HDA-2 protein in synovial tissues from patients with RA shown at different magnifications. Double labeling indicates that synovial macrophages do not express HDA-2 protein. Color for CD68 was developed with nitroblue tetrazolium/BCIP. Boxed areas show higher magnification views of selected nuclei. (Original magnification ϫ 100 in a–d and f; ϫ 50 in e; ϫ 400 in boxed areas in a–c, e, and f.) See Figure 1 for definitions. stream of NF-␬B and other related transcription factors We report here for the first time an overall status in RA. of histone hyperacetylation within RA synovium due to HISTONE DEACETYLASE/ACETYLASE ACTIVITY IN RA AND OA 1093

low activity levels of total HDA enzymes, which was 9. Wolffe AP. Transcriptional control: sinful repression. Nature 1997;387:16–7. further underpinned by reduced protein expression of 10. De Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van HDAs 1 and 2. We conclude that the observed reduction Kuilenburg AB. Histone deacetylases (HDACs): characterization in the activity levels of class I HDAs contributes to the of the classical HDAC family. Biochem J 2003;370(Pt 3):737–49. 11. Barnes PJ, Adcock IM, Ito K. Histone acetylation and deacetyla- pathogenesis of RA, probably by activation of proin- tion: importance in inflammatory lung diseases. Eur Respir J flammatory transcription factors. These findings have 2005;25:552–63. potential implications for the development of novel, 12. Ito K, Ito M, Elliott WM, Cosio B, Caramori G, Kon OM, et al. Decreased histone deacetylase activity in chronic obstructive molecule-targeted therapies against inflammatory dis- pulmonary disease. N Engl J Med 2005;352:1967–76. eases of the joint. 13. Chen LF, Greene WC. Assessing acetylation of NF-␬B. Methods 2005;36:368–75. 14. Adcock IM, Cosio B, Tsaprouni L, Barnes PJ, Ito K. Redox AUTHOR CONTRIBUTIONS regulation of histone deacetylases and glucocorticoid-mediated inhibition of the inflammatory response. Antioxid Redox Signal Dr. Huber had full access to all of the data in the study and 2005;7:144–52. takes responsibility for the integrity of the data and the accuracy of the 15. Granet C, Maslinski W, Miossec P. Increased AP-1 and NF-␬B data analysis. activation and recruitment with the combination of the proinflam- Study design. Huber, Brock, J. H. W. Distler, R. E. Gay, S. Gay, matory cytokines IL-1␤, tumor necrosis factor ␣ and IL-17 in O. Distler, Ju¨ngel. rheumatoid synoviocytes. Arthritis Res Ther 2004;6:R190–8. Acquisition of data. Huber, Brock, Hemmatazad, Giger, Moritz, 16. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Trenkmann, Kolling. Cooper NS, et al. The American Rheumatism Association 1987 Analysis and interpretation of data. Huber, S. Gay, O. Distler, Ju¨ngel. revised criteria for the classification of rheumatoid arthritis. Manuscript preparation. Huber, J. H. W. Distler, R. E. Gay, Moch, Arthritis Rheum 1988;31:315–24. Michel, S. Gay, O. Distler, Ju¨ngel. 17. Ito K, Lim S, Caramori G, Cosio B, Chung KF, Adcock IM, et al. Statistical analysis. Huber. A molecular mechanism of action of theophylline: induction of histone deacetylase activity to decrease inflammatory gene expression. Proc Natl Acad SciUSA2002;99:8921–6. REFERENCES 18. Huang BH, Laban M, Leung CH, Lee L, Lee CK, Salto-Tellez M, et al. Inhibition of histone deacetylase 2 increases apoptosis and 1. Urnov FD, Wolffe AP. Chromatin remodeling and transcriptional p21Cip1/WAF1 expression, independent of histone deacetylase 1. activation: the cast (in order of appearance). Oncogene 2001;20: Cell Death Differ 2005;12:395–404. 2991–3006. 19. Barnes PJ. How corticosteroids control inflammation: Quintiles 2. Ito K, Adcock IM. Histone acetylation and histone deacetylation. Prize Lecture 2005. Br J Pharmacol 2006;148:245–54. Mol Biotechnol 2002;20:99–106. 20. Chung YL, Lee MY, Wang AJ, Yao LF. A therapeutic strategy 3. Ito K, Hanazawa T, Tomita K, Barnes PJ, Adcock IM. Oxidative uses histone deacetylase inhibitors to modulate the expression of stress reduces histone deacetylase 2 activity and enhances IL-8 genes involved in the pathogenesis of rheumatoid arthritis. Mol gene expression: role of tyrosine nitration. Biochem Biophys Res Ther 2003;8:707–17. Commun 2004;315:240–5. 21. Blanchard F, Chipoy C. Histone deacetylase inhibitors: new drugs 4. Marwick JA, Kirkham PA, Stevenson CS, Danahay H, Giddings J, for the treatment of inflammatory diseases? Drug Discov Today Butler K, et al. Cigarette smoke alters chromatin remodeling and 2005;10:197–204. induces proinflammatory genes in rat lungs. Am J Respir Cell Mol 22. Ashburner BP, Westerheide SD, Baldwin AS Jr. The p65 (RelA) Biol 2004;31:633–42. subunit of NF-␬B interacts with the histone deacetylase (HDAC) 5. Beato M. Chromatin structure and the regulation of gene expres- corepressors HDAC1 and HDAC2 to negatively regulate gene sion: remodeling at the MMTV promoter. J Mol Med 1996;74: expression. Mol Cell Biol 2001;21:7065–77. 711–24. 23. Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, Barnes PJ, 6. Beato M, Eisfeld K. Transcription factor access to chromatin. et al. Histone deacetylase 2-mediated deacetylation of the glu- Nucleic Acids Res 1997;25:3559–63. cocorticoid receptor enables NF-␬B suppression. J Exp Med 2005. 7. Perry M, Chalkley R. Histone acetylation increases the solubility E-pub ahead of print. of chromatin and occurs sequentially over most of the chromatin: 24. Place RF, Noonan EJ, Giardina C. HDAC inhibition prevents a novel model for the biological role of histone acetylation. J Biol NF-␬ B activation by suppressing proteasome activity: down- Chem 1982;257:7336–47. regulation of proteasome subunit expression stabilizes I ␬ B ␣. 8. Ura K, Kurumizaka H, Dimitrov S, Almouzni G, Wolffe AP. Biochem Pharmacol 2005;70:394–406. Histone acetylation: influence on transcription, nucleosome mo- 25. Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial bility and positioning, and linker histone-dependent transcrip- fibroblasts: key players in rheumatoid arthritis [review]. Rheuma- tional repression. EMBO J 1997;16:2096–107. tology (Oxford) 2006;45:669–75. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1094–1105 DOI 10.1002/art.22450 © 2007, American College of Rheumatology

Analysis of Vascular Gene Expression in Arthritic Synovium by Laser-Mediated Microdissection

Atsushi Hashimoto,1 Ingo H. Tarner,1 Rainer M. Bohle,2 Andreas Gaumann,3 Mirko Manetti,4 Oliver Distler,5 Ju¨rgen Steinmeyer,6 Ann-Kristin Ulfgren,7 Andreas Schulz,2 Steffen Gay,5 Ulf Mu¨ller-Ladner,1 and Elena Neumann1

Objective. In rheumatoid arthritis (RA), forma- DNA binding/differentiation 2 (Id2), and CD82 in RA tion of new blood vessels is necessary to meet the and OA synovial microvasculature and synovial lining. nutritional and oxygen requirements of actively prolif- Results. Expression of Id2 mRNA was signifi- erating synovial tissue. The aim of this study was to cantly lower in RA synovial vessels compared with OA whereas expression of ,(0.0011 ؍ analyze the specific synovial vascular expression pro- synovial vessels (P .(0.0433 ؍ files of several angiogenesis-related genes as well as VEGFR-1 was significantly higher in RA (P CD82 in RA compared with osteoarthritis (OA), using No differences were observed for the other parameters. laser-mediated microdissection (LMM). At the protein level, no statistically significant differ- Methods. LMM and subsequent real-time poly- ences were observed for any parameter, although Id2 ,However .(0.0952 ؍ merase chain reaction were used in combination with levels were 2.5-fold lower in RA (P immunohistochemical analysis for area-specific analy- the number of synovial blood vessels and the number of sis of messenger RNA (mRNA) and protein expression VEGFR-2–expressing blood vessels were significantly of vascular endothelial growth factor (VEGF), VEGF higher in RA compared with OA. Conclusion. receptor 1 (VEGFR-1), VEGFR-2, hypoxia-inducible Our results underscore the impor- factor 1␣ (HIF-1␣), HIF-2␣, platelet-derived growth tance of area-specific gene expression analysis in study- factor receptor ␣ (PDGFR␣), PDGFR␤, inhibitor of ing the pathogenesis of RA and support LMM as a robust tool for this purpose. Of note, our results indicate that previously described differences between RA Dr. Tarner’s work was supported in part by the German and OA in the expression of angiogenic molecules are Research Foundation (Deustsche Forschungsgemeinschaft grant attributable to higher total numbers of synovial and Ta297). Dr. Mu¨ller-Ladner’s work was supported in part by the German Research Foundation (Deustsche Forschungsgemeinschaft vascular cells expressing these molecules in RA rather grant Mu1383). than higher expression levels in the individual cells. 1Atsushi Hashimoto, MD, Ingo H. Tarner, MD, Ulf Mu¨ller- Ladner, MD, Elena Neumann, PhD: Justus-Liebig-University of Gies- sen, Giessen, Germany, and Kerckhoff-Klinik, Bad Nauheim, Ger- Rheumatoid arthritis (RA) is an autoimmune many; 2Rainer M. Bohle, MD, Andreas Schulz, MD: Justus-Liebig- disease characterized by chronic inflammation of multi- University of Giessen, Giessen, Germany; 3Andreas Gaumann, MD: ple joints, but numerous details regarding its pathogen- University Hospital Regensburg, Regensburg, Germany; 4Mirko Manetti, PhD: Justus-Liebig-University of Giessen, Giessen, Ger- esis still need to be clarified. Rheumatoid articular many, Kerckhoff-Klinik, Bad Nauheim, Germany, and University of inflammation is characterized by excessive growth of Florence, Florence, Italy; 5Oliver Distler, MD, Steffen Gay, MD: activated synovial tissue, which invades and ultimately University Hospital Zurich, Zurich, Switzerland; 6Ju¨rgen Steinmeyer, PhD: University Hospital of Giessen and Marburg, Giessen, Germany; destroys articular cartilage and subchondral bone (1,2). 7Ann-Kristin Ulfgren, PhD: Karolinska Hospital, Stockholm, Sweden. In general, increased vascular proliferation of activated Drs. Hashimoto and Tarner contributed equally to this work. synovial tissue and expression of angiogenetic factors Address correspondence and reprint requests to Ingo H. Tarner, MD, Department of Medicine and Rheumatology, Justus- can be observed in RA (3–5). Liebig-University of Giessen, Division of Rheumatology and Clinical In addition, recent work in animal models sug- Immunology, Kerckhoff-Klinik, Benekestrasse 2-8, D-61231 Bad Nau- gests that angiogenesis is a key mechanism in the heim, Germany. E-mail: [email protected]. Submitted for publication February 14, 2006; accepted in pathogenesis of RA. For example, administration of revised form December 12, 2006. antibodies directed against vascular endothelial growth

1094 GENE ANALYSIS OF SYNOVIAL VASCULATURE 1095

factor (VEGF), the main mediator of angiogenesis, this study, we analyzed the synovial expression profile of delayed the onset and diminished the severity of murine distinct genes specifically related to angiogenesis, includ- collagen-induced arthritis (CIA) and also inhibited an- ing the genes for VEGF, VEGF receptor 1 (VEGFR-1; giogenesis in the joints of mice with CIA (6,7). Gene also known as flt-1), VEGFR-2 (also known as kinase transfer studies using angiostatin (8) or a soluble Tie2 insert domain receptor), hypoxia-inducible factor 1␣ receptor (9) in CIA demonstrated that the development (HIF-1␣), HIF-2␣ (3), platelet-derived growth factor of arthritis can be inhibited and the severity of estab- receptor ␣ (PDGFR␣), PDGFR␤ (18–20), and inhibitor lished disease can be diminished. Similarly, the irrevers- of DNA binding/differentiation 2 (Id2) (21) in synovial ible methionine aminopeptidase-2 inhibitors TNP-470 tissue samples obtained from patients with RA or OA. and PPI-2458 have antiangiogenic and thus arthritis- In addition, we examined vascular expression of CD82 attenuating properties in animal models and human (also known as KAI1), a recently identified gene that is synovial tissue in vitro and in vivo (10,11). known to act as a suppressor of tumor metastasis and as For a better understanding of the role of angio- a bridging molecule between lymphocytes and matrix genic processes in the pathogenesis of RA, it is necessary metalloproteinase inducers. In previous experiments, we to examine the respective genes and their differential detected expression of CD82 in synovial fibroblasts (22), expression in RA synovium. A commonly used method but the role of CD82 in angiogenesis in RA synovium of investigating differential gene expression is the com- has not yet been determined. parison of messenger RNA (mRNA) or protein expres- Using LMM and optimizing the technique for sion in RA synovial tissue and a control disease, e.g., these nonmalignant entities, we were able to select small osteoarthritis (OA), a degenerative disease of articular areas of vascular and adjacent perivascular cells from cartilage. However, this method has certain limitations. RA and OA synovium for quantitative analysis of First, the number of a given type of cell varies in each mRNA expression. Protein expression and localization tissue sample. Second, synovial tissue can be subdivided of candidate genes in synovium were confirmed by into different areas, in particular synovial lining and immunohistochemical analysis. sublining but also the so-called cartilage–pannus junction in arthritis. Moreover, we previously demonstrated that the distribution of distinct cell types involved in the PATIENTS AND METHODS pathogenesis of arthritis as well as that of different genes Tissue sections. Synovial tissue samples were obtained varies substantially between these areas (12). Blood from patients with RA and patients with OA who were vessels, for instance, are observed primarily in synovial undergoing joint surgery (synovectomy or joint replacement) sublining, not adjacent to the zone of cartilage destruc- at the Departments of Orthopedics, University of Regensburg tion (1). and University of Giessen. All experiments were performed Therefore, a more refined high-resolution tech- after approval by the local ethics committee, and informed consent was obtained from all patients. All patients with RA nique is required for the detection of differential gene met the American College of Rheumatology (ACR; formerly, expression in specific cell populations or in certain areas the American Rheumatism Association) 1987 revised criteria or compartments of synovial tissue. With regard to the for the classification of RA (23). Samples were placed in role of angiogenesis in the development of RA, a embedding medium (TissueTek OCT; VWR Scientific Prod- specific analysis of differential gene expression in syno- ucts, San Diego, CA) and immediately snap-frozen in liquid nitrogen. All cryoblocks were stored at Ϫ80°C until analyzed vial blood vessels is of particular interest. For this further. Glass slides were membrane-mounted (PEN mem- purpose, the laser-mediated microdissection (LMM) brane; P.A.L.M. Microlaser Technologies, Bernried, Ger- method has recently been adopted to obtain tissue many) and coated with 0.1% poly-L-lysine in sterile, diethyl samples exclusively from specific regions of interest. pyrocarbonate–treated H2O under RNase-free conditions. ␮ This novel technique has been used with considerable Cryosections (8 m) were fixed in 5% acetic acid/95% ethanol, stained with hematoxylin, and dehydrated in ascending alcohol success in the fields of oncology (13), embryology (14), concentrations. nephrology (15), and endocrinology (16) and has most LMM. LMM was performed using a Robot- recently also been introduced in the field of rheumatol- MicroBeam laser microscope (P.A.L.M. Microlaser Technol- ogy (12,17). ogies) as described previously (12,24). Briefly, the vascular Thus far, LMM technology has not been applied areas were selected, positioned, and cut out using a focused, pulsed laser beam. Dissected areas were collected on a micro- to the molecular analysis of synovial vessels, the regula- scope with a cannula (273⁄4-gauge) into the microcentrifuge tion of angiogenesis, and the genes expressed specifically tube containing RLT lysis buffer (RNeasy Micro Kit; Qiagen, in and around synovial microvasculature. Therefore, in Hilden, Germany) and lysed by vortexing. 1096 HASHIMOTO ET AL

Table 1. Conditions and primer sequences for real-time polymerase chain reaction*

MgCl2 Product Annealing concentration, Target length, bp temperature, °C mM Primer (5Ј–3Ј) Description 18S rRNA 187 50–59 3 CGGCTACCACATCCAAGGAA 20 mer GCTGGAATTACCGCGGCTGC 20 mer CD82 176 50 3 CCGGCAACAGGACCCAGAGT 21 mer CCGGCACAAGCAGATGGACA 21 mer Id2 55 55 3.5 AGGAATTGCCCAATCTAAGC 20 mer TATCACGCTCCACCTTTGAA 20 mer VEGF 104 56 3 GGGGCTGCTGCAATGACGAG 19 mer TCCTATGTGCTGGCCTTGGTGA 20 mer VEGFR-1 115 56 3 GCATATGGTATCCCTCAACCTACAA 25 mer CATCCAGGATAAAGGACTCTTCATTAT 27 mer VEGFR-2 132 56 3 ATGGGAACCGGAACCTCACTAT 22 mer TCTTTTCCTGGGCACCTTCTAT 22 mer PDGFR␣ 144 56 4 GGCACGCCGCTTCCTGATA 19 mer GCCCTCCACGGTACTCCTGTC 21 mer PDGFR␤ 108 59 3.5 TTCCTGTCTCTCTGCTGCTACCTG 24 mer ATCATCAAAGGAGCGGATCGAG 22 mer HIF-1␣ 167 52 3 TAGCCGAGGAAGAACTATGAAC 22 mer TTGATGGGTGAGGAATGG 18 mer HIF-2␣ 135 57 3 GGAGAACAGCAAGAGCAGGT 20 mer GGCAGCAGGTAGGACTCAAA 20 mer *rRNA ϭ ribosomal RNA; Id2 ϭ inhibitor of DNA binding/differentiation 2; VEGF ϭ vascular endothelial growth factor; VEGFR-1 ϭ VEGF receptor 1; PDGFR␣ ϭ platelet-derived growth factor receptor ␣; HIF-1␣ ϭ hypoxia-inducible factor 1␣.

To determine the minimum amount of cells necessary ing: all extracted RNA, 50 mM Tris HCl (pH 8.3) at 25°C, 50

to obtain enough RNA for a stable real-time quantitative mM KCl, 1 mM MgCl2, 0.5 mM spermidine, 1 mM dithiothre- polymerase chain reaction (PCR), vessels containing a defined itol, 1 mM each dNTP (Roche, Mannheim, Germany), 1 ␮ number of cells were microdissected from RA synovial tissue A260-unit random primer (Roche), 1.6 units/ l RNase inhibi- and used for RNA isolation, complementary DNA (cDNA) tor (Roche), and 1.3 units/␮l AMV reverse transcriptase synthesis, and real-time PCR. The area of interest was circled (Promega, Mannheim, Germany). Conditions for the cDNA using the tissue-marking function of the P.A.L.M. software, transcription were as follows: preannealing at 25°C for 10 and the number of cells in the vascular area was counted. To minutes, extension at 42°C for 60 minutes, and reverse tran- establish real-time PCR with the lasered material, 1,000, 2,000, scription (RT) inactivation at 99°C for 5 minutes. The resulting 5,000, and 10,000 cells were obtained. Total RNA was isolated, cDNA was stored at Ϫ20°C until further analyzed. and real-time PCR for CD82, which showed the weakest Real-time PCR. We used real-time PCR to compare expression of all genes analyzed, was performed. For quanti- mRNA expression of each gene in synovial vessels from fication with real-time PCR, it is recommended not to use an patients with RA or OA. Real-time PCR was performed using amplification crossing point over 40 cycles. Applying this rule, the LightCycler system (Roche) with SYBR Green detection we found that, for quantification purposes, cell numbers and melting curve analysis. The LightCycler system amplifies Ͼ5,000 were sufficient for quantification of CD82 RNA (data target nucleic acid and monitors the development of PCR not shown). Therefore, cell numbers of 8,000 (corresponding product by measuring fluorescence after each cycle (denatur- to areas of ϳ5mm2, depending on cell density and vessel size) ation, annealing, and extension) (25). The oligonucleotide were chosen for all experiments to ensure reliable quantifica- primers are listed in Table 1. As an endogenous control, 18S tion within the detection range of the PCR for all samples ribosomal RNA (rRNA) was measured. The PCR mixture ␮ measured. All other genes analyzed were more abundant in the contained 2 l of the generated cDNA or distilled H2O (for analyzed samples and therefore were within an appropriate negative control), 0.5 ␮M of each sense and antisense primers, quantification range of the real-time PCR. Efficiencies (E) of 10 ␮lof2ϫ QuantiTect SYBR Green PCR Master Mix all real-time PCRs reached the required values of E ϭ 2 Ϯ 0.05 (including HotStar Taq DNA polymerase, reaction buffer, ϭ Ϫ1/slope (calculated as E 10 ). dNTP mix, and SYBR Green I) (Qiagen), and MgCl2. The RNA extraction and cDNA synthesis. RNA was ex- final reaction volume was increased up to 20 ␮l by adding the

tracted by silica gel binding using the RNeasy Micro Kit required volume of distilled H2O. (Qiagen). To remove the remaining genomic DNA, total RNA Essentially, amplification was performed according to was treated using the RNase-free DNase Set (Qiagen) for 30 a standard protocol recommended by the manufacturer. For minutes at room temperature. Extraction was performed ac- each primer, the optimal annealing temperature in an ampli-

cording to the protocol described by the manufacturer, and fication cycle and the concentration of MgCl2 in the PCR RNA was eluted with 12 ␮l of RNase-free water. Complemen- mixture were determined individually (Table 1), and quantifi- tary DNA was synthesized in a mixture containing the follow- cation was confirmed using LightCycler software. Calculation GENE ANALYSIS OF SYNOVIAL VASCULATURE 1097

of the crossing point was done automatically by the second sured in 5 RA and 5 OA samples, normalized against derivative maximum method of LightCycler version 3 software. background, and subsequently normalized against the values Levels of mRNA were normalized with the level of endoge- obtained for RA, which were set as baseline (reference nous 18S rRNA. For comparison of the sample groups, the value ϭ 1). relative mRNA levels were subsequently normalized against In addition, synovial vascular density was evaluated in the values obtained for RA, which were set as baseline each of the 5 samples of RA synovium and the 5 samples of (reference value ϭ 1). OA synovium that were used for immunohistochemical analy- Immunohistochemical analysis. Snap-frozen sections sis. Blood vessels were stained specifically using an antibody ␮ (6 m) of synovial tissue from patients with RA or OA were against type IV collagen, and digital images of 4 fields of vision fixed in acetone and covered with 2% normal goat serum per sample were obtained, at a magnification of ϫ200. The (Jackson ImmunoResearch, West Grove, PA) in Tris–NaCl number of vessels per image was counted by 2 independent buffer to block nonspecific binding. The slides were then assessors, and the mean vascular density per square millimeter washed in Tris–NaCl buffer and incubated at room tempera- was determined for each sample. The number of VEGF– and ture for 60 minutes with anti-Id2 antibody (Santa Cruz Bio- technology, Santa Cruz, CA) at a 1:200 dilution, anti-VEGF VEGFR-2–expressing vessels was determined by the same antibody (BD PharMingen, Heidelberg, Germany) at a 1:50 method, and ratios of the number of VEGF-expressing vessels dilution, anti–VEGFR-1 antibody (Abcam, Cambridge, UK) versus the total number of vessels and the number of VEGFR- at a 1:50 dilution, anti–VEGFR-2 antibody (R&D Systems, 2–expressing vessels versus the total number of vessels were Wiesbaden, Germany) at a 1:50 dilution, anti–HIF-1␣ anti- calculated. body (Abcam) at a 1:8,000 dilution, anti–HIF-2␣ antibody Statistical analysis. All results are expressed as the (Abcam) at a 1:1,000 dilution, anti-CD82 antibody (Santa Cruz mean Ϯ SD. Data from real-time PCR were analyzed by the Biotechnology) at a 1:100 dilution, or anti–type IV collagen Mann-Whitney U test using GraphPad Prism statistical soft- antibody (DakoCytomation, Hamburg, Germany) at a 1:50 ware (GraphPad Software, San Diego, CA). Values for the dilution. Slides were rinsed in Tris–NaCl buffer and incubated intensity of immunohistochemical staining were obtained from for 60 minutes with a biotinylated secondary antibody, biotin defined region-of-interest analyses, as described, and analyzed polyclonal anti-rabbit immunoglobulin (for Id-2 and CD82) by the Mann-Whitney U test using GraphPad Prism statistical (BD PharMingen) at a 1:200 dilution, or biotin polyclonal software. Vascular density was determined for RA and OA anti-mouse immunoglobulin (for VEGFR-1) (BD PharMin- samples as described above and also was analyzed by the gen) at a 1:100 dilution. Incubation with streptavidin– Mann-Whitney U test using GraphPad Prism software. P horseradish peroxidase (Jackson ImmunoResearch) at a 1:600 values less than 0.05 were considered significant. dilution for 45 minutes was followed by color development with aminoethylcarbazole (AEC) substrate (Vector, Burlin- game, CA) at room temperature. Color development was RESULTS stopped under microscopic examination by washing with water. Slides stained with anti-VEGF, anti–VEGFR-2, and anti–type Patients. The study group comprised 12 patients IV collagen antibodies were rinsed in phosphate buffered with RA, all of whom met the ACR criteria for the saline, and endogenous peroxidase activity was blocked by classification of RA (23), and 12 patients with OA 0.3% hydrogen peroxide in methanol. The Histofine system (Nichirei, Tokyo, Japan), which contains secondary anti-goat (Table 2). The mean age of patients with RA was 63.8 antibodies (for VEGFR-2) or anti-mouse antibodies (for years (median 63.5 years, range 34–83 years), and the VEGF and type IV collagen), was used for development, with mean age of patients with OA was 64 years (median 60.5 subsequent color development using AEC as described above. years, range 52–76 years). Among patients with RA, the The slides were mounted in an aqueous mounting medium (Aquatex; Merck, Darmstadt, Germany). female-to-male ratio was 2:1, and the mean disease Analysis of immunohistochemical staining intensity. duration was 11.7 years (median 8.5 years, range 1–40 The intensity of immunohistochemical staining for Id2, years). Among patients with OA, the female-to-male ␣ ␣ VEGFR-1, HIF-1 , HIF-2 , and CD82 was analyzed using ratio was 10:2. For obvious clinical reasons, an exact image analysis software (Scion Image for Windows; Scion, Frederick, MD). The Scion Image program is based on the determination of disease duration was not possible. NIH image program for Macintosh, which has been estab- We examined area-specific mRNA and corre- lished as a useful tool for image analysis in medical sciences sponding protein expression of Id2, VEGF, VEGFR-1, and was used as previously described (26). Briefly, digital VEGFR-2, HIF-1␣, HIF-2␣, PDGFR␣, PDGFR␤, and micrograph data of immunohistochemistry slides were im- ported from the microscope-mounted digital imaging system as CD82 in the vessels of synovial tissue samples from a Scion Image file for analysis of staining intensity. A repre- patients with RA and patients with OA. The expression sentative region of interest was then selected from within the of mRNA was analyzed by LMM (Figure 1) and subse- stained wall of each vessel to be analyzed. In addition, a region quent real-time PCR. Protein expression was analyzed of interest in an unstained area outside the vasculature was defined and measured as background. For quantification of by immunohistochemistry. For comparison, we also an- staining intensity in the lining layer, regions of interest of alyzed the immunohistochemical staining of synovial 10,000 ␮m2 were defined. Thus, staining intensity was mea- lining and sublining samples in order to detect differ- 1098 HASHIMOTO ET AL

Table 2. Characteristics of the patients from whom synovial specimens were used for analysis of mRNA and protein expression* Patient/ Disease Rheumatoid CRP, ESR, Vascular density, age/sex Disease duration, years factor status mg/liter mm/hour mean Ϯ SD mm2 1/52/F RA 7 Negative 5.04 7 128.16 Ϯ 24.18 2/34/F RA 12 Positive 0.38 42 124.82 Ϯ 17.76 3/59/M RA 25 Positive 1.0 3 80.77 Ϯ 11.41 4/72/F RA 16 Not determined 6.28 35 Not determined† 5/63/M RA 6 Not determined 3.62 40 244.31 Ϯ 18.3 6/55/M RA 40 Not determined 0.32 10 68.09 Ϯ 37.28 7/64/F RA 12 Positive 3.43 50 96.79 Ϯ 11.2 8/76/F RA 3 Negative 0.9 13 112.81 Ϯ 22.06 9/61/F RA 7 Negative 0.84 12 118.15 Ϯ 13.33 10/79/F RA 10 Positive 4.4 73 106.8 Ϯ 13.61 11/68/F RA 1 Not determined 0.62 10 84.77 Ϯ 9.6 12/83/M RA 1 Not determined 0.32 10 Not determined† 13/76/F OA Undetermined Not determined 0.34 4 64.08 Ϯ 16.89 14/55/F OA Undetermined Not determined 0.47 27 Not determined† 15/71/F OA Undetermined Not determined 1.38 13 105.47 Ϯ 19.19 16/57/F OA Undetermined Not determined 2.36 16 Not determined† 17/52/M OA Undetermined Not determined 0.92 14 71.42 Ϯ 19.18 18/57/F OA Undetermined Not determined 0.45 8 71.42 Ϯ 29.6 19/76/M OA Undetermined Not determined 0.34 5 90.78 Ϯ 30.98 20/64/F OA Undetermined Not determined 0.77 5 87.44 Ϯ 26.73 21/57/F OA Undetermined Not determined 1.38 10 83.44 Ϯ 17.36 22/76/F OA Undetermined Not determined 0.71 24 73.43 Ϯ 7.06 23/56/F OA Undetermined Not determined 1.47 17 60.74 Ϯ 24.89 24/71/F OA Undetermined Not determined 0.54 8 85.44 Ϯ 25.75 * CRP ϭ C-reative protein; ESR ϭ erythrocyte sedimentation rate (Westergren method); RA ϭ rheumatoid arthritis; OA ϭ osteoarthritis. † Because of limited sample size. ences in expression that are specific to the synovial used for analysis. Expression of Id2 mRNA was signifi- microvasculature. cantly lower in RA synovial vessels compared with OA Analysis of gene expression in blood vessels from synovial vessels (0.9999 Ϯ 0.5979 versus 4.064 Ϯ 2.265; RA and OA synovium by LMM and real-time PCR. At P ϭ 0.0011) (Figure 2a). In contrast, expression of least 10 samples each of RA and OA synovium were VEGFR-1 mRNA was significantly higher in RA syno-

Figure 1. Laser-mediated microdissection using a hematoxylin-stained cryosection of rheumatoid arthritis synovial tissue. a, A defined area of the vasculature was marked using P.A.L.M. microdissector software. b, The preselected area was microdissected with a guided laser beam. (Original magnification ϫ 100.) GENE ANALYSIS OF SYNOVIAL VASCULATURE 1099

Figure 2. Relative mRNA expression of a, inhibitor of DNA binding/ differentiation 2 (Id2), b, vascular endothelial growth factor receptor 1 (VEGFR-1), and c, CD82 in synovial microvasculature of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). For comparison, the detected amounts were normalized against endogenous 18S ribosomal RNA and subsequently normalized against the values obtained for RA, which were set as baseline (reference value ϭ 1). Bars show the mean and SD.

vial vessels than in OA synovial vessels (1.0 Ϯ 0.3911 RA (0.999 Ϯ 0.3934), although this difference did not versus 0.6255 Ϯ 0.2363; P ϭ 0.0433) (Figure 2b). No reach statistical significance (P ϭ 0.0952). VEGFR-1 significant differences between RA and OA in mRNA protein expression could also be observed in both RA expression were detected for VEGF (P ϭ 0.7959), and OA synovial vessels (Figure 3). However, digital VEGFR-2 (P ϭ 0.7959), HIF-1␣ (P ϭ 0.5338), HIF-2␣ image analysis revealed no marked difference in the (P ϭ 0.1255), PDGFR␣ (P ϭ 0.2176), and PDGFR␤ intensity of VEGFR-1 staining between RA and OA (P ϭ 0.6305) (results not shown). Analysis of CD82 (1.0 Ϯ 0.2805 versus 1.011 Ϯ 0.4016; P ϭ 1.0). revealed a clear tendency toward higher expression in Protein expression of VEGF and VEGFR-2 was OA vessels (1.665 Ϯ 1.011) compared with RA vessels strong in both RA and OA vessels (Figure 3), but digital (0.9999 Ϯ 0.6408), but this difference did not reach image analysis revealed no statistically significant differ- statistical significance (P ϭ 0.0999) (Figure 2c). ences for either molecule (for VEGF, 1.0 Ϯ 0.3298 in Immunohistochemical analysis of protein ex- RA and 1.013 Ϯ 0.2525 in OA [P ϭ 1.0]; for VEGFR-2, pression in blood vessels from RA synovium and OA 1.0 Ϯ 0.2165 in RA and 1.158 Ϯ 0.1277 in OA [P ϭ synovium. In order to confirm the differential expression 0.3095]). Of note, VEGFR-2 expression was limited to of Id2 and VEGFR-1 mRNA in RA synovium and OA endothelial cells. synovium at the protein level, immunostaining was per- Protein expression of both HIF-1␣ (for RA, formed using specific antibodies. For each immunostain- 1.0 Ϯ 0.1735; for OA, 0.8371 Ϯ 0.1811 [P ϭ 0.4206]) and ing, 5 RA and 5 OA synovial samples were used. HIF-2␣ (for RA, 1.0 Ϯ 0.4962; for OA, 0.7229 Ϯ 0.2454 In accordance with the results of the real-time [P ϭ 0.3939]) did not differ in the synovial vasculature PCR experiments, Id2 expression was stronger in syno- (results not shown). CD82 was readily detected in the vial vessels from patients with OA compared with that in vessels of both RA synovium and OA synovium (Figure synovial vessels from patients with RA (Figure 3). 3), and the intensity of CD82 staining was similar in both Digital image analysis showed a 2.5-fold higher mean sample groups (for RA, 1.0 Ϯ 0.2396; for OA, 1.244 Ϯ intensity of Id2 staining in OA (2.463 Ϯ 1.154) versus 0.2104 [P ϭ 0.0952]). 1100 HASHIMOTO ET AL

differences in protein expression between the synovium as a whole and the synovial vessels. Therefore, the expression of Id2, VEGF, VEGFR-1, VEGFR-2, HIF- 1␣, HIF-2␣, and CD82 protein was also analyzed in the synovial lining and sublining layers.

Figure 3. Photomicrographs showing protein expression in the synovial microvasculature of patients with RA and patients with OA, by immunohistochemical analysis. Differential expression was observed for Id2 (original magnification ϫ 200). See Figure 2 for definitions.

Immunohistochemical analysis of protein expression in the lining layer of RA synovium and OA Figure 4. Photomicrographs showing protein expression in the synovial lining layers of patients with RA and patients with OA, by synovium. Because previous studies on the expression of immunohistochemical analysis. Differential expression was observed angiogenesis-related molecules have analyzed samples for Id2 and CD82 (original magnification ϫ 100). See Figure 2 for of whole synovium, we intended to investigate potential definitions. GENE ANALYSIS OF SYNOVIAL VASCULATURE 1101

Figure 5. Comparison of histologic features of rheumatoid arthritis (RA) synovium and osteoarthritis (OA) synovium. a, Photomicrographs showing differences in synovial lining thickness and cellularity as an indicator of inflammatory activity (by hematoxylin and eosin [H&E] staining), differences in the number and density of synovial vessels (by type IV collagen staining), differences in the number of vascular endothelial growth factor (VEGF)–expressing cells, and differences in the number and density of VEGF receptor 2 (VEGFR-2)– expressing vessels (original magnification ϫ 100). b, Results of statistical analysis showing differences between RA and OA synovium in the number of total vessels and VEGFR-2–expressing vessels.

Results of immunohistochemical analysis (Figure RA compared with OA. Thus, visual comparison of 4) revealed strong expression of Id2 in the synovial lining protein staining by immunohistochemistry can be mis- of RA samples, which was significantly higher than that leading, and digital image analysis was applied again, in in OA synovial lining (1.0 Ϯ 0.3839 versus 0.4094 Ϯ order to obtain objective values for comparison. 0.1217; P ϭ 0.0079). VEGFR-1 protein expression could Consistent with previous reports (3,27,28), we also be observed in both RA and OA synovial lining observed expression of both HIF-1␣ and HIF-2␣ in the layers (Figure 4), but no difference was detected by synovial lining layer, whereas expression in the synovial digital image analysis (0.9998 Ϯ 0.4980 for RA and sublining layer was very sparse (data not shown). How- 1.373 Ϯ 0.7816 for OA; P ϭ 0.3095). Likewise, the ever, digital image analysis revealed no difference be- staining intensity for VEGF expression (Figure 4) did tween RA and OA in the expression levels of either not differ between RA and OA synovial lining (1.0 Ϯ molecule (for HIF-1␣, 0.9999 Ϯ 0.3347 versus 0.7984 Ϯ 0.642 versus 0.9463 Ϯ 0.434; P ϭ 1.0). However, due to 0.1852; P ϭ 0.3095; for HIF-2␣, 1.0 Ϯ 0.5934 versus the increased thickness of RA synovial lining, the num- 0.5279 Ϯ 0.2470; P ϭ 0.0931). CD82 staining was also ber of VEGF-expressing cells far exceeded that of OA detected in the lining layer of both RA synovium and synovial lining (Figures 4 and 5a). In contrast to what OA synovium (Figure 4), but digital image analysis was observed in the synovial vasculature, VEGFR-2 revealed significantly stronger expression in RA com- (Figure 4) was not detectable in synovial lining cells pared with OA (0.9999 Ϯ 0.3829 versus 0.4764 Ϯ 0.3937; obtained from patients with either disease (P ϭ 0.8413). P ϭ 0.0317). Hematoxylin and eosin staining (Figure 5a) illus- In our RA and OA samples, the finding of similar trated the strongly hyperplastic synovial lining layer in expression levels of the key angiogenic molecules VEGF 1102 HASHIMOTO ET AL

and VEGFR-2 in mRNA and protein analyses raised the ital image analysis confirmed the interesting observation question of whether there is any meaningful difference that in the vascular compartment there is no difference in angiogenesis between the two diseases. Therefore, we between RA and OA in the expression levels of VEGF, performed additional immunohistochemistry studies us- VEGFR-2, the HIF proteins, the PDGF receptors, and ing an antibody against type IV collagen that facilitates CD82. Of note, this analysis also revealed that the selective staining of blood vessels. Quantitative analysis difference in mRNA expression for VEGFR-1 and Id2 revealed a significantly higher number of total vessels in did not translate to protein expression, although a RA synovium compared with OA synovium (116.5 Ϯ 2.5-fold higher level of Id2 protein was detected in the 50.16 versus 79.37 Ϯ 24.09 vessels/mm2; P Ͻ 0.0001), OA vasculature, which is noteworthy and consistent with indicating more productive angiogenesis in RA (Figures the mRNA analysis. 5a and b). Of interest, the number of VEGFR-2– In order to investigate potential differences in expressing (and thus presumably VEGF-responsive) ves- gene and protein expression patterns between the syno- sels per square millimeter (Figures 5a and b) was also vial vasculature and surrounding synovial tissue, immu- found to be significantly higher in RA than in OA nohistochemical staining of the synovial lining and sub- (71.16 Ϯ 28.17 versus 35.24 Ϯ 14.14; P Ͻ 0.0001), lining layers was conducted. Again, consistent with the consistent with an important role for VEGF in RA above-mentioned findings, no difference between RA angiogenesis. and OA could be detected for VEGF and its receptors in terms of protein expression. There was, however, no discernable expression of VEGFR-2 in the synovial DISCUSSION lining cells, in contrast to the blood vessels. This clear The majority of novel therapeutic strategies that distribution of VEGFR-2, which appears to be limited to are currently being tested and introduced into clinical vascular endothelial cells in our samples, is consistent practice target distinct molecules and mechanisms of with previous reports on the expression pattern of this disease within the inflamed joints of patients with ar- molecule under physiologic conditions (29) and in RA thritides. Thus, it is of utmost interest to be able to (30). In a study of OA, however, Ikeda and coworkers identify such molecules and molecular mechanisms spe- (31) described the absence of VEGFR-2 mRNA expres- cifically, precisely, and reliably. This requires high- sion, which is in contrast to our findings of vascular resolution analysis techniques, which thus provide a high expression of VEGFR-2 mRNA as well as protein in all degree of specificity. 12 of the OA tissue samples analyzed. The reason for Such an approach, LMM, was used in this study this discrepancy remains to be elucidated, but it most to selectively analyze the expression of several genes likely is attributable to different levels of sensitivity known to be involved in angiogenesis in the vasculature between RT-PCR of whole synovial samples (as used by of RA and OA synovium. Of the 9 genes analyzed in this Ikeda et al) and our quantitative real-time PCR of setting, only two genes, Id2 and VEGFR-1, showed a selectively microdissected microvasculature. significant difference in expression between RA and OA Our results appear also to contradict those of synovial vessels by real-time PCR. In contrast, no signif- earlier studies, which showed significantly higher levels icant difference was observed for VEGF, VEGFR-2, of VEGF protein in RA compared with OA synovial HIF-1␣, HIF-2␣, PDGFR␣, PDGFR␤, and CD82.In fluid (30,32). Likewise, Hollander et al (27) demon- addition, using the method of comparing amplification strated that HIF-1␣ was more abundant in RA synovium crossing points, as described in Patients and Methods, than in OA synovium. However, none of those studies we were able to define the minimal (3,000) and optimal analyzed the expression of VEGF or HIF-1␣ specifically (5,000–8,000) numbers of vascular cells (endothelial on the cellular level. Increased levels of VEGF in RA cells and smooth muscle cells) to be excised by LMM in synovial fluid could be caused by either increased pro- order to ensure reproducible and valid real-time PCR tein expression by individual synovial cells or by a higher results (Figure 2). These results are consistent with those total number of VEGF-producing cells. As illustrated by from our previous study on area-specific gene expression Figure 5, the present data support the hypothesis that in nonmalignant rheumatic diseases such as RA (12). the higher total cellularity of RA synovium in compari- In a second step, we analyzed the degree of son with OA synovium determines the elevated amounts correlation between this selective, quantitative analysis of VEGF in RA synovial fluid, whereas the individual of mRNA expression and the corresponding protein cellular expression levels are similar in both diseases. expression. Immunohistochemistry with subsequent dig- Analogous to this finding, the difference in expression of GENE ANALYSIS OF SYNOVIAL VASCULATURE 1103

synovial HIF-1␣ that was observed in the study by lining compared with that in OA synovial lining. Of Hollander et al (27) is attributable to a difference in the note, we also confirmed distinct expression of CD82 in number of HIF-1␣–positive cells in their samples (as the vasculature of the synovial sublining. Interestingly, determined by immunohistochemistry) and was not similar expression of CD82 mRNA and CD82 protein caused by a difference in cellular protein expression could be demonstrated in RA and OA microvasculature, levels, because comparative quantitative analyses of suggesting a potential common role for CD82 in angio- mRNA and protein expression were not performed. genesis. Moreover, a dense staining pattern in a thickened CD82 is a member of the transmembrane 4 synovial lining layer can create the impression of higher superfamily, which also includes CD9, CD63, and CD81. protein expression levels, as illustrated by the VEGF It was originally identified as a metastasis-suppressor staining shown in Figure 5a. Objective determination of gene for prostate cancer (34) and other malignancies. In staining intensity by digital image analysis, however, analogy to particular effects reported in tumor cell facilitates the elucidation of such discrepancies. studies, CD82 could have a regulatory function in ar- Thus, it is tempting to hypothesize that either the thritic synovium, aiming at suppression of proliferation stimuli for the formation of new synovial vessels are of mesenchymal and/or endothelial cells. In tumor cell equally intense in RA and OA, or that in both diseases lines, expression of CD82 also reduced the proteolytic the stimulated cells express the proangiogenic factors activity of pericellular urokinase-type plasminogen acti- VEGF and HIF at a maximal rate that does not differ vator (35) and decreased adhesion to the matrix constit- between the two diseases. Both assumptions would uent laminin (36), both of which are also important underscore the importance of (neo)angiogenesis in the mechanisms operative in RA pathophysiology. pathogenesis of RA and OA. Id2 is a member of the family of Id proteins In this scenario, the markedly increased cellular- (Id1–Id4), which function as inhibitors of DNA binding ity of RA synovium would afford an excess in the total and inhibitors of differentiation and as such impede amount of proangiogenic factors in comparison with OA cellular differentiation, induce cellular proliferation, and and thus result in more efficient angiogenesis. This have the ability to inhibit apoptosis (37). With regard to assumption is supported by the significantly higher num- angiogenesis, Id proteins can mediate proliferation, ber of VEGFR-2–expressing (and thus VEGF- transmigration, and tube formation of endothelial cells responsive) vessels in RA synovium and, accordingly, (38,39) as well as proliferation of vascular smooth muscle a significantly higher total number of vessels in our cells (40,41). Appropriate stimuli for this effect include study. This finding matches the results reported by ␤ Giatromanolaki et al (33), who also observed an ele- VEGF (39), transforming growth factor (39,42), HIF-1 vated number of VEGFR-2–expressing vessels in RA. (43), and bone morphogenetic proteins (44). Interestingly, the total number of vessels did not differ Thus far, little is known about the role of Id between RA and OA in that study. This discrepancy may proteins in arthritis angiogenesis, because the majority be explained, at least in part, by differences in RA of results have been derived from studies on tumor disease activity as described by Ikeda et al (31), who angiogenesis. In RA and OA, expression of Id proteins observed significantly higher vascular density in synovial has been reported for Id1, Id2, and Id3. Of these, Id1 samples that were characterized by the expression of the and Id3 were localized predominantly in vascular endothelial cells, and expression levels were higher in RA mitogenic VEGF165 isoform (31). In addition to analyzing the classic players in than in OA (45). In contrast, vascular Id2 expression in angiogenesis, VEGF, VEGF receptors, and HIF, we our study was clearly, although not significantly, higher analyzed the specific expression pattern of two other in OA. It can be hypothesized that the expression of Id2 molecules, Id2 and CD82, the exact roles of which in in vascular epithelium could be of relatively greater angiogenesis are a subject of great interest. CD82 was importance for the angiogenic processes operative in included in our current analysis because its high expres- OA compared with RA. sion in RA had been detected in a recent study by our Of interest, the synovial expression pattern of Id2 group (22) using high-resolution DNA array analysis, distant from the vasculature in our study is consistent but hitherto no specific function or role of CD82 in with hypoxia-induced up-regulation in synovial fibro- arthritis could be assigned. The current immunohisto- blasts and synovial macrophages, as described in previ- chemistry results confirmed our previous findings of ous studies (21,46). The significantly higher expression markedly up-regulated CD82 expression in RA synovial of Id2 in RA synovial lining is also consistent with the 1104 HASHIMOTO ET AL

proliferative and invasive nature of the activated syno- factor prevents collagen-induced arthritis and ameliorates estab- vium in RA. lished disease in mice. Biochem Biophys Res Commun 2001;281: 562–8. In summary, we have established and optimized 8. Takahashi H, Kato K, Miyake K, Hirai Y, Yoshino S, Shimada T. the combination of LMM and subsequent validation by Adeno-associated virus vector-mediated anti-angiogenic gene real-time PCR and immunohistochemistry for the ana- therapy for collagen-induced arthritis in mice. Clin Exp Rheuma- tol 2005;23:455–61. lysis of specific mRNA and protein expression in RA 9. Chen Y, Donnelly E, Kobayashi H, DeBusk LM, Lin PC. Gene and OA synovial microvasculature. The technique therapy targeting the Tie2 function ameliorates collagen-induced proved to be feasible, sensitive, and reliable for the arthritis and protects against bone destruction. Arthritis Rheum 2005;52:1585–94. overall goal of analyzing area-specific gene expression in 10. Nagashima M, Tanaka H, Takahashi H, Tachihara A, Tanaka K, a complex, nonmalignant rheumatic disease. Moreover, Ishiwata T, et al. Study of the mechanism involved in angiogenesis the data demonstrate that previously described differ- and synovial cell proliferation in human synovial tissues of patients with rheumatoid arthritis using SCID mice. Lab Invest 2002;82: ences between RA and OA in the protein levels of 981–8. VEGF and HIF-1␣ are not caused by higher expression 11. Bernier SG, Lazarus DD, Clark E, Doyle B, Labenski MT, levels in the individual cells but by a higher total number Thompson CD, et al. A methionine aminopeptidase-2 inhibitor, of cells expressing these molecules in RA. This under- PPI-2458, for the treatment of rheumatoid arthritis. Proc Natl Acad SciUSA2004;101:10768–73. lines the relevance of an area-specific, high-resolution, 12. Judex M, Neumann E, Lechner S, Dietmaier W, Ballhorn W, quantitative approach as compared with total tissue Grifka J, et al. Laser-mediated microdissection facilitates analysis analysis for elucidating potential disease-specific molec- of area-specific gene expression in rheumatoid synovium. Arthritis Rheum 2003;48:97–102. ular mechanisms. 13. Lechner S, Muller-Ladner U, Renke B, Scholmerich J, Ruschoff J, Kullmann F. Gene expression pattern of laser microdissected colonic crypts of adenomas with low grade dysplasia. Gut 2003;52: AUTHOR CONTRIBUTIONS 1148–53. Dr. Tarner had full access to all of the data in the study and 14. Imamichi Y, Koebernick K, Wedlich D. Prospects for tissue- takes responsibility for the integrity of the data and the accuracy of the specific analysis of gene expression in Xenopus embryos through data analysis. laser-mediated microdissection of histological sections. Pathol Res Study design. Hashimoto, Tarner, Bohle, Ulfgren, Schulz, Mu¨ller- Pract 2003;199:381–9. Ladner, Neumann. 15. Fries JW, Roth T, Dienes HP, Weber M, Odenthal M. A novel Acquisition of data. Hashimoto, Tarner, Bohle, Gaumann, Manetti, evaluation method for paraffinized human renal biopsies using Steinmeyer, Ulfgren, Mu¨ller-Ladner, Neumann. quantitative analysis of microdissected glomeruli and VCAM-1 as Analysis and interpretation of data. Hashimoto, Tarner, Bohle, Gau- marker of inflammatory mesangial cell activation. Nephrol Dial mann, Manetti, Distler, Schulz, Gay, Mu¨ller-Ladner, Neumann. Transplant 2003;18:710–6. Manuscript preparation. Hashimoto, Tarner, Gaumann, Manetti, 16. Hong SH, Nah HY, Lee JY, Gye MC, Kim CH, Kim MK. Analysis Distler, Gay, Mu¨ller-Ladner, Neumann. of estrogen-regulated genes in mouse uterus using cDNA microar- Statistical analysis. Hashimoto, Tarner. ray and laser capture microdissection. J Endocrinol 2004;181: Specimen collection. Steinmeyer. 157–67. 17. Judex M, Neumann E, Gay S, Muller-Ladner U. Laser-mediated microdissection as a tool for molecular analysis in arthritis. REFERENCES Methods Mol Med 2004;101:93–105. 18. Distler O, Neidhart M, Gay RE, Gay S. The molecular control of 1. Tarner IH, Harle P, Muller-Ladner U, Gay RE, Gay S. The angiogenesis. Int Rev Immunol 2002;21:33–49. different stages of synovitis: acute vs chronic, early vs late and 19. Shibuya M. Structure and function of VEGF/VEGF-receptor non-erosive vs erosive [review]. Best Pract Res Clin Rheumatol system involved in angiogenesis. Cell Struct Funct 2001;26:25–35. 2005;19:19–35. 20. Raines EW. PDGF and cardiovascular disease. Cytokine Growth 2. Muller-Ladner U, Pap T, Gay RE, Neidhart M, Gay S. Mecha- Factor Rev 2004;15:237–54. nisms of disease: the molecular and cellular basis of joint destruc- 21. Kurowska-Stolarska M, Distler J, Pap T, Jungel A, Michel BA, tion in rheumatoid arthritis. Nat Clin Pract Rheumatol 2005;1: Simmen BR, et al. The inhibitor of differentiation-2 (Id-2) induced 102–10. by hypoxia promotes bone degradation in rheumatoid arthritis 3. Distler JH, Wenger RH, Gassmann M, Kurowska M, Hirth A, Gay (RA) [abstract]. Arthritis Rheum 2004;50 Suppl 9:S655. S, et al. Physiologic responses to hypoxia and implications for 22. Neumann E, Kullmann F, Judex M, Justen HP, Wessinghage D, hypoxia-inducible factors in the pathogenesis of rheumatoid ar- Gay S, et al. Identification of differentially expressed genes in thritis [review]. Arthritis Rheum 2004;50:10–23. rheumatoid arthritis by a combination of complementary DNA 4. Pap T, Distler O. Linking angiogenesis to bone destruction in array and RNA arbitrarily primed–polymerase chain reaction. arthritis [editorial]. Arthritis Rheum 2005;52:1346–8. Arthritis Rheum 2002;46:52–63. 5. Gaber T, Dziurla R, Tripmacher R, Burmester GR, Buttgereit F. 23. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,

Hypoxia inducible factor (HIF) in rheumatology: low O2! See what Cooper NS, et al. The American Rheumatism Association 1987 HIF can do! Ann Rheum Dis 2005;64:971–80. revised criteria for the classification of rheumatoid arthritis. 6. Lu J, Kasama T, Kobayashi K, Yoda Y, Shiozawa F, Hanyuda M, Arthritis Rheum 1988;31:315–24. et al. Vascular endothelial growth factor expression and regulation 24. Lechner S, Muller-Ladner U, Neumann E, Dietmaier W, Welsh J, of murine collagen-induced arthritis. J Immunol 2000;164:5922–7. Scholmerich J, et al. Use of simplified transcriptors for the analysis 7. Sone H, Kawakami Y, Sakauchi M, Nakamura Y, Takahashi A, of gene expression profiles in laser-microdissected cell popula- Shimano H, et al. Neutralization of vascular endothelial growth tions. Lab Invest 2001;81:1233–42. GENE ANALYSIS OF SYNOVIAL VASCULATURE 1105

25. O’Mahony J, Hill C. A real time PCR assay for the detection and down-regulating integrin ␣6-mediated cell adhesion. J Biol Chem quantitation of Mycobacterium avium subsp. paratuberculosis 2005;280:3346–54. using SYBR Green and the Light Cycler. J Microbiol Methods 37. Pagliuca A, Gallo P, De Luca P, Lania L. Class A helix-loop-helix 2002;51:283–93. proteins are positive regulators of several cyclin-dependent kinase 26. Mize RR. Quantitative image analysis for immunohistochemistry inhibitors’ promoter activity and negatively affect cell growth. and in situ hybridization. J Neurosci Methods 1994;54:219–37. Cancer Res 2000;60:1376–82. 27. Hollander AP, Corke KP, Freemont AJ, Lewis CE. Expression of 38. Aitkenhead M, Wang SJ, Nakatsu MN, Mestas J, Heard C, hypoxia-inducible factor 1␣ by macrophages in the rheumatoid Hughes CC. Identification of endothelial cell genes expressed in synovium: implications for targeting of therapeutic genes to the an in vitro model of angiogenesis: induction of ESM-1, ␤ig-h3, and inflamed joint. Arthritis Rheum 2001;44:1540–4. NrCAM. Microvasc Res 2002;63:159–71. 28. Giatromanolaki A, Sivridis E, Maltezos E, Athanassou N, Papa- 39. Sakurai D, Tsuchiya N, Yamaguchi A, Okaji Y, Tsuno NH, Kobata zoglou D, Gatter KC, et al. Upregulated hypoxia inducible fac- T, et al. Crucial role of inhibitor of DNA binding/differentiation in tor-1␣ and -2␣ pathway in rheumatoid arthritis and osteoarthritis. the vascular endothelial growth factor-induced activation and Arthritis Res Ther 2003;5:R193–201. angiogenic processes of human endothelial cells. J Immunol 29. Shibuya M. Vascular endothelial growth factor (VEGF)-Receptor 2004;173:5801–9. 2: its biological functions, major signaling pathway, and specific 40. Matsumura ME, Lobe DR, McNamara CA. Contribution of the helix-loop-helix factor Id2 to regulation of vascular smooth muscle ligand VEGF-E [review]. Endothelium 2006;13:63–9. cell proliferation. J Biol Chem 2002;277:7293–7. 30. Fava RA, Olsen NJ, Spencer-Green G, Yeo KT, Yeo TK, Berse B, 41. Kumar MS, Hendrix JA, Johnson AD, Owens GK. Smooth muscle et al. Vascular permeability factor/endothelial growth factor ␤-actin gene requires two E-boxes for proper expression in vivo (VPF/VEGF): accumulation and expression in human synovial and is a target of class I basic helix-loop-helix proteins. Circ Res fluids and rheumatoid synovial tissue. J Exp Med 1994;80:341–6. 2003;92:840–7. 31. Ikeda M, Hosoda Y, Hirose S, Okada Y, Ikeda E. Expression of 42. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, vascular endothelial growth factor isoforms and their receptors ten Dijke P. Balancing the activation state of the endothelium via Flt-1, KDR, and neurophilin-1 in synovial tissues of rheumatoid two distinct TGF-␤ type I receptors. EMBO J 2002;21:1743–53. arthritis. J Pathol 2000;191:426–33. 43. Lofstedt T, Jogi A, Sigvardsson M, Gradin K, Poellinger L, 32. Koch AE, Harlow LA, Haines GK, Amento EP, Unemori EN, Pahlman S, et al. Induction of ID2 expression by hypoxia-inducible Wong WL, et al. Vascular endothelial growth factor: a cytokine factor-1: a role in dedifferentiation of hypoxic neuroblastoma cells. modulating endothelial function in rheumatoid arthritis. J Immu- J Biol Chem 2004;279:39223–31. nol 1994;152:4149–56. 44. Valdimarsdottir G, Goumans MJ, Rosendahl A, Brugman M, Itoh 33. Giatromanolaki A, Sivridis E, Athanassou N, Zois E, Thorpe PE, S, Lebrin F, et al. Stimulation of Id1 expression by bone morpho- Brekken RA, et al. The angiogenic pathway ‘vascular endothelial genetic protein is sufficient and necessary for bone morphogenetic growth factor/flk-1(KDR)-receptor’ in rheumatoid arthritis and protein-induced activation of endothelial cells. Circulation 2002; osteoarthritis. J Pathol 2001;94:101–8. 106:2263–70. 34. Dong JT, Lamb PW, Rinker-Schaeffer CW, Vukanovic J, Ichikawa 45. Sakurai D, Yamaguchi A, Tsuchiya N, Yamamoto K, Tokunaga K. T, Isaacs JT, et al. KAI1, a metastasis suppressor gene for prostate Expression of ID family genes in the synovia from patients with cancer on human chromosome 11p11.2. Science 1995;268:884–6. rheumatoid arthritis. Biochem Biophys Res Commun 2001;284: 35. Bass R, Werner F, Odintsova E, Sugiura T, Berditchevski F, Ellis 436–42. V. Regulation of urokinase receptor proteolytic function by the 46. Burke B, Giannoudis A, Corke KP, Gill D, Wells M, Ziegler- tetraspanin CD82. J Biol Chem 2005;280:14811–8. Heitbrock L, et al. Hypoxia-induced gene expression in human 36. He B, Liu L, Cook GA, Grgurevich S, Jennings LK, Zhang XA. macrophages: implications for ischemic tissues and hypoxia-regu- Tetraspanin CD82 attenuates cellular morphogenesis through lated gene therapy. Am J Pathol 2003;163:1233–43. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1106–1117 DOI 10.1002/art.22493 © 2007, American College of Rheumatology

LIGHT Up-Regulated on B Lymphocytes and Monocytes in Rheumatoid Arthritis Mediates Cellular Adhesion and Metalloproteinase Production by Synoviocytes

Young Mo Kang, So Young Kim, Jin Hee Kang, Seung Woo Han, Eon Jeong Nam, Hee Soo Kyung, Jae Yong Park, and In San Kim

Objective. To study the expression of LIGHT molecules, which were efficiently inhibited by dexameth- (tumor necrosis factor superfamily 14) and herpesvirus asone. LIGHT-mediated up-regulation of MMPs and entry mediator (HVEM; tumor necrosis factor receptor intercellular adhesion molecule 1 was blocked by inhib- superfamily 14) in rheumatoid arthritis (RA) and to itors of NF-␬B and JNK, whereas up-regulation of determine the regulatory role of LIGHT on the effector vascular cell adhesion molecule 1 was blocked by inhib- functions of fibroblast-like synoviocytes (FLS). itors of phosphatidylinositol 3-kinase, as well as NF-␬B. Methods. The expression of LIGHT and HVEM Conclusion. These data suggest that binding of was assessed by immunohistochemical staining of syno- LIGHT with its receptors may play a role in the vial tissue and by flow cytometric analysis of mono- progression of inflammation within rheumatoid syno- nuclear cells. The presence of HVEM and lymphotoxin vium, especially by mediating the interactions between ␤ receptor was measured by reverse transcriptase– infiltrating inflammatory cells and stromal cells. These polymerase chain reaction and by flow cytometry. The findings thus emphasize the relevance of LIGHT as a regulation of effector molecules, including matrix met- potential therapeutic target in RA. alloproteinases (MMPs) and adhesion molecules, was evaluated. The adhesiveness of FLS was determined by Rheumatoid synovitis is characterized by infiltra- adhesion assay. tion of T cells, B cells, and macrophages, which may Results. HVEM was detected in most cell types interact with each other and with resident cells that within rheumatoid synovial tissue, while only a few cells proliferate to form a hyperplastic lining layer via direct were positive for LIGHT. In RA patients, LIGHT ex- cell–cell contact and secretion of inflammation media- pression was significantly up-regulated only in CD20؉ tors such as cytokines (1). Tumor necrosis factor (TNF)– B cells and monocytes, whereas the mean fluorescence intensity of HVEM was down-regulated in mononuclear related cytokines may be involved in the progression of cells. The stimulation of FLS with LIGHT resulted in inflammation by mediating the interaction between im- the production of MMPs and the expression of adhesion mune cells and parenchymal cells such as fibroblast-like synoviocytes (FLS) and providing a tissue-specific pattern to inflammatory injury (2,3). The TNF superfamily Supported by the Basic Research Program of the Korea (TNFSF) of cytokines, which is currently recognized as Science and Engineering Foundation (grant R01-2003-000-10887-0) and the Brain Korea 21 Project in 2006. consisting of more than 40 distinct ligand–receptor Young Mo Kang, MD, PhD, So Young Kim, MS, Jin Hee systems, activates signaling pathways for cell survival, Kang, MS, Seung Woo Han, MD, Eon Jeong Nam, MD, PhD, Hee Soo death, and differentiation of lymphoid and nonlymphoid Kyung, MD, PhD, Jae Yong Park, MD, PhD, In San Kim, MD, PhD: Kyungpook National University School of Medicine, Daegu, Republic tissue (4). Significant cross-utilization of ligands and of Korea. receptors among the TNFSF members has been ob- Address correspondence and reprint requests to Young Mo Kang, MD, PhD, Division of Rheumatology, Department of Internal served in various systems. The results of controlled Medicine, Kyungpook National University Hospital, Samduk 2-Ga, clinical trials, which showed that approximately one- Junggu, Daegu 700-721, Republic of Korea. E-mail: ymkang@ third of patients with rheumatoid arthritis (RA) did not knu.ac.kr. ␣ Submitted for publication June 30, 2006; accepted in revised respond to anti-TNF therapy, suggest that other TN- form December 21, 2006. FSF members may be involved (3,5).

1106 ROLE OF LIGHT IN RA 1107

LIGHT (TNFSF14), which is active in either MATERIALS AND METHODS membrane-anchored or secreted form, is expressed in Tissue from patients. To analyze the expression of activated lymphocytes and dendritic cells, and binds to LIGHT and HVEM in lymphocytes and monocytes, heparin- recognize 3 different members of the TNF receptor ized PB was collected from 15 patients with RA (3 men and 12 (TNFR) superfamily, including lymphotoxin ␤ receptor women) and 9 healthy controls (3 men and 6 women). RA was (LT␤R), herpesvirus entry mediator (HVEM), and de- defined according to the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 re- coy receptor 3 (6,7). Among the 3 receptors, HVEM is vised criteria (23). Patients with RA ranged in age from 27 to widely expressed in cells of the immune system such as T 74 years (mean Ϯ SD 52.8 Ϯ 15.0 years). Healthy controls and B cells, natural killer (NK) cells, dendritic cells, and ranged in age from 24 to 48 years (mean Ϯ SD 33.3 Ϯ 8.4 endothelial cells (8,9), while LT␤R is found on fibro- years). All RA patients received nonsteroidal antiinflamma- blasts, monocytic cells, endothelial cells, as well as tory drugs; 12 patients received methotrexate, and 12 patients received other disease-modifying antirheumatic drugs. RA epithelial and stromal cells (10). The biologic functions patients had a mean Ϯ SD disease duration of 7.5 Ϯ 9.2 years, of these receptors are often cell context specific due an erythrocyte sedimentation rate of 49.79 Ϯ 36.49 mm/hour, to their expression by a variety of cell types (11). Re- and a C-reactive protein level of 4.18 Ϯ 4.52 mg/dl. All donors sults of previous studies indicate that LIGHT is a potent had no recent history of infection for 1 month prior to sample collection. Synovial fluid was prepared during therapeutic T cell costimulatory molecule via interaction with arthrocentesis. Synovial tissue was obtained from 7 RA pa- HVEM that is expressed in T cells, while other costimu- tients and 5 osteoarthritis (OA) patients at the time of total latory molecules mediate interaction between T cells joint replacement. Tonsil tissue (kindly provided by Dr. Jung and antigen-presenting cells (APCs) (12–14). Consti- Soo Kim, Kyungpook National University) was obtained during a tonsilloadenoidectomy. This study was approved by the tutive expression of LIGHT in T cells causes the activa- Kyungpook National University Hospital Institutional Review tion and expansion of peripheral T cells, which subse- Board, and all donors of blood and synovial fluid provided quently leads to the breakdown of peripheral tolerance written informed consent. and the spontaneous development of autoimmune dis- Isolation of mononuclear cells and FLS. Mononuclear cells were isolated by Ficoll-Hypaque (Amersham Biosciences, eases (15,16). Uppsala, Sweden) density-gradient centrifugation of heparin- LIGHT, with lymphotoxins and TNF, forms an ized venous blood (peripheral blood mononuclear cells integrated signaling network necessary for efficient in- [PBMCs]) and synovial fluid (synovial fluid mononuclear cells nate and adaptive immune responses (3), and may be [SFMCs]). FLS were isolated by enzymatic dispersion of synovial tissue, as previously described (24). FLS at passages involved in the pathogenesis of RA. Prophylactic treat- 4–8 were used for the experiments. ment with the LT␤R-Ig fusion protein has been shown Antibodies and reagents. Monoclonal antibodies to to block the induction of collagen-induced arthritis in CD3 (UCHT-1), CD14 (TU¨ K4), CD68 (EBM II), and biotin- mice by reducing the production of anti–type II collagen ylated rabbit anti-mouse Ig were purchased from Dako (Glostrup, Denmark), recombinant human cytokines, includ- antibodies, supporting the notion that the LT/LIGHT ing LIGHT, interleukin-1␤ (IL-1␤), and TNF␣, and monoclo- system has an important role in RA (17). The interaction nal antibodies against LIGHT (115520), LT␤R (71319), between LT␤/LIGHT and LT␤R has been shown to play HVEM (94801), and vascular cell adhesion molecule 1 a crucial role in ectopic lymphoid organogenesis, which (VCAM-1) (BBIG-V1 [4B2]) were obtained from R&D Sys- tems (Minneapolis, MN), monoclonal antibodies to MMP-1 is observed in chronic inflammatory foci in autoimmune (41-1E5), MMP-3 (55-2A4), and tissue inhibitor of metallo- diseases, such as RA (18–21). In a previous study, proteinases 1 (TIMP-1) (NaTIA2) were purchased from LIGHT expressed in the synovial tissue of RA patients Chemicon (Temecula, CA), and the monoclonal antibody to induced the production of chemokines, cytokines, and GAPDH (mAbcam 9484) was obtained from Abcam (Cam- bridge, UK). The monoclonal antibody to intercellular adhe- matrix metalloproteinase 9 (MMP-9) from macrophage sion molecule 1 (ICAM-1) was a gift from Dr. Chang Ho Jun cell lines (22). The cellular source of LIGHT in RA, (Kyungpook National University School of Medicine). Phyco- however, has yet to be elucidated. erythrin (PE)–labeled antibodies against CD3 (SK7) and ␸ In the present study, we sought evidence regard- CD14 (M P9), and fluorescein isothiocyanate (FITC)–labeled ␤ goat anti-mouse antibody were purchased from BD Bio- ing the involvement of the LT R/LIGHT/HVEM inter- sciences (San Jose, CA), and PE-labeled antibody against action in RA by examining LIGHT- or HVEM- CD20 (MEM-7) was purchased from Dinona (Seoul, Korea). expressing cells in synovial tissue and peripheral blood NF-␬B pathway inhibitors, including Bay 11-7082 and sul- (PB). We further examined the functional consequences fasalazine, were purchased from Calbiochem (Darmstadt, Ger- many), and inhibitors of MEK (PD98059), JNK (SP600125), of stimulating FLS with LIGHT, by evaluating the effect p38 MAPK (SB203580), and phosphatidylinositol 3-kinase (PI on MMP production and cellular adhesiveness. 3-kinase; LY294002) were obtained from EMD Biosciences 1108 KANG ET AL

(Darmstadt, Germany). Dexamethasone and cyclosporin A blots were then incubated for 2 hours with horseradish were obtained from Sigma (St. Louis, MO). peroxidase–conjugated goat anti-mouse antibody (Amersham Immunohistochemical staining. Biopsy tissue was em- Biosciences) and were visualized using an enhanced chemilu- bedded in OCT compound. Sections of tissue were incubated minescence detection system (SuperSignal kit; Pierce, Rock- with the primary antibodies for 30 minutes and then with ford, IL). The intensity of the band signals in exposed film was biotinylated secondary antibody for 30 minutes. Slides were determined by densitometric scanning and analyzed using NIH developed using Vectastatin Elite ABC reagent (Vector, Bur- Image J software, version 1.36. lingame, CA) and 3,3Ј-diaminobenzidine (Dako), and were Adhesion assay. FLS were cultured in 96-well culture 4 counterstained with hematoxylin. Control sections were stained plates at 1.5 ϫ 10 cells/well and were stimulated with recom- with irrelevant primary isotype–specific IgG antibodies. binant human LIGHT or with TNF␣ for 24 hours. PB lympho- Flow cytometry. PBMC and SFMC quantities were cytes (PBLs) were prepared after removing adherent cells from ␮ adjusted to 1 ϫ 107 cells/ml in complete media. Cells (5 ϫ PBMCs and were labeled with 5 M carboxyfluorescein suc- 105/ml) were incubated with unconjugated primary antibodies cinimidyl ester (CFSE; Molecular Probes, Eugene, OR). PBLs ϫ 4 against LIGHT and HVEM at 4°C for 30 minutes, followed by (5 10 cells/well) were irradiated with 4,000 rads and FITC-conjugated goat anti-mouse Ig at 4°C for 30 minutes. cocultured on stimulated FLS monolayers. Nonadherent PBLs Phenotypes of mononuclear cells were assigned using PE- were removed by gentle washing with phosphate buffered conjugated antibodies to CD3, CD20, and CD14 (BD Bio- saline prewarmed to 37°C. The number of adherent PBLs was sciences). Each flow cytometry experiment included cells in- estimated by counting CFSE-stained cells in 5 high-power cubated with fluorochrome-conjugated IgG (R&D Systems). fields in each well, using ultraviolet fluorescence microscopy FLS were incubated under adherent conditions in medium (Leica Microsystems, Wetzlar, Germany), and photomicro- alone or in the presence of stimulants for 24 hours at 37°C graphs of adherent PBLs on the FLS monolayer were prepared when indicated. Cells were incubated with primary antibodies by confocal laser microscopy (Bio-Rad, Richmond, CA). at 4°C for 30 minutes and then with FITC-conjugated goat Statistical analysis. Results are expressed as the Ϯ anti-mouse Ig at 4°C for 30 minutes. Cytometric data were mean SEM. The statistical significance of the differences acquired using a flow cytometer (FACSCalibur; BD Bio- between groups was calculated using Student’s t-test. P values sciences) and analyzed with WinMDI software (developed by less than 0.05 were considered statistically significant. Joseph Trotter, Scripps Research Institute, La Jolla, CA). Reverse transcriptase–polymerase chain reaction (RT- RESULTS PCR). Total RNA was extracted from FLS. Complementary DNA was obtained from total RNA using oligo(dT) and avian Expression of LIGHT and HVEM in rheumatoid myeloblastosis virus reverse transcriptase (Roche, Indianapo- synovitis. To evaluate the expression pattern of LIGHT lis, IN), and amplified with primers (Bioneer, Hørsholm, Denmark) as follows: for HVEM, sense 5Ј-CTT-GAG-GCT- and HVEM in rheumatoid synovitis, immunohistochem- GGT-GCT-GTA-TC-3Ј and antisense 5Ј-CTT-CAC-ACG- ical staining was performed on synovial tissue obtained ATA-ACC-TGG-AC-3Ј (133 bp); for LT␤R, sense 5Ј- from 7 patients with RA and 5 patients with OA. In RA ATA-ACA-TAG-GCG-CTC-CGC-TGA-3Ј and antisense 5Ј- synovial tissue, HVEM was detected in most types of AGT-AGA-GGT-AAT-AGA-GGC-CG-3Ј (134 bp); for MMP-1, sense 5Ј-GGG-AGC-AAA-CAC-ATC-TGA-CC-3Ј and antisense 5Ј-TCC-CGA-TGA-TCT-CCC-CTG-AC-3Ј (184 bp); for MMP-3, sense 5Ј-GGC-TGT-ATG-AAG-GAG- AGG-CTG-3Ј and antisense 5Ј-TGG-TCC-CTG-TTG-TAT- CCT-TTG-3Ј (181 bp); for TIMP-1, sense 5Ј-GAG-ACA- CCA-GAG-AAC-CCA-CC-3Ј and antisense 5Ј-GGT-GTA- GAC-GAA-CCG-GAT-G-3Ј (278 bp); and for GAPDH, sense 5Ј-GGA-GTC-AAC-GGA-TTT-GGT-CG-3Ј and antisense 5Ј- GAC-GGT-GCC-ATG-GAA-TTT-GC-3Ј (162 bp). Amplifi- cation was conducted for 35 cycles at an annealing temperature of 60°C for 20 seconds, and PCR products were separated by electrophoresis using 2% agarose gel. Western blot analysis. To prepare proteins secreted from the FLS, culture supernatants were precipitated with trichloroacetic acid. To prepare whole cell lysate, FLS were lysed in a buffer containing 10 mmoles/liter HEPES (pH 7.9), 10 mmoles/liter KCl, 0.1 mmole/liter EGTA, 0.1 mmole/liter EDTA, 1 mmole/liter dithiothreitol, 1 ␮g/ml aprotinin, 1 ␮g/ml Figure 1. Immunohistochemical detection of herpesvirus entry medi- leupeptin, 1 ␮g/ml pepstatin, and 1 mM phenylmethylsulfonyl ator (HVEM) in synovial tissue from patients with rheumatoid arthri- fluoride (sodium deoxycholate, sodium dodecyl sulfate, Non- tis (RA) and osteoarthritis (OA). Expression of HVEM, CD14, and idet P40). CD68 was detected in RA synovial tissue (A, C, and E), but not in OA Equal amounts of protein were used for each experi- synovial tissue (B, D, and F). As a negative control, nonimmunized ment. Proteins were separated on 12% sodium dodecyl isotype control antibody was used for staining (G and H). Expression sulfate–polyacrylamide gels and transferred to nitrocellulose of HVEM and CD20 was also examined in tonsil tissue (I and J). membranes, which were probed with specific antibodies. The (Hematoxylin counterstained; original magnification ϫ 200.) ROLE OF LIGHT IN RA 1109

Figure 2. Expression of LIGHT and herpesvirus entry mediator (HVEM) on the surface of mononuclear cells in healthy controls (HCs) and rheumatoid arthritis (RA) patients. A, Flow cytometry showing LIGHT expression in peripheral blood lymphocytes (PBLs). B, Percentage of LIGHT-positive lymphocytes in PB from 7 controls and 9 RA patients. Values are the ,ϭ P Ͻ 0.05 versus controls. C, Flow cytometry showing LIGHT expression in CD20ϩ B cells in PB. D ء .mean and SEM Representative histograms showing LIGHT expression in CD3ϩ and CD3Ϫ lymphocytes after incubation of control PBLs with phorbol myristate acetate (PMA; 1 ng/ml) and ionomycin (1 ␮g/ml) and analysis by flow cytometry. E, Percentage of ϭ ء .LIGHT-positive cells among CD14ϩ monocytes in PB from controls and RA patients. Values are the mean and SEM P Ͻ 0.05 versus controls. F, Expression levels (mean fluorescence intensity [MFI]) of HVEM in CD3ϩ and CD3Ϫ lymphocytes and CD14ϩ PB mononuclear cells (PBMCs) from controls and RA patients, and in synovial fluid mononuclear ϭ P Ͻ 0.05 versus control PBMCs. G, HVEM expression ء .cells (SFMCs) from RA patients. Values are the mean and SEM in cultured CD3ϩ T lymphocytes before and at various times after stimulation with PMA and ionomycin. cells within the lining and sublining layers (Figure 1A), and immature dendritic cells (13,25,26), and is tightly although the staining intensity was not as strong as that regulated in T cells following activation (27). We ex- of CD68 and CD14 (Figures 1C and F). In tonsil tissue, plored whether LIGHT expression is up-regulated in a secondary lymphoid organ, HVEM was detected in lymphocytes and monocytes of RA patients. Within the almost all types of cells except for a part of B cells within lymphocyte compartment, a small proportion of lympho- the dark zone of secondary follicles (Figures 1I and J). cytes was positive for LIGHT molecules on the surface, In contrast to HVEM, LIGHT was only sparsely ex- and the number of LIGHT-expressing lymphocytes was pressed within the synovial tissue of RA patients and was significantly higher in patients with RA compared with not detected in the synovial tissue of OA patients healthy controls (mean Ϯ SEM 8.03 Ϯ 0.9 versus 5.69 Ϯ (results not shown). 0.68; P Ͻ 0.05) (Figures 2A and B). Although previous Regulation of expression of LIGHT and HVEM studies performed using artificial systems showed that in lymphocytes and monocytes of RA patients. LIGHT the expression of LIGHT was up-regulated in T cells expression is found in activated lymphocytes, NK cells, after activation (25), CD3ϩ T cells in PB of healthy 1110 KANG ET AL

Figure 3. Expression of herpesvirus entry mediator (HVEM) transcripts in fibroblast-like synoviocytes (FLS). Primary FLS were established from synovial tissue obtained from rheumatoid arthritis (RA) patients. A, Transcripts of HVEM and lymphotoxin ␤ receptor (LT␤R) were amplified after reverse transcription of total RNA isolated from confluent FLS from 4 RA patients. N ϭ negative control. B, Expression of HVEM and LT␤R was examined by flow cytometry using antibodies against the respective molecules. Data are expressed as the number of cells plotted as a function of fluorescence intensity and are representative of 3 independent experiments. Broken lines represent the negative control; solid lines represent the surface expression of each receptor. controls or RA patients did not have LIGHT molecules more frequent in synovial fluid compared with the PB of on the surface. Most lymphocytes expressing LIGHT on RA patients (data not shown). the surface were confined to CD20ϩ B cells (Figure 2C). HVEM messenger RNA and surface protein are We then evaluated the expression of LIGHT in constitutively expressed in PB T cells, B cells, and lymphocytes after stimulation with phorbol myristate monocytes (9,27), and are down-regulated on the sur- acetate (PMA; 1 ng/ml) and ionomycin (1 ␮g/ml) and face of T cells after activation (25). In healthy controls found that LIGHT was up-regulated on T lymphocytes and RA patients, considerable levels of HVEM were within 24 hours. LIGHT expression was induced earlier seen on the surface of mononuclear cells. Lymphocytes in non–T lymphocytes after stimulation (Figure 2D). and monocytes both constitutively expressed HVEM These findings were consistent with the results of a on the cell surface. HVEM expression levels in CD3ϩ previous study (25). The percentage of LIGHT- and CD3Ϫ lymphocytes and monocytes from PB, how- expressing monocytes was also significantly higher in the ever, were lower in RA patients than in healthy controls. PB of RA patients compared with that of healthy The mean fluorescence intensity of HVEM was further controls (mean Ϯ SEM 28.01 Ϯ 6.26 versus 17.90 Ϯ 6.38; down-regulated in SFMCs of RA patients, which was P Ͻ 0.05) (Figure 2E). LIGHT-positive monocytes were most conspicuous in CD3Ϫ lymphocytes (Figure 2F). ROLE OF LIGHT IN RA 1111

Figure 4. Induction of matrix metalloproteinase (MMP) production from fibroblast-like synoviocytes (FLS) by LIGHT. A, FLS were incubated with recombinant human LIGHT (100 ng/ml) for the indicated periods, and transcripts of MMP-1, MMP-3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) were amplified with specific primers. B and C, Western blot analysis of MMP-1, MMP-3, and TIMP-1 was performed with culture supernatants, and their relative band densities were determined. FLS were incubated for 24 hours with recombinant human LIGHT (100 ng/ml), interleukin-1␤ (IL-1␤) (ϩϭ0.1 ng/ml, ϩϩ ϭ 10 ng/ml), and tumor necrosis factor ␣ (TNF␣)(ϩϭ0.1 ng/ml, ϩϩ ϭ 10 ng/ml), either alone or in combination (B), or were stimulated with LIGHT (100 ng/ml) in combination with IL-1␤ in the presence or absence of dexamethasone (Dexam; 100 nM) or cyclosporin A (CSA; 1 ␮g/ml) (C). Graphs in C show the mean and SD optical ϭ P Ͻ 0.05 versus FLS stimulated with LIGHT and IL-1␤ without ء .density from at least 3 independent experiments dexamethasone.

Stimulation of cultured PBLs with PMA (1 ng/ml) and tion system in RA (2,24,28). We hypothesized that the ionomycin (1 ␮g/ml) decreased the expression of HVEM FLS is a target parenchymal cell of LIGHT, based on the in the T cell (Figure 2G) and non–T cell populations findings that HVEM is widely distributed on synovial within 6 hours. These results demonstrate that the lining and sublining layers (Figure 1). HVEM transcripts pathogenesis of RA involves the up-regulation of were constitutively expressed in FLS isolated from 4 RA LIGHT and the down-regulation of HVEM on mono- patients (Figure 3A). Surface phenotype analyses of FLS nuclear cells. showed reactivity to the anti-HVEM and anti-LT␤R HVEM and LT␤R expression in FLS. LT/LIGHT antibodies (Figure 3B). pathways provide essential communication links be- Production of MMP-1, MMP-3, and TIMP-1 tween lymphocytes and the surrounding stromal and from FLS induced by LIGHT. It has been suggested that parenchymal cells via interaction with LT␤R and MMPs are the most important matrix-degrading en- HVEM (3). FLS are important resident cells within the zymes responsible for the destruction of cartilage in RA synovial membrane and could function as the amplifica- (1). LIGHT has been shown to induce MMP-9 in 1112 KANG ET AL

Figure 5. Regulation of cellular adhesion molecules (intercellular adhesion molecule 1 [ICAM-1] and vascular cell adhesion molecule 1 [VCAM-1]) and adhesiveness of FLS by LIGHT. A, FLS surface expression of ICAM-1 was determined by flow cytometry. Results shown are representative of 3 independent experiments. B and C, Following incubation, cultures were harvested and whole cell lysates were subjected to Western blot analysis. FLS were incubated with LIGHT (100 ng/ml) alone or in combination with IL-1␤ (0.1 ng/ml) and tumor necrosis factor ␣ (0.1 ng/ml) for 24 hours in serum-free Dulbecco’s modified Eagle’s medium (B), or were treated with dexamethasone (100 nM) or CSA (1 ␮g/ml) in the presence of LIGHT (100 ng/ml) and 2 different concentrations of IL-1␤ (ϩϭ0.1 ng/ml, ϩϩ ϭ 10 ng/ml) (C). Molecular size markers are shown in B. Graphs show the ϭ P Ͻ 0.05 versus FLS stimulated with LIGHT and IL-1␤ without ء .mean and SD optical density (OD) from at least 3 independent experiments dexamethasone. D, FLS in a confluent layer were stimulated with LIGHT or TNF␣ for 24 hours, followed by coculture with carboxyfluorescein succinimidyl ester–labeled peripheral blood lymphocytes (PBLs; green) and examination by confocal microscopy. Representative photomicrographs are shown (original magnification ϫ 20). E, FLS were pretreated with the indicated stimulants, and the number of PBLs adherent to FLS was determined by counting the number of green fluorescent cells in 5 random fields per culture. PBLs were prepared from different donors in each experiment. Values are the mean and SD of at least 3 independent experiments using 2 FLS lines derived from different donors. See Figure 4 for other definitions. macrophage cell lines (22). To determine whether TIMP-1 was increased in an additive manner. LIGHT LIGHT stimulates the production of MMPs from FLS, did not demonstrate any additional effects on TNF␣- transcripts of MMP-1, MMP-3, and TIMP-1 from FLS induced production (Figure 4B). Interferon-␥ inhibited were analyzed. When exposed to LIGHT, the transcripts the production of MMP-1, MMP-3, and TIMP-1 that were up-regulated within 4 hours (Figure 4A). Culture was induced by LIGHT (results not shown). To deter- supernatants from stimulated FLS were examined in mine whether therapeutic agents can modulate the order to measure the production of these proteins. induction of MMPs and TIMP-1 by LIGHT, FLS were Stimulation with LIGHT induced the secretion of treated with dexamethasone (100 nM) and cyclosporin A MMPs and TIMP-1 in a dose-dependent manner (re- (1 ␮g/ml). The production of MMP-1, MMP-3, and sults not shown). TIMP-1, induced by LIGHT in combination with IL-1␤, When compared with IL-1␤ and TNF␣, a higher was efficiently inhibited by dexamethasone but not by concentration of LIGHT was required to induce MMPs. cyclosporin A (Figure 4C). These results indicate that When FLS were stimulated with LIGHT in combination LIGHT induces the production of MMPs from FLS in with IL-1␤ (0.1 ng/ml), the production of MMPs and combination with IL-1␤. ROLE OF LIGHT IN RA 1113

Up-regulation of expression of cell adhesion mol- Signaling pathways of FLS activation by LIGHT. ecules (CAMs) in FLS. In RA, CAMs within the hyper- The 2 main receptors for LIGHT, HVEM and LT␤R, plastic synovial lining may be a critical regulator of directly interact with TNFR-associated factor 2 synovial lining organization and may modulate the par- (TRAF2) and TRAF5, which leads to gene induction by ticipation of FLS in synovial inflammation (29). To the activation of NF-␬B or the JNK/activator protein 1 determine whether LIGHT could induce the expression (AP-1) pathway (31,32). To determine whether LIGHT of CAMs in FLS, flow cytometry and Western blot stimulation of the MMP/TIMP system is mediated by assays were performed. ICAM-1, which is constitutively activation of the NF-␬B or AP-1 pathways, FLS were expressed in FLS, was up-regulated after stimulation pretreated with NF-␬B inhibitors, including Bay 11- with LIGHT (Figures 5A and B). LIGHT showed an 7082, or inhibitors of MEK (PD98059) and JNK additive effect on the IL-1␤–induced up-regulation of (SP600125) before stimulation with LIGHT. Inhibition ␤ ICAM-1. When LIGHT or IL-1 (0.1 ng/ml) was used to of the NF-␬B pathway by Bay 11-7082 significantly stimulate FLS, VCAM-1 expression was weakly induced. attenuated the production of MMP-1 and TIMP-1 in- ␤ Costimulation with LIGHT and IL-1 increased duced by LIGHT stimulation in FLS (Figure 6A). VCAM-1 expression by 3–4-fold compared with that PD98059 and SP600125 also inhibited LIGHT-induced observed in FLS treated with one of these cytokines MMP-1 and TIMP-1 synthesis, whereas a p38 MAPK individually (Figure 5B). inhibitor (SB203580) and a PI 3-kinase inhibitor We examined the modulation of LIGHT-induced (LY294002) did not (Figures 6A and B). NF-␬B and CAM expression by therapeutic agents. Dexamethasone MEK inhibitors together had no additional effect on reversed the expression of ICAM-1 and VCAM-1 to the MMP-1 and TIMP-1 production compared with the level of that observed with pretreated FLS. When FLS NF-␬B inhibitor alone. ␤ were stimulated with LIGHT and a higher dose of IL-1 Up-regulation of adhesion molecules appears to (10 ng/ml), the effect of dexamethasone on the expres- involve both transcriptional and posttranscriptional sion of ICAM-1 and VCAM-1 was divergent, in that mechanisms. The promoters of ICAM-1 and VCAM-1 VCAM-1 expression was inhibited to pretreatment lev- include binding motifs for the transcriptional factors els but ICAM-1 expression was not. Cyclosporin A did NF-␬B and AP-1 (33). Both ICAM-1 and VCAM-1, not inhibit the expression of CAMs induced by LIGHT induced by LIGHT, were efficiently inhibited by Bay ␤ and IL-1 (Figure 5C). 11-7082 (Figures 6A and C). Expression of ICAM-1 was The potential role of CAMs in the recruitment of efficiently inhibited by the JNK inhibitor, but not by inflammatory cells into the synovial sublining may in- PD98059, SB203580, or LY294002. Consistent with pre- volve interaction between inflammatory cells and mes- viously reported findings (34), these results indicated enchymal cells (29,30). To investigate the biologic con- that LIGHT activates both the NF-␬B and the JNK sequences of altered expression of CAMs in FLS, we pathways in FLS. Although the induction of VCAM-1 to examined the interaction of PBLs with unstimulated and pretreatment levels was also inhibited by Bay 11-7082, LIGHT-stimulated FLS monolayers. PBLs, prepared by inhibition of JNK did not influence the production of removing adherent cells from PBMCs, consisted of VCAM-1. LY294002 and PD98059 efficiently blocked 84.6% T cells and 4.6% B cells and were labeled with VCAM-1 production induced by LIGHT (Figure 6A). CFSE. FLS in confluent layers were stimulated with 1 These data imply that LIGHT activates differential ng/ml or 100 ng/ml of LIGHT. The number of CFSE- signaling pathways, in addition to the common pathway labeled adherent PBLs was quantified by fluorescence of NF-␬B, in inducing effector molecules. microscopy after coculture with conditioned FLS for 1 hour at 37°C. Stimulation with LIGHT increased the number of PBLs adherent to FLS, in a dose-dependent DISCUSSION manner. Exposure of FLS to 100 ng/ml of LIGHT Previous studies have shown that LIGHT func- resulted in an almost 2-fold increase in the number of tions as a costimulatory molecule on T cells by inducing adherent PBLs versus controls (mean Ϯ SD 41.43 Ϯ 9.80 proliferation, expression of activation markers, and cy- versus 25.03 Ϯ 4.47; P ϭ 0.007) (Figure 5E). These tokine production (8,12,14). An in vitro study using results demonstrate that LIGHT plays a role in the purified T cells from healthy donors revealed that, after progression of inflammation by regulating cell–cell con- activation of T cells, LIGHT expression increases from tact between FLS and leukocytes via the regulation of baseline undetectable levels (25). Transgene expression CAM expression. of LIGHT in T cells resulted in enhanced Th1 cytokine 1114 KANG ET AL

Figure 6. Effects of signaling pathway inhibitors on LIGHT-induced expression of MMPs and cell adhesion molecules. Confluent FLS were pretreated with DMSO (control medium) or inhibitors of signaling pathways, followed by treatment with LIGHT (100 ng/ml). Proteins were purified from culture supernatants or whole cell lysates after another 24 hours of incubation and were measured by Western blot analysis, using antibodies against MMP-1, MMP-3, TIMP-1, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and GAPDH. A, FLS were pretreated with DMSO, Bay 11-7082 (Bay; 5 ␮M), sulfasalazine (SSZ; 400 ␮M), or PD98059 (10 ␮M), alone or in combination. Results of a representative Western blot experiment are shown. Graphs show the mean and SD optical ϭ P Ͻ 0.05 versus FLS stimulated with LIGHT alone. B, FLS ء .density (OD) from at least 3 independent experiments were pretreated with DMSO, SP600125 (10 ␮M), SB203580 (10 ␮M), or LY294002 (10 ␮M). Results of a representative experiment are shown. C, Flow cytometric analysis of the surface expression of ICAM-1 by FLS pretreated with the indicated inhibitors before stimulation with LIGHT (100 ng/ml). Representative histograms indicate ICAM-1 on FLS compared with isotype staining (broken lines). See Figure 4 for other definitions. activity in mucosal T cells and an increase in the number LIGHT-positive lymphocytes in both groups were of activated T cells (15,16). In the present study, how- CD20ϩ B cells. ever, the expression of LIGHT was not up-regulated in Another source of LIGHT among PBMCs was T cells in the PB or synovial fluid of RA patients. When CD14ϩ monocytes, and the frequency of LIGHT- we stimulated PBLs isolated from healthy donors with positive monocytes also increased in the PB and synovial PMA and ionomycin, LIGHT expression increased both fluid of RA patients. Although the specific functions of in T cells and in non–T cells. The discrepancy between LIGHT-expressing cells in RA have not been investi- the results from artificial culture systems and those from gated, the cells may activate T lymphocytes by providing RA patients may be explained by the absence, in the a costimulatory signal via LIGHT–HVEM interaction, inflammatory milieu of RA, of the potent stimuli used in since one of the common features of LIGHT-positive the in vitro study. The proportion of LIGHT-expressing cells is their role as APCs. Alternatively, they may B lymphocytes was significantly higher in RA patients mediate interactions of infiltrating leukocytes and stro- compared with healthy controls, and nearly all of the mal cells in the synovium via the binding of LIGHT to ROLE OF LIGHT IN RA 1115

LT␤R or HVEM, which was widely expressed in nearly LIGHT has overlapping patterns of ligand– all types of cells within rheumatoid synovial tissue receptor binding with 3 closely related ligands, including (Figure 1). LT␣,LT␤, and TNF (39). In vitro studies showed that Of the 3 receptors for LIGHT, HVEM is mainly receptor binding of LT␤ and TNF resulted in the expressed in hemopoietic tissue, while LT␤R is primarily attraction and localization of leukocytes to areas of expressed in cells of stromal origin (9,11). Through inflammation by the activation of genes involved in interaction with HVEM, LIGHT and LT␣ (another proinflammatory reactions, including chemokines and ligand for HVEM) may activate T cells, B cells, and CAMs (24,40). The present data showed that LIGHT, monocytes in RA patients. Activation of T cells has been alone and in combination with proinflammatory cyto- shown to be followed by a rapid decrease of HVEM kines, induces the expression of ICAM-1 and VCAM-1 expression (14,25). Expression levels of HVEM may be from FLS and efficiently facilitates the adhesion of T directly down-modulated by the binding of LIGHT (25). and B lymphocytes. Lower levels of HVEM expression in T cells, B cells, and One of the cardinal features of rheumatoid syno- monocytes of RA patients, as shown in the present vitis is the recruitment and retention of inflammatory study, may reflect the activation of mononuclear cells by cells in the sublining layer, which sometimes organize direct cell–cell contact and by soluble mediators, which into complex lymphoid structures, including lymphocyte are abundant in rheumatoid synovitis. aggregates or ectopic germinal centers (30,41). Expres- The LT/LIGHT system is part of a complex sion of ICAM-1 and VCAM-1 in FLS, up-regulated by communication system linking lymphocytes and sur- LIGHT, can help retain cells in the synovial tissue by rounding parenchymal and stromal cells, including FLS, binding to integrins. FLS have been shown to promote B in synovial tissue. The present study revealed that in lymphocyte survival via a VCAM/␣4␤1 integrin– cultured FLS, HVEM is constitutively expressed in dependent mechanism, which induces expression of the addition to LT␤R, and this is consistent with the results antiapoptotic molecule Bcl-xL (42). ICAM-1 and of a previous study (24). Although HVEM and LT␤R VCAM-1 expressed in FLS may contribute to T cell have been shown to be differentially expressed according activation by supporting a costimulatory signal. Through to cell type, dendritic cells (27), human umbilical vein the enhanced retention, increased cell survival, and endothelial cells (35), and certain tumor cells (26) activation and proliferation of inflammatory cells, me- display both receptors. Stimulation of LIGHT may diated by up-regulated expression of CAMs, LIGHT trigger the activation of FLS by the engagement of either may contribute to the perpetuation of synovial inflam- receptor. Further studies are necessary to elucidate mation. which receptor plays a dominant role in the LIGHT- HVEM and LT␤R contain cytoplasmic domains induced activation of FLS. that engage TRAF adapters (11), 2 of which, TRAF2 FLS are the main cellular compartment that and TRAF5, are involved in the activation of NF-␬Bor causes cartilage degradation by the release of proteolytic JNK/AP-1 pathways (32). It has been shown that enzymes, including a broad range of MMPs. Among MMP-1 and MMP-3 genes have regulatory elements in these MMPs, MMP-1 and MMP-3 are regarded as common on their 5Ј-flanking promoter regions that may important mainly for their role in the destruction of be activated by transcription factors such as AP-1 and joints in RA. In this study, we showed that the produc- NF-␬B (43–46). LIGHT-induced MMP production was tion of MMP-1 and MMP-3 in FLS was significantly efficiently blocked by an NF-␬B inhibitor (Bay 11-8082) up-regulated by LIGHT. It has been reported that the and a JNK inhibitor (SP600125), indicating that both the biosynthesis of MMP-1 and MMP-3 is up-regulated by NF-␬B pathway and the JNK/AP-1 pathway may be cytokines such as IL-1, TNF␣, epidermal growth factor, critical in regulating MMP induction from primary FLS and platelet-derived growth factor in a variety of cells by LIGHT. In contrast to the MMP/TIMP-1 system, the (36–38). We found that LIGHT-induced production of signaling pathways for ICAM-1 and VCAM-1, stimu- MMPs from FLS was up-regulated in a synergistic lated by LIGHT, were divergent. NF-␬B is critical for manner with IL-1␤ at a lower dose, but not with TNF␣. the expression of ICAM-1 and VCAM-1, since deletion The synergistic effect was not observed when FLS were or mutation of the NF-␬B sites abolishes cytokine- stimulated with LIGHT and IL-1␤ at a higher dose, inducible transcription of these molecules (47). suggesting that LIGHT-induced up-regulation of MMPs Although the promoters of ICAM-1 and may be mediated, at least in part, by induction of VCAM-1 contain binding motifs for AP-1, LIGHT- synthesis of IL-1␤. induced VCAM-1 expression was not inhibited by JNK 1116 KANG ET AL

inhibitors. LIGHT modulated VCAM-1 expression in identified member of the tumor necrosis factor receptor superfam- FLS via the PI 3-kinase pathway, as well as via the ily with a wide tissue distribution and involvement in lymphocyte activation. J Biol Chem 1997;272:14272–6. NF-␬B pathway. Morel et al (48) showed that PI 10. Browning JL, Miatkowski K, Sizing I, Griffiths D, Zafari M, 3-kinase, as well as NF-␬B, is involved in IL-18–induced Benjamin CD, et al. Signaling through the lymphotoxin ␤ receptor VCAM-1 expression in RA FLS. Although the down- induces the death of some adenocarcinoma tumor lines. J Exp Med 1996;183:867–78. stream transcription factor that may be activated by PI 11. Granger SW, Rickert S. LIGHT-HVEM signaling and the regu- 3-kinase remains unidentified, AP-1 and STAT-3 might lation of T cell-mediated immunity [review]. Cytokine Growth be candidates because these transcription factors are Factor Rev 2003;14:289–96. 12. Tamada K, Shimozaki K, Chapoval AI, Zhu G, Sica G, Flies D, et already known to be activated by PI 3-kinase (49,50). al. Modulation of T-cell-mediated immunity in tumor and graft- Our data indicate that differential transcriptional regu- versus-host disease models through the LIGHT co-stimulatory lation mechanisms are present for the induction of pathway. Nat Med 2000;6:283–9. ␤ 13. Tamada K, Shimozaki K, Chapoval AI, Zhai Y, Su J, Chen SF, et effector molecules via LT R/LIGHT/HVEM interac- al. LIGHT, a TNF-like molecule, costimulates T cell proliferation tion. and is required for dendritic cell-mediated allogeneic T cell In conclusion, our data show that LT␤R/LIGHT/ response. J Immunol 2000;164:4105–10. 14. Harrop JA, McDonnell PC, Brigham-Burke M, Lyn SD, Minton J, HVEM interaction may be involved in the progression Tan KB, et al. Herpesvirus entry mediator ligand (HVEM-L), a of inflammation in rheumatoid synovium, especially by novel ligand for HVEM/TR2, stimulates proliferation of T cells mediating the interaction between infiltrating inflamma- and inhibits HT29 cell growth. J Biol Chem 1998;273:27548–56. 15. Shaikh RB, Santee S, Granger SW, Butrovich K, Cheung T, tory cells and resident cells such as FLS. Strategies Kronenberg M, et al. Constitutive expression of LIGHT on T cells aimed at blocking LIGHT–receptor interaction may leads to lymphocyte activation, inflammation, and tissue destruc- provide a new tool for the treatment of RA. tion. J Immunol 2001;167:6330–7. 16. Wang J, Lo JC, Foster A, Yu P, Chen HM, Wang Y, et al. The regulation of T cell homeostasis and autoimmunity by T cell- AUTHOR CONTRIBUTIONS derived LIGHT. J Clin Invest 2001;108:1771–80. 17. Fava RA, Notidis E, Hunt J, Szanya V, Ratcliffe N, Ngam-Ek A, Dr. Y. M. Kang had full access to all of the data in the study et al. A role for the lymphotoxin/LIGHT axis in the pathogenesis and takes responsibility for the integrity of the data and the accuracy of murine collagen-induced arthritis. J Immunol 2003;171:115–26. of the data analysis. 18. Wang J, Foster A, Chin R, Yu P, Sun Y, Wang Y, et al. The Study design. Y. M. Kang, Nam, I. S. Kim. complementation of lymphotoxin deficiency with LIGHT, a newly Acquisition of data. S. Y. Kim, J. H. Kang, Han, Park, Kyung. discovered TNF family member, for the restoration of secondary Analysis and interpretation of data. Y. M. Kang, Park, I. S. Kim. lymphoid structure and function. Eur J Immunol 2002;32:1969–79. Manuscript preparation. Y. M. Kang, S. Y. Kim, J. H. Kang. 19. Takemura S, Braun A, Crowson C, Kurtin PJ, Cofield RH, Statistical analysis. Y. M. Kang, Han. O’Fallon WM, et al. Lymphoid neogenesis in rheumatoid synovitis. J Immunol 2001;167:1072–80. 20. Kang YM, Zhang X, Wagner UG, Yang H, Beckenbaugh RD, REFERENCES Kurtin PJ, et al. CD8 T cells are required for the formation of ectopic germinal centers in rheumatoid synovitis. J Exp Med 1. Firestein GS. Evolving concepts of rheumatoid arthritis [review]. 2002;195:1325–36. Nature 2003;423:356–61. 21. Drayton DL, Ying X, Lee J, Lesslauer W, Ruddle NH. Ectopic 2. McInnes IB, Leung BP, Liew FY. Cell-cell interactions in synovi- LT␣␤ directs lymphoid organ neogenesis with concomitant expres- tis: interactions between T lymphocytes and synovial cells [review]. sion of peripheral node addressin and a HEV-restricted sulfo- Arthritis Res 2000;2:374–8. transferase. J Exp Med 2003;197:1153–63. 3. Ware CF. Network communications: lymphotoxins, LIGHT, and 22. Kim WJ, Kang YJ, Koh EM, Ahn KS, Cha HS, Lee WH. LIGHT TNF [review]. Annu Rev Immunol 2005;23:787–819. is involved in the pathogenesis of rheumatoid arthritis by inducing 4. Ware CF. The TNF superfamily [review]. Cytokine Growth Factor the expression of pro-inflammatory cytokines and MMP-9 in Rev 2003;14:181–4. macrophages. Immunology 2005;114:272–9. 5. Olsen NJ, Stein CM. New drugs for rheumatoid arthritis [review]. 23. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, N Engl J Med 2004;350:2167–79. Cooper NS, et al. The American Rheumatism Association 1987 6. Mauri DN, Ebner R, Montgomery RI, Kochel KD, Cheung TC, revised criteria for the classification of rheumatoid arthritis. Yu GL, et al. LIGHT, a new member of the TNF superfamily, and Arthritis Rheum 1988;31:315–24. lymphotoxin ␣ are ligands for herpesvirus entry mediator. Immu- 24. Braun A, Takemura S, Vallejo AN, Goronzy JJ, Weyand CM. nity 1998;8:21–30. Lymphotoxin ␤–mediated stimulation of synoviocytes in rheuma- 7. Yu KY, Kwon B, Ni J, Zhai Y, Ebner R, Kwon BS. A newly toid arthritis. Arthritis Rheum 2004;50:2140–50. identified member of tumor necrosis factor receptor superfamily 25. Morel Y, Schiano de Colella JM, Harrop J, Deen KC, Holmes SD, (TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem 1999; Wattam TA, et al. Reciprocal expression of the TNF family 274:13733–6. receptor herpes virus entry mediator and its ligand LIGHT on 8. Harrop JA, Reddy M, Dede K, Brigham-Burke M, Lyn S, Tan KB, activated T cells: LIGHT down-regulates its own receptor. J Im- et al. Antibodies to TR2 (herpesvirus entry mediator), a new munol 2000;165:4397–404. member of the TNF receptor superfamily, block T cell prolifera- 26. Zhai Y, Guo R, Hsu TL, Yu GL, Ni J, Kwon BS, et al. LIGHT, a tion, expression of activation markers, and production of cyto- novel ligand for lymphotoxin ␤ receptor and TR2/HVEM induces kines. J Immunol 1998;161:1786–94. apoptosis and suppresses in vivo tumor formation via gene trans- 9. Kwon BS, Tan KB, Ni J, Oh KO, Lee ZH, Kim KK, et al. A newly fer. J Clin Invest 1998;102:1142–51. ROLE OF LIGHT IN RA 1117

27. Morel Y, Truneh A, Sweet RW, Olive D, Costello RT. The TNF signaling pathways and target genes [review]. Immunol Rev 2004; superfamily members LIGHT and CD154 (CD40 ligand) costimu- 202:49–66. late induction of dendritic cell maturation and elicit specific CTL 40. Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C, et activity. J Immunol 2001;167:2479–86. al. The lymphotoxin-␤ receptor induces different patterns of gene 28. Muller-Ladner U, Gay RE, Gay S. Activation of synoviocytes expression via two NF-␬B pathways. Immunity 2002;17:525–35. [review]. Curr Opin Rheumatol 2000;12:186–94. 41. Sweeney SE, Firestein GS. Rheumatoid arthritis: regulation of 29. Agarwal SK, Brenner MB. Role of adhesion molecules in synovial synovial inflammation [review]. Int J Biochem Cell Biol 2004;36: inflammation. Curr Opin Rheumatol 2006;18:268–76. 372–8. 30. Weyand CM, Kang YM, Kurtin PJ, Goronzy JJ. The power of the 42. Hayashida K, Shimaoka Y, Ochi T, Lipsky PE. Rheumatoid third dimension: tissue architecture and autoimmunity in rheuma- arthritis synovial stromal cells inhibit apoptosis and up-regulate toid arthritis [review]. Curr Opin Rheumatol 2003;15:259–66. Bcl-xL expression by B cells in a CD49/CD29-CD106-dependent 31. Hsu H, Solovyev I, Colombero A, Elliott R, Kelley M, Boyle WJ. mechanism. J Immunol 2000;164:1110–6. ATAR, a novel tumor necrosis factor receptor family member, 43. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of signals through TRAF2 and TRAF5. J Biol Chem 1997;272: collagenase (MMP-1, MMP-13) genes in arthritis: integration of 13471–4. complex signaling pathways for the recruitment of gene-specific 32. Marsters SA, Ayres TM, Skubatch M, Gray CL, Rothe M, transcription factors [review]. Arthritis Res 2002;4:157–64. 44. Brenner DA, O’Hara M, Angel P, Chojkier M, Karin M. Pro- Ashkenazi A. Herpesvirus entry mediator, a member of the tumor longed activation of jun and collagenase genes by tumour necrosis necrosis factor receptor (TNFR) family, interacts with members of factor-␣. Nature 1989;337:661–3. the TNFR-associated factor family and activates the transcription 45. Conca W, Kaplan PB, Krane SM. Increases in levels of procolla- factors NF-␬B and AP-1. J Biol Chem 1997;272:14029–32. genase messenger RNA in cultured fibroblasts induced by human 33. Carter RA, Wicks IP. Vascular cell adhesion molecule 1 (CD106): recombinant interleukin 1␤ or serum follow c-jun expression and a multifaceted regulator of joint inflammation [review]. Arthritis are dependent on new protein synthesis. J Clin Invest 1989;83: Rheum 2001;44:985–94. 1753–7. 34. Kim YS, Nedospasov SA, Liu ZG. TRAF2 plays a key, nonredun- 46. Aupperle KR, Bennett BL, Boyle DL, Tak PP, Manning AM, ␤ dant role in LIGHT-lymphotoxin receptor signaling. Mol Cell Firestein GS. NF-␬B regulation by I␬B kinase in primary fibro- Biol 2005;25:2130–7. blast-like synoviocytes. J Immunol 1999;163:427–33. 35. Chang YH, Hsieh SL, Chao Y, Chou YC, Lin WW. Proinflamma- 47. Shu HB, Agranoff AB, Nabel EG, Leung K, Duckett CS, Neish ␤ tory effects of LIGHT through HVEM and LT R interactions in AS, et al. Differential regulation of vascular cell adhesion mole- cultured human umbilical vein endothelial cells. J Biomed Sci cule 1 gene expression by specific NF-␬B subunits in endothelial 2005;12:363–75. and epithelial cells. Mol Cell Biol 1993;13:6283–9. 36. Dayer JM, de Rochemonteix B, Burrus B, Demczuk S, Dinarello 48. Morel JC, Park CC, Woods JM, Koch AE. A novel role for CA. Human recombinant interleukin 1 stimulates collagenase and interleukin-18 in adhesion molecule induction through NF␬B and prostaglandin E2 production by human synovial cells. J Clin Invest phosphatidylinositol (PI) 3-kinase-dependent signal transduction 1986;77:645–8. pathways. J Biol Chem 2001;276:37069–75. 37. Frisch SM, Ruley HE. Transcription from the stromelysin pro- 49. Reddy SA, Huang JH, Liao WS. Phosphatidylinositol 3-kinase in moter is induced by interleukin-1 and repressed by dexametha- interleukin 1 signaling: physical interaction with the interleukin 1 sone. J Biol Chem 1987;262:16300–4. receptor and requirement in NF␬B and AP-1 activation. J Biol 38. Delany AM, Brinckerhoff CE. Post-transcriptional regulation of Chem 1997;272:29167–73. collagenase and stromelysin gene expression by epidermal growth 50. Pfeffer LM, Mullersman JE, Pfeffer SR, Murti A, Shi W, Yang factor and dexamethasone in cultured human fibroblasts. J Cell CH. STAT3 as an adapter to couple phosphatidylinositol 3-kinase Biochem 1992;50:400–10. to the IFNAR1 chain of the type I interferon receptor. Science 39. Schneider K, Potter KG, Ware CF. Lymphotoxin and LIGHT 1997;276:1418–20. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1118–1124 DOI 10.1002/art.22496 © 2007, American College of Rheumatology

Bone Erosions and Bone Marrow Edema as Defined by Magnetic Resonance Imaging Reflect True Bone Marrow Inflammation in Rheumatoid Arthritis

Esther Jimenez-Boj,1 Iris No¨bauer-Huhmann,1 Beatrice Hanslik-Schnabel,1 Ronald Dorotka,1 Axel-Hugo Wanivenhaus,1 Franz Kainberger,1 Siegfried Trattnig,1 Roland Axmann,2 Wayne Tsuji,3 Sonja Hermann,2 Josef Smolen,1 and Georg Schett4

Objective. To investigate the pathologic nature of increasing water content, which appears as bright signal features termed “bone erosion” and “bone marrow enhancement on STIR MRI sequences. MRI bone mar- edema” (also called “osteitis) on magnetic resonance row edema was recorded based on the finding of inflam- imaging (MRI) scans of joints affected by rheumatoid matory infiltrates, which were less dense than those of arthritis (RA). MRI bone erosions and localized more centrally in the Methods. RA patients scheduled for joint replace- joint. These lesions were either isolated or found in ment surgery (metacarpophalangeal or proximal inter- contact with MRI bone erosions. phalangeal joints) underwent MRI on the day before Conclusion. MRI bone erosions and MRI bone surgery. The presence and localization of bone erosions marrow edema are due to the formation of inflamma- and bone marrow edema as evidenced by MRI (MRI tory infiltrates in the bone marrow of patients with RA. bone erosions and MRI bone marrow edema) were This emphasizes the value of MRI in sensitively detect- ؍ documented in each joint (n 12 joints). After surgery, ing inflammatory tissue in the bone marrow and dem- sequential sections from throughout the whole joint onstrates that the inflammatory process extends to the were analyzed histologically for bone marrow changes, bone marrow cavity, which is an additional target and these results were correlated with the MRI findings. structure for antiinflammatory therapy. Results. MRI bone erosion was recorded based on bone marrow inflammation adjacent to a site of cortical Chronic synovitis in the context of rheumatoid bone penetration. Inflammation was recorded based on arthritis (RA) leads to pathologic changes in adjacent either invading synovial tissue (pannus), formation of structures, such as the articular cartilage, the cortical lymphocytic aggregates, or increased vascularity. Fat- bone surface, and the underlying bone marrow. Knowl- rich bone marrow was replaced by inflammatory tissue, edge of this complex destructive process is predominantly driven by findings of radiographic examinations, Supported by the Austrian Ministry of Sciences (START prize award to Dr. Schett). which have identified local bone erosions as well as joint 1Esther Jimenez-Boj, MD, Iris No¨bauer-Huhmann, MD, Bea- space narrowing as key monitoring parameters in RA trice Hanslik-Schnabel, MD, Ronald Dorotka, MD, Axel-Hugo (1,2). From these findings it is apparent that inflamed Wanivenhaus, MD, Franz Kainberger, MD, Siegfried Trattnig, MD, Josef Smolen, MD: Medical University of Vienna, Vienna, Austria; synovial tissue has the capacity to invade neighboring 2Roland Axmann, MD, Sonja Hermann, MD: University of Erlangen- structures such as cartilage and bone. It has been 3 Nuremberg, Erlangen, Germany; Wayne Tsuji, MD: Amgen, Inc., particularly difficult, however, to unravel the histopatho- Thousand Oaks, California; 4Georg Schett, MD: Medical University of Vienna, Vienna, Austria, and University of Erlangen-Nuremberg, logic nature of these changes, since usual methods to Erlangen, Germany. assess and/or to surgically treat synovitis, such as biopsy Dr. Tsuji holds stock or stock options in Amgen. Address correspondence and reprint requests to Georg or synovectomy, target the synovial tissue itself but do Schett, MD, Department of Internal Medicine III and Institute for not yield insight into changes in cartilage, bone, or bone Clinical Immunology, University of Erlangen-Nuremberg, Erlangen marrow. It is therefore not surprising that information 91054, Germany. E-mail: [email protected]. Submitted for publication October 3, 2006; accepted in re- about the structural and functional correlates of radio- vised form December 21, 2006. graphic findings in RA is scarce and is driven by findings

1118 MRI BONE EROSION AND BONE MARROW EDEMA IN RA 1119

in specimens obtained at joint replacement surgery, phalangeal, and 1 fourth proximal phalangeal bone) from 3 which usually occurs late in the disease course. Thus, key patients (all women, ages 43, 56, and 61 years) with longstand- features of local bone erosions in RA have only recently ing RA (disease duration 8, 14, and 24 years) were assessed. All patients fulfilled the American College of Rheumatology been described, revealing that these lesions are popu- (formerly, the American Rheumatism Association) criteria for lated by osteoclasts, which have the capacity to degrade the classification of RA (11). Patients were scheduled for joint bone (3,4). replacement surgery because of chronic and persistent pain, Improvements in imaging strategies, in particu- joint swelling, and impaired range of motion in the target joint. All patients were being treated with methotrexate (15 mg/ lar, technical developments in magnetic resonance im- week) and low-dose glucocorticoids (5 mg/day). Surgical pro- aging (MRI), have provided insight into the complexity cedures were performed according to the methods described of joint destruction in RA (5,6). Thus, MRI scanning has by Swanson (12), consisting of resection of the affected meta- extended our knowledge of RA by allowing direct visu- carpal or proximal phalangeal heads followed by implantation alization of synovial inflammation and by depicting the of a silicone spacer (NeuFlex; DePuy Orthopaedics, Warsaw, IN) (13). Written informed consent was obtained for all invasion of inflammation into bone and bone marrow procedures. very early in the disease. Importantly, MRI scans show Magnetic resonance imaging. MRI was performed signal changes, which extend into the bone marrow with a 1.5T MR scanner (Philips, Nijmegen, The Netherlands), cavity and are linked either to cortical bone destruction using a flexible surface coil (flex medium) to obtain coronal STIR sequences. The acquisition parameters were as follows: (“MRI bone erosion”) or to more diffuse changes in the repetition time (TR) 1,200 msec, echo time (TE) 14 msec, 2 bone marrow space (“MRI bone marrow edema” or averages, field of view (FOV) 200 mm, matrix 1,926 ϫ 256 “MRI osteitis”). The latter lesions have also been de- pixels, slice thickness 3 mm, interslice gap 0.3 mm, scan time 2 scribed in osteoarthritis, ankylosing spondylitis, and sys- minutes 18 seconds. In addition, coronal T1-weighted se- temic lupus erythematosus (7–9). Both lesions exhibit quences were obtained (TR 450 msec, TE 13.8 msec, 2 averages, FOV 200 mm, matrix 1,926 ϫ 256 pixels, slice signal characteristics consistent with increased water thickness 3 mm, interslice gap 0.3 mm, scan time 2 minutes 18 content (5), distinguishing these lesions from the fatty seconds). MRI bone erosions were defined based on hyperin- bone marrow of the extremities. tensity on STIR sequences and hypointensity on T1-weighted The morphologic nature of bone marrow changes sequences, in direct contact with cortical bone and with well-defined margins and apparent destruction of the cortical in RA, however, has not been well investigated. The lack bone barrier. MRI bone marrow edema was identified as of easy access to bone marrow from RA patients is a hyperintense lesions on STIR sequences, with less clearly logistic challenge and has so far prevented clear defini- defined margins and intact trabecular structures (5). tion of the structural correlates of MRI changes. How- Histologic examination. After resection, the localiza- ever, recent histologic investigations using specimens tion (MCP or PIP joint; second, third, fourth, or fifth digit), side (left or right), and dorsal-palmar plane of each joint were obtained at joint replacement surgery have shown that documented. To ascertain an orientation of the histologic fat-rich bone marrow can indeed be focally replaced by sections identical to that of the MRI, the 3-dimensional inflammatory synovial tissue, which invades the cortical orientation of the joint had to be documented (Figure 1). In bone, penetrates the cortical barrier, and exposes the accordance with the MR images, joints were cut in the coronal bone marrow to inflammatory triggers, leading in par- axis. This was ascertained by defining the distal-proximal orientation by the resection rim and the cartilage, respectively, ticular to B cell–rich cellular aggregates (10). the lateral-medial orientation by the side of the joint (left or In the present study, we aimed to define the right hand), and the dorsal-palmar orientation by marking the nature of bone marrow edema in RA. We studied RA dorsal rim with a suture. patients scheduled for total joint replacement of the After explantation, the specimen was immediately proximal interphalangeal (PIP) and metacarpophalan- placed into 0.9% NaCl, fixed in 4.0% formalin, and decalcified in 14% EDTA (Sigma, St. Louis, MO). Paraffin-embedded geal (MCP) joints. These joints were scanned by MRI on joints were then cut into 2 equal-sized pieces along the coronal the day before surgery and subsequent histologic pro- plane. Both pieces were used to cut sequential sections (2 ␮m) cessing. This allowed investigation of the histopathologic every 50 ␮m, directed to the dorsal rim of the joint in 1 piece nature of bone marrow changes in RA as depicted by and to the palmar rim in the other. For each joint ϳ70 serial MRI. sections were analyzed. All sections were stained with hematoxylin and eosin and analyzed quantitatively for the degree of bone marrow alterations, using a histomorphometric technique PATIENTS AND METHODS with an Axioskop 2 microscope (Zeiss, Marburg, Germany) and the OsteoMeasure Analysis System (Osteometrics, Deca- Patients. Twelve different joints (heads of 2 second tur, GA) (14). The area covered by bone (cortical plus metacarpal, 3 third metacarpal, 2 fourth metacarpal, 2 fifth trabecular), normal bone marrow, and bone marrow with mild metacarpal, 1 second proximal phalangeal, 1 third proximal cellular infiltration (Ͻ50% inflammatory infiltrates per tissue 1120 JIMENEZ-BOJ ET AL

bright on the STIR sequences but dark on the T1- weighted images, reflecting increased water content and decreased fat content. Origin of bone erosions seen on MRI. Analysis of corresponding histologic sections showed that bone erosions seen on MRI were due to localized replacement of bone marrow fat by accumulated inflammatory cells adjacent to a broken cortical bone barrier. Cortical bone is actually only a very thin barrier (ϳ0.25 mm in width) between the synovium and the bone marrow. A perfo- ration of this layer enabled the accumulation of inflammatory tissue, in the form of either synovial inflammatory tissue or lymphocytic B cell–rich aggregates within the marrow space, appearing as bone erosions. In fact, only a small portion of the MRI lesion that was designated bone erosion represented true structural damage Figure 1. Ascertainment of coronal-plane magnetic resonance images (MRIs) and histologic sections. Metacarpophalangeal and proximal of bone, since inflammation affects the bone marrow interphalangeal joints were analyzed in the coronal plane by MRI after penetration through the cortical barrier. scanning, as well as by histologic examination of serial sections (black Figure 2 shows an example of 2 histologic sec- bars). To precisely define the orientation of sections, all 3 dimensions tions of a second metacarpal head from a patient with were documented: the distal-proximal orientation was based on the RA, as well as matched MR images with STIR and distal localization of the articular cartilage, the dorsal-palmar axis was identified through labeling with a suture placed at the dorsal rim of the T1-weighted sequences. MRI lesions were characterized joint head (red box) directly after explantation, and the lateral-medial as a clearly demarcated zone of hyperintense signal axis was defined based on knowledge of whether the explants came within normal hypointense marrow on STIR images, and from the left or the right hand. Serial sections were obtained at a hypointense signal on T1-weighted sections. The his- intervals of 50 ␮m, allowing identification of the exact localization of the respective section within the dorsal-palmar axis. tologic correlate was identified as local bone marrow inflammation and accumulation of blood vessels at these sites, which were closely linked to a break in the adjacent area; intact trabecular structure) or severe infiltration (Ͼ50% cortical bone. inflammatory infiltrates per tissue area; trabeculae destroyed) Origin of bone marrow edema seen on MRI. were recorded. MRI and histomorphometric data were inter- More diffuse MRI signal alterations in the bone marrow preted by 2 independent observers (EJ-B and IN-H), under of patients with RA are considered to indicate bone blinded conditions. marrow edema or osteitis. As was seen with the above- mentioned lesions, they appeared bright on STIR se- RESULTS quences, indicating increased water but lower fat con- Colocalization of cellular infiltrates with bone tent. Similar to the findings in MRI bone erosions, the marrow edema seen on MRI. To better understand the histopathologic correlate of MRI bone marrow edema/ processes causing the pathologic changes seen on MRI osteitis was infiltration of the bone marrow by inflam- in patients with RA, we performed a serial histologic matory tissue. This lends more credence to the term analysis of sections from throughout the entire finger “osteitis” rather than “bone marrow edema.” Thus, all joint of patients who had undergone joint replacement lesions that appeared bright on STIR sequences (and surgery. STIR MRI sequences obtained on the day dark on T1-weighted sequences) and were localized before surgery were compared with histologic sections. within the cortical bone layer were due to inflammatory In all 12 joints analyzed, bone marrow changes were infiltrates in the bone marrow, regardless of whether evident on MRI scans as well as in histologic sections. these lesions were attached to the endosteum and asso- Bone lesions seen on MRI were designated as erosions ciated with cortical penetration (MRI bone erosion) or when they were localized close to cortical bone and were more diffusely located within the marrow space associated with synovitis, whereas the more diffuse (MRI bone marrow edema/osteitis). lesions in the bone marrow were designated bone mar- Distribution of bone marrow changes. Normal row edema or osteitis. Both types of lesion appeared bone marrow is dominated by adipocytes, with occa- MRI BONE EROSION AND BONE MARROW EDEMA IN RA 1121

Figure 2. T1-weighted (A) and STIR (B) magnetic resonance images (MRIs) and corresponding histologic sections at low and high magnification (C and D), of a second metacarpal head from a patient with rheumatoid arthritis. MRI bone erosion is defined based on penetration of cortical bone and localized bone marrow inflammation, depicted as a circumscribed area of hypointense signal at the medial circumference on the T1-weighted image in the upper row (arrowhead) within normal hyperintense bone marrow. This corresponds to findings on the STIR image in the upper row, where the erosion is seen as a clearly demarcated zone of hyperintense signal within normal hypointense marrow at this site (arrowhead). A blood vessel and inflammatory tissue next to the junction zone of the joint (arrowheads) are seen in the corresponding histologic images in the upper row. The MR and histologic images in the lower row show a more palmar view of the same joint, with clear signs of invasion of inflammatory tissue into subchondral bone and bone marrow seen on the STIR MRI (arrowhead). The corresponding histologic section shows cortical penetration at this site, with inflammatory tissue invading the bone marrow space and replacing fatty tissue (arrowheads). (Original magnification ϫ 10 in C; ϫ 50 in D.)

sional interspersed stromal cells. Mild infiltration of and palmar rims of the bone marrow cavity, reflecting bone marrow was characterized by a decreased number their close interaction with synovial tissue penetrating of adipocytes in favor of hematopoietic cells infiltrating through the cortical barrier into the bone marrow. the bone marrow (Ͻ50% infiltrates/tissue area [grade I Grade II lesions were absent in the center of the bone lesion]). Severe infiltration of bone marrow was recorded marrow. Grade I lesions, in contrast, were localized at based on findings of either synovial pannus–like tissue the center and palmar areas of the joint, colocalizing within the cortical lining, lymphocytic aggregates, or blood with lesions appearing as MRI bone marrow edema/ vessels associated with inflammatory infiltrates almost osteitis. Areas of mild infiltration of the bone marrow completely replacing bone marrow fat (grade II lesion). (grade I lesions) were generally more prevalent (by Most areas of the more diffuse lesions reflecting ϳ4-fold) than areas with more severe changes (grade II MRI bone marrow edema/osteitis were composed of lesions) (Figures 3C and D). Our findings indicated that grade I lesions, with some interspersed grade II lesions. STIR MRI sequences can depict mild inflammatory In contrast, peripheral lesions reflecting MRI bone infiltrates in the bone marrow, which are commonly erosions were almost exclusively dense infiltrates corre- termed bone marrow edema/osteitis, as well as dense sponding to grade II lesions. In accordance with this, bone marrow infiltrates associated with penetration of grade II lesions were localized peripherally at the dorsal cortical bone, termed bone erosions. 1122 JIMENEZ-BOJ ET AL

Figure 3. Different localization patterns of mild and severe bone marrow inflammation. Magnetic resonance imaging (MRI) bone marrow edema is defined based on bone marrow inflammation. A, STIR MRI of the third metacarpal head, showing signal enhancement reflecting bone marrow edema. The image on the right is a close-up of the boxed area in the image on the left. B, Histologic section corresponding to the boxed area in the right image shown in A, demonstrating inflammatory infiltrates in the bone marrow at the site of the MRI lesion. C, Higher-magnification views of the boxed areas in B, showing normal bone marrow containing adipocytes (left portion of C, corresponding to the boxed area in the upper right of B), mild infiltration with hematopoietic cells, reflecting a grade I lesion (middle portion of C, corresponding to the boxed area in the middle of B), and strong infiltration (grade II lesion) with almost complete replacement of fatty tissue by inflammatory tissue (right portion of C, corresponding to the boxed area in the lower left of B). D, Graph depicting the findings in Ͼ70 serial coronal sections from the metacarpal head, showing that grade II lesions are localized peripherally at the dorsal and palmar rims where bone erosions are present, whereas grade I lesions are distributed more centrally. (Original magnification ϫ 20 in B; ϫ 200 in C.)

DISCUSSION focally increased water content in the bone marrow, Magnetic resonance imaging not only allows vi- suggesting that bone marrow fat is replaced by water, or sualization and quantification of synovitis, but has also structures containing more water and less fat than enabled more detailed viewing of the pathologic changes normal bone marrow. They appear dark (low signal of neighboring bone, cartilage, and bone marrow. Local- intensity) on T1-weighted MRIs, whereas they are bright ized MRI changes in close association with the cortical (high signal intensity) on STIR MRI sequences (5). bone are termed bone erosions, whereas more diffuse The fact that MR techniques have revealed pro- changes in the bone marrow are termed bone marrow found changes in a previously uncharacterized compart- edema or osteitis (5,6). These changes originate from ment of the rheumatoid joint, beneath the inflamed MRI BONE EROSION AND BONE MARROW EDEMA IN RA 1123

surface, is of particular interest. MRI is increasingly used investigated is a strong indicator that true inflammation in the monitoring of RA patients who are receiving is the cause of MRI lesions in the bone marrow. The immunomodulatory therapies, including biologic agents, second limitation of the study, lack of inclusion of in both clinical trials and daily clinical practice. Among patients with early disease, was unavoidable since, for the radiographic changes observed on the MRIs, the obvious reasons, finger joint replacement surgery is not anatomic basis of synovitis is very well characterized and the treatment of choice in early arthritis. Thus, we the structural nature of local bone erosion has been cannot exclude the possibility that MRI bone marrow recently defined (3,10,15,16). In contrast, little informa- changes in early disease, which can be reversible, have a tion has been available on the nature of bone marrow different structural correlate than the lesions found in changes found in RA joints but not in normal joints. This advanced disease as investigated in this study. is due to 1) the apparent difficulty in assessing this In summary, the present results show that MRI particular joint region, which is not accessible via syno- bone erosions as well as MRI bone marrow edema/ vial biopsy or synovectomy, and 2) the scientific focus on osteitis reflect bone marrow inflammation. This indi- joint pathology at the outside, but not the inside, of the cates that, in addition to the synovial membrane, juxta- cortical bone barrier (10). articular parts of the bone marrow are inflamed in RA, The cortical bone barrier, which separates the suggesting active involvement of this compartment in the synovial compartment from the bone marrow compart- inflammatory process. These findings reveal a previously ment, is only a very thin layer. The vast majority of the uncharacterized component of the pathophysiology of lesion termed bone erosion on MRI scans and the whole RA. lesion termed bone marrow edema/osteitis is clearly localized within this cortical bone layer. This suggests ACKNOWLEDGMENTS that MRI can depict pathologic changes in the bone marrow beneath the inflamed joint. The present study We thank Ivana Mikulic and Birgit Tuerk for excellent reveals that these lesions are due to the replacement of technical assistance. bone marrow fat by an inflammatory infiltrate resem- bling a sterile “osteitis” or “osteomyelitis,” rather than a AUTHOR CONTRIBUTIONS true edema. Dr. Schett had full access to all of the data in the study and Dense bone marrow infiltrates were found at the takes responsibility for the integrity of the data and the accuracy of the data analysis. periphery of the bone marrow, where adjacent cortical Study design. Jimenez-Boj, No¨bauer-Huhmann, Dorotka, Waniven- bone had been fenestrated by synovial inflammatory haus, Kainberger, Tsuji, Smolen, Schett. tissue (MRI bone erosions). These lesions were com- Acquisition of data. Jimenez-Boj, No¨bauer-Huhmann, Hanslik- Schnabel, Dorotka, Wanivenhaus, Trattnig, Axmann, Hermann, posed of dense infiltrates consisting of 1) synovial in- Schett. flammatory tissue invading the bone marrow, 2) lympho- Analysis and interpretation of data. Jimenez-Boj, No¨bauer-Huhmann, cytic infiltrates emerging at the interface between Hanslik-Schnabel, Kainberger, Trattnig, Axmann, Hermann, Smolen, Schett. synovial tissue and bone marrow fat, and 3) blood vessels Manuscript preparation. Jimenez-Boj, Dorotka, Tsuji, Smolen, Schett. close to inflammatory infiltrates. Thus, MRI bone ero- Statistical analysis. Jimenez-Boj, Schett. sions not only show penetration of the cortical barrier, Operations. Hanslik-Schnabel, Dorotka, Wanivenhaus. but are largely due to inflammatory changes in the neighboring bone marrow. MRI bone marrow edema REFERENCES was also due to inflammatory infiltrates, but infiltration 1. Van der Heijde DM. Radiographic imaging: the ‘gold standard’ was less severe and localized to more central regions of for assessment of disease progression in rheumatoid arthritis. the bone marrow. Rheumatology (Oxford) 2000;39:9–16. This study had limitations due to the small num- 2. Sharp JT, Young DY, Bluhm GB, Brook A, Brower AC, Corbett M, et al. How many joints in the hands and wrists should be ber of patients investigated and the focus on late-stage included in a score of radiologic abnormalities used to assess disease. The number of patients was small due to the low rheumatoid arthritis? Arthritis Rheum 1985;28:1326–35. frequency of surgical replacement of finger joints, the 3. Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS, Goldring SR. Identification of cell types responsible for bone improved control of disease by pharmacologic methods, resorption in rheumatoid arthritis and juvenile rheumatoid arthri- and the complexity of the histologic analysis (Ͼ70 tis. Am J Pathol 1998;152:943–51. sections per joint for histomorphometric analysis). How- 4. Schett G, Hayer S, Zwerina J, Redlich K, Smolen J. Mechanisms of disease: the link between RANKL and arthritic bone disease. ever, the fact that MRI lesions corresponded to histo- Nat Clin Pract Rheum 2005;1:47–54. logic signs of bone marrow inflammation in all 12 joints 5. Ostergaard M, Peterfy C, Conaghan P, McQueen F, Bird P, 1124 JIMENEZ-BOJ ET AL

Ejbjerg B, et al. OMERACT Rheumatoid Arthritis Magnetic haus A, Chott A, et al. Interaction between synovial inflammatory Resonance Imaging Studies: core set of MRI acquisitions, joint tissue and bone marrow in rheumatoid arthritis. J Immunol pathology definitions, and the OMERACT RA-MRI scoring 2005;175:2579–88. system. J Rheumatol 2003;30:1385–6. 11. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, 6. McQueen FM, Benton N, Perry D, Crabbe J, Robinson E, Cooper NS, et al. The American Rheumatism Association 1987 Yeoman S, et al. Bone edema scored on magnetic resonance revised criteria for the classification of rheumatoid arthritis. imaging scans of the dominant carpus at presentation predicts Arthritis Rheum 1988;31:315–24. radiographic joint damage of the hands and feet six years later in 12. Swanson AB. Silicone rubber implants for replacement of arthritis patients with rheumatoid arthritis. Arthritis Rheum 2003;48: or destroyed joints in the hand. Surg Clin North Am 1968;48: 1814–27. 1113–27. 7. Zanetti M, Bruder E, Romero J, Hodler J. Bone marrow edema 13. Erdogan A, Weiss AP. NeuFlex silastic implant in metacarpophal- pattern in osteoarthritic knees: correlation between MR imaging angeal arthroplasty. Orthopade 2003;32:789–93. and histologic findings. Radiology 2000;215:835–40. 14. Deleuran BW, Chu CQ, Field M, Brennan FM, Mitchell T, 8. Appel H, Loddenkemper C, Grozdanovic Z, Ebhardt H, Feldmann M, et al. Localization of tumor necrosis factor receptors Dreimann M, Hempfing A, et al. Correlation of histopathological in the synovial tissue and cartilage–pannus junction in patients findings and magnetic resonance imaging in the spine of patients with rheumatoid arthritis: implications for local actions of tumor with ankylosing spondylitis. Arthritis Res Ther 2006;8:R143. necrosis factor ␣. Arthritis Rheum 1992;35:1170–8. 9. Boutry N, Hachulla E, Flipo RM, Cortet B, Cotton A. MR 15. Bromley M, Woolley DE. Chondroclasts and osteoclasts at sub- imaging findings in hands in early rheumatoid arthritis: compari- chondral sites of erosion in the rheumatoid joint. Arthritis Rheum son with those in systemic lupus erythematosus and primary 1984;27:968–75. Sjogren syndrome. Radiology 2006;241:320–1. 16. Goldring SR. Bone and joint destruction in rheumatoid arthritis: 10. Jimenez-Boj E, Redlich K, Turk B, Hanslik-Schnabel B, Waniven- what is really happening? J Rheumatol 2002;65:44–8. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1125–1133 DOI 10.1002/art.22504 © 2007, American College of Rheumatology

Risk of Serious Bacterial Infections Among Rheumatoid Arthritis Patients Exposed to Tumor Necrosis Factor ␣ Antagonists

Jeffrey R. Curtis,1 Nivedita Patkar,1 Aiyuan Xie,1 Carolyn Martin,2 Jeroan J. Allison,1 Michael Saag,1 Deborah Shatin,2 and Kenneth G. Saag1

Objective. To evaluate the risk of serious bacterial models evaluated time-dependent infection risks associ- infections associated with tumor necrosis factor ␣ ated with TNF␣ antagonists. (TNF␣) antagonists among rheumatoid arthritis (RA) Results. Hospital medical records with claims- patients. identified suspected bacterial infections were abstracted ␣among RA patients who received TNF (187 ؍ Methods. A retrospective cohort study of US RA (n -observation time 3,894 person ;2,393 ؍ patients enrolled in a large health care organization antagonists (n person-years). Over a 4,846 ;2,933 ؍ identified patients who received either TNF␣ antago- years) or MTX (n nists or methotrexate (MTX). Administrative data were median followup time of 17 months, the rate of hospi- used to identify hospitalizations with possible bacterial talization with a confirmed bacterial infection was 2.7% infections; corresponding medical records were ab- among the patients treated with TNF␣ antagonists stracted and reviewed by infectious disease specialists compared with 2.0% among the patients treated with for evidence of definite infections. Proportional hazards MTX only. The multivariable-adjusted hazard ratio (HR) of infection among the patients who received TNF␣ antagonists was 1.9 (95% confidence interval Supported by the Maryland Chapter of the Arthritis Founda- [95% CI] 1.3–2.8) compared with patients who received tion, the Agency for Healthcare Research and Quality (grant HS- 10389), and the NIH (grant K24-AR-052361-01 from the National MTX only. The incidence of infections was highest Institute of Arthritis and Musculoskeletal and Skin Diseases and grant within 6 months after initiating TNF␣ antagonist ther- AR-47512-03). 1Jeffrey R. Curtis, MD, MPH, Nivedita Patkar, MD, MSPH, apy (2.9 versus 1.4 infections per 100 person-years; Aiyuan Xie, MD, MS, Jeroan J. Allison, MD, MS, Michael Saag, MD, multivariable-adjusted HR 4.2, 95% CI 2.0–8.8). Kenneth G. Saag, MD, MSc: University of Alabama at Birmingham; Conclusion. The multivariable-adjusted risk of 2Carolyn Martin, MSW, Deborah Shatin, PhD: Center for Health Care Policy and Evaluation, Eden Prairie, Minnesota. hospitalization with a physician-confirmed definite bac- Dr. Michael Saag has received grants and/or research support terial infection was ϳ2-fold higher overall and 4-fold (less than $10,000 each) from Achillion Pharmaceutica, Boehringer- higher in the first 6 months among patients receiving Ingelheim, Gilead Sciences, GlaxoSmithKline, Merck, Panacos, Pfizer/ ␣ Agouron, Progenics, Roche, Serono, Tibotec, Trimeris, and Vertex. TNF antagonists versus those receiving MTX alone. He has received consulting fees (less than $10,000 each) from Achill- RA patients were at increased risk of serious infections, ion Pharmaceutica, Avexa, Boehringer-Ingelheim, Bristol-Myers irrespective of the method used to define an infectious Squibb, Gilead Sciences, GlaxoSmithKline, Merck, Monogram Bio- sciences, Panacos, Pfizer/Agouron, Progenics, Roche, Tanox, Tibotec/ outcome. Patients and physicians should vigilantly Virco, Trimeris, and Vertex. He has received speaking fees (less than monitor for signs of infection when using TNF␣ antag- $10,000 each) from all of the above. He has received consulting fees onists, particularly shortly after treatment initiation. and/or speaking fees (more than $10,000 each) from Bristol-Myers Squibb and GlaxoSmithKline. Dr. Kenneth G. Saag has received consulting fees (less than $10,000) from Amgen (data safety monitor- Tumor necrosis factor ␣ (TNF␣) antagonists are ing board). Address correspondence and reprint requests to Kenneth G. used in a rapidly growing number of patients with Saag, MD, MSc, University of Alabama at Birmingham, Center for rheumatoid arthritis (RA), and their use is expanding in Education and Research on Therapeutics, UAB FOT 840, 510 20th other conditions, including inflammatory bowel disease, Street South, Birmingham AL 35294. E-mail: [email protected]. Submitted for publication September 30, 2006; accepted in psoriasis, psoriatic arthritis, ankylosing spondylitis, and revised form January 4, 2007. juvenile inflammatory arthritis. However, enthusiasm

1125 1126 CURTIS ET AL

for the increasing use of these agents has been tempered mandated that each individual have received an infusion or by concern regarding adverse events, including infec- filled a prescription for a TNF␣ antagonist (e.g., etanercept, tions, which are common in patients with serious inflam- infliximab, or adalimumab) or filled at least 3 prescriptions for MTX. Those who received a TNF␣ antagonist were the matory disorders (1) and may be related either to the exposed cohort, and those who received MTX only were the underlying disease itself or to the medications used for unexposed/comparator cohort. We required that the unex- treatment (2). Results from many short-term, random- posed cohort receive MTX because it is the most commonly ized controlled trials (RCTs) of selected RA patients prescribed nonbiologic DMARD in the US (24). We did not ␣ include RA patients who received less-aggressive DMARD suggest that TNF antagonists increase the risk of regimens (e.g., hydroxychloroquine only) in order to avoid infection minimally, if at all (3–6), although an increased substantial heterogeneity in the comparator cohort. risk of infection has been observed in some (7,8). In light of clinical trial data showing that TNF␣ However, based on reports of spontaneous adverse antagonists have greater efficacy when combined with MTX than when used as monotherapy, we anticipated that a majority events and data from case series, concern has arisen that ␣ ␣ of those who received TNF antagonists would also take MTX TNF antagonists may increase the risk of infection concomitantly, and thus, our analyses focused on the risk of beyond that observed with older antiinflammatory and infections of a TNF␣ antagonist–based regimen compared immunosuppressive agents (9–12). with an MTX-based regimen. For this reason, we allowed Recent data from studies examining the use of patients in either cohort to receive other nonbiologic DMARDs. We also examined the pharmacy claims for oral these agents in special populations such as RA patients glucocorticoid use and converted those other than prednisone undergoing surgery suggest an increased risk of infec- to prednisone-equivalent dosages. We examined the fill dates tion (13). In other cohorts of RA patients as well as in of each TNF␣ antagonist and MTX prescription and consid- a meta-analysis of RCTs, TNF␣ antagonists were as- ered patients to be at risk for infections for 6 months after each sociated with up to a 3-fold increased risk of infection filled prescription. The date of first exposure to the TNF␣ antagonist or compared with patients receiving traditional disease- the third filled MTX prescription was defined as each person’s modifying antirheumatic drug (DMARD) therapy index date. Exposed patients thus had just begun TNF␣ (14,15), but this conclusion is not consistent with other antagonist therapy, and unexposed patients were already re- study results that indicate no increased risk (16–21). The ceiving ongoing MTX therapy. Thus, we were able to test hypotheses about the risk of initiating TNF␣ antagonist ther- limited duration of followup and differences in the rigor apy compared with individuals already receiving MTX or of the criteria used to diagnose infections and in the “background” DMARDs. Individuals who switched TNF␣ composition and comorbidity profile of the patients not antagonists were treated similarly to those treated with only 1 exposed to TNF␣ antagonists may have contributed to TNF␣ antagonist and were assigned to the exposed cohort. the variability in results between studies. Potential confounders and covariates of interest were examined in the administrative data in the 6 months prior to each To address clinical uncertainties about the risk of member’s index date. Individuals diagnosed as having human serious bacterial infections among patients who receive immunodeficiency virus, organ transplantation, or malignancy TNF␣ antagonists, we conducted a population-based, were excluded from the study. retrospective cohort study of US RA patients enrolled in Identification and classification of infections. Among the study population, we identified hospitalizations that oc- a large health care organization. To circumvent the curred after the member’s index date that had at least 2 limitations of past studies, we assessed the time- ICD-9-CM codes for bacterial infections. We required at least dependent risk of definite infections using a study period 2 ICD-9-CM codes for each hospitalization in order to improve of Ͼ1 year and required high diagnostic certainty for all the specificity of our initial case-finding strategy; at least 1 of infections. We hypothesized that TNF␣ antagonists these codes had to be based on a face-to-face encounter with a physician. Additional ICD-9-CM codes could originate from would increase the risk of hospitalization with a bacterial a claim for a diagnostic test or procedure (e.g., laboratory test infection compared with the use of methotrexate (MTX) or a radiograph). Codes for the infections could be in any alone. position in the claims data, indicating that the individual may have been hospitalized expressly for the purposes of treating PATIENTS AND METHODS the infection or may have been treated for an infection incidental to a hospitalization for another reason. We required Study population. After Institutional Review Board that infections be identified within 6 months of the most recent approval, we used the medical and pharmacy administrative exposure to the TNF␣ antagonist or MTX in order to maintain claims of a large US health care organization (22) between a biologically plausible link with the medication exposure. May 1998 and December 2003 to identify RA patients Ն18 Using a pilot-tested data collection tool, trained nurses years old. We required at least 2 International Classification of abstracted the medical records of members of the national RA Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) study cohort with a suspected bacterial infection based upon diagnosis codes for RA (714.x, excluding 714.3) (23) and also administrative claims data. Nurses abstracted only the medical TNF␣ ANTAGONISTS AND RISK OF INFECTION 1127

Table 1. Characteristics of the RA patients, by drug exposure group* TNF␣ antagonist MTX only (n ϭ 2,393 persons, (n ϭ 2,933 persons, 3,894 person-years) 4,846 person-years) P† Demographics Age, mean Ϯ SD years 50 Ϯ 12 55 Ϯ 13 Ͻ0.0001 Female 1,751 (73) 2,146 (73) NS US region of residence Midwest 1,034 (43) 1,605 (55) Northeast 152 (6) 143 (5) South 1,043 (44) 984 (33) West 164 (7) 201 (7) Health services utilization Year of first study observation Ͻ0.0001 1998–1999 405 (17) 648 (22) 2000–2001 1,009 (42) 1,294 (44) 2002–2003 958 (40) 978 (33) Duration of observation, mean Ϯ SD months 20 Ϯ 15 20 Ϯ 16 NS Insurance type Ͻ0.0001 Commercial 2,199 (92) 2,291 (78) Medicare 190 (8) 642 (22) Age Ͻ65 years (e.g., disabled) 60 114 Age Ͼ65 years 130 528 Medicaid 4 (0) 0 No. of face-to-face physician visits, mean Ϯ SD‡ 7.3 Ϯ 5.1 6.9 Ϯ 4.6 Ͻ0.001 Selected medical conditions‡ Severe RA§ 76 (3) 59 (2) Ͻ0.01 Joint surgery or deformity¶ 64 (3) 37 (1) Ͻ0.001 Prior bacterial infection 200 (8) 205 (7) NS Coronary artery disease (including CABG, angioplasty) 285 (12) 435 (15) Ͻ0.01 Chronic obstructive pulmonary disease or asthma 198 (8) 275 (9) NS Diabetes mellitus 199 (8) 285 (10) NS Kidney disease# 19 (1) 28 (1) NS Decubitus ulcer 38 (2) 31 (1) NS No. of comorbidities, mean Ϯ SD** 0.7 Ϯ 1.1 0.9 Ϯ 1.2 Ͻ0.0001 Selected drug use‡ TNF␣ antagonist NA – Infliximab 792 (33) Etanercept 1,201 (50) Adalimumab 118 (5) Ն1 TNF␣ antagonist 282 (12) Methotrexate 1,677 (70) 2,933 (100) Ͻ0.0001 Mean prednisone-equivalent dosage None (referent) 1,061 (44) 1,290 (44) NS Յ5 mg/day 252 (10) 294 (10) 5–10 mg/day 348 (15) 418 (14) Ͼ10 mg/day 732 (31) 931 (32) * Except where indicated otherwise, values are the number (%). RA ϭ rheumatoid arthritis; TNF␣ ϭ tumor necrosis factor ␣; MTX ϭ methotrexate; CABG ϭ coronary artery bypass graft; NA ϭ not applicable. † Specific P values are shown if Ͻ0.10; P values Ͼ0.10 are reported as not significant (NS). ‡ Assessed in the 6 months prior to the index date. § Defined by administrative claims for rheumatoid lung disease, rheumatoid carditis, inflammatory eye disease, or Felty’s syndrome. ¶ Defined by administrative claims for joint arthrodesis, fusion, replacement, or deformity. # Includes nephritis, nephrotic syndrome, and acute or chronic renal failure. ** Defined by the sum of unique diagnoses found in the administrative claims data (range 0–21). records of the first hospitalization with a suspected infection the admission medication list for each hospitalization was not for each individual because of the concern that subsequent abstracted by the nurses. Based on their clinical judgment, hospitalizations with infections were not independent events. these physicians determined whether the medical record con- Two infectious disease physicians who were unaware of the tained sufficient data to support a diagnosis of a “definite” study hypothesis and TNF␣ antagonist exposure status inde- bacterial infection. Discordance was resolved by consensus pendently reviewed the data from each abstracted medical with a third physician reviewer (JRC or KGS). The physicians’ record. To facilitate the blinding of the reviewing physicians, assessment of a “definite” bacterial infection was the primary 1128 CURTIS ET AL

end point for our analysis. A secondary analysis examined the Among the patients exposed to TNF␣ antagonists, half risk of infection in the first 6 months after initiation of TNF␣ received etanercept, and a majority of the remainder antagonist therapy. received infliximab. Slightly more than half of the study As an alternate method for classifying infections, we then conducted an evidence-based literature review to develop cohort received prednisone, and the pattern of pred- de novo case definitions for 17 groups of common bacterial nisone use was similar between the exposed and unex- infections. In conjunction with a multi-institutional panel of posed groups. The median duration of observation fol- expert infectious disease physicians who did not participate in lowing the index date was 17 months and was similar for the review of the abstracted medical charts (author MS; Nenad both groups. Auramovski, University of Alabama at Birmingham, Ari Ro- bicsek, Brigham and Women’s Hospital, Boston, MA), we After identifying suspected infections using the developed case definitions that incorporated clinical, micro- administrative claims data, we obtained and abstracted biologic, and radiologic results relevant to the diagnosis of 187 of 217 desired medical records from health care each bacterial infection. The case definitions (available upon facilities throughout the US (86% response rate). The request) were constructed to be highly specific and have high characteristics of the 30 patients for whom we were not positive predictive values and therefore required a high degree of certainty in diagnosis (25). For this reason, microbiologic or able to abstract medical records were similar to those for other objective data were required for a majority of the whom we did abstract the records, in terms of age, sex, infections except those that are usually clinically diagnosed RA characteristics, health service use, and comorbidities (e.g., cellulitis). (data not shown), and were approximately balanced We then compared the frequency, crude risk, and between the cohort exposed to TNF␣ antagonists (n ϭ adjusted risk of hospitalization with a bacterial infection using 4 methods of classifying infections, increasing from the most 17 records not abstracted) and the unexposed cohort sensitive to the most specific, as follows: 1) administrative (n ϭ 13 records not abstracted). Because we were not claims data alone, 2) a physician reviewer’s diagnosis of an able to review these 30 medical records, these potential “empirically treated” or “definite” infection, 3) a physician cases were excluded from the primary analysis, although reviewer’s diagnosis of a “definite” infection, and 4) objective, they were included in the sensitivity analysis based on prespecified, evidence-based case definitions. Statistical analysis. Cox proportional hazards models administrative data. were used to compare the risk of hospitalization with a Using data from the 187 abstracted medical physician-confirmed “definite” bacterial infection among the records, the infectious disease physicians independently cohort exposed to TNF␣ antagonists compared with the unex- reviewed the abstracted medical records, and agreement posed cohort. Observations were censored for each patient at (kappa value) on the diagnosis of a “definite” infection the time of diagnosis of the first hospitalization with a “definite” bacterial infection, disenrollment from the health care prior to adjudication was 0.7 (95% confidence interval organization, or the end of the study. The proportional hazards [95% CI] 0.6–0.8). Table 2 shows the overall rate and assumption was satisfied over the entire study period except immediately following initiation of TNF␣ antagonist therapy, Table 2. Types of physician-confirmed “definite” bacterial infections when it was greater, and thus, results from the secondary during hospitalization* analysis are useful to address risk in this time period. ␣ All survival models were constructed according to the TNF methods advocated by Hosmer and Lemeshow (26). Variables antagonist MTX-only of high biologic relevance (i.e., infection in the 6 months before patients patients the index date) were forced into multivariable models. For No. (%) of patients with any infection 65 (2.7) 58 (2.0) other baseline variables, a bivariate P value of less than 0.20 Site-specific infections, no. was required for model entry, and an adjusted P value of less Pneumonia/empyema 25 23 than 0.05 was required for a variable to remain in the model. Cellulitis/soft tissue 23 17 Analyses were performed using SAS software, version 9.1 Bacteremia/sepsis 7 8 (SAS Institute, Cary, NC). Kidney/urinary tract 8 10 Postoperative 7 5 Device-associated 6 4 RESULTS Septic arthritis 4 4 A description of the study cohort, stratified by Gastroenteritis 1 5 Abdominal abscess 1 2 TNF␣ antagonist exposure status, is presented in Table Osteomyelitis 1 3 1. As shown, those who received TNF␣ antagonists were Bacterial sinusitis 3 0 younger, had more physician visits, and were more likely Diverticulitis 0 1 Total† 86 82 to have extraarticular features of RA. Seventy percent of the group exposed to TNF␣ antagonists received MTX * No cases of meningitis or endocarditis were observed, although our case identification strategy included these infections. See Table 1 for in the 6 months prior to their index date; another 5% definitions. received MTX after the index date (data not shown). † Some individuals had Ͼ1 infection during a hospitalization. TNF␣ ANTAGONISTS AND RISK OF INFECTION 1129

Table 3. Factors associated with hospitalization with a “definite” bacterial infection* Crude HR (95% CI) Adjusted HR (95% CI) TNF␣ antagonist treatment 1.39 (0.97–1.98) 1.94 (1.32–2.83) Demographics Age (5-year increments) 1.22 (1.14–1.31) 1.14 (1.03–1.27) Female sex (referent to male) 0.86 (0.58–1.27) NS US region of residence NS Midwest 0.89 (0.60–1.31) West 0.64 (0.25–1.61) Northeast 2.22 (1.24–3.97) South (referent) 1.0 Health services utilization Year of first study observation NS 1998–1999 1.11 (0.67–1.84) 2000–2001 0.82 (0.52–1.31) 2002–2003 (referent) 1.0 Insurance type Medicare Age Ͻ65 years (e.g., disabled) 2.82 (1.42–5.61) 2.88 (1.41–5.86) Age Ͼ65 years 2.83 (1.89–4.22) 1.88 (1.01–3.32) Commercial (referent) 1.0 1.0 No. of face-to-face physician visits† 1.07 (1.05–1.10) 1.04 (1.01–1.07) Selected medical conditions† Prior infection 1.98 (1.19–3.31) 1.46 (0.85–2.51) Severe RA‡ 1.07 (0.34–3.35) NS Coronary artery disease 2.57 (1.72–3.83) NS Chronic obstructive pulmonary disease or asthma 2.68 (1.71–4.18) 1.90 (1.19–3.04) Joint surgery or deformity§ 2.30 (0.94–5.62) NS Diabetes mellitus 2.60 (1.66–4.06) 1.75 (1.10–2.78) Kidney disease¶ 6.84 (3.01–15.5) 3.23 (1.35–7.73) Decubitus ulcer 6.35 (3.22–12.52) 3.05 (1.50–6.19) No. of comorbidities# 1.50 (1.31–1.68) NS Selected drug use† Mean prednisone-equivalent dosage None (referent) 1.0 1.0 Յ5 mg/day 1.64 (0.90–2.98) 1.49 (0.82–2.72) 5–10 mg/day 1.56 (0.90–2.70) 1.46 (0.84–2.54) Ͼ10 mg/day 1.92 (1.26–2.91) 1.85 (1.21–2.85) Any parenteral glucocorticoid 1.28 (0.88–1.88) NS *HRϭ hazard ratio; 95% CI ϭ 95% confidence interval (see Table 1 for other definitions). † Assessed in the 6 months prior to the index date. ‡ Includes rheumatoid lung disease, rheumatoid carditis, inflammatory eye disease, and Felty’s syndrome. § Includes joint arthrodesis, fusion, replacement, or contracture. ¶ Includes nephritis, nephrotic syndrome, and acute or chronic renal failure. # Defined by the sum of unique diagnoses found in the administrative claims data (range 0–21).

various sites and types of bacterial infections during the to TNF␣ antagonists (for the exposed group) or MTX entire study period. There were 65 infections among (for the unexposed group) to a confirmed infection was 2,393 exposed persons (2.7%) in the group exposed to Ͻ30 days in both groups, and 93% of all confirmed TNF␣ antagonists, and there were 58 infections among infections occurred within 90 days of the most recent 2,933 persons (2.0%) in the unexposed cohort during the exposure to the drug of interest. In the first 6 months entire study period (number needed to harm ϭ 143). A after the index date, there were 32 infections in the majority of the infections were pneumonias, followed by exposed cohort (incidence of 2.9 infections per 100 cellulitis and soft tissue infections, and kidney/urinary person-years) compared with 19 infections in the unex- tract infections. There was no significant difference in posed cohort (incidence of 1.4 infections per 100 person- the length of hospitalization among patients who were years). exposed to TNF␣ antagonists versus those who received Table 3 shows the factors associated with hospi- MTX. talization with a definite bacterial infection. After mul- The median time from the most recent exposure tivariable adjustment for the factors shown in Table 1, 1130 CURTIS ET AL

Table 4. Impact of varying the definition of serious bacterial infection on the risk estimate of infections associated with TNF␣ antagonists* Most sensitive definition

Physician-assessed Most specific definition, Infection case definition (n ϭ 217 Administrative claims data “definite” or met criteria of presumptive cases identified using alone (ICD-9-CM codes “empirically treated” Physician-assessed prespecified, evidence-based administrative claims data) for infections) (possible) infection “definite” infection† case definition for infection Proportion of persons determined 100 85‡ 66‡ 35‡ to have been hospitalized with an infection using the case definition Unadjusted HR (95% CI) of 1.1 (0.9–1.5) 1.1 (0.8–1.5) 1.4 (1.0–2.0) 1.6 (1.0–2.6) infection associated with TNF␣ antagonist use Adjusted HR (95% CI) of infection 1.6 (1.1–2.4) 1.7 (1.2–2.6) 1.9 (1.3–2.8) 2.3 (1.2–4.3) associated with TNF␣ antagonist use§ * TNF␣ ϭ tumor necrosis factor ␣; ICD-9-CM ϭ International Classification of Diseases, Ninth Revision, Clinical Modification; HR ϭ hazard ratio; 95% CI ϭ 95% confidence interval. † Detailed results are shown in Table 3. ‡ Denominator used to calculate proportions was the number of abstracted medical records (n ϭ 187). § Covariates used for the multivariable models are shown in Table 3.

there was an almost 2-fold increased risk of hospitaliza- talization with a bacterial infection associated with tion with a bacterial infection among those who received TNF␣ antagonist therapy increased. TNF␣ antagonists. Older individuals, those with more physician visits, and those with several comorbidities, DISCUSSION including chronic obstructive pulmonary disease/asthma, diabetes mellitus, and kidney disease, were also associ- TNF␣ antagonists have provided substantial ben- ated with hospitalization with a bacterial infection. Be- efit to many RA patients in controlling disease symp- cause most of these risk factors were more prevalent in toms and progression. Despite their potential benefits, the MTX cohort, the multivariable-adjusted hazard es- over a 20-month period we observed a 2.7% rate of timates were greater than the unadjusted results. All hospitalization with a confirmed serious bacterial infec- doses of prednisone were associated with trends toward tion among individuals treated with these agents com- an increased risk of infection, although only an average pared with a 2.0% rate among those who received MTX daily dose of Ͼ10 mg was statistically significant. An only. After multivariable adjustment accounting for age interaction term between TNF␣ antagonist and MTX and a number of other important comorbidities, we use was forced into the regression model but was not observed an ϳ2-fold increased risk of infections among significant (data not shown). Likewise, there was no RA patients who received TNF␣ antagonists compared significant interaction with glucocorticoids. In a sep- with those who received only MTX. Of substantial arate but similar multivariable model, the adjusted concern, the adjusted risk of infection increased 4-fold in hazard ratio of infections in the first 6 months after the 6 months following initiation of TNF␣ antagonist the index date among patients who received TNF␣ therapy. Pneumonia was the most common type of antagonists compared with MTX controls was 4.2 infection and accounted for more than one-third of (95% CI 2.0–8.8). cases. As has been reported previously for RA patients Table 4 shows results of the analyses examining (2,17,27), older age, pulmonary disease, and diabetes the impact of varying the definition of an infection from mellitus were independently associated with the risk of a a more sensitive definition (administrative data without bacterial infection. The magnitude of the risk of infec- medical record review by a physician) to a more specific tion associated with dosages of prednisone Ͼ10 mg/day definition (prespecified, evidence-based criteria). As the was similar to that associated with the TNF␣ antago- specificity of the case definition for the infection im- nists. The unique aspects of our study include a median proved, fewer cases met the criteria for an infection (i.e., duration of followup Ͼ1 year, minimal exclusion criteria sensitivity decreased), and the estimated risk for hospi- yielding a large RA cohort representative of patients TNF␣ ANTAGONISTS AND RISK OF INFECTION 1131

treated in a large US health care organization, and high ies compared with controls 1.9 [95% CI 1.2–2.9]). Fi- diagnostic certainty in confirming infections. nally, our results are consistent with those of a long-term In contrast to our results, other observational study of the safety of anakinra that found a similarly studies have not found increased rates of serious bacte- increased incidence of serious infections in anakinra- rial infections associated with the TNF␣ antagonists. treated patients, particularly for individuals who re- Wolfe and colleagues (17) found no increased risk for ceived glucocorticoid therapy (28). hospitalization with pneumonia among a biologic- Our study was based on a cohort of RA patients exposed cohort of comparable size to ours. Ascertain- enrolled in a large health care organization treated ment of pneumonias in that study was made using a throughout the US in diverse practice settings, with only combination of patient self-report, hospital records, minimal exclusions. The median observation time was outpatient physician reports, administrative claims data, ϳ1.5 years, up to a maximum of 5 years, which was and mortality records. A potential explanation for the longer than past RCTs and most observational studies. discordance with our findings may be in the definition of The requirement for at least 2 infection diagnosis codes pneumonia that Wolfe and colleagues used. We re- in the initial claims-based screening procedure and high quired a physician-assessed “definite” diagnosis of pneu- diagnostic certainty in confirming infections likely ex- monia, intentionally excluding those cases with less cluded many of the conditions that mimic signs and certainty in diagnosis. A similar explanation may ac- symptoms of bacterial infections (e.g., noninfectious count for the greater validity of our risk estimates for exacerbations of bronchitis or asthma). High back- confirmed bacterial infections associated with TNF␣ ground rates of noninfectious conditions that are unre- antagonist therapy compared with results from an early lated to drug exposures of interest might serve to mask study of a large cohort of RA patients enrolled in a US the true risk of infection associated with TNF␣ antago- managed care plan that showed no increased risk of nist therapy. infections associated with biologics (16). In that study, Our results must be interpreted in the context of biologics included both TNF␣ antagonists and anakinra our study design. First, we explicitly restricted our (an interleukin-1 antagonist that may have a different attention to bacterial infections only and did not study risk profile), and infections were defined using an ICD- opportunistic infections such as tuberculosis. In addition 9-CM code for an infection during a hospitalization to their lower incidence, some of these atypical infec- without medical record confirmation. tions are diagnosed and treated in the outpatient setting While our results were somewhat similar to those and would not require hospitalization. We abstracted of a prospective, observational German cohort of 1,529 only the first hospital medical record for each patient patients who received etanercept, infliximab, anakinra, and therefore did not examine repeated infections. or traditional DMARD therapy (15), our study was of However, this approach limited concern that patients longer duration and included almost 3 times the number with infections might discontinue use of biologic agents, of patients exposed to TNF␣ antagonists. In that study, which could bias our results if the remaining individuals by Listing and colleagues, the risk of serious infections in the exposed cohort were healthier than those who was 2.7–2.8 times higher in patients who received the 3 originally started in the cohort (depletion of suscep- biologic response modifiers, which is comparable to our tibles). We also required at least 2 diagnosis codes for risk estimate of 1.9. Consistent with our study, pneumo- infections in the claims data; only 1 code had to be from nias and cellulitis/soft tissue infections were the most a face-to-face physician visit and the other(s) may have common bacterial infections, but not all adverse events come from a diagnostic test or procedure (e.g., labora- in that patient cohort led to hospitalization. tory test or a radiograph). This requirement allowed us Further evidence consistent with our findings to focus on more severe infections but may have missed comes from a meta-analysis of RCTs of RA patients who milder infections or those receiving minimal workup. received TNF␣ antagonist antibodies (14). In that ana- Another limitation is that we were not able to lysis, the infectious end points were investigator- quantify the dose of the TNF␣ antagonist received, diagnosed infections that were not systematically de- which, particularly for infliximab, may be clinically im- fined or adjudicated. Nevertheless, despite data sources portant (29). Although we lacked infliximab dosage and analysis methods significantly different from those data, a study examining medication use among RA used in our study, our risk estimates were comparable to patients using the same data source and similar inclusion those results (odds ratio for nongranulomatous infec- criteria as ours found that adult patients were receiving tions in patients treated with TNF␣ antagonist antibod- 3–4 mg/kg and that fewer than half had any dosage 1132 CURTIS ET AL

escalation over a 2.5-year period (30). Thus, it would AUTHOR CONTRIBUTIONS seem that most RA patients in our study received a dose Drs. Curtis and K. Saag had full access to all of the data in the very close to the standard 3 mg/kg approved by the US study and take responsibility for the integrity of the data and the accuracy of the data analysis. Food and Drug Administration and by the European Study design. Curtis, Martin, Allison, M. Saag, Shatin, K. Saag, Larry Agency for the Evaluation of Medicinal Products. It is Moreland (nonauthor; University of Alabama at Birmingham). possible that physicians have a lower threshold for Acquisition of data. Curtis, Patkar, Martin, Shatin, K. Saag, Margaret Burgess (nonauthor; Center for Health Care Policy and Evaluation). hospitalization for suspected infections in patients Analysis and interpretation of data. Curtis, Patkar, Martin, Allison, M. treated with TNF␣ antagonists, although the robustness Saag, Shatin, K. Saag. of our findings of various case definitions for infections Manuscript preparation. Curtis, Patkar, Martin, Allison, M. Saag, Shatin, K. Saag. should attenuate this concern. Statistical analysis. Curtis, Xie, Allison, Shatin, K. Saag. Although we used the administrative claims data Project coordination. Patkar. to adjust for a variety of conditions indicative of greater Review of medical charts. Cynthia King (nonauthor; University of Alabama at Birmingham), Paul Perry (nonauthor; University of Ala- RA disease severity (e.g., Felty’s syndrome) and general bama at Birmingham). medical comorbidities, neither administrative data nor hospital medical records provide information on RA- REFERENCES specific markers of disease activity and damage. We therefore cannot exclude residual confounding by indi- 1. Doran MF, Crowson CS, Pond GR, O’Fallon WM, Gabriel SE. Frequency of infection in patients with rheumatoid arthritis com- cation (31). However, our comparator group of RA pared with controls: a population-based study. Arthritis Rheum patients filling at least 3 prescriptions for MTX and our 2002;46:2287–93. analytical control for a number of important confound- 2. Doran MF, Crowson CS, Pond GR, O’Fallon WM, Gabriel SE. Predictors of infection in rheumatoid arthritis. Arthritis Rheum ers minimizes this concern. Furthermore, the MTX-only 2002;46:2294–300. cohort had a higher burden of comorbidities that might 3. Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, et al, for the ATTRACT Study Group. Infliximab (chimeric predispose to bacterial infections, as shown in Table 1. anti-tumour necrosis factor ␣ monoclonal antibody) versus pla- Finally, because we focused on RA patients with at least cebo in rheumatoid arthritis patients receiving concomitant meth- 2 diagnosis codes, we may have excluded some patients otrexate: a randomised phase III trial. Lancet 1999;354:1932–9. 4. Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleisch- with milder or early RA. However, this should have no mann RM, Fox RI, et al. A trial of etanercept, a recombinant impact on the internal validity of the study. tumor necrosis factor receptor:Fc fusion protein, in patients with In conclusion, we found a 2–4-fold increase in the rheumatoid arthritis receiving methotrexate. N Engl J Med 1999; 340:253–9. risk of hospitalization with a serious bacterial infection 5. Keystone EC, Kavanaugh AF, Sharp JT, Tannenbaum H, Hua Y, compared with traditional RA therapies, which was Teoh LS, et al. Radiographic, clinical, and functional outcomes of proportionally greater shortly after beginning therapy treatment with adalimumab (a human anti–tumor necrosis factor ␣ monoclonal antibody) in patients with active rheumatoid arthritis with TNF antagonists. RA patients were at increased receiving concomitant methotrexate therapy: a randomized, pla- risk of serious infections, irrespective of the method cebo-controlled, 52-week trial. Arthritis Rheum 2004;50:1400–11. used to define an infectious outcome. Older age, diabe- 6. Genovese MC, Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff MH, et al. Etanercept versus methotrexate in patients tes mellitus, and preexisting pulmonary disease place with early rheumatoid arthritis: two-year radiographic and clinical patients at particular risk. Our large cohort of RA outcomes. Arthritis Rheum 2002;46:1443–50. patients, with minimal exclusion criteria and with study 7. Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pavelka K, van Vollenhoven R, et al. The PREMIER study: a multicenter, physicians confirming infections with a high degree of randomized, double-blind clinical trial of combination therapy certainty and reliability, increases the confidence that with adalimumab plus methotrexate versus methotrexate alone or our results are valid and generalizable to other settings. adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Ar- The dramatic efficacy of TNF␣ antagonist therapy for thritis Rheum 2006;54:26–37. most RA patients needs to be balanced against the 8. StClair EW, van der Heijde DM, Smolen JS, Maini RN, Bathon JM, Emery P, et al, for the Active-Controlled Study of Patients potential harm of an increased risk of infection associ- Receiving Infliximab for the Treatment of Rheumatoid Arthritis ated with these agents, albeit with the recognition that of Early Onset Study Group. Combination of infliximab and the absolute difference in the number of infections was methotrexate therapy for early rheumatoid arthritis: a randomized, controlled trial. Arthritis Rheum 2004;50:3432–43. relatively low (0.7 infections per 100 patients). Patients 9. Colombel JF, Loftus EV Jr, Tremaine WJ, Egan LJ, Harmsen WS, and physicians should vigilantly monitor for signs of Schleck CD, et al. The safety profile of infliximab in patients with Crohn’s disease: the Mayo clinic experience in 500 patients. infection when prescribing these medications, particu- Gastroenterology 2004;126:19–31. larly shortly after their initiation. 10. Kroesen S, Widmer AF, Tyndall A, Hasler P. Serious bacterial TNF␣ ANTAGONISTS AND RISK OF INFECTION 1133

infections in patients with rheumatoid arthritis under anti-TNF-␣ 21. Dixon WG, Watson K, Lunt M, Hyrich KL, Silman AJ, Symmons therapy. Rheumatology (Oxford) 2003;42:617–21. DP, et al. Rates of serious infection, including site-specific and 11. Baghai M, Osmon DR, Wolk DM, Wold LE, Haidukewych GJ, bacterial intracellular infection, in rheumatoid arthritis patients Matteson EL. Fatal sepsis in a patient with rheumatoid arthritis receiving anti–tumor necrosis factor therapy: results from the treated with etanercept. Mayo Clin Proc 2001;76:653–6. British Society for Rheumatology Biologics Register. Arthritis 12. Neven N, Vis M, Voskuyl AE, Wolbink GJ, Nurmohamed MT, Rheum 2006;54:2368–76. Dijkmans BA, et al. Adverse events in patients with rheumatoid 22. Shatin D, Rawson N, Stergachis A. UnitedHealth Group. In: arthritis treated with infliximab in daily clinical practice [letter]. Strom B, editor. Pharmacoepidemiology. Sussex (UK): John Ann Rheum Dis 2005;64:645–6. Wiley; 2005. 13. Giles JT, Bartlett SJ, Gelber AC, Nanda S, Fontaine K, Ruffing V, 23. MacLean CH, Louie R, Leake B, McCaffrey DF, Paulus HE, et al. Tumor necrosis factor inhibitor therapy and risk of serious Brook RH, et al. Quality of care for patients with rheumatoid postoperative orthopedic infection in rheumatoid arthritis. Arthri- arthritis. JAMA 2000;284:984–92. tis Rheum 2006;55:333–7. 24. Kremer JM. Rational use of new and existing disease-modifying 14. Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL, agents in rheumatoid arthritis [review]. Ann Intern Med 2001;134: Montori V. Anti-TNF antibody therapy in rheumatoid arthritis 695–706. and the risk of serious infections and malignancies: systematic 25. Patkar N, Teng GG, Curtis JR, et al. Reliability and validity of review and meta-analysis of rare harmful effects in randomized evidence-based case definitions of serious bacterial infections controlled trials [published erratum appears in JAMA 2006;295: among rheumatoid arthritis patients [abstract]. Arthritis Rheum 2482]. JAMA 2006;295:2275–85. 2005;52:4094. 15. Listing J, Strangfeld A, Kary S, Rau R, von Hinueber U, Stoy- 26. Hosmer JD, Lemeshow S. Applied survival analysis: regression anova-Scholz M, et al. Infections in patients with rheumatoid modeling of time to event data. New York: John Wiley and Sons; arthritis treated with biologic agents. Arthritis Rheum 2005;52: 1999. 3403–12. 27. Saag KG, Koehnke R, Caldwell JR, Brasington R, Burmeister LF, 16. Smitten A, Simon T, Hochberg M, Suissa S. Rates of infection in Zimmerman B, et al. Low dose long-term corticosteroid therapy in rheumatoid arthritis [abstract]. Arthritis Rheum 2004;50 Suppl rheumatoid arthritis: an analysis of serious adverse events. Am J 9:S478. Med 1994;96:115–23. 17. Wolfe F, Caplan L, Michaud K. Treatment for rheumatoid 28. Fleischmann RM, Tesser J, Schiff MH, Schechtman J, Burmester arthritis and the risk of hospitalization for pneumonia: associations GR, Bennett R, et al. Safety of extended treatment with anakinra with prednisone, disease-modifying antirheumatic drugs, and in patients with rheumatoid arthritis. Ann Rheum Dis 2006;65: anti–tumor necrosis factor therapy. Arthritis Rheum 2006;54: 1006–12. 628–34. 29. Westhovens R, Yocum D, Han J, Berman A, Strusberg I, Geusens 18. Den Broeder A, Schraven T, De Jong E, De Rooij. Infectious P, et al, for the START Study Group. The safety of infliximab, complications in elective surgery in RA patients in the anti-TNF combined with background treatments, among patients with era: a retrospective study [abstract]. Ann Rheum Dis 2005;64 rheumatoid arthritis and various comorbidities: a large, ran- Suppl III:60. domized, placebo-controlled trial. Arthritis Rheum 2006;54: 19. Talwalkar SC, Grennan DM, Gray J, Johnson P, Hayton MJ. 1075–86. Tumour necrosis factor ␣ antagonists and early postoperative 30. Harley CR, Frytak JR, Tandon N. Treatment compliance and complications in patients with inflammatory joint disease under- dosage administration among rheumatoid arthritis patients receiv- going elective orthopaedic surgery [letter]. Ann Rheum Dis 2005; ing infliximab, etanercept, or methotrexate. Am J Manag Care 64:650–1. 2003;9 Suppl 6:S136–43. 20. Schneeweiss S, Setoguchi S, Weinblatt ME, et al. Anti-TNF-␣ 31. Salas M, Hofman A, Stricker BH. Confounding by indication: an therapy and the risk of severe bacterial infections. Pharmacoepi- example of variation in the use of epidemiologic terminology. demiol Drug Saf 2006;15(S28). Am J Epidemiol 1999;149:981–3. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1134–1144 DOI 10.1002/art.22458 © 2007, American College of Rheumatology

Aberrant Expression of BAFF by B Lymphocytes Infiltrating the Salivary Glands of Patients With Primary Sjo¨gren’s Syndrome

Capucine Daridon, Vale´rie Devauchelle, Pascal Hutin, Rozenn Le Berre, Christine Martins-Carvalho, Boutahar Bendaoud, Maryvonne Dueymes, Alain Saraux, Pierre Youinou, and Jacques-Olivier Pers

Objective. To identify the cells that produce BAFF thermore, this cytokine was shown to be functional, in in the salivary glands of patients with primary Sjo¨gren’s that epithelial cell–bound BAFF extended the survival of syndrome (SS), and to analyze BAFF receptor expres- normal B cells, but cell-free BAFF released in the sion by local T and B lymphocytes. supernatants did not. Methods. We used 3 methods to identify the Conclusion. These experiments establish that in source of BAFF: in situ hybridization of the transcripts primary SS, BAFF is produced not only by epithelial for BAFF combined with staining of membrane mark- cells and T cells but also by B cells. The expression of ers, regular and real-time reverse transcription– receptors for BAFF would thus allow these receptors to polymerase chain reaction (RT-PCR) of cultured epithe- participate in an autocrine pattern of self-stimulation. lial cells, and RT-PCR of sorted single-cell T and B lymphocytes eluted from salivary glands. Cells express- Sjo¨gren’s syndrome (SS) is an autoimmune epi- ing TACI, BCMA, and B lymphocyte stimulator receptor thelitis (1) that may develop against a background of 3 (BR-3) were disclosed by combining each specific connective tissue disease as secondary SS (2) or alone as staining of the receptors with each specific staining of primary SS. In primary SS (3), lymphocyte foci are the cells. The function of BAFF generated by epithelial scattered over the salivary glands, and germinal centers cells on B lymphocytes was determined in short-term (GCs) are annexed to their epithelial cells. It is interest- cocultures. ing that similar structures may be encountered in normal Results. Transcripts for BAFF were seen in epi- salivary glands (4). thelial cells and infiltrating T lymphocytes and, for the Although B cells represent as few as 10–20% of first time, were detected in local B cells. It is interesting infiltrating mononuclear cells, major breakthroughs in that BR-3 was present on these B cells but not on T cells. the study of B cells have occurred (5), such as the In contrast, TACI and, to a lesser degree, BCMA were dissection of B cell developmental stages, the profiling observed on transitional B lymphocytes, whereas T of B cell–related gene expression using microarray tech- lymphocytes were devoid of receptors for BAFF. Fur- nology, and the discovery of 2 ligands for the tumor necrosis factor receptor family: one is a proliferation- Supported by a grant from the Association Franc¸aise du Gougerot-Sjo¨gren et des syndromes secs. inducing ligand, and the other is a B cell–activating Capucine Daridon, BSc, Vale´rie Devauchelle, MD, PhD, factor (BAFF). New evidence has thus sparked a great Pascal Hutin, MD, Rozenn Le Berre, MD, PhD, Christine Martins- deal of interest in the possibility that B cells play a Carvalho, MD, Boutahar Bendaoud, PharmD, Maryvonne Dueymes, MD, PhD, Alain Saraux, MD, PhD, Pierre Youinou, MD, DSc, central role in the pathogenesis of primary SS (6,7). Jacques-Olivier Pers, DDS, PhD: Brest University Medical School B cells are more heterogeneous than originally Hospital, Brest, France. Address correspondence and reprint requests to Pierre Youi- assumed. When newly emigrated into secondary lym- nou, MD, DSc, Laboratory of Immunology, Brest University Medical phoid organs, they give rise to transitional type I and School Hospital, BP824, F-29609 Brest, France. E-mail: youinou@ type II (TII) B cells, of which the majority enter the univ-brest.fr. Submitted for publication June 30, 2006; accepted in revised GCs, and a minority differentiate into marginal-zone B form December 19, 2006. cells (8). At this stage, the relative expression levels of

1134 THERAPEUTIC EFFICACY OF BAFF ANTAGONISTS IN AUTOIMMUNE DISORDERS 1135

IgD and CD38 (9) have generated a model for mature B PATIENTS AND METHODS (Bm) cell homeostasis. Naive Bm1 cells are primed as Ј Patients, control subjects, and cell lines. Salivary gland Bm2 cells, become GC founder Bm2 cells, differentiate biopsy specimens were obtained from 18 patients (3 men and into centroblast Bm3 and centrocyte Bm4 cells, and 15 women; ages 32–77 years), all of whom fulfilled the converge on memory Bm5 or plasma cells. American-European Consensus Group criteria for primary SS BAFF is critically involved in the life span of B (33) and had a focus score of Ն3 (34). The disease was limited cells (10), most notably those that are autoreactive (11), to the exocrine glands in 5 patients, and the remaining 13 patients had at least 1 extraglandular complication. Patients and thereby in the production of self antibodies and did not have associated lymphoma, nor were they receiving subsequently in the development of primary SS in steroids or immunosuppressive drugs. After the study, 15 of BAFF-transgenic mice (12). Consistent with this model these 18 patients were enrolled in a trial with anti-CD20 are elevated levels of BAFF in the serum (13), saliva monoclonal antibody (mAb) (35,36). Control samples con- (14), and salivary glands (12,15) of patients with primary sisted of 15 salivary gland specimens from patients who did not meet the criteria for SS, although they had described sicca SS. Thus far, cell sources of BAFF include macrophages symptoms and, as such, had undergone a salivary gland biopsy. (16), dendritic cells (17), stromal cells (18), neutrophils Tonsils were obtained from 6 children undergoing routine (19), synoviocytes (20), astrocytes (21), and activated T tonsillectomy; tonsils were obtained from children because we cells (22). With one exception (23), previous studies did not have access to tonsils from elderly control subjects. Potentially purulent tonsils were excluded. have failed to detect BAFF expression in normal resting All of these individuals or their parents gave informed or activated B cells (16,24–26). consent, and the study was approved by the Ethics Committee With regard to the salivary glands of patients with at the Brest University Medical School. Embryonic kidney 293 primary SS, BAFF is highly expressed in aggregates of cells served as negative controls for BAFF (31), Daudi B cells leukocytes but also on epithelial cells and TII and served as positive controls for cell-bound BAFF, and U937 cells served as positive controls for soluble BAFF (all from marginal-zone B cells (27). Of note, serum levels of American Type Culture Collection, Manassas, VA). BAFF are proportional to the focus score, and high Immunofluorescence staining of salivary glands. After concentrations are associated with the development of an overnight incubation at 4°C in phosphate buffered saline GCs (28). (PBS) containing 15% sucrose, the specimens were embedded in OCT compound (Miles, Naperville, IL) and snap-frozen in There are 3 distinct receptors for BAFF (29–31): isopentane. Serial 4-␮m–thick sections were mounted onto TACI, BCMA, and B lymphocyte stimulator receptor 3 poly-L-lysine–coated slides. All mAb were obtained from Beck- (BR-3). BR-3 is expressed in all mature B cells and a man Coulter (Villepinte, France), unless otherwise specified. subset of activated T cells. In contrast to BR-3, TACI In the first step of these double-staining experiments, goat and BCMA are each restricted to subsets of B cells. For anti-human BAFF antibody (PeproTech, Rocky Hill, NJ) was combined with either anti-CD19, anti-CD3, or anticytokeratin example, TII B cells and activated B cells harbor TACI, mAb. Alexa Fluor 594 cholera toxin B (Molecular Probes, provided they remain outside of the GCs. In contrast, Eugene, OR) was used to stain the membranes of 293 cells. plasma cells and B cells bear BCMA once they have After a 40-minute incubation and 3 washes in PBS, the second entered the GCs, while 50% of T cells can be induced to step combined tetramethylrhodamine isothiocyanate (TRITC)– conjugated donkey anti-mouse polyclonal antibody (pAb) with express TACI by ionomycin and phorbol ester (32). fluorescein isothiocyanate (FITC)–conjugated donkey anti- However, we do not know which cells synthesize BAFF goat pAb (Jackson ImmunoResearch, West Grove, PA) in PBS within the salivary glands and what effects are exerted containing 2% donkey serum. After another 3 washes, the locally. tissues were fixed with 4% cold paraformaldehyde, washed in PBS, and coverslipped (Vector, Burlingame, CA). Images were The aim of the present study was to address these obtained using an Ar/Kr laser and analyzed with a Leica concerns. First, we established that constitutive synthesis TCS-NT confocal imaging system (Leica, Westlar, Germany). of BAFF by epithelial cells does not characterize salivary Neither mouse IgG (developed with FITC-conjugated donkey glands from patients with primary SS inasmuch as BAFF anti-mouse pAb) nor goat IgG (developed with TRITC- was also detected in the salivary glands of normal conjugated donkey anti-goat pAb) produced background fluorescence. To ensure that the goat anti-BAFF pAb was specific individuals. Next, transcripts for BAFF were observed in for BAFF, 20 ␮g of this antibody was mixed with 20 ␮gof individual T lymphocytes (which was predictable) but BAFF (PeproTech) prior to being added to the sections. also in individual B lymphocytes (which was not pre- To determine the binding of BAFF to the receptors, ␮ ␮ dicted). Finally, based on the presence of BR-3 on all B 10 g of BAFF was incubated with 2.5 g biotin–N- hydroxysuccinimide (biotin-NHS) (Sigma, St. Louis, MO) in cells and that of TACI on subgroups of B cells, the 0.1M sodium borate buffer, pH 8.8, for 4 hours. The prepara- cell-bound form of BAFF, but not its cell-free form, ␮ ␮ tion was then incubated with 2 lof0.1M NH4Cl per 2.5 gof impacted B lymphocytes but not T lymphocytes. biotin-NHS for another 10 minutes and centrifuged at 4,500g 1136 DARIDON ET AL

Table 1. Oligonucleotides used as primers for the amplification of cDNA in reverse transcription–polymerase chain reaction (RT-PCR), quantitative RT-PCR, nested RT-PCR, and in situ hybridization Primers Oligonucleotide sequences RT-PCR BAFF nested sense 5Ј-CAGGGTCCAGAAGAAACAGTCAC-3Ј BAFF nested antisense 5Ј-TAGATGTCCCATGGCGTAGGTC-3Ј GAPDH sense 5Ј-CTTAGCACCCCTGGCCAAGG-3Ј GAPDH antisense 5Ј-CTTACTCCTTGGAGGCCATG-3Ј Quantitative RT-PCR 18S sense 5Ј-GCCGCTAGAGGTGAAATTCTTG-3Ј 18S antisense 5Ј-CATTCTTGGCAAATGCTTTCG-3Ј BAFF sense 5Ј-GTCTGGTGACTTTGTTTCGATGTATT-3Ј BAFF antisense 5Ј-TTCATCTCCTTCTTCCAGTTTTGC-3Ј In situ hybridization BAFF antisense 5Ј-TTGCAGCCAGCAGCTTTCCGTCTTTGGAGGATCGGACAGAGGGGCTTT-3Ј BAFF sense 5Ј-AAAGCCCCTCTGTCCGATCCTCCAAAGACGGAAAGCTGCTGGCTGCAA-3Ј Single-cell CD79␤ sense 5Ј-CAGCACCTTGGCACAGCTGAAGCAG-3Ј CD79␤ antisense 5Ј-GGGGCTGGAGACAAATGGCAGCT-3Ј CD79␤ nested sense 5Ј-TCATCGTGCCTATCTTCCTGCTGCTG-3Ј CD79␤ nested antisense 5Ј-CCAGACCGCAGCGTCACTATGTCCTC-3Ј CD3␧ sense 5Ј-GCTACCCCAGAGGAAGCAAACCAG-3Ј CD3␧ antisense 5Ј-GAACGATTCTCTCTCGTGGGGTCC-3Ј CD3␧ nested sense 5Ј-CTGGGGGCTTGCTGCTGCTGGTTTAC-3Ј CD3␧ nested antisense 5Ј-CCGGATGGGCTCATAGTCTGGGTTGG-3Ј BAFF sense 5Ј-GAGAAGGCAACTCCAGTCAGAAC-3Ј BAFF antisense 5Ј-CCCATGGCGTAGGTCTTATCA-3Ј BAFF nested sense 5Ј-CAGGGTCCAGAAGAAACAGTCAC-3Ј BAFF nested antisense 5Ј-TAGATGTCCCATGGCGTAGGTC-3Ј for 3 hours. Biotinylated BAFF was adjusted to 200 ␮g/ml and X-100 (Sigma) in lysis buffer (20 mM Tris HCl, pH 7.5, 140 applied to the sections for 30 minutes, in combination with mM NaCl, 1 mM EDTA with 1 mM phenylmethylsulfonyl either anti-CD19, anti-CD3, or anticytokeratin mAb. BAFF fluoride, 10 ␮g/ml aprotinin, and 1 mM sodium orthovana- was revealed by FITC–streptavidin, and mAb were developed date). The protein concentration was measured using the with TRITC-conjugated donkey anti-mouse IgG pAb. To Micro BCA Protein Assay kit (Pierce, Rockford, IL). Super- localize the 3 BAFF receptors, anti-CD19 or anti-CD3 mouse natants of salivary gland epithelial cells, 293 cells, and U937 mAb were combined with either rabbit anti–BR-3 (ProSci, cells were resolved by 13% sodium dodecyl sulfate– Poway, CA), goat anti-BCMA (R&D Systems, Minneapolis, polyacrylamide gel electrophoresis and transferred to a poly- MN), or goat anti-TACI pAb (PeproTech). These antibodies vinylidene difluoride membrane (Bio-Rad, Hercules, CA). were developed with either TRITC-conjugated donkey anti- After 3 washes with PBS, the unbound sites were blocked for mouse, FITC-conjugated donkey anti-rabbit (Jackson Immuno- 1 hour with 0.05% Tween 20 in 5% nonfat milk. The mem- Research), or FITC-conjugated donkey anti-goat pAb. brane was probed with 1 ␮g/ml anti-BAFF mAb (R&D Primary culture of epithelial cells. Long-term salivary Systems) or antiactin mAb (Abcam, Cambridge, UK). This gland epithelial cell lines were established from tissue biopsy binding was revealed by biotinylated goat anti-mouse IgG specimens from patients and control subjects (37). Upon (Amersham, Orsay, France), stained with horseradish removal, these were placed in a 3:1 mixture of Ham’s F-12 peroxidase–labeled streptavidin (Jackson ImmunoResearch), medium and Dulbecco’s modified Eagle’s medium (Gibco, and read by enhanced chemiluminescence (ECL). Paisley, UK) supplemented with 2.5% fetal bovine serum BAFF transcript analysis. Total messenger RNA (FBS) and 10 ng/ml epidermal growth factor (EGF), 0.4 ␮g/ml (mRNA) was extracted from salivary glands, cultured salivary hydrocortisone, 0.5 ␮g/ml insulin, and antibiotics. The salivary gland epithelial cells, 293 cells and U937 cells, and purified gland lobules were then cut into small fragments and incubated with random hexamer (Table 1) and 25 units of reverse in medium that was replaced every 3 days. Cell outgrowth transcriptase (RT) using the SuperScript First-Strand system began 3 weeks later, and the epithelial origin of cells was (Invitrogen, Rockville, MD). After a 5-minute denaturation at ascertained by morphology and specific markers. Salivary 94°C and the addition of 2.5 units Taq polymerase, comple- gland epithelial cells were used once they reached 70–80% mentary DNA (cDNA) was amplified. The first round of confluency. Fifteen salivary gland epithelial cell lines were polymerase chain reaction (PCR) consisted of 5 cycles, as derived from the patients and another 15 from the control follows: 30 seconds at 94°C for denaturation, 60 seconds at subjects. 61°C for annealing, and 60 seconds at 72°C for extension. The Immunoblot analysis. After 3 washes in Hanks’ bal- second round consisted of 35 cycles, as follows: 30 seconds at anced salt solution, 107 salivary gland epithelial cells, 293 cells, 94°C, 60 seconds at 56°C, and 60 seconds at 72°C with a final or U937 cells were incubated for 1 hour at 4°C with 1% Triton extension step at 72°C for 10 minutes. RT-PCR products were THERAPEUTIC EFFICACY OF BAFF ANTAGONISTS IN AUTOIMMUNE DISORDERS 1137

Figure 1. Binding of BAFF to cells in the infiltrate of salivary glands from patients with primary Sjo¨gren’s syndrome. Sections were stained with biotinylated BAFF, and staining was revealed by fluorescein isothiocyanate–conjugated streptavidin, followed by counterstaining with mouse monoclonal antibody to CD20 (top row) or CD3 (bottom row), both of which were developed with tetramethylrhodamine isothiocyanate– conjugated donkey anti-mouse antibody. The overlay of green-stained BAFF with red-stained B cells (but not T cells) is shown as yellow.

resolved on 2% agarose gels, and their intensity was normal- 1ϫ blocking buffer (Roche Diagnostics, Meylan, France) with ized to GAPDH. 0.1% Triton X-100, incubated with FITC-conjugated donkey Quantitative PCR was also applied with 20-␮l mixtures anti-DIG pAb (Roche) overnight at 4°C, and examined by containing 50 ng of template cDNA, 1ϫ SYBER Green PCR confocal microscopy. master mix, and 300 nM of each primer. The transcripts were Single-cell sorting protocol. Salivary gland samples cloned using the GeneAmp 5700 Sequence Detection System from 3 patients were homogenized with a glass grinder and (Applied Biosystems, Foster City, CA). Amplification com- incubated in RPMI 1640 containing 10% FBS with 1 unit/ml prised 1 cycle at 50°C for 2 minutes, 1 cycle at 95°C for 10 collagenase and Dispase (Sigma), for 15 minutes at 37°C. After minutes, and 40 cycles at 95°C for 15 seconds and at 60°C for the washes, the cells were incubated with FITC-conjugated

60 seconds. The number of threshold cycles (Ct) was compared anti-CD19 and phycoerythrin (PE)–conjugated anti-CD3 Ϫ⌬ using the 2 Ct method, with ribosomal 18S RNA as an mAb. Individual CD19ϩ cells or CD3ϩ cells were sorted into internal control. PCR tubes containing 10 ␮l of RT buffer, 5 ␮M random In situ hybridization and immunofluorescence stain- hexamer, 0.5ϫ RT buffer, 0.01M DTT, and 0.5 mM dNTP ing on frozen sections. Biopsy specimens from patients and (Promega, Madison, WI), using a sorter outfitted with the control subjects were cut into 12-␮m–thick sections, fixed with Autoclone single-cell deposition unit (Beckman Coulter), as 4% paraformaldehyde in 0.1% diethyl pyrocarbonate (DEPC) described previously in detail (38). RNA conversion from for 15 minutes at 4°C, washed 3 times with 0.1% DEPC, and individual cells was conducted with SuperScript II RT (Invitro- incubated for 8 minutes at 4°C with either anti-CD19, antigen) for 150 minutes at 42°C and 10 minutes at 70°C. CD79b, CD3, or anticytokeratin mAb. After 3 washes in 0.1% DEPC, CD3, GAPDH, and BAFF cDNA were subjected to nested the slides were incubated for 8 minutes at 4°C with TRITC- RT-PCR, with 5 cycles of 30 seconds at 94°C, 1 minute at 56°C, conjugated donkey anti-mouse pAb, washed 3 times, incubated and 1 minute at 72°C in the first round of PCR. The second in 100% ethanol for 5 minutes, and air dried. They were then round consisted of 40 cycles of 30 seconds at 94°C, 1 minute at incubated overnight at 37°C with 200 ng/ml digoxigenin 56°C, and 1 minute at 72°C with a final extension at 72°C for 10 (DIG)–labeled probe mix in 60 ␮lof4ϫ saline–sodium citrate minutes. Preparations contaminated with genomic DNA were (SSC), 0.5ϫ Denhardt’s solution, 50% formamide, 0.2 gm/ml excluded. dextran sulfate, 10 mM dithiothreitol (DTT), 40 ␮g/ml DNA, Culture of lymphocytes. Six tonsillar cell suspensions and 40 ␮g/ml transfer RNA. After hybridization, the slides were enriched in B cells by rosetting with sheep erythrocytes were washed twice in 2ϫ SSC and twice in 1ϫ SSC, blocked in and Ficoll-Hypaque centrifugation. Among the PE–cyanin 1138 DARIDON ET AL

Figure 2. B lymphocyte stimulator receptor 3 (BR-3), TACI, and BCMA expression in salivary glands from patients with primary Sjo¨gren’s syndrome. Sections were stained with either rabbit anti–BR-3, goat anti-TACI, or goat anti-BCMA antibodies, and staining was revealed by fluorescein isothiocyanate–conjugated donkey anti-rabbit or anti-goat antibodies. Mouse anti-CD19 or anti-CD3 monoclonal antibodies were directed to B and T cells, respectively, and were developed with tetramethylrhodamine isothiocyanate–conjugated donkey anti-mouse antibodies.

7–conjugated CD19ϩ B cells, Bm1 cells were sorted as PE– BAFF with red-stained lymphocytes (shown as yellow) ϩ Ϫ conjugated IgD and PE–cyanin 5–conjugated CD38 . These suggested that B cells, but not T cells, retained BAFF. were cultured on plates coated with 8 ␮g/cm2 type I collagen, in basic medium for keratinocytes, with 10 ng/ml EGF, 0.4 To strengthen this finding, we explored expres- ␮g/ml hydrocortisone, 0.5 ␮g/ml insulin, 25 ␮g/ml bovine sion of the BAFF receptors. BR-3 was seen on a pituitary extract (Sigma), 10% FBS, and antibiotics. The majority of B cells, and TACI and BCMA were observed cultures were completed with or without salivary gland epithe- on a minority of B cells (Figure 2). We previously lial cells from patients, and/or with or without supernatant of described these B cells that were positive for TACI their salivary gland epithelial cells. One aliquot of Bm1 cells was seeded at 106/ml with 5 ␮g/ml anti-BAFF mAb, cultured and/or for BCMA as TII B cells (27,39). In contrast, T for 24 hours, and stained with FITC-conjugated annexin V and cells did not carry any of the 3 receptors for BAFF propidium iodide before being analyzed. Live cells were de- (Figure 2). Thus, in sharp contrast to T lymphocytes, B fined as negative for both annexin V and propidium iodide. lymphocytes in the salivary glands of patients with Statistical analysis. All results are expressed as the mean Ϯ SD. Comparisons were made using the Mann- primary SS are potentially sensitive to BAFF. Whitney U test for unpaired data. Increased level of BAFF in the salivary glands of patients with primary SS. Transcripts for BAFF were observed in the total lysate from the salivary glands of RESULTS the 12 patients tested and, to a lesser degree, in that Expression of BR-3, TACI, and BCMA in T and from salivary glands of the 12 control subjects tested B lymphocytes. BAFF-sensitive cells were detected (Figure 3A, and results not shown). Quantitative RT- through the binding of BAFF and were recognized as B PCR (Figure 3B) demonstrated that levels were higher and T cells by the expression of CD20 and CD3, in patients than in control subjects (11.37 Ϯ 0.96 versus respectively (Figure 1). The overlay of green-stained 2.83 Ϯ 0.73; P Ͻ 0.05). Immunofluorescence revealed THERAPEUTIC EFFICACY OF BAFF ANTAGONISTS IN AUTOIMMUNE DISORDERS 1139

Figure 3. BAFF expression in salivary glands from patients with primary Sjo¨gren’s syndrome. U937 cell line cells served as positive controls for BAFF, and 293 cell line cells served as negative controls. A, Representative conventional reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of total lysate from patient and control salivary glands. B, Representative quantitative RT-PCR analysis. ⌬ ϭ Bars show the mean and SD. Ct difference in threshold cycle. C–F, Salivary glands were stained with goat anti-human BAFF antibody, and staining was revealed by fluorescein isothiocyanate–conjugated donkey anti-goat antibody. This staining was combined with mouse monoclonal antibodies to either cytokeratin (C and F), CD3 (D), or CD19 (E) and was developed with tetramethylrhodamine isothiocyanate (TRITC)–conjugated donkey anti-mouse antibody. F, The binding of goat anti- human BAFF antibody to BAFF was inhibited by previous incubation of the antibody with BAFF. G, Results obtained using Daudi cell line cells and 293 cell line cells as positive and negative controls, respectively, for BAFF; 293 cells were localized using TRITC-conjugated cholera toxin B (CTB). intense BAFF staining on ductal epithelial cells (Figure confirmed to be specific for BAFF by inhibition of 3C) but also on T cells (Figure 3D) and even on B cells staining by prior incubation with BAFF (Figure 3F). As (Figure 3E). Of importance, the anti-BAFF pAb was expected, Daudi cells were positive for BAFF (Figure 1140 DARIDON ET AL

Figure 4. Production of transcripts for BAFF by epithelial cells and B and T lymphocytes in salivary glands from patients with primary Sjo¨gren’s syndrome. Tetramethylrhodamine isothiocyanate–conjugated monoclonal antibodies to either cytokeratin (A and B), CD3 (C), or CD19 (D–F) were combined with in situ hybridization of the transcripts for BAFF plus fluorescein isothiocyanate–conjugated rabbit anti-digoxigenin antibody. The sense probe served as a negative control for BAFF (B), and oligo(dT) served as a positive control for total mRNA staining (F).

3G). In contrast, 293 cells were localized by cholera toxin sophisticated techniques were required to identify B and were negative for BAFF (Figure 3G). The expo- BAFF-manufacturing cells. First, in situ hybridization of sure of BAFF on the plasma membrane suggests that it the transcripts for BAFF was combined with immuno- comes from epithelial cells, T cells, and B cells. It may, fluorescence staining of cytokeratin to prove that the however, be possible that BAFF has been inserted into BAFF antisense probe bound to epithelial cells (Figure its receptors on these cells. 4A) but the sense probe did not (Figure 4B). Next, the Production of BAFF by epithelial cells. Given the lysates of salivary gland epithelial cells and their super- aforementioned inconsistency in previous reports, 2 natants were blotted. Cell-bound BAFF was visualized THERAPEUTIC EFFICACY OF BAFF ANTAGONISTS IN AUTOIMMUNE DISORDERS 1141

Figure 5. Identification of BAFF on cultured epithelial cells and detection of transcripts for BAFF in single-cell–sorted B and T lymphocytes eluted from salivary glands from patients with primary Sjo¨gren’s syndrome (SS). A, Representative analysis of salivary gland epithelial cells isolated from 12 patients and 12 control subjects. Patient and control salivary gland epithelial cell lysates and supernatants (sBAFF) were probed with anti-BAFF and antiactin antibodies. U937 cell line cells and 293 cell line cells served as positive and negative controls, respectively. B, Nested reverse transcriptase–polymerase chain reactions for CD79b, CD3, and BAFF. Fifty individual CD3ϩ or CD19ϩ cells were sorted after elution from the salivary glands of 3 patients, and transcripts were converted from single cells. Eight cells from patient 1 are shown in the left column, and 8 cells from patient 2 are shown in the right column. C, Impact of BAFF from pathologic epithelial cells on normal B lymphocytes. Salivary gland epithelial cells from 6 patients were cocultured with sorted Bm1 cells from 6 normal tonsils. Tonsillar Bm1 cells were cultured in either medium (open bar), supernatant of salivary gland epithelial cells (shaded bar), supernatant of salivary gland epithelial cells plus salivary gland epithelial cells themselves (hatched bar), or supernatant of salivary gland epithelial cells plus salivary gland epithelial cells themselves plus anti-BAFF monoclonal antibodies (solid bar) for 24 hours. Survival of Bm1 cells was defined as annexin V and propidium iodide negativity. Bars show the mean and SD. D, Overview of BAFF-related processes in primary SS salivary glands. BR3 ϭ B lymphocyte stimulator receptor 3. in patient and control extracts, and cell-free BAFF was (13,14,39) was not sensitive enough (1 ng/ml) to detect observed in patient but not control supernatants (Figure this cytokine. 5A). Taken together, these results indicate that BAFF is Synthesis of BAFF by salivary gland T cells, but up-regulated in epithelial cells from patients with pri- also B cells, in primary SS. The in situ hybridization mary SS. However, the enzyme-linked immunosorbent technique detected transcripts for BAFF in T cells assay (ELISA) that we developed to assess BAFF (Figure 4C), but their expression was weak in B cells 1142 DARIDON ET AL

(Figures 4D and E). This was not ascribed to an artefact for its local production have never been unequivocally attributable to the deterioration of transcripts, because recognized. The expression of BAFF on their plasma the cells were stained with oligo(dT) (Figure 4F). How- membrane has been taken as evidence that, like ductal ever, the evidence that malignant B cells express BAFF epithelial cells, infiltrating T cells secrete the cytokine (23,25) prompted us to use the most sensitive technique (12,15,40), but the presence of BAFF could equally to detect BAFF. Single-cell–sorted T and B lymphocytes result from its insertion into the receptors. from 4 different salivary glands were examined (Figure Our RT-PCRs of the lysates confirm the pres- 5B). The presence of mRNA for CD3 but not CD79b, ence of transcripts for BAFF in the salivary glands of and for CD79b but not CD3, proved that these individ- patients with primary SS but also in those of control ual cells were T lymphocytes or B lymphocytes, respec- subjects. Furthermore, BAFF was seen on ductal epithe- tively. We observed that not only 80.9 Ϯ 2.5% of T cells lial cells of salivary gland tissue from control subjects produced BAFF (which was predictable) but also that and was detected in the supernatant of their cultured 69.2 Ϯ 3.6% of B cells did so (which was not predict- salivary gland epithelial cells. This finding would appear able). Synthesis of BAFF by B cells, together with the to be at odds with previous reports that BAFF is lacking membrane expression of its receptors, point to an autoin the supernatant of cultured epithelial cells. Such crine BAFF response in the salivary glands of patients divergences might simply reflect improved sensitivity of with primary SS. the current techniques compared with those used previ- Impact of BAFF from pathologic epithelial cells ously. For example, immunofluorescence staining of on normal B lymphocytes. Among tonsillar B cells, Bm1 frozen sections is more precise than histochemical ana- have the highest level of membrane expression of BR-3 lysis of paraffin-embedded tissues in discerning BAFF, (39). Therefore, based on IgD and CD38 expression, Western blots using ECL are more sensitive than ELISA Bm1 cells were sorted from 6 tonsils, and each of the in detecting BAFF in culture supernatants, and anti- resulting suspensions was distributed into 5 aliquots. The BAFF mAb are more specific than anti-BAFF pAb in first was analyzed for purity, the second was cultured identifying this cytokine in tissue sections. alone, the third was cultured with a supernatant of The increased expression of BAFF by epithelial salivary gland epithelial cells, the fourth was cultured cells activates autoreactive B cells in primary SS. BAFF with a supernatant of salivary gland epithelial cells plus was found on the plasma membrane of salivary gland the salivary gland epithelial cells themselves, and the epithelial cell line cells but also in their culture super- fifth was cultured in the same manner as the fourth, but natants. The demonstration that BAFF was secreted with 5 ␮g/ml anti-BAFF mAb (Figure 5C). After a reinforces our previous finding that BAFF was shed in 24-hour incubation, the percentage of annexin VϪ/ saliva (14). However, this observation implies also that propidium iodideϪ cells (i.e., live cells) was determined. epithelial cell membrane–bound BAFF is more anti- The percentage of live Bm1 cells was modestly increased apoptotic than its soluble counterpart on B cells. One with the supernatant (23.0 Ϯ 3.3% of the cells were alive explanation is the requirement of crosslinking of cell- in medium alone, compared with 27.3 Ϯ 3.7% in epithe- bound BAFF by cell-bound BR-3. Similar findings have lial cell supernatant), suggesting that soluble BAFF was recently been reported in T cells (41), and BAFF has efficient. This increase became significant when salivary been described on the membrane of malignant B cells gland epithelial cells were added to the culture (up to but not in the environment (23). T cells have been 59.2 Ϯ 4.5%; P Ͻ 0.05 versus supernatant) and was claimed to be associated with BAFF (12,15,40), but our abrogated by anti-BAFF mAb (31.9 Ϯ 5.0%; P Ͻ 0.05 findings raise the issue that BAFF binds to T cells. versus salivary gland epithelial cells alone). These results There is also controversy regarding which cell established the efficacy of epithelial cell membrane– subsets express receptors for BAFF. The BAFF pAb bound BAFF (Figure 5D). Collectively, they showed that we used revealed TACI on activated T cells (29), that salivary gland epithelial cells favor the survival of but we failed to confirm this observation with several adjacent B cells by virtue of membrane-bound BAFF anti-BAFF mAb. BR-3 was also present on activated T rather than soluble BAFF. cells in the blood but was absent on infiltrating T cells in the salivary glands. The implication of these results is that, at least in this particular environment, BAFF DISCUSSION impacts exclusively B cells. For unknown reasons, Despite large amounts of BAFF in the salivary autoantigen-driven B cells seem to be an exquisite target glands of patients with primary SS, the cells responsible for BAFF (11). THERAPEUTIC EFFICACY OF BAFF ANTAGONISTS IN AUTOIMMUNE DISORDERS 1143

Also of interest is our finding that salivary gland ACKNOWLEDGMENTS B cells synthesize BAFF. In this respect, BAFF has We extend our gratitude to Roger Budd for editorial previously been shown to be made, but not secreted, by assistance and also thank Cindy Se´ne´ and Simone Forest for malignant B cells (23). An attractive possibility is that secretarial assistance. the expression of BAFF on B cells confers a survival advantage to leukemic and autoimmune B cells through AUTHOR CONTRIBUTIONS an autocrine loop. Dysregulated BAFF expression on B Dr. Youinou had full access to all of the data in the study and cells might even be the triggering event in autoimmune takes responsibility for the integrity of the data and the accuracy of the disorders. As opposed to B cells, neutrophils stimulated data analysis. with granulocyte colony-stimulating factor or Study design. Pers, Youinou. ␥ Acquisition of data. Daridon, Hutin, Le Berre, Martins-Carvalho, interferon- contain transcripts for BAFF but do not Bendaoud, Dueymes. express BAFF protein (42). This suggests that the mech- Analysis and interpretation of data. Daridon, Youinou, Pers. anisms to process and/or to transport BAFF are not the Manuscript preparation. Youinou, Pers. same in different cell types and across the subsets of B Statistical analysis. Devauchelle, Saraux. cells (43). The salivary glands of BAFF-transgenic mice REFERENCES display an excess of marginal-zone B cells that express 1. Moutsopoulos HM. Sjo¨gren’s syndrome: autoimmune epithelitis. high levels of BR-3 and are potentially pathogenic Clin Immunol Immunopathol 1994;72:162–5. (12,27). Conversely, in BR-3–knockout mice, there is a 2. Moutsopoulos HM, Webber BL, Vlagopoulos TP, Chused TM, Decker JL. Differences in the clinical manifestations of sicca paucity of marginal-zone B cells, owing to an upstream syndrome in the presence and absence of rheumatoid arthritis. accumulation of TII B cells (44). This receptor is essen- Am J Med 1979;66:733–6. tial for B cells to evolve from TII cells to mature B cells. 3. Tzioufas AG, Youinou P, Moutsopoulos HM. Sjo¨gren’s syndrome. In: Maddison PJ, Isenberg DA, Woo P, Glass DN, editors. Oxford The combination of BR-3 and the B cell receptor (BCR) textbook of rheumatology. 2nd ed. Oxford: Oxford University renders BR-3 dispensable for the development of Press; 1998. p. 1301–17. marginal-zone B cells (45) and their expression of CD21 4. Radfar L, Kleiner DE, Fox PC, Pillemer SR. Prevalence and clinical significance of lymphocytic foci in minor salivary glands of and CD23 (46). Ligation of the BCR up-regulates the healthy volunteers. Arthritis Rheum 2002;47:520–4. expression of BR-3, and the coupling of BCR signaling 5. Dorner T, Lipsky PE. Abnormalities of B-cell phenotype, immu- with BR-3 would be restricted to TII and mature B cells noglobulin gene expression and the emergence of autoimmunity in Sjo¨gren’s syndrome. Arthritis Res 2002;4:360–71. (47). 6. Youinou P, Daridon C, Steinfeld S, Pers JO. A case for B cells in We also discerned a minority of TACI- and the pathogenesis of Sjo¨gren’s syndrome. Trends Immunol. In BCMA-expressing B cells in the salivary glands of our press. 7. Youinou P, Devauchelle V, Hutin P, Le Berre R, Saraux A, Pers patients with primary SS. TACI appears prominent on JO. A conspicuous role for B cells in Sjo¨gren’s syndrome [review]. TII and mature B cells (47) because TACI-knockout Clin Rev Allergy Immunol. In press. mice have normal mature B cells and marginal-zone B 8. Martin F, Kearney JF. Marginal-zone B cells [review]. Nat Rev Immunol 2002;2:233–5. cells. However, we (27) and other investigators (48) have 9. Pascual V, Liu YJ, Magalski A, de Bouteiller O, Banchereau J, noticed that a proportion of memory B cells are housed Capra JD. Analysis of somatic mutation in five B-cell subsets of in the salivary glands. Hence, one may speculate that human tonsil. J Exp Med 1994;180:329–39. 10. Do RK, Hatada E, Lee H, Tourigny MR, Hilbert D, Chen-Kiang once B cells differentiate into memory B cells and S. Attenuation of apoptosis underlies B-lymphocyte stimulator plasma cells, TACI and BCMA are up-regulated while, enhancement of humoral immune response. J Exp Med 2000;192: in turn, BR-3 is down-regulated (43). Human plasma 953–64. 11. Thien M, Phan TG, Gardam S, Amesbury M, Basten A, Mackay F, cells derive from BCMA-expressing memory B cells, and et al. Excess BAFF rescues self-reactive B cells from peripheral long-lived plasma cells are lacking in BCMA-knockout deletion and allows them to enter forbidden follicular and mar- mice (49). TACI and BCMA might thus expand the ginal zone niches. Immunity 2004;20:785–98. 12. Groom J, Kalled SL, Cutler AH, Olson C, Woodcock SA, Schnei- population of plasma cells, resulting in the local produc- der P, et al. Association of BAFF/BLyS overexpression and altered tion of autoantibodies. B-cell differentiation with Sjo¨gren’s syndrome. J Clin Invest 2002; In conclusion, this study is the first to demon- 109:59–68. 13. Pers JO, Daridon C, Devauchelle V, Jousse S, Saraux A, Jamin C, strate this unique autocrine pathway related to apoptosis et al. BAFF overexpression is associated with autoantibody proin primary SS. The results indicate that BAFF antago- duction in autoimmune diseases. Ann N Y Acad Sci 2005;1050: nists, such as anti-BAFF antibodies or decoy receptors 34–9. 14. Pers JO, d’Arbonneau F, Devauchelle-Pensec V, Saraux A, Pen- (50), may have therapeutic efficacy in patients with such nec YL, Youinou P. Is periodontal disease mediated by salivary autoimmune disorders. BAFF in Sjo¨gren’s syndrome? Arthritis Rheum 2005;52:2411–4. 1144 DARIDON ET AL

15. Lavie F, Miceli-Richard C, Quillard J, Roux S, Leclerc P, Mariette teria for Sjo¨gren’s syndrome: a revised version of the European X. Expression of BAFF (BLyS) in T cells infiltrating labial salivary criteria proposed by the American-European Consensus Group. glands from patients with Sjo¨gren’s syndrome. J Pathol 2004;202: Ann Rheum Dis 2002;61:554–8. 496–502. 34. Daniels TE. Labial salivary gland biopsy in Sjo¨gren’s syndrome: 16. Nardelli B, Belvedere O, Roschke V, Moore PA, Olsen HS, assessment as a diagnostic criterion in 362 suspected cases. Arthri- Migone TS, et al. Synthesis and release of B-lymphocyte stimulator tis Rheum 1984;27:147–56. from myeloid cells. Blood 2001;97:198–204. 35. Devauchelle V, Morvan J, Pennec Y, Pers JO, Jamin C, Jousse S, 17. Craxton A, Magaletti D, Ryan EJ, Clark EA. Macrophage- and et al. Rituximab (anti-CD20) in the treatment of primary Sjo¨gren’s dendritic cell-dependent regulation of human B-cell proliferation syndrome: results of an open-label study [abstract]. Ann Rheum requires the TNF family ligand BAFF. Blood 2003;101:4464–71. Dis 2005;64 Suppl III:iii309. 18. Gorelik L, Gilbride K, Dobles M, Kalled SL, Zondman D, Scott 36. Devauchelle V, Pennec Y, Morvan J, Pers JO, Daridon C, Jousse ML. Normal B cell homeostasis requires B-cell activation factor S, et al. Improvement of Sjo¨gren’s syndrome after two infusions of production by radiation-resistant cells. J Exp Med 2003;198: rituximab (anti-CD20). Arthritis Rheum. In press. 937–47. 37. Dimitriou ID, Kapsogeorgou EK, Abu-Helu RF, Moutsopoulos 19. Scapini P, Nardelli B, Nadali G, Calzetti F, Pizzolo G, Montecucco HM, Manoussakis MN. Establishment of a convenient system for C, et al. G-CSF-stimulated neutrophils are a prominent source of the long-term culture and study of non-neoplastic human salivary functional BLyS. J Exp Med 2003;197:297–302. gland epithelial cells. Eur J Oral Sci 2002;110:21–30. 20. Okata J, Zvaifler NJ, Nishio M, Boyle DL, Kalled SL, Carson DA, 38. Hillion S, Saraux A, Youinou P, Jamin C. Expression of RAGs in et al. Synoviocytes of mesenchymal origin express functional B peripheral B cells outside germinal centers is associated with the cell-activating factor of the TNF family in response to proinflam- expression of CD5. J Immunol 2005;174:5553–61. matory cytokines. J Immunol 2005;174:864–70. 39. D’Arbonneau F, Pers JO, Devauchelle V, Pennec Y, Saraux A, 21. Krumbholz M, Theil D, Derfuss T, Rosenwald A, Schrader F, Youinou P. BAFF-induced changes in B cell antigen Monoranu CM, et al. BAFF is produced by astrocytes and receptor–containing lipid rafts in Sjo¨gren’s syndrome. Arthritis up-regulated in multiple sclerosis lesions and primary central Rheum 2006;54:115–26. nervous system lymphoma. J Exp Med 2005;201:195–200. 40. Szodoray P, Jellestad S, Teague MO, Jonsson R. Attenuated 22. Schneider P, Mackay F, Steiner V, Hofmann K, Bodmer JL, apoptosis of B cell activating factor-expressing cells in primary Holler N, et al. BAFF, a novel ligand of the tumor necrosis factor Sjo¨gren’s syndrome. Lab Invest 2003;83:357–65. family, stimulates B cell growth. J Exp Med 1999;189:1747–56. 41. Ng LG, Sutherland AP, Newton R, Qian F, Cachero TG, Scott 23. Kern C, Cornuel JF, Billard C, Tang R, Rouillard D, Stenou V, et ML, et al. B-cell-activating factor belonging to the TNF family al. Involvement of BAFF and APRIL in the resistance to apoptosis (BAFF)-R is the principal BAFF receptor facilitating BAFF of B-CLL through an autocrine pathway. Blood 2004;103:679–88. costimulation of circulating T and B cells. J Immunol 2004;173: 24. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, et 807–17. al. BLyS: member of the tumor necrosis factor family and B- 42. Sasaki Y, Casola S, Kutok JL, Rajewsky K, Schmidt-Supprian M. lymphocyte stimulator. Science 1999;285:260–3. TNF family member B cell-activating factor (BAFF) receptor- 25. Novak AJ, Bram RJ, Kay NE, Jelinek DF. Aberrant expression of dependent and -independent roles for BAFF in B-cell physiology. B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: J Immunol 2004;173:2245–52. a mechanism for survival. Blood 2002;100:2973–9. 43. Hsu BL, Harless SM, Lindsley RC, Hilbert DM, Cancro MP. BLyS 26. Mackay F, Browning JL. BAFF: a fundamental survival factor for enables survival of transitional and mature B cells through distinct B cells. Nat Rev Immunol 2002;2:465–75. mediators. J Immunol 2002;168:5993–6. 27. Daridon C, Pers JO, Devauchelle V, Martins-Carvalho C, Hutin P, 44. Schneider P, Takatsuka H, Wilson A, Mackay F, Tardivel A, Lens Pennec YL, et al. Identification of transitional type II B cells in S, et al. Maturation of marginal zone and follicular B cells requires salivary glands of patients with Sjo¨gren’s syndrome. Arthritis B-cell activating factor of the tumor necrosis factor family and is Rheum 2006;54:2280–8. independent of B cell maturation antigen. J Exp Med 2001;194: 28. Jonsson MV, Szodoray P, Jellestad S, Jonsson R, Skarstein K. 1691–7. Association between circulating levels of the novel TNF family 45. Smith SH, Cancro MP. Cutting edge: B cell receptor signals members APRIL and BAFF and lymphoid organization in pri- regulate BLyS receptor levels in mature B cells and their imme- mary Sjo¨gren’s syndrome. J Clin Immunol 2005;25:187–99. diate progenitors. J Immunol 2003;170:5820–3. 29. Wu Y, Bressette D, Carrell JA, Kaufman T, Feng P, Taylor K, et 46. Gorelik L, Cutler AH, Thill G, Miklasz SD, Shea DE, Ambrose C, al. Tumor necrosis factor (TNF) receptor superfamily member et al. BAFF regulates CD21/35 and CD23 expression independent TACI is a high affinity receptor for TNF family members APRIL of its B-cell survival function. J Immunol 2004;172:762–6. and BLyS. J Biol Chem 2000;275:35478–85. 47. Zhang X, Park CS, Yoon SO, Li L, Hsu YM, Ambrose C, et al. 30. Shu HB, Johnson H. B-cell maturation protein is a receptor for the BAFF supports human B-cell differentiation in the lymphoid TNF family member TALL-1. Proc Natl Acad SciUSA2000;97: follicles through distinct receptors. Int Immunol 2005;17:779–88. 9156–61. 48. Hansen A, Odendahl M, Reiter K, Jacobi AM, Feist E, Scholze J, 31. Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, et al. Diminished peripheral blood memory B cells and accumu- et al. BAFF-R, a newly identified TNF receptor that specifically lation of memory B cells in the salivary glands of patients with interacts with BAFF. Science 2001;293:2108–11. Sjo¨gren’s syndrome. Arthritis Rheum 2002;46:2160–71. 32. Von Bulow GU, Bram RJ. NF-AT activation induced by a 49. O’Connor BP, Raman VS, Erickson LD, Cook WJ, Weaver LK, CAML-interacting member of the TNF receptor superfamily. Ahonen C, et al. BCMA is essential for the survival of long-lived Science 1997;278:138–41. bone marrow plasma cells. J Exp Med 2004;199:91–8. 33. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alex- 50. St.Clair EW, Tedder TF. New prospects for autoimmune disease ander EL, Carsons SE, et al, and the European Study Group on therapy: B cells on deathwatch [editorial]. Arthritis Rheum 2006; Classification Criteria for Sjo¨gren’s Syndrome. Classification cri- 54:1–9. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1145–1151 DOI 10.1002/art.22453 © 2007, American College of Rheumatology

Interferon-␥ Regulates Susceptibility to Collagen-Induced Arthritis Through Suppression of Interleukin-17

Cong-Qiu Chu,1 David Swart,2 Dina Alcorn,2 Joel Tocker,2 and Keith B. Elkon1

Objective. The enhanced expression of experimen- WT B6 mice. Administration of the anti–IL-17 mAb tal arthritis in the absence of interferon-␥ (IFN␥) attenuated arthritis in DBA/1 mice and almost com- suggests that IFN␥ suppresses arthritis. Interleukin-17 pletely prevented expression of arthritis in IFN␥-KO B6 (IL-17) is a pivotal T cell cytokine in arthritis, and in mice. vitro studies have indicated that IFN␥ suppresses IL-17 Conclusion. These results indicate that sensitivity production. We undertook this study to test the hypothe- of IFN␥-deficient B6 mice to CIA is associated with high sis that resistance to collagen-induced arthritis (CIA) in IL-17 production and that this cytokine is required for C57BL/6 (B6) mice is regulated by IFN␥-mediated expression of arthritis in this strain. suppression of IL-17. Methods. Wild-type (WT) B6 mice, IFN␥- Despite the powerful inflammatory effect of cy- knockout (KO) B6 mice, and DBA/1 mice were immu- tokines such as tumor necrosis factor ␣ (TNF␣) pro- nized with type II collagen (CII) in Freund’s complete duced by the innate immune system, abundant evidence adjuvant (CFA). Lymphocytes from immunized mice indicates that T cells are required for initiation and/or were analyzed for cytokine production ex vivo by intra- chronicity both in human rheumatoid arthritis (RA) and cellular staining or restimulation with CII and enzyme- in mouse models of RA (1–3). Interleukin-17 (IL-17) linked immunosorbent assays. In vivo blockade of IL-17 has emerged as a critical T cell cytokine in the patho- was achieved with an anti–IL-17 monoclonal antibody genesis of human RA and several mouse models of this (mAb). disease (for review, see ref. 4). IL-17 promotes inflam- Results. CII restimulation of T cells from CII/ mation through enhancing the production of other CFA-immunized mice resulted in an ϳ5-fold increase in proinflammatory cytokines, IL-1 and TNF␣, by mono- IL-17 production in IFN␥-KO B6 mice compared with cytes (5) and has a synergistic effect in TNF␣-induced WT B6 mice. Neutralization of IFN␥ increased IL-17 IL-1, IL-6, and IL-8 synthesis by synovial fibroblasts production in WT B6 mice, and neutralization of IL-4 (6,7). In addition, IL-17 is implicated in cartilage de- had a synergistic effect. Interestingly, the prototypical struction and bone resorption (8,9). A necessary role for CIA-susceptible strain DBA/1 also demonstrated a high this cytokine has been demonstrated in several experi- IL-17 and a low IFN␥ cytokine profile compared with mental models of arthritis by attenuation of murine arthritis with IL-17 neutralization by anti–IL-17 antibod- Supported by the NIH (grant AR-050772). Dr. Chu’s work ies or with mice genetically deficient in IL-17 or IL-17 was supported by an NIH T32 training grant and by the Richard and receptor (IL-17R) (10–12). Linda Carson Fellowship Grant. 1 IL-17 is produced by activated memory/effector Cong-Qiu Chu, MD, PhD, Keith B. Elkon, MD: University ϩ of Washington, Seattle; 2David Swart, BS, Dina Alcorn, BS, Joel T cells. Recent studies suggest that CD4 T cells Tocker, PhD: Amgen, Seattle, Washington. producing IL-17 represent a distinct subpopulation of T Mr. Swart, Ms Alcorn, and Dr. Tocker own stock and/or hold helper cells that have been designated Th17 (13,14). At stock options in Amgen. Address correspondence and reprint requests to Keith B. least in vitro, IL-17 production is negatively regulated by Elkon, MD, Division of Rheumatology, University of Washington, both interferon-␥ (IFN␥) and IL-4 (13,14). One of us 1959 NE Pacific Avenue, Box 356428, Seattle, WA 98195. E-mail: and other investigators previously reported that a lack of [email protected]. ␥ Submitted for publication September 26, 2006; accepted in IFN paradoxically enhanced autoimmunity in diseases revised form December 12, 2006. thought to be mediated by Th1, such as experimental

1145 1146 CHU ET AL

autoimmune encephalomyelitis (EAE) and collagen- 96-well plates in Dulbecco’s modified Eagle’s medium contain- induced arthritis (CIA) (15–18). In CIA, IFN␥ defi- ing 10% FCS. Mouse IL-17 (10 ng/ml; R&D Systems) or ␣ ciency renders the normally resistant C57BL/6 (B6) IL-17F (300 ng/ml; Amgen) in combination with human TNF ␥ (0.5 ng/ml; R&D Systems) was added to the culture. In some strain susceptible to disease (16–18), and lack of IFN experiments, IL-17 or IL-17F and TNF␣ were preincubated for or signaling through the IFN␥ receptor enhances the 45 minutes with M210 (30 ␮g/ml) or mouse IL-17R-Fc fusion severity of arthritis in susceptible strains such as DBA/1 protein (30 ␮g/ml; Amgen) prior to adding to the culture. Cells (19,20). Lack of IFN␥ was thought to promote disease were cultured for 24 hours, and supernatant was assayed for KC by ELISA (R&D Systems) according to the manufacturer’s through lack of activation-induced cell death and up- instructions. regulation of IL-1 (15,17). In the present study, we Induction of CIA and anti–IL-17 mAb treatment. tested a different hypothesis, that increased susceptibil- Native bovine CII was provided by Dr. Marie Griffiths (Uni- ity to arthritis is explained by loss of IFN␥-mediated versity of Utah, Salt Lake City, UT), and native chicken CII suppression of IL-17. was obtained from Sigma-Aldrich (St. Louis, MO). CII was dissolved in 0.1M acetic acid and emulsified in Freund’s complete adjuvant (CFA; Difco, Detroit, MI) containing 5 MATERIALS AND METHODS mg/ml of killed Mycobacterium tuberculosis H37Ra. Mice were injected intradermally at the base of the tail with 100 ␮g CII in Mice. Wild-type (WT) B6 mice, IFN␥-knockout (KO) CFA and boosted with an intraperitoneal (IP) injection of 100 B6 mice, and DBA/1 mice were obtained from The Jackson ␮g CII on day 21. For in vivo anti–IL-17 mAb treatment in Laboratory (Bar Harbor, ME) and Taconic (Germantown, DBA/1 mice, arthritic mice were randomized into 3 groups and NY) and kept in a modified specific pathogen–free facility. treated with phosphate buffered saline, normal rat IgG, or Mice were used at age 8–10 weeks under a protocol approved anti–IL-17 mAb (M210) at 100 ␮g IP every other day. For by the Institutional Animal Care and Use Committee. IFN␥-KO B6 mice, on the day of initial immunization, mice Cytokines and antibodies to cytokines. Rat anti-mouse were injected IP with 200 ␮g anti–IL-17 mAb (M210) or IL-17 monoclonal antibody (mAb) (clone M210, IgG2a) was normal rat IgG2a weekly for a total of 4 doses. Arthritis was generated at Amgen (Seattle, WA) by immunization with assessed using a scoring system as described (16). Mice were recombinant mouse IL-17. M210 was shown to bind to IL-17 killed on day 56, and the limbs were removed. Joint pathology specifically, but not to TNF␣, IL-1␤, IL-6, or IFN␥, in enzyme- was evaluated on decalcified hematoxylin and eosin–stained linked immunosorbent assays (ELISAs) using M210 as a sections (16). capture antibody (data not shown). Furthermore, M210 did Flow cytometry. Intracellular cytokine staining was not block TNF␣ cytotoxicity in L929 cells (data not shown). performed using an intracellular staining kit (BD PharMin- Fine specificity of M210 was tested by its ability to neutralize gen). Lymphocytes from CII-immunized mice were stimulated IL-17 family members in a bioassay that measures IL-17 with phorbol myristate acetate (0.05 ␮g/ml) and ionomycin (0.5 stimulation of keratinocyte-derived chemokine (KC) secretion ␮g/ml) in the presence of GolgiStop solution (BD PharMin- by fibroblasts (21). We used the following cytokines and gen) for 6 hours and stained with fluorescein isothiocyanate– antibodies to cytokines for ELISA and cell culture: recombi- conjugated anti-CD4, allophycocyanin-conjugated anti-IFN␥ nant IL-17, purified rat anti-mouse IL-17, and biotinylated (clone XMG1.2; BioLegend), and phycoerythrin-conjugated goat anti-mouse IL-17 (R&D Systems, Minneapolis, MN); anti–IL-17 (clone TC11-18H10.1; BD PharMingen) and ana- biotinylated anti-mouse IL-4, IL-6, and IL-12/IL-23 (p40) and lyzed on a FACSCanto (BD Biosciences, San Jose, CA). purified anti-mouse IL-12 (p35) and TNF␣ (BioLegend, San Statistical analysis. Results were analyzed with Stu- Diego, CA); purified anti-mouse IL-6 and recombinant IFN␥ dent’s t-test. (BD PharMingen, San Diego, CA); biotinylated anti-mouse IFN␥ (Caltag, South San Francisco, CA); anti-mouse IL-23 (p19), biotinylated rabbit anti-mouse TNF␣, functional grade RESULTS purified anti-mouse IFN␥, and recombinant IL-4, IL-23, IL-6, IL-12, and IFN␥ (eBioscience, San Diego, CA); and anti- Antigen-specific and dose-dependent exagger- mouse IL-4 (National Cancer Institute Biological Resources ated T cell IL-17 response to CII in IFN␥-KO B6 mice. Branch Preclinical Repository, Rockville, MD). Since recent studies have shown that IFN␥ suppresses Ex vivo cell culture and ELISAs for cytokines. Inguinal IL-17 production in vitro (13,14) and IL-17 is intimately lymph nodes and spleen were harvested 10–14 days postimmu- associated with inflammatory responses in several exper- nization, and single-cell suspensions were prepared. Cells were cultured in RPMI 1640 containing 10% fetal calf serum (FCS) imental models of arthritis (for review, see ref. 4), we at 5 ϫ 106/ml in round-bottomed 96-well plates in the presence tested the hypothesis that susceptibility to CIA in of type II collagen (CII). Cytokines or mAb to cytokines were IFN␥-KO B6 mice was due to lack of repression of IL-17 added to the culture for 72 hours. Cytokines in supernatants production. We therefore induced CIA in WT B6 and were quantified by ELISA according to the manufacturers’ IFN␥-KO B6 mice and compared T cell IL-17 produc- instructions. IL-17 bioassay. NIH3T3 cells derived from mouse tion immediately ex vivo and following restimulation embryonic fibroblasts (American Type Culture Collection, Man- with CII in vitro. assas, VA) were plated at 1 ϫ 105/well in round-bottomed CII immunization of IFN␥-KO B6 mice induced IFN␥ SUPPRESSES IL-17–MEDIATED ARTHRITIS 1147

susceptible mouse strain DBA/1 also showed a higher proportion of IL-17–producing T cells and a lower proportion of IFN␥-producing T cells compared with WT B6 mice. Ex vivo restimulation of T cells from IFN␥-KO B6 mice primed with CII in vivo revealed an ϳ5-fold increase in IL-17 production compared with WT B6 mice (Figure 1b). Since IL-17 production was only detected in IFN␥-KO B6 mice that were immunized with CFA/CII and restimulated with CII, these re-

Figure 1. Increased interleukin-17 (IL-17) produced by CD4ϩ T cells from interferon-␥–knockout C57BL/6 mice (IFN␥-KO B6 mice) in response to type II collagen (CII) immunization. Wild-type (WT) B6 mice (B6), IFN␥-KO B6 mice (B6-IFN-␥ KO), and DBA/1 mice were injected with Freund’s complete adjuvant (CFA) containing buffer alone or bovine CII, as described in Materials and Methods. Ten days postimmunization, inguinal lymph nodes and spleen were harvested and examined for inflammatory cytokine production. a, Cells obtained immediately ex vivo were stimulated with phorbol myristate acetate/ ionomycin for 6 hours, and intracellular production of IL-17 and IFN␥ by CD4ϩ T cells was detected by flow cytometry. Results are representative of 4–6 mice analyzed in each group. b, Mononuclear cells were cultured with medium (Med) alone, ovalbumin (OVA), or CII for 72 hours. IL-17 production in the supernatant was measured by Figure 2. IFN␥ suppression of IL-17 production by T cells from enzyme-linked immunosorbent assay (ELISA). c, CII was added at IFN␥-KO B6 mice in response to CII. a, Spleen and lymph node ␮ ␮ concentrations ranging from 0 g/ml to 100 g/ml. Results shown in b mononuclear cells from immunized IFN␥-KO B6 mice were cultured and c are presented as the mean and SD and are pooled from 6–9 mice with CII (100 ␮g/ml) as described in Figure 1, except that IFN␥ was in each group. d, Mononuclear cells were restimulated with CII as added at the indicated concentrations (in ng/ml) at the beginning of above. IL-6 in the supernatant was quantified by ELISA. Results are culture, and IL-17 was quantified on day 3. b, To determine the effect presented as the mean and SD and are pooled from 4–6 mice in each of neutralization of IFN␥ on IL-17 production in vitro, mononuclear ϭ ϭ ءء ϭ ϭ ء group. P 0.04 versus WT B6 mice; P 0.013 versus WT cells from immunized WT B6 mice were cultured as described in ϭ B6 mice. PBS phosphate buffered saline. Figure 1, except that anti-IFN␥ monoclonal antibody (mAb) was added to the culture at the indicated concentrations (in ␮g/ml). c, In vivo–primed lymphocytes from WT B6 mice were stimulated with CII ϩ in the presence of 10 ␮g/ml anti-IFN␥,10␮g/ml anti–IL-4, or a a 2–3-fold higher proportion of IL-17–producing CD4 combination of mAb. Results shown in a–c are presented as the mean T cells compared with WT B6 mice when analyzed and SD and are pooled from 3 experiments (n ϭ 6 mice in each group). immediately ex vivo (Figure 1a). The prototypical CIA- See Figure 1 for other definitions. 1148 CHU ET AL

Figure 3. Suppression of arthritis by blockade of IL-17 in vivo. a, NIH3T3 cells were cultured in tumor necrosis factor ␣ (0.5 ng/ml)–supplemented medium in the presence of IL-17 (10 ng/ml) or IL-17F (300 ng/ml) for 24 hours. Anti–IL-17 monoclonal antibody (M210; 30 ␮g/ml) or IL-17 receptor-Fc fusion protein (IL-17R-Fc; 30 ␮g/ml) was preincubated with IL-17 or IL-17F prior to adding to the culture. Keratinocyte-derived chemokine (KC) was measured by ELISA. b–d, DBA/1 and IFN␥-KO B6 mice were immunized with CII in CFA and boosted with an intraperitoneal (IP) injection of CII on day 21. b, DBA/1 mice were treated with M210 (100 ␮g/mouse IP, n ϭ 20), control rat IgG (100 ␮g/mouse IP, n ϭ 6), or PBS (n ϭ 15) every other day after the onset of arthritis. Severity of arthritis was monitored using a scoring system as described elsewhere (16). c, Clinical score and cumulative incidence of arthritis in IFN␥-KO B6 mice. On the day of immunization, IFN␥-KO B6 mice were treated with M210 (200 ␮g/mouse IP; n ϭ 10) or with normal rat IgG2a (n ϭ 10) followed by weekly injections for a total of 4 doses. Mice were followed up for 56 days for development of arthritis. Results shown in a–c are presented as the mean and SD. d, Hematoxylin and eosin–stained sections from the hind paws of IFN␥-KO B6 mice obtained 56 days postimmunization. A and B, Sections from rat IgG2a–treated mice showing massive synovitis, erosion, and joint destruction. C and D, Sections from anti–IL-17–treated mice, representative of paws with no (C) or minimal (D) inflammation. See Figure 1 for other definitions.

sponses were antigen specific and were also dose depen- Synergistic action of IFN␥ and IL-4 to suppress dent (Figure 1c). The increased IL-17 cytokine response the IL-17 response to CII. Since we proposed that was not accompanied by enhanced production of all enhanced IL-17 production in IFN␥-KO B6 mice was inflammatory cytokines in IFN␥-KO B6 mice: no differ- caused by loss of IFN␥ suppression, we added IFN␥ ences were observed in IL-12 or TNF␣ responses (data back to T cell restimulation cultures and quantified not shown), whereas IL-6 production was significantly IL-17 in the supernatants. As shown in Figure 2a, IL-17 higher in IFN␥-KO B6 mice (Figure 1d). production was almost completely suppressed by IFN␥. IFN␥ SUPPRESSES IL-17–MEDIATED ARTHRITIS 1149

To determine the effect of suppression of IFN␥ on IL-17 nation of control IgG2a-injected joints showed extensive production by WT B6 mice in the restimulation assay, we synovitis, pannus formation, marginal erosion, cartilage added a neutralizing anti-IFN␥ mAb during the 3-day destruction, and joint architectural changes (Figure 3d, culture period. Neutralization of IFN␥ in vitro resulted parts A and B) which are typically seen in CIA (16). In in a statistically significant increase in IL-17 production, contrast, most joints from anti–IL-17–treated animals although the IL-17 concentrations were lower than in had minimal changes (Figure 3d, part C) or a very mild IFN␥-KO B6 mice (Figure 2b). Since IL-4 has also been synovitis in joints showing clinical inflammation (Figure implicated in IL-17 suppression, we compared the ef- 3d, part D). These results indicated that IL-17 is neces- fects of neutralization of IFN␥, neutralization of IL-4, sary for the expression of arthritis in IFN␥-deficient B6 and neutralization of both cytokines together. Despite a mice. lack of effect of neutralizing IL-4 alone and detection of Association of heightened IL-17 production in very low levels of IL-4 in T cell restimulation cultures CIA-susceptible DBA/1 mice with low IFN␥ responses to (not shown), neutralization of both cytokines had a CII. DBA/1 mice have previously been shown to pro- striking synergistic effect (Figure 2c). duce high levels of IL-17 in response to CII (22). We Suppression of arthritis by in vivo treatment with observed that, like IFN␥-KO B6 mice, DBA/1 mice had anti–IL-17 mAb. We first examined the fine specificity a higher proportion of CD4ϩ T cells producing IL-17 of the anti–IL-17 mAb, M210, using a bioassay that but markedly fewer IFN␥-producing T cells compared quantifies IL-17 stimulation of KC secretion by fibro- with WT B6 animals when studied ex vivo. Furthermore, blasts (21). As shown in Figure 3a, M210 completely highly significant differences in IL-17 and IFN␥ produc- blocked IL-17–mediated KC production, but not IL- tion were observed between DBA/1 and WT B6 mice 17F–mediated KC production, by the murine fibroblast upon restimulation in vitro (Figures 1a and c). cell line NIH3T3. The IL-17–neutralizing capacity of the anti–IL-17 mAb M210 was equivalent to that of the DISCUSSION IL-17R-Fc fusion protein. This demonstrates a high degree of specificity of M210 and its ability to neutralize In this study, we demonstrated that lack of IFN␥ IL-17 bioactivity. Since IL-17–neutralizing antibodies in a normally resistant strain of mouse leads to height- have previously been shown to be effective in the ened IL-17 responses to CII immunization, and that treatment of CIA in DBA/1 mice (10), we further tested neutralization of IL-17 almost entirely prevents expres- M210 for functional activity in this arthritis model in sion of disease. This strongly suggests that conversion vivo. As shown in Figure 3b, M210 significantly attenu- from CIA resistance to susceptibility is explained by the ated progression of arthritis even when administered release of IL-17 from IFN␥-mediated suppression. Our after disease onset, consistent with previous studies findings are consistent with those of Murphy et al (23), using polyclonal anti–IL-17 antibodies (10). who reported that IL-12 (p35)–deficient mice on a B6 or ␥ ϫ IFN -KO B6 mice produced higher levels of (B6 129)F2 background developed CIA and had IL-17 in response to immunization with CII in CFA as increased numbers of IL-17–producing T cells. It is of compared with WT B6 mice. To determine whether interest that exaggerated IL-17 responses were associ- IL-17 was necessary for the development of CIA, we ated with increased IL-6, but not TNF␣, production in determined whether neutralization of IL-17 with the vitro. Since IL-6 may be both downstream and, together anti–IL-17 mAb M210 could prevent CIA in IFN␥-KO with transforming growth factor ␤, upstream of IL-17 B6 mice. These mice were injected with 200 ␮gof (24–26), and since IL-17–producing cells also secrete anti–IL-17 mAb or control rat IgG2a on the day of CII IL-6 (27), a positive feedback loop may be in operation immunization, followed by weekly injections of mAb for in arthritis. a total of 4 doses. Development of arthritis was followed It was reported that both IFN␥ and IL-4 can for 56 days after initial immunization and scored using a suppress IL-17 production in vitro (13,14). Our ex vivo system described previously (16). Eighty percent of mice data indicate that in WT B6 mice, IFN␥ was the treated with normal rat IgG2a developed arthritis with a dominant T cell cytokine response to CII in CFA, typical clinical course with a mean onset on day 29 after whereas IL-4 was only weakly detected. Nevertheless, initial immunization (Figure 3c), as observed previously IFN␥ and IL-4 together had a synergistic effect on (16). In contrast, only 20% of the mice in the anti–IL- suppression of CII-specific IL-17 production during CII 17–treated group developed arthritis, and this was mild restimulation in vitro. The suppressive effect of IFN␥ in and had a delayed onset (Figure 3c). Histologic exami- vivo also appears to be dominant in CIA, as mentioned 1150 CHU ET AL

above, and in myelin oligodendrocyte glycoprotein ACKNOWLEDGMENTS peptide–induced EAE, in which IFN␥-KO mice devel- We would like to thank Drs. Chris Wilson and Jacques oped a fatal disease, with 50% mortality as compared Peschon for discussion and Daniel Kim for technical assistance. with no mortality in IFN␥-intact mice (15). Since IFN␥ may actually be required for IL-4 production by Th2 AUTHOR CONTRIBUTIONS cells (28,29), a possible reduction of both IL-17– Dr. Elkon had full access to all of the data in the study and suppressing cytokines may explain the striking effect on takes responsibility for the integrity of the data and the accuracy of the disease severity when IFN␥ is deficient. In contrast, B6 data analysis. mice genetically deficient in IL-4 are resistant to CIA Study design. Chu, Tocker, Elkon. (18), and IFN␥-KO B6 mice that were treated with Acquisition of data. Chu, Swart, Alcorn. Analysis and interpretation of data. Chu, Tocker, Elkon. anti–IL-4 mAb appeared to be resistant to CIA, leading Manuscript preparation. Chu, Tocker, Elkon. to the suggestion that IL-4 may actually promote CIA Statistical analysis. Chu. (18). Based on the results reported here and the known ␥ suppression of IFN by IL-4, perhaps a lack of IL-4 REFERENCES leads to increased IFN␥, resulting in suppression of IL-17. However, it should be noted that high concentra- 1. Kouskoff V, Korganow AS, Duchatelle V, Degott C, Benoist C, Mathis D. Organ-specific disease provoked by systemic autoimmu- tions of exogenous IL-4 or systems overexpressing IL-4 nity. Cell 1996;87:811–22. were reported to suppress arthritis (30,31), and this 2. Brand DD, Kang AH, Rosloniec EF. The mouse model of effect was associated with decreased IL-17 in arthritic collagen-induced arthritis. Methods Mol Med 2004;102:295–312. 3. Bluestone JA, St.Clair EW, Turka LA. CTLA4Ig: bridging the joints (31). basic immunology with clinical application. Immunity 2006;24: A particularly interesting observation in the 233–8. present study was that high IL-17 production by T cells 4. Lubberts E, Koenders MI, van den Berg WB. The role of T cell interleukin-17 in conducting destructive arthritis: lessons from in response to CII restimulation in DBA/1 mice was animal models. Arthritis Res Ther 2005;7:29–37. associated with low IFN␥ secretion. It was recently 5. Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He reported that DBA/1-derived natural killer (NK) T cells Y, Zhang M, et al. IL-17 stimulates the production and expression ␥ of proinflammatory cytokines, IL-␤ and TNF-␣, by human macro- have defective production of IFN and IL-4 in re- phages. J Immunol 1998;160:3513–21. sponse to stimulation with a natural agonist, ␣- 6. Katz Y, Nadiv O, Beer Y. Interleukin-17 enhances tumor necrosis galactosylceramide, as compared with NK T cells from factor ␣–induced synthesis of interleukins 1, 6, and 8 in skin and synovial fibroblasts: a possible role as a “fine-tuning cytokine” in B6 mice (32). This defect occurred despite equivalent inflammation processes. Arthritis Rheum 2001;44:2176–84. numbers of NK T cells. These findings are consistent 7. Miossec P. IL-17 in rheumatoid arthritis: a new target for treat- with the idea that a relative lack of IFN␥, possibly in ment or just another cytokine? Joint Bone Spine 2004;71:87–90. 8. Martel-Pelletier J, Mineau F, Jovanovic D, Di Battista JA, Pelle- conjunction with IL-4, may contribute to disease suscep- tier JP. Mitogen-activated protein kinase and nuclear factor ␬B tibility to CIA in DBA/1 mice. together regulate interleukin-17–induced nitric oxide production Previous studies have demonstrated the relative in human osteoarthritic chondrocytes: possible role of transacti- ␥ vating factor mitogen–activated protein kinase–activated protein deficiency of IFN (33,34), but abundant IL-17 (for kinase (MAPKAPK). Arthritis Rheum 1999;42:2399–409. review, see ref. 4), in RA patients. CD4ϩ T cells from 9. LeGrand A, Fermor B, Fink C, Pisetsky DS, Weinberg JB, Vail RA patients produce low levels of IFN␥ in response to TP, et al. Interleukin-1, tumor necrosis factor ␣, and interleukin-17 synergistically up-regulate nitric oxide and prostaglandin E2 pro- IL-12 or IL-12/IL-18 stimulation (35), although it reduction in explants of human osteoarthritic knee menisci. Arthritis mains to be determined whether this defect is primary or Rheum 2001;44:2078–83. secondary to suppression by other proinflammatory 10. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersse- ␥ laar L, Coenen-de Roo CJ, Joosten LA, et al. Treatment with a cytokines, since the IFN response to IL-12 or IL-18 was neutralizing anti-murine interleukin-17 antibody after the onset of partially restored by TNF␣ blockade (35). As with the collagen-induced arthritis reduces joint inflammation, cartilage low IFN␥–producing mouse strains we studied in RA destruction, and bone erosion. Arthritis Rheum 2004;50:650–9. ␥ 11. Nakae S, Nambu A, Sudo K, Iwakura Y. Suppression of immune patients, reduced IFN responses to CII or other joint- induction of collagen-induced arthritis in IL-17-deficient mice. derived antigens may allow excessive IL-17 production J Immunol 2003;171:6173–7. that, in turn, drives chronic inflammation. Further stud- 12. Koenders MI, Kolls JK, Oppers-Walgreen B, van den Bersselaar ␥ L, Joosten LA, Schurr JR, et al. Interleukin-17 receptor deficiency ies are required to better understand how IFN regu- results in impaired synovial expression of interleukin-1 and matrix lates CD4ϩ T cell production of IL-17 during arthritis metalloproteinases 3, 9, and 13 and prevents cartilage destruction development. Therapeutic intervention designed to re- during chronic reactivated streptococcal cell wall–induced arthri- ␥ tis. Arthritis Rheum 2005;52:3239–47. direct T cell cytokine production from IL-17 to IFN 13. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, may prove to be beneficial in RA patients. Murphy KM, et al. Interleukin 17-producing CD4ϩ effector T IFN␥ SUPPRESSES IL-17–MEDIATED ARTHRITIS 1151

cells develop via a lineage distinct from the T helper type 1 and 2 genic effector TH17 and regulatory T cells. Nature 2006;441: lineages. Nat Immunol 2005;6:1123–32. 235–8. 14. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al. 25. Harrington LE, Mangan PR, Weaver CT. Expanding the effector A distinct lineage of CD4 T cells regulates tissue inflammation by CD4 T-cell repertoire: the Th17 lineage. Curr Opin Immunol producing interleukin 17. Nat Immunol 2005;6:1133–41. 2006;18:349–56. 15. Chu CQ, Wittmer S, Dalton DK. Failure to suppress the expansion 26. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. of the activated CD4 T cell population in interferon ␥-deficient TGF␤ in the context of an inflammatory cytokine milieu supports mice leads to exacerbation of experimental autoimmune enceph- de novo differentiation of IL-17-producing T cells. Immunity alomyelitis. J Exp Med 2000;192:123–8. 2006;24:179–89. 16. Chu CQ, Song Z, Mayton L, Wu B, Wooley PH. IFN␥ deficient 27. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, C57BL/6 (H-2b) mice develop collagen induced arthritis with Sedgwick JD, et al. IL-23 drives a pathogenic T cell population predominant usage of T cell receptor V␤6 and V␤8 in arthritic that induces autoimmune inflammation. J Exp Med 2005;201: joints. Ann Rheum Dis 2003;62:983–90. 233–40. ␥ 17. Guedez YB, Whittington KB, Clayton JL, Joosten LA, van de Loo 28. Wensky A, Marcondes MC, Lafaille JJ. The role of IFN- in the FA, van den Berg WB, et al. Genetic ablation of interferon-␥ production of Th2 subpopulations: implications for variable Th2- up-regulates interleukin-1␤ expression and enables the elicitation mediated pathologies in autoimmunity. J Immunol 2001;167: 3074–81. of collagen-induced arthritis in a nonsusceptible mouse strain. 29. Bocek P Jr, Foucras G, Paul WE. Interferon ␥ enhances both in Arthritis Rheum 2001;44:2413–24. vitro and in vivo priming of CD4ϩ T cells for IL-4 production. J 18. Ortmann RA, Shevach EM. Susceptibility to collagen-induced Exp Med 2004;199:1619–30. arthritis: cytokine-mediated regulation. Clin Immunol 2001;98: 30. Morita Y, Yang J, Gupta R, Shimizu K, Shelden EA, Endres J, et 109–18. al. Dendritic cells genetically engineered to express IL-4 inhibit 19. Manoury-Schwartz B, Chiocchia G, Bessis N, Abehsira-Amar O, murine collagen-induced arthritis. J Clin Invest 2001;107:1275–84. Batteux F, Muller S, et al. High susceptibility to collagen-induced ␥ 31. Lubberts E, Joosten LA, Chabaud M, van den Bersselaar L, arthritis in mice lacking IFN- receptors. J Immunol 1997;158: Oppers B, Coenen-De Roo CJ, et al. IL-4 gene therapy for 5501–6. collagen arthritis suppresses synovial IL-17 and osteoprotegerin 20. Vermeire K, Heremans H, van de Putte M, Huang S, Billiau A, ligand and prevents bone erosion. J Clin Invest 2000;105:1697–710. ␥ Matthys P. Accelerated collagen-induced arthritis in IFN- recep- 32. Miellot A, Zhu R, Diem S, Boissier MC, Herbelin A, Bessis N. tor-deficient mice. J Immunol 1997;158:5507–13. Activation of invariant NK T cells protects against experimental 21. Numasaki M, Lotze MT, Sasaki H. Interleukin-17 augments tumor rheumatoid arthritis by an IL-10-dependent pathway. Eur J Im- necrosis factor-␣-induced elaboration of proangiogenic factors munol 2005;35:3704–13. from fibroblasts. Immunol Lett 2004;93:39–43. 33. Firestein GS, Alvaro-Gracia JM, Maki R. Quantitative analysis of 22. Lubberts E, Joosten LA, Oppers B, van den Bersselaar L, cytokine gene expression in rheumatoid arthritis. J Immunol Coenen-de Roo CJ, Kolls JK, et al. IL-1-independent role of IL-17 1990;144:3347–53. in synovial inflammation and joint destruction during collagen- 34. Raza K, Falciani F, Curnow SJ, Ross EJ, Lee CY, Akbar AN, et al. induced arthritis. J Immunol 2001;167:1004–13. Early rheumatoid arthritis is characterized by a distinct and 23. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClana- transient synovial fluid cytokine profile of T cell and stromal cell han T, Kastelein RA, et al. Divergent pro- and antiinflammatory origin. Arthritis Res Ther 2005;7:R784–95. roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp 35. Kawashima M, Miossec P. Effect of treatment of rheumatoid Med 2003;198:1951–7. arthritis with infliximab on IFN ␥, IL4, T-bet, and GATA-3 24. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. expression: link with improvement of systemic inflammation and Reciprocal developmental pathways for the generation of patho- disease activity. Ann Rheum Dis 2005;64:415–8. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1152–1163 DOI 10.1002/art.22452 © 2007, American College of Rheumatology

Blockade of the Interleukin-21/Interleukin-21 Receptor Pathway Ameliorates Disease in Animal Models of Rheumatoid Arthritis

Deborah A. Young,1 Martin Hegen,1 Hak Ling Margery Ma,1 Matthew J. Whitters,1 Leo M. Albert,1 Leslie Lowe,1 Mayra Senices,1 Paul W. Wu,1 Barbara Sibley,1 Yelena Leathurby,1 Tom P. Brown,2 Cheryl Nickerson-Nutter,1 James C. Keith, Jr.,1 and Mary Collins1

Objective. Interleukin-21 (IL-21) is a T cell– induced arthritis. Nonspecific IgG1 levels were de- derived cytokine that modulates T cell, B cell, and creased in response to treatment. The levels of IL-6 natural killer cell responses. In this study, the effects of mRNA in the paws and the serum IL-6 levels were blocking IL-21 were examined in 2 rodent models of decreased after treatment with IL-21R.Fc. IFN␥ mRNA rheumatoid arthritis (RA) to determine whether IL-21 levels were increased in the paws, and the addition of contributes to their pathologic processes. IL-21R.Fc to collagen-activated lymph node cultures Methods. DBA/1 mice were immunized with bo- enhanced the levels of IFN␥. Collagen-specific spleen vine type II collagen and then treated with murine IL-21 cell responses in IL-21R.Fc–treated mice were observed receptor Fc fusion protein (IL-21R.Fc), which was ini- as reduced levels of IFN␥ and increased levels of IL-6. tiated after the onset of arthritis symptoms in 10% of the Treatment of Lewis rats with IL-21R.Fc after induction cohort. The mice were assessed 3 times per week for of adjuvant-induced arthritis resulted in reversal of signs of disease, including histologic features as well as disease signs and improvements in histologic para- serum cytokine, Ig, and cytokine messenger RNA meters. (mRNA) levels in the paws. In a separate experiment, Conclusion. These findings demonstrate a patho- Lewis rats were immunized with Freund’s complete genic role for IL-21 in animal models of RA, and adjuvant followed by administration of IL-21R.Fc at the support consideration of IL-21 as a therapeutic target in peak of inflammation in the joints. Rats were assessed human RA. daily for histologic features and for scoring of arthritis severity. In addition, the effects of IL-21R.Fc on the T cells have been implicated in pathologic pro- ␥ ␥ production of interferon- (IFN ) by T cells were cesses in animal models of rheumatoid arthritis (RA), by examined. producing potentially inflammatory cytokines and by Results. Treatment of DBA/1 mice with IL-21R.Fc providing help for T cell–dependent autoantibody re- reduced the clinical and histologic signs of collagen- sponses. Recent clinical data on the effects of abatacept, a reagent targeting the B7/CD28 costimulatory pathway, Supported by Wyeth Research. support the role of T cells in the pathogenesis of RA (1). 1Deborah A. Young, PhD, Martin Hegen, PhD, Hak Ling Margery Ma, PhD, Matthew J. Whitters, BS, Leo M. Albert, BVMsc, T cells may contribute to human disease by driving Leslie Lowe, BA, Mayra Senices, BS, Paul W. Wu, BA, Barbara Sibley, pathogenic autoantibody responses, consistent with the AB, Yelena Leathurby, BS, Cheryl Nickerson-Nutter, PhD, James C. success of B cell–depleting strategies in RA. In addition, Keith, Jr., DVM, PhD, Mary Collins, PhD: Wyeth Research, Cam- bridge, Massachusetts; 2Tom P. Brown, DVM, MS, PhD, DACVP, T cells may contribute to disease by producing inflam- Wyeth Research, Andover, Massachusetts. matory cytokines. Drs. Young, Hegen, and Ma, Mr. Whitters, Mr. Albert, Ms Interleukin-21 (IL-21), a member of the type I Lowe, Ms Senices, Mr. Wu, Ms Sibley, and Drs. Nickerson-Nutter, Keith, and Collins hold stock options in Wyeth. cytokine family, is related most closely to IL-2 and IL-15 Address correspondence and reprint requests to Deborah A. (2). IL-21 is made by activated CD4ϩ T cells and Young, PhD, Wyeth Research, 200 Cambridge Park Drive, Cam- regulates the T cell, B cell, and natural killer (NK) cell bridge, MA 02140. E-mail: [email protected]. Submitted for publication January 10, 2006; accepted in responses (3,4). Mice deficient in IL-21 receptor (IL- revised form December 12, 2006. 21R) have normal T cell and NK cell development and

1152 BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1153

do not develop spontaneous autoimmune disease, sug- NKTFINDLLAQLRRRLPAKRTGNKQRHAMAKCPSCD- gesting a role for IL-21 that is distinct from that of IL-2 LYEKKTPKEFLERLKWLLQKMIHQHLS. Transfection of IL-21R Fc fusion protein (IL-21R.Fc). or IL-15 (5,6). IL-21R–deficient mice have deficits in Murine IL-21R.Fc was constructed by polymerase chain reac- humoral immunity (6), consistent with a nonredundant tion (PCR), in which amino acids 1–235 of mouse IL-21R were role for IL-21 in B cell responses. In addition, multiple linked, with a GSGS linker, to mouse IgG2a-Fc domains studies indicate that IL-21 can potentiate antitumor containing mutations, which allowed us to minimize Fc binding responses, supporting a potential role for IL-21 as a and complement fixation (18). This was followed by subcloning into a DHFR expression vector and transfection into CHO proinflammatory cytokine (7–9). cells (19,20). Peak fractions from a protein A affinity column The receptor complex for IL-21 is composed of were pooled and buffer exchanged into phosphate buffered IL-21R, which specifically binds IL-21, together with the saline. The protein was Ͼ95% pure as determined by sodium common cytokine receptor ␥-chain (10,11). In contrast dodecyl sulfate–polyacrylamide gel electrophoresis. BAF-3 proliferation assays. BAF-3 cells (2 ϫ 104) to the cytokine, IL-21R is expressed on multiple cell expressing amino-terminal FLAG-tagged IL-21R were incu- types in the immune system (2,5,12), including T cells, B bated for 3 days with murine IL-21 (594-ML-010 [R&D cells, NK cells, macrophages, and dendritic cells (13,14). Systems, Minneapolis, MN] or recombinant murine IL-21) and ␮ In the present study, we evaluated the role of the murine IL-21R.Fc in 96-well plates, and pulsed with 0.5 Ci 3H-thymidine for 5 hours. The cells were then harvested and IL-21 pathway in 2 animal models of autoimmune counted. arthritis. Rat adjuvant-induced arthritis (AIA) is a T Analysis of cytokines in response to collagen. Ten- cell–dependent inflammatory arthritis (15,16). The dis- week-old male DBA/1 mice were immunized with 100 ␮g/ml ease can be modulated by depletion of T cells or by the bovine type II collagen (Chondrex, Seattle, WA) in Freund’s complete adjuvant (CFA), and draining lymph nodes were administration of antiinflammatory compounds. Mouse harvested on day 10. Lymph node cells (5 ϫ 106 cells/ml) were collagen-induced arthritis (CIA) is dependent on both T cultured with 100 ␮g/ml heat-denatured bovine type II colla- cells and B cells for pathologic development (17). Our gen and either 100 ␮g/ml isotype control IgG2a antibody or results indicate that IL-21 contributes to the develop- murine IL-21R.Fc. Spleens and draining lymph nodes were also harvested at the end of the studies and stimulated in ment of both AIA and CIA, and that therapeutic culture with type II collagen. Cytokines were measured by inhibition of IL-21 correlates with modulation of serum multiplex array analysis (Pierce Biotechnology, Rockford, IL) cytokine levels and improvements in disease scores. after 72 hours. These findings suggest a novel mechanism by which T Cytokine messenger RNA (mRNA) analysis of mouse paws. RNA was isolated from individual homogenized mouse cells can contribute to pathogenesis in animal models paws (n ϭ 5) using RNeasy Mini kits (Qiagen, Chatsworth, of RA. CA) following the manufacturer’s instructions. Purified RNA was treated with DNase (Ambion, Austin, TX) and adjusted to a concentration of 20 ng/␮l before mRNA analysis by quanti- MATERIALS AND METHODS tative PCR on an ABI PRISM 7300HT sequence detection system (Applied Biosystems, Foster City, CA) using primer Animals. The Institutional Animal Care and Use Com- pairs and probes specific for GAPDH, IL-6, tumor necrosis mittee approved all of the animal procedures performed. All factor ␣ (TNF␣), interferon-␥ (IFN␥), and IL-21R, at opti- animals were serologically negative for common pathogens. mized primer and probe concentrations. Expression of mRNA DBA/1LacJ mice were obtained from The Jackson Laboratory in individual paws was normalized to the expression of (Bar Harbor, ME). Twelve-week-old male Lewis rats were GAPDH in each sample, with results expressed as the relative obtained from Charles River (Wilmington, MA). Animals units of target mRNA. were housed according to standard procedures and maintained Rat T cell proliferation. Splenic T cells from male under a standard regimen of food and water ad libitum. Sprague-Dawley rats were purified to 91% CD3ϩ using rat T Rat IL-21 complementary DNA (cDNA). IL-21 cDNA cell–enrichment columns (RTCC-5; R&D Systems) and incu- was amplified using KOD Hot Start (Novagen, Madison, WI) bated on plates coated with 1 ␮g/ml anti–rat CD3 antibody using PW485 (GTACAAAAAAGCAGGCTCCACCATG- (554829; BD PharMingen, San Diego, CA) in 0.1% condi- GAGAGGACCCTTGTCTGTC) and PW486 (ACTTTGTA- tioned medium from mock-transfected or rat IL-21 cDNA– CAAGAAAGCTGGGTCTAGGAAAGATGCTGATGA- transfected COS cells. Murine IL-21R.Fc or isotype control ATC). A gel-purified fragment was reamplified using PW469 was titered into the cultures. On day 3, 0.5 ␮Ci of 3H- (GGGGACAAGTTTGTACAAAAAAGCAGGCTCCAC- thymidine was added for 5 hours, and cells were harvested and CATG) and PW482 (GGGGACCACTTTGTACAAGAAA- counted. GCTGGGT). The rat IL-21 coding region matched that of the Rat spleen cell cytokines. Spleen cells (8 ϫ 106) from rat Genomic Assembly (GenBank 26007405_16). The pre- Sprague-Dawley rats were grown in Dulbecco’s modified Ea- dicted amino acid sequence is MERTLVCLIL- gle’s medium plus 10% fetal calf serum in 9.1-cm2 wells for 3 IFLGTVAHKSSPQRPDHLLIRLRHLMDIVEQLKIYEND- days, and then incubated in 4 ␮g/ml concanavalin A (Con A) LDPELLTAPQDVKGQCEHEAFACFQKAKLKPSNTGN- (C-2010; Sigma, St. Louis, MO) with either 10 ␮g/ml or 1 1154 YOUNG ET AL

Figure 1. Inhibitory actions of mouse interleukin-21 (IL-21) receptor Fc fusion protein (mIL-21R.Fc) on IL-21–dependent proliferation and on interferon-␥ (IFN␥) production. A, Proliferation of BAF-3 cells transfected with mIL-21R cDNA, in response to increasing doses of mIL-21. B, Neutralization of mouse IL-21 bioactivity by mIL-21R.Fc. IL-21R–expressing BAF-3 cells were cultured with increasing levels of soluble IL-21R.Fc or control antibody in the presence of 80 pM mouse IL-21. The calculated 50% inhibitory concentration of mIL-21R.Fc is 2.9 nM. C, Enhanced production of IFN␥ from collagen-primed lymph node cells by blockade with mIL-21R.Fc. The cells were isolated from DBA/1 mice primed for collagen-induced arthritis on day 10 and restimulated in vitro with collagen in the presence of mIL-21R.Fc or control Ig, followed by measurement of IFN␥ levels in the conditioned media. D, Neutralization of proliferation of rat T cells in response to rat IL-21 and anti-CD3 by mIL-21R.Fc. An IgG2a antibody (anti–Elmeria tenella) was used as control. Results were compared with the proliferative responses to anti-CD3 plus 0.1% conditioned media from COS cells transfected with rat IL-21 (rIL21 0.1% CM const). E, Enhancement of production of IFN␥ from concanavalin A (Con A)–stimulated rat splenocytes by mIL-21R.Fc. Results are the mean Ϯ SD.

␮g/ml murine IL-21R.Fc or 10 ␮g/ml control Ig. The levels of malities and articular cartilage abnormalities and scored as IFN␥ were measured by enzyme-linked immunosorbent assay previously described (21–24). (ELISA) (R1F00; R&D Systems). Induction of CIA in mice. Bovine type II collagen Induction of AIA in rats. Immunization with CFA (lot (Chondrex) was dissolved in 0.01N acetic acid and emulsified 084H8800; Sigma-Aldrich, Bornem, Belgium) was used to in an equal volume of CFA containing 1 mg/ml of heat-killed induce AIA in rats (21,22). The study treatment was then Mycobacterium tuberculosis (Sigma). Arthritis was induced in initiated on day 9 after induction of arthritis, when the joints 10-week-old male DBA/1LacJ mice (25). Semitherapeutic were maximally inflamed. Rats were administered a mouse treatment was started in the mice after priming and boosting IgG2a control (anti–Elmeria tenella, HB 8389 2.03.7; American with type II collagen. The treatment, either murine IL-21R.Fc, Type Culture Collection, Rockville, MD), murine IL-21R.Fc, murine TNFRII.Fc, or control mouse IgG2a (anti–E tenella, murine TNF receptor type II Fc fusion protein (TNFRII.Fc), HB 8389 2.03.7; American Type Culture Collection), was or saline on alternate days. Arthritis severity was scored daily initiated when 10% of the mice exhibited clinical signs of (21,22) for 9 days, after which the rats were killed and the hind disease. Experiments contained 14 mice per group and were limbs fixed in 10% buffered formalin. For histopathologic performed 3 times. analysis, the tarsal joints were decalcified, embedded in paraf- Arthritis severity was scored visually 3 times per week, fin, and stained with hematoxylin and eosin or Saffranin O–fast beginning 3 weeks after the primary type II collagen immuni- green. Each stained section was evaluated for synovial abnor- zation (25). Paws were processed for histologic assessment and BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1155

stained with hematoxylin and eosin (Sigma). Each stained section was evaluated for synovial hyperplasia, inflammatory cell infiltrates, cartilage damage, pannus formation, bone erosion, fibrillation, and ankylosis. Histopathologic scores were assigned using previously described methods (25). The arthritis severity score for each paw was weighted based on the number of joints per paw receiving a specific score. IgG antibody levels against the immunogen were measured by ELISA (25). The anti–type II collagen concentrations (in units/ml) were determined by reference to a standard curve generated from 1:2 serial dilutions of a standard CIA serum. Statistical analysis. All histologic features between groups were analyzed using SuperANOVA (Abacus Concepts, Berkeley, CA) with Duncan’s new multiple-range post hoc testing. Disease severity, histopathologic scores, and serum anti–type II collagen IgG levels were compared between groups, using Student’s t-test. Disease incidence was analyzed with Fisher’s exact test. Results are expressed as the mean Ϯ SD. P values less than 0.05 were considered significant.

RESULTS Neutralization of IL-21 bioactivity and modulation of IFN␥ production by IL-21R.Fc. Mouse CIA is a model of RA that is mediated by both cellular and humoral immune responses (17). Immunization of susceptible DBA/1 mice with bovine type II collagen results in an RA-like disease of the joint, characterized by inflammatory cell infiltrates, swelling, and, ultimately, cartilage and bone damage. Th1 cells have been shown to play a role in driving the initial immune response, which is followed by pathogenic anti–type II collagen antibody production. Cytokines, such as TNF␣ and IL-6, also play a role in CIA, underscoring the shared mech- Figure 2. Reductions in disease severity scores in mouse paws with anisms with human RA (26,27). collagen-induced arthritis via blockade of the interleukin-21 (IL-21) pathway. DBA/1 mice were immunized with type II collagen/Freund’s To examine the effects of blocking IL-21–IL-21R complete adjuvant and boosted on day 21 with type II collagen/ interactions on lymphoid cell responses, we used a Freund’s incomplete adjuvant. Murine IL-21 receptor Fc fusion pro- fusion protein (mouse soluble IL-21R.Fc) containing the tein (IL-21R.Fc), murine soluble tumor necrosis factor receptor type II extracellular domain of mouse IL-21R fused to the Fc fusion protein (m.s.TNFRII.Fc), or Ig control was administered at ␮ ␮ constant region of mouse IgG2a, which bears a mutation 200 g/mouse or 400 g/mouse 3 times per week, beginning on day 0, which was the time at which 10% of the mice exhibited disease signs. that prevents binding to Fc receptors. BAF-3 cells, which Mice were dosed for 4 weeks. Results are the mean Ϯ SEM arthritis contain endogenous ␥–common chain, were engineered severity scores, representative of 3 independent experiments, for A, the to express the IL-21R. As shown in Figure 1A, IL-21R– 200 ␮g treatment group and B, the 400 ␮g treatment group (n ϭ 14 in ϭ P Յ 0.05 versus the murine isotype control, by 2-tailed ء .(expressing BAF-3 cells exhibited vigorous proliferation each ϭ in response to increasing doses of IL-21. Treatment with t-test. IP intraperitoneal. the IL-21R.Fc fusion protein, but not an isotype- matched control antibody, effectively neutralized the IL-21–mediated proliferation of IL-21R–expressing antibody. No changes in the proliferative responses to BAF-3 cells (Figure 1B). type II collagen were observed (results not shown). In experiments utilizing DBA/1 mice immunized IL-21 has been shown to specifically modulate with bovine type II collagen in CFA to induce arthritis, the production of IFN␥ from naive T cells (28). Since the draining lymph nodes were harvested on day 10. IFN␥ modulates the severity of disease in CIA, condi- Lymph node cells were restimulated in vitro with type II tioned media from these cultures were assayed for collagen in the presence of IL-21R.Fc or isotype control production of IFN␥. Specific enhancement of IFN␥ 1156 YOUNG ET AL

production was observed in cultures in which IL-21R.Fc model an early stage of RA in which the disease was added during antigen restimulation (Figure 1C). process had already been initiated. DBA/1 mice were These results indicate that IL-21 can modulate IFN␥ immunized with type II collagen in CFA. Twenty-one production by T cells primed with collagen in vivo. days later, the mice were boosted with type II collagen To study the role of IL-21 in rat AIA, cDNA was in Freund’s incomplete adjuvant. IL-21R.Fc was ad- isolated for assessment of rat IL-21 and used to express ministered to the experimental cohort of mice when recombinant rat IL-21 protein in COS cells. Prolifera- 10% of the mice began to exhibit clinical signs of tion of rat splenic T cells in response to plate-bound disease. anti-CD3 was enhanced by addition of rat IL-21, in that Administration of IL-21R.Fc resulted in reduced the addition of 0.1% conditioned medium from trans- disease severity scores (Figures 2A and B). Moreover, fected 293 cells augmented the anti-CD3–induced base- we observed a greater reduction in disease severity with line proliferation from 2,250 counts per minute (mock the higher dose of IL-21R.Fc (400 ␮g), such that the transfection) to 150,000 cpm. Thus, rat IL-21 is a potent animals receiving the higher dose showed significantly costimulus for the proliferation of unprimed rat T cells, less disease on several days compared with the 400 ␮g which are characterized by suboptimal stimulation control–treated animals. The lower dose of IL-21R.Fc through the T cell receptor (TCR). Addition of the (200 ␮g) produced a significant difference in disease mouse IL-21R.Fc fusion protein to this assay effectively severity scores only at the last time point of the study, as neutralized the enhanced proliferation of rat T cells compared with the respective control group. As a posi- occurring in response to rat IL-21 (Figure 1D). tive control, mouse soluble TNFRII.Fc was adminis- We also examined the effect of IL-21R.Fc on rat tered, and the results indicated a profound amelioration T cells stimulated with Con A. As observed with primed of disease, which is consistent with the role of TNF␣ as mouse T cells, incubation of Con A–stimulated rat T an effector-phase cytokine (Figure 2A). cells with IL-21R.Fc resulted in no alteration in prolif- Histologic scoring of the paws at the end of the eration (results not shown). However, measurement of experiment reflected the clinical findings, in that the IFN␥ production in these cultures revealed that treat- histologic severity score was reduced significantly after ment with IL-21R.Fc resulted in enhanced IFN␥ pro- administration of soluble mouse IL-21R.Fc, although duction (Figure 1E). the analysis did not reveal a dose-response effect (Table Reduction in the severity of CIA following IL- 1). A treatment regimen with mouse soluble TNFRII.Fc 21R.Fc administration. We examined the effects of also resulted in a significant reduction in histologic IL-21R.Fc in a semitherapeutic model of CIA in severity scores (Table 1). which animals were treated after priming and boosting At the end of each experiment, we examined with type II collagen so that both cellular and humoral serum cytokine levels in mice primed for CIA and immune responses were ongoing. Our rationale was to subsequently treated with soluble mouse IL-21R.Fc or

Table 1. Histologic assessment of paws and serum interleukin-6 (IL-6) levels after treatment in mice with collagen-induced arthritis* Histologic severity score, Paws with grade 0 Serum IL-6, mean Treatment group mean Ϯ SEM† arthritis, % Ϯ SEM pg/ml Untreated 3.25 Ϯ 0.84 17.9 – Ig control 200 ␮g 3.29 Ϯ 1.09 16.1 917.3 Ϯ 132.2 400 ␮g 3.46 Ϯ 1.01 12.5 846.1 Ϯ 113.9 Mouse soluble TNFRII.Fc 1.98 Ϯ 1.40‡ 50.0‡ 204.8 Ϯ 21.7‡ (200 ␮g) Mouse soluble IL-21R.Fc 200 ␮g 2.18 Ϯ 1.28‡ 44.6‡ 182.1 Ϯ 34.1‡ 400 ␮g 2.89 Ϯ 0.92‡ 26.8 311.8 Ϯ 55.1‡ * Front and rear paws were harvested from mice at the end of the experiment for histologic evaluation. The histologic severity score, ranging from 0 to 4, was assessed for each paw as detailed in Materials and Methods. TNFRII ϭ tumor necrosis factor receptor type II; IL-21R ϭ interleukin-21 receptor. † For each treatment group, 56 paws were scored, except for the 200 ␮g mouse soluble IL-21R.Fc group, in which 52 paws were scored. ‡ P Ͻ 0.05 versus the same dose of Ig control. BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1157

Figure 3. Alterations in cytokine mRNA and collagen-specific antibody levels in mouse paws with collagen-induced arthritis, and spleen cell cytokine levels at the end of the study, after treatment with murine interleukin-21 (IL-21) receptor Fc fusion protein (mIL-21R.Fc). A, Messenger RNA was isolated from homogenized paws of mice treated with 400 ␮g of control Elmeria tenella or mIL-21R.Fc or 200 ␮g of murine soluble tumor necrosis factor ␣ receptor type II Fc fusion protein (msTNFRII.Fc). Levels of each indicated cytokine were determined by quantitative mRNA analysis, with results expressed as the mean Ϯ SD of 5 mice per group. B, Collagen-specific IgG1and IgG2a levels were determined in individual mice from both the 400 ␮g E tenella control and IL-21R.Fc groups, by enzyme-linked immunosorbent assay (ELISA). Total serum IgG1 levels were also measured. Bars show the mean. C, Collagen-induced cytokines from spleen cells were determined by harvesting spleens at the end of the study from the 400 ␮g E tenella control, IL-21R.Fc, and 200 ␮g msTNFRII.Fc groups, which were then cultured with collagen and assessed by multiplex ;ϭ P Ͻ 0.05 versus 400 ␮g isotype control antibody, by 2-tailed t-test. RU ϭ relative units ء .ELISA. Results are the mean Ϯ SD of 5 mice per group IFNg ϭ interferon-␥; GMCSF ϭ granulocyte–macrophage colony-stimulating factor. 1158 YOUNG ET AL

control Ig in the semitherapeutic regimen. We observed Reversal of rat AIA by blockade of the IL-21 statistically significant decreases in IL-6 levels (Table 1). pathway. Rat AIA is a disease initiated by polyclonal T In addition, we observed increased serum levels of IFN␥ cell responses to heat-killed M tuberculosis in CFA in in some of the mice primed for CIA and treated with male Lewis rats (15,16). The disease is characterized by IL-21R.Fc, consistent with our in vitro results, although a multiorgan inflammatory response, which includes an these changes did not reach statistical significance (re- arthritic response in the paws that occurs 8–14 days after sults not shown). immunization. Inflammatory infiltrates are found in the The levels of mRNA for several inflammatory spleen, liver, bone marrow, meninges, skin, and eyes. cytokines were determined by quantitative PCR of ho- The disease resolves after several months. Examination mogenized paws harvested at the end of the study. of the joints during maximal disease reveals infiltration Treatment with IL-21R.Fc resulted in significantly lower of neutrophils, monocytes, and lymphocytes. In addition, levels of IL-6 mRNA when compared with the levels in synovial pannus formation, proteoglycan depletion, and control-treated animals. Expression of mRNA for IL- cartilage erosion are observed. 21R and TNF␣ also appeared lower, but the changes We evaluated the effect of administration of were not statistically significant. In contrast, levels of mouse soluble IL-21R.Fc on rat AIA. Arthritis was IFN␥ mRNA in the paws were significantly increased in induced in male Lewis rats, and clinical signs of arthritis response to IL-21R.Fc treatment (Figure 3A). We were were allowed to develop. The tarsal joints were fully unable to detect mRNA for IL-21 itself (results not swollen by day 8. Starting on day 9, the rats were given shown). IL-21R.Fc or IgG control 3 times per week. In the first Development of CIA is dependent on the gener- experiment (Figure 4A), doses of 1 mg/kg and 3 mg/kg of ation of antibodies to bovine type II collagen that are IL-21R.Fc resulted in partial reduction of swollen joint scores over time, whereas no reduction in swollen joint cross-reactive to mouse type II collagen, and IL-21 is scores was observed in the controls. The response was important in the regulation of Ig production, specifically dose dependent, suggesting that additional reagent IgG1 and IgE responses (5,6). We therefore examined might be needed to fully abrogate disease. the levels of serum IgG1 and IG2a antibodies to type II In a second experiment (Figure 4B), the dose of collagen and the total IgG1 levels in the mice after IL-21R.Fc was increased to 6 mg/kg, and full ameliora- treatment with IL-21R.Fc (Figure 3B). Levels of anti– tion of clinical signs was observed. After completion of collagen IgG1 and anti–collagen IgG2a were slightly the experiment, the rats were killed and the tarsal joints lower, but not significantly different, in the IL-21R.Fc– were obtained for histologic analysis. As shown in Table treated mice when compared with the control-treated 2, features of synovitis and Mankin scores of articular mice. Similarly, IgG2b levels were not significantly dif- cartilage damage were significantly reduced with IL- ferent between control and treated mice (results not 21R.Fc. These data indicate that IL-21 contributes to the shown). However, total IgG1 levels were significantly pathologic features of AIA, even after the disease is fully lower in the IL-21R.Fc–treated group. We could not established. Inhibition of IL-21 in this model resulted in detect antigen-specific IgE in the serum of these mice a reduction in inflammatory cell infiltrates and a de- (results not shown). crease in cartilage erosion in the joints. To determine the cytokine phenotype of lym- The efficacy of IL-21/IL-21R inhibition by IL- phoid populations in response to treatment, we cultured 21R.Fc was compared in this model with inhibition of draining lymph node cells or splenocytes with collagen, the TNF pathway by mouse soluble TNFRII.Fc. Block- and examined the levels of several cytokines. No signif- ade of either cytokine pathway resulted in a dose- icant differences were observed in collagen-restimulated dependent amelioration of the clinical signs of arthritis, lymph node cell cultures in response to treatment (re- indicating that both cytokines play a key role in this sults not shown). In splenocyte cultures, however, we disease. Similar histologic scores were obtained after observed higher levels of IFN␥, IL-2, and granulocyte– treatment in each case (Table 2), indicating that block- macrophage colony-stimulating factor and lower levels ade of either cytokine pathway is sufficient to reverse the of IL-6 and IL-17 in IL-21R.Fc–treated mice compared clinical and histologic disease features in AIA. with control mice. Mice treated with murine soluble Representative photomicrographs of the paws TNFRII.Fc secreted significantly lower levels of IL-6 of rats with AIA treated with vehicle, mouse soluble compared with the control group (Figure 3C). TNFRII.Fc, or IL-21R.Fc in this experiment are BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1159

Figure 4. Effects of alternate-day treatment with vehicle, IgG, murine interleukin-21 receptor Fc fusion protein (IL-21R.Fc) or murine tumor necrosis factor receptor type II Fc fusion protein (muTNFR.Fc) on the clinical signs of joint inflammation in adjuvant-induced arthritis, beginning 9 days after adjuvant injection of rats. Results are the mean Ϯ SD joint scores. A, In a first experiment, the dose-response effects of all 3 proteins were compared with those of saline vehicle injection. Clinical signs of joint inflammation were decreased in a dose-dependent manner by mouse IL-21R.Fc or muTNFRII.Fc, while IgG produced no discernible effect (n ϭ 4 per group). B, In a second experiment, a higher dose of mouse IL-21R.Fc was compared with IgG or muTNFR.Fc. The effects of mouse IL-21R.Fc at this dose were similar to those of muTNFR.Fc (n ϭ 6 per group). 1160 YOUNG ET AL

Table 2. Histologic scores for rat AIA* PBS vehicle, IgG, Murine TNFR.Fc, IL-21R.Fc, Histologic feature (score range) 1 ml/kg 6.0 mg/kg 3.0 mg/kg 6.0 mg/kg Synovial structure (0–3) 2.92 Ϯ 0.21 2.83 Ϯ 0.26 1.08 Ϯ 0.49† 1.33 Ϯ 0.68† Fibroplasia (0–3) 2.67 Ϯ 0.41 2.50 Ϯ 0.45 0.58 Ϯ 0.49† 0.67 Ϯ 0.61† Inflammatory cells (0–3) 2.92 Ϯ 0.21 2.92 Ϯ 0.21 0.67 Ϯ 0.61† 1.00 Ϯ 0.63† Pannus (0–2) 1.83 Ϯ 0.41 2.00 Ϯ 00Ϯ 0† 0 Ϯ 0† Total synovitis score (0–11) 10.33 Ϯ 0.75 10.25 Ϯ 0.52 2.33 Ϯ 1.51† 3.00 Ϯ 1.73† Cartilage structure (0–6) 3.42 Ϯ 0.38 3.08 Ϯ 0.20 1.08 Ϯ 0.38† 0.92 Ϯ 0.38† Cartilage cells (0–6) 2.83 Ϯ 0.26 2.75 Ϯ 0.42 0.33 Ϯ 0.41† 0.67 Ϯ 0.52† Safranin O–fast green staining (0–4) 3.00 Ϯ 0.00 2.67 Ϯ 0.26‡ 0.92 Ϯ 0.38§ 1.58 Ϯ 0.20† Tidemark integrity (0–1) 0 0 0 0 Total Mankin score (0–14) 9.25 Ϯ 0.52 8.50 Ϯ 0.63 2.33 Ϯ 0.98† 3.00 Ϯ 0.63† * Values are the mean Ϯ SD histologic scores for synovitis and Mankin scores for cartilage changes in tarsal joints from animals with adjuvant-induced arthritis (AIA) treated as indicated, beginning on day 8 after injection of Freund’s complete adjuvant. TNFR ϭ tumor necrosis factor receptor; IL-21R ϭ interleukin-21 receptor. † P Ͻ 0.05 versus phosphate buffered saline (PBS) vehicle and IgG groups. ‡ P Ͻ 0.05 versus PBS vehicle group. § P Ͻ 0.05 versus all other groups. shown in Figure 5. When compared with IgG-treated resulted in amelioration of disease progression. Block- specimens, both IL-21R.Fc and mouse soluble ade of IL-21 was correlated with enhanced IFN␥ pro- TNFRII.Fc reduced all of the parameters of synovial duction and suppression of serum IL-6 levels. Thus, the inflammation and all of the components of the Man- IL-21–IL-21R interaction defines a new pathway con- kin score for articular cartilage lesions. In the vehicle- tributing to the inflammatory arthritis response in these treated animals, the articular cartilage was damaged models. and in some cases was completely destroyed. In these Both the rat AIA model and the mouse CIA areas, hyperplastic synoviocytes and an evolving pan- model are driven by pathogenic T cell responses. Patho- nus reaction invaded the articular cartilage region. genicity has been associated with the production of Extensive proliferation of the synovial membrane and inflammatory cytokines, including TNF␣, IL-6, and pannus resulted in villus formation that filled the joint IL-17 (29–31). Herein we identified IL-21 as a new T cell space. cytokine that is contributing to the pathologic processes In animals treated with either mouse soluble of both CIA and AIA. In both models, inhibition of the IL-21R.Fc or mouse soluble TNFRII.Fc, significant re- cytokine using a soluble mouse IL-21R.Fc fusion protein ductions in articular cartilage damage were found, and resulted in a reduction of the clinical signs of disease in the development of pannus was greatly attenuated. animals in which the disease process had already been Moderate synoviocyte proliferation still occurred, but minimal villus formation was seen. The beneficial effects initiated. This implicates IL-21 as having a role in the of mouse soluble IL-21R.Fc and mouse soluble TNFRII.Fc effector phase of both diseases. on reducing the synovial and articular cartilage lesions IL-21 can enhance the proliferative response of were similar in the AIA model. naive mouse T cells to TCR stimulation (5). Similarly, in the present study, the addition of IL-21 to rat T cells stimulated with anti-CD3 resulted in enhanced prolifer- DISCUSSION ation. This suggests that IL-21 may contribute to the We have explored the role of the T cell–derived initial expansion of pathogenic T cell populations. How- cytokine IL-21 in pathology development in animal ever, inhibition of IL-21 using soluble IL-21R.Fc had no models of RA. In these experiments, we found that effect on the proliferative responses of previously IL-21 contributes to disease in both CIA and AIA, each primed mouse T cells or Con A–activated rat T cells, of which is a distinct animal model of RA. Administra- suggesting that IL-21 is not the predominant cytokine tion of IL-21R.Fc, a potent neutralizing reagent for driving the expansion of primed T cells. IL-21, resulted in reversal of clinical disease in a rat The complete reversal of disease and histologic model of AIA. Administration of IL-21R.Fc to mice changes observed in the AIA model of arthritis suggest with CIA after the onset of disease symptoms also that IL-21 is necessary for the prolonged accumulation BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1161

macrophage-mediated events has not been well described. In mice, IL-21 can contribute to the expansion of the macrophage lineage (32). In RA patients, expression of IL-21R has been described on synovial macrophages and fibroblasts, suggesting that these cell types may potentially participate in IL-21–mediated effects (33). In addition, IL-21, which is made by activated CD4ϩ T cells, may be contributing to the expansion, activation, or recruitment of inflammatory cells, either within the peripheral compartment or directly in the joints. B cells are required for disease in CIA, since mice deficient in B cells are resistant to CIA induction (34). Production of type II collagen–reactive antibodies is necessary for disease induction (35), and arthritis can be induced by transfer of mixtures of type II collagen– reactive antibodies (36). However, production of antibodies to type II collagen is not sufficient to induce disease, since disease can be suppressed in the presence of anti–type II collagen antibody responses (25). IL-21 is required for the induction of IgG responses, as shown by the reduced IgG responses of IL-21R–deficient mice (6). We did not detect a significant difference in anti–type II collagen antibody titers in those experiments in which IL-21R.Fc was administered after type II collagen boosting, suggesting that anti–type II collagen antibody production had already proceeded past the IL-21– dependent step. Pathogenic antibodies in CIA are associated with the IgG2a and IgG2b isotypes, which are less profoundly affected than IgG1 in IL-21R–deficient mice (6). It may be that IgG2a responses in our experiments were being driven by IL-21–independent mechanisms. However, we also did not see any significant changes in the levels of Figure 5. Photomicrographs of the tarsal joints from rats in the second adjuvant-induced arthritis experiment. A, Tarsal joint from a IgG1 or IgG2b anti–type II collagen antibody titers. vehicle-treated rat, showing loss of articular cartilage (arrow), hyper- Thus, the mechanism of disease amelioration in these plasia of the synovial membrane (H), and organization of the aerolar CIA studies is independent of modulation of anti–type connective tissue to produce pannus (P). B, Tarsal joint from a murine II collagen antibody responses and may share a common tumor necrosis factor receptor type II Fc fusion protein–treated rat, pathway with that observed in AIA. The lack of com- showing normal articular cartilage (C), hyperplasia of the synovial membrane, minimal villus formation, and normal aerolar connective plete disease amelioration in CIA may reflect the con- tissue (AC). C, Tarsal joint from an interleukin-21 receptor Fc fusion tribution of the anti–type II collagen antibodies to protein–treated rat, showing slight articular cartilage damage from disease, which are still present in these treated animals. overlying, hyperplastic synovial membrane and minimal organization In both CIA and AIA, the initiating pathogenic T of the aerolar connective tissue (O). (Hematoxylin and eosin stained; cell response is predominantly a Th1 response. How- original magnification ϫ 200.) ever, the role of INF␥, the canonical Th1 cell cytokine, does not correlate with disease susceptibility and ap- of inflammatory cells in the joints. AIA in the Lewis rat pears to have a protective role in CIA (37–39). Recent is mediated by migration of activated T cells and neu- studies examining the distinct roles of IL-12 and IL-23 trophils into the joints of immunized animals (15,16). have raised questions about the role of Th1 cells in CIA Activated macrophages are also thought to contribute to (40). Interestingly, IL-21 has been shown to inhibit the the disease. The role of the IL-21 pathway in differentiation of naive T cells into Th1 cells that 1162 YOUNG ET AL

produce IFN␥ (28). The production of IFN␥ from Study design. Young, Hegen, Ma, Albert, Senices, Wu, Nickerson- reactivated type II collagen–primed mouse lymph node Nutter, Keith. Acquisition of data. Hegen, Ma, Whitters, Albert, Lowe, Senices, Wu, T cells or Con A–activated rat T cells was enhanced in Sibley, Leathurby, Brown, Keith. the presence of IL-21R.Fc, and mRNA for IFN␥ was Analysis and interpretation of data. Young, Hegen, Ma, Whitters, also reduced in the paws of mice isolated after treatment Albert, Lowe, Senices, Wu, Brown, Nickerson-Nutter, Keith, Collins. Manuscript preparation. Young, Hegen, Ma, Albert, Lowe, Senices, with IL-21R.Fc. These results suggest that IL-21R.Fc Brown, Keith, Collins. may be affecting disease by modulation of IFN␥. Mice Statistical analysis. Ma, Senices, Keith. deficient in IL-21R have enhanced IFN␥ responses after priming for a delayed-type hypersensitivity response ROLE OF THE STUDY SPONSOR (28). Experiments in IFN␥-deficient mice might help to This study was designed and carried out by staff members of elucidate whether the protective effect of IL-21R.Fc is Wyeth Research; these members are all authors of the manuscript. All authors independently reviewed and contributed to the manuscript mediated through IFN␥. during its development, agreed to submit the manuscript, and ap- Serum IL-6 levels were decreased consistently in proved the content of the submitted manuscript. The study was multiple experiments with IL-21 or TNF␣ blockade. supported by Wyeth Research, and decisions on study design, data collection, data analysis, and interpretation of the data were made by Expression of IL-6 and TNF␣ mRNA in TaqMan ana- Wyeth Research scientists. lyses of the diseased paws was also reduced in IL- 21R.Fc–treated mice. A decrease in serum IL-6 levels REFERENCES has also been observed in therapeutic CIA studies in 1. Kremer JM, Dougados M, Emery P, Durez P, Sibilia J, Shergy W, which IL-17 was inhibited (29). IL-21 has been reported et al. Treatment of rheumatoid arthritis with the selective costimu- to promote production of IL-17 from activated human T lation modulator abatacept: twelve-month results of a phase IIb, cells (41).Thus, blockade of IL-21 in CIA may function, double-blind, randomized, placebo-controlled trial. Arthritis Rheum 2005;52:2263–71. in part, by modulation of IL-6–driven inflammation, 2. Parrish-Novak J, Dillon SR, Nelson A, Hammond A, Sprecher C, either directly or through down-regulation of TNF␣ or Gross JA, et al. Interleukin 21 and its receptor are involved in NK IL-17. IL-6 has a pathogenic role in CIA (26,31,42) and cell expansion and regulation of lymphocyte function. Nature 2000;408:57–63. is a critical inflammatory cytokine in RA. Blockade of 3. Parrish-Novak J, Foster DC, Holly RD, Clegg CH. Interleukin-21 IL-6R has demonstrated efficacy in clinical trials of RA and the IL-21 receptor: novel effectors of NK and T cell responses. (43). J Leukocyte Biol 2002;72:856–63. 4. Collins M, Whitters MJ, Young DA. IL-21 and IL-21 receptor: a Our data show a role for IL-21 in 2 models of RA new cytokine pathway modulates innate and adaptive immunity. in which therapeutic intervention was started after onset Immunol Res 2003;28:131–40. of the disease process. These data identify IL-21 as a 5. Kasaian MT, Whitters MJ, Carter LL, Lowe LD, Jussif JM, Deng B, et al. IL-21 limits NK cell responses and promotes antigen- new T cell cytokine contributing to the pathogenesis of specific T cell activation: a mediator of the transition from innate animal models of RA. The regulation of IFN␥ by IL-21 to adaptive immunity. Immunity 2002;16:559–69. further suggests that IL-21 may have a role in determin- 6. Ozaki K, Spolski R, Feng CG, Qi CF, Cheng J, Sher A, et al. A critical role for IL-21 in regulating immunoglobulin production. ing the type of Th cell response generated in these Science 2002;298:1630–4. autoimmune models. Blockade of IL-21 resulted in 7. Ma HL, Whitters MJ, Konz RF, Senices M, Young DA, Grusby down-regulation of IL-6, a cytokine that is implicated in MJ, et al. IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are the pathologic processes of both CIA and RA, demon- independent of IFN-␥. J Immunol 2003;171:608–15. strating that IL-21 contributes to the production of IL-6 8. Smyth MJ, Wallace ME, Nutt SL, Yagita H, Godfrey DI, Hay- in animal models of arthritis. These data support the akawa Y. Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med 2005;201: need for further evaluation of the role of IL-21 in human 1973–85. RA. 9. Moroz A, Eppolito C, Li Q, Tao J, Clegg CH, Shrikant PA. IL-21 enhances and sustains CD8ϩ T cell responses to achieve durable tumor immunity: comparative evaluation of IL-2, IL-15, and IL-21. ACKNOWLEDGMENTS J Immunol 2004;173:900–9. 10. Asao H, Okuyama C, Kumaki S, Ishii N, Tsuchiya S, Foster D, et We thank Qiuna Bi for assistance with the anti- al. Cutting edge: the common ␥-chain is an indispensable subunit collagen ELISAs, and Neil Wolfman, Beatriz Carreno, and of the IL-21 receptor complex. J Immunol 2001;167:1–5. Kyri Dunussi-Joannopoulos for helpful discussions. 11. Habib T, Senadheera S, Weinberg K, Kaushansky K. The common ␥ chain is a required signaling component of the IL-21 receptor AUTHOR CONTRIBUTIONS and supports IL-21-induced cell proliferation via JAK3. Biochem- istry 2002;41:8725–31. Dr. Collins had full access to all of the data in the study and 12. Ozaki K, Kikly K, Michalovich D, Young PR, Leonard WJ. takes responsibility for the integrity of the data and the accuracy of the Cloning of a type I cytokine receptor most related to the IL-2 data analysis. receptor ␤ chain. Proc Natl Acad SciUSA2000;97:11439–44. BLOCKADE OF INTERLEUKIN-21 IN RA ANIMAL MODELS 1163

13. Brandt K, Bulfone-Paus S, Foster DC, Ruckert R. Interleukin-21 29. Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersse- inhibits dendritic cell activation and maturation. Blood 2003;102: laar L, Coenen-de Roo CJ, Joosten LA, et al. Treatment with a 4090–8. neutralizing anti-murine interleukin-17 antibody after the onset of 14. Brandt K, Bulfone-Paus S, Jenckel A, Foster DC, Paus R, Ruckert collagen-induced arthritis reduces joint inflammation, cartilage R. Interleukin-21 inhibits dendritic cell-mediated T cell activation destruction, and bone erosion. Arthritis Rheum 2004;50:650–9. and induction of contact hypersensitivity in vivo. J Invest Dermatol 30. Mori L, Iselin S, De Libero G, Lesslauer W. Attenuation of 2003;121:1379–82. collagen-induced arthritis in 55-kDa TNF receptor type 1 15. Holmdahl R, Lorentzen JC, Lu S, Olofsson P, Wester L, Holm- (TNFR1)-IgG1-treated and TNFR1-deficient mice. J Immunol berg J, et al. Arthritis induced in rats with nonimmunogenic 1996;157:3178–82. adjuvants as models for rheumatoid arthritis. Immunol Rev 2001; 31. Takagi N, Mihara M, Moriya Y, Nishimoto N, Yoshizaki K, 184:184–202. 16. Van Eden W, Waksman BH. Immune regulation in adjuvant- Kishimoto T, et al. Blockage of interleukin-6 receptor ameliorates induced arthritis: possible implications for innovative therapeutic joint disease in murine collagen-induced arthritis. Arthritis Rheum strategies in arthritis [review]. Arthritis Rheum 2003;48:1788–96. 1998;41:2117–21. 17. Holmdahl R, Bockermann R, Backlund J, Yamada H. The molec- 32. Wang G, Tschoi M, Spolski R, Lou Y, Ozaki K, Feng C, et al. In ular pathogenesis of collagen-induced arthritis in mice: a model vivo antitumor activity of interleukin 21 mediated by natural killer for rheumatoid arthritis. Ageing Res Rev 2002;1:135–47. cells. Cancer Res 2003;63:9016–22. 18. Steurer W, Nickerson PW, Steele AW, Steiger J, Zheng XX, 33. Jungel A, Distler JH, Kurowska-Stolarska M, Seemayer CA, Seibl R, Strom TB. Ex vivo coating of islet cell allografts with murine Forster A, et al. Expression of interleukin-21 receptor, but not CTLA4/Fc promotes graft tolerance. J Immunol 1995;155: interleukin-21, in synovial fibroblasts and synovial macrophages of 1165–74. patients with rheumatoid arthritis. Arthritis Rheum 2004;50:1468–76. 19. Kaufman RJ. Selection and coamplification of heterologous genes 34. Svensson L, Jirholt J, Holmdahl R, Jansson L. B cell-deficient in mammalian cells. Methods Enzymol 1990;185:537–66. mice do not develop type II collagen-induced arthritis. Clin Exp 20. Kaufman RJ. Vectors used for expression in mammalian cells. Immunol 1998;111:521–6. Methods Enzymol 1990;185:487–511. 35. Brand DB, Kang AH, Rosloniec EF. Immunopathogenesis of 21. Keith JC Jr, Albert LM, Leathurby Y, Follettie M, Wang L, collagen arthritis. Springer Semin Immunopathol 2003;25:3–18. Borges-Marcucci L, et al. The utility of pathway selective estrogen 36. Terato K, Hasty KA, Reife RA, Cremer MA, Kang AH, Stuart ␬ receptor ligands that inhibit nuclear factor- B transcriptional JM. Induction of arthritis with monoclonal antibodies to collagen. activity in models of rheumatoid arthritis. Arthritis Res Ther J Immunol 1992;148:2103–8. 2005;7:R427–38. 37. Jacob CO, Holoshitz J, van der Meide P, Strober S, McDevitt HO. 22. Keith JC Jr, Sainz IM, Isordia-Salas I, Pixley RA, Leathurby Y, Heterogeneous effects of IFN-␥ in adjuvant arthritis. J Immunol Albert LM, et al. A monoclonal antibody against kininogen 1989;142:1500–5. reduces inflammation in the HLA-B27 transgenic rat. Arthritis 38. Guedez YB, Whittington KB, Clayton JL, Joosten LA, van de Loo Res Ther 2005;7:R769–76. FA, van den Berg WB, et al. Genetic ablation of interferon-␥ 23. Poole AR, Coombs RR. Rheumatoid-like joint lesions in rabbits up-regulates interleukin-1␤ expression and enables the elicitation injected intravenously with bovine serum. Int Arch Allergy Appl of collagen-induced arthritis in a nonsusceptible mouse strain. Immunol 1977;54:97–113. Arthritis Rheum 2001;44:2413–24. 24. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and 39. Vermeire K, Heremans H, van de Putte M, Huang S, Billiau A, metabolic abnormalities in articular cartilage from osteo-arthritic ␥ human hips. II. Correlation of morphology with biochemical and Matthys P. Accelerated collagen-induced arthritis in IFN- recep- metabolic data. J Bone Joint Surg Am 1971;53:523–37. tor-deficient mice. J Immunol 1997;158:5507–13. 25. Hegen M, Sun L, Uozumi N, Kume K, Goad ME, Nickerson- 40. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClana- Nutter CL, et al. Cytosolic phospholipase A2␣-deficient mice are han T, Kastelein RA, et al. Divergent pro- and antiinflammatory resistant to collagen-induced arthritis. J Exp Med 2003;197: roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp 1297–302. Med 2003;198:1951–7. 26. Sasai M, Saeki Y, Ohshima S, Nishioka K, Mima T, Tanaka T, et 41. Hoeve MA, Savage ND, de Boer T, Langenberg DM, de Waal al. Delayed onset and reduced severity of collagen-induced arthri- Malefyt R, Ottenhoff TH, et al. Divergent effects of IL-12 and tis in interleukin-6–deficient mice. Arthritis Rheum 1999;42: IL-23 on the production of IL-17 by human T cells. Eur J Immunol 1635–43. 2006;36:661–70. 27. Williams RO, Feldmann M, Maini RN. Anti-tumor necrosis factor 42. Alonzi T, Fattori E, Lazzaro D, Costa P, Probert L, Kollias G, et ameliorates joint disease in murine collagen-induced arthritis. al. Interleukin 6 is required for the development of collagen- Proc Natl Acad SciUSA1992;89:9784–8. induced arthritis. J Exp Med 1998;187:461–8. 28. Wurster AL, Rodgers VL, Satoskar AR, Whitters MJ, Young DA, 43. Nishimoto N, Yoshizaki K, Miyasaka N, Yamamoto K, Kawai S, Collins M, et al. Interleukin 21 is a T helper cell 2 cytokine that Takeuchi T, et al. Treatment of rheumatoid arthritis with human- specifically inhibits the differentiation of naive Th cells into ized anti–interleukin-6 receptor antibody: a multicenter, double- interferon ␥-producing Th1 cells. J Exp Med 2002;196:969–77. blind, placebo-controlled trial. Arthritis Rheum 2004;50:1761–9. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1164–1174 DOI 10.1002/art.22495 © 2007, American College of Rheumatology

A Tumor Necrosis Factor Receptor Loop Peptide Mimic Inhibits Bone Destruction to the Same Extent as Anti–Tumor Necrosis Factor Monoclonal Antibody in Murine Collagen-Induced Arthritis

Hiroaki Saito,1 Takefumi Kojima,2 Mariko Takahashi,2 William C. Horne,3 Roland Baron,3 Teruo Amagasa,2 Keiichi Ohya,2 and Kazuhiro Aoki2

Objective. The cyclic peptide WP9QY disease was not delayed by the peptide. The inhibitory (YCWSQYLCY) was designed to mimic the most critical effect of WP9QY on inflammation was definitely weaker tumor necrosis factor ␣ (TNF␣) recognition loop on than that of anti-TNF antibody. Microfocal computed TNF receptor I, and it prevents interactions of TNF␣ tomography analyses, however, revealed that WP9QY with its receptor. We undertook this study to compare blocked CIA-induced bone destruction at the knee joints the effects of the WP9QY peptide on collagen-induced to the same extent as did anti-TNF antibody. In addi- arthritis (CIA) in mice with those of anti-TNF␣ mono- tion, WP9QY inhibited synovial pannus infiltration and clonal antibody. reduced osteoclast number. Furthermore, inhibition of Methods. CIA was induced by primary and sec- CIA-induced systemic bone loss by WP9QY was more ondary immunizations. Osmotic minipumps were im- apparent than that by anti-TNF antibody. planted in the backs of all mice on the day of the Conclusion. The TNF␣ antagonist WP9QY would booster injection (day 21), and vehicle, anti-TNF anti- be a useful template for the development of small body (4 mg/kg/day), or WP9QY peptide (2 mg/kg/day or molecular inhibitors to prevent both inflammatory bone 4 mg/kg/day) was continuously infused until the mice destruction and systemic bone loss in rheumatoid ar- were killed (day 40). Thereafter, clinical, radiographic, thritis. and histologic assessments were performed. Results. WP9QY treatment inhibited CIA- Rheumatoid arthritis is an inflammatory disease induced increases in the arthritis score, but onset of caused by autoimmune response, and it is aggravated by excessive bone resorption at peripheral joints (1,2). Dr. Ohya’s work was supported by the Ministry of Education, Cytokines such as tumor necrosis factor ␣ (TNF␣), Culture, Sports, Science, and Technology of Japan (grant 16209053). interleukin-1 (IL-1), and IL-6 are thought to be impor- Dr. Aoki’s work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grants 16390530 and tant molecules responsible for the clinical onset of 17659584). rheumatoid arthritis (3). TNF␣ is thought to play a 1Hiroaki Saito, MS: Tokyo Medical and Dental University, crucial role in rheumatoid arthritis since it enhances Tokyo, Japan, and Yale University School of Medicine, New Haven, Connecticut; 2Takefumi Kojima, DDS, PhD, Mariko Takahashi, BS, production of inflammatory disease–related molecules Teruo Amagasa, DDS, PhD, Keiichi Ohya, DDS, PhD, Kazuhiro Aoki, such as IL-1 or IL-6 in serum and synovial fluid (4,5), DDS, PhD: Tokyo Medical and Dental University, Tokyo, Japan; and neutralization therapies using anti-TNF␣ monoclo- 3William C. Horne, PhD, Roland Baron, DDS, PhD: Yale University School of Medicine, New Haven, Connecticut. nal antibody or soluble TNF receptor (TNFR) have Drs. Baron and Aoki receive royalties through Yale Univer- proved to be a successful strategy for ameliorating both sity for a patent on a method for inhibiting osteoclastogenesis. inflammation and joint destruction in rheumatoid arthri- Address correspondence and reprint requests to Kazuhiro Aoki, DDS, PhD, Department of Hard Tissue Engineering (Pharma- tis (6,7). cology), Graduate School, Tokyo Medical and Dental University, Newly developed peptide inhibitors, designated 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail: kazu. aromatically modified exocyclic analogs (8), were de- [email protected]. Submitted for publication February 9, 2006; accepted in signed to interfere with the receptor–ligand complex and revised form December 21, 2006. have been shown to block CD4–class II major histocom-

1164 PROPERTIES OF ANTI-TNF␣ PEPTIDE IN MURINE CIA 1165

patibility complex interactions, inhibiting T cell activa- dermally injected at the base of the tail with 200 ␮g bovine type tion (8), and to prevent human immunodeficiency virus II collagen in 0.05M acetic acid emulsified in CFA. Twenty-one (HIV) infection by blocking the binding of CD4 to days after the primary immunization, the mice were boosted in the same way. The day of the first immunization was desig- gp120, HIV type 1 envelope glycoprotein (9). The nated day 0. The first signs of arthritis onset occurred on day peptide inhibitor of the aromatically modified exocyclic 5 after the second immunization. analog type has a cyclical structure which increases the Treatment with the WP9QY peptide. The WP9QY efficiency of binding to the targeted protein because of peptide was dissolved in phosphate buffered saline–buffered the enhancement of the structural stability by its struc- 10% DMSO. Alzet osmotic minipumps (Model 2001; Alza, Palo Alto, CA) were prepared according to the manufacturer’s ture (8). instructions. On day 21, the mice were anesthetized with This aromatically modified exocyclic analog– injections of medetomidine hydrochloride (0.5 mg/kg; Meiji- based approach, in which the molecular weight of an seika, Tokyo, Japan) and ketamine hydrochloride (50 mg/kg; inhibitor is reduced (10), was applied in developing a Sankyo, Tokyo, Japan). A 1-cm incision was made in the skin, new TNF␣ inhibitor, the WP9QY peptide (YC- and the osmotic minipumps filled with 10% DMSO (vehicle), WP9QY peptide (2 mg/kg/day or 4 mg/kg/day), or anti-TNF WSQYLCY) investigated in the present study. Based on antibody (4 mg/kg/day) were subcutaneously implanted. The x-ray crystallographic information about the TNF␤– osmotic minipumps were replaced on day 28 and day 35, and TNFRI complex (11) and about TNF␣ itself (12), the infusions were continued until the mice were killed (on day critical binding site on TNFRI was clarified. The aro- 40). The mice (6–7 per group) were divided into 5 groups, as matically modified exocyclic analog type of TNF inhib- follows: 1) nonimmunized mice receiving vehicle (10% DMSO) (vehicle-treated normal mice), 2) immunized mice itor was then designed to mimic the 3-dimensional receiving vehicle (10% DMSO) (vehicle-treated mice with structure of the critical region, interfering with TNF␣– CIA), 3) immunized mice receiving 2 mg/kg/day WP9QY TNFR interactions (10). peptide (2 mg WP9QY–treated mice with CIA), 4) immunized We performed a comparative study of the effect mice receiving 4 mg/kg/day WP9QY peptide (4 mg WP9QY– of WP9QY peptide administration in mice with treated mice with CIA), and 5) immunized mice receiving 4 mg/kg/day anti-TNF␣ monoclonal antibody (4 mg anti-TNF– collagen-induced arthritis (CIA), using anti-TNF anti- treated mice with CIA). body as a positive control to clarify the effects of the Clinical assessment of arthritis. To determine the WP9QY peptide on inflammatory bone resorption. We arthritis score, 2 independent observers examined the mice show that WP9QY peptide at the same dose as that of daily from the day of the second immunization. The day of anti-TNF antibody inhibits inflammation only weakly, arthritis onset was considered the day that erythema and/or swelling were first observed. The severity of arthritis was but has an effect on CIA-induced bone destruction graded on a 0–3 scale as previously described (14). The criteria similar to that of anti-TNF antibody. Furthermore, for the grading were as follows: 0 ϭ normal; 1 ϭ slight swelling complete inhibition of CIA-induced systemic bone loss, and/or erythema; 2 ϭ pronounced edematous swelling; and which is not apparent with anti-TNF antibody, occurs in 3 ϭ deformed paw or joint with ankylosis and joint rigidity. WP9QY peptide–treated mice. Each paw was graded, and the 4 scores were summed so that the maximum possible score was 12 per mouse. Incidence was expressed as the percentage of mice with an arthritis score Ն1. MATERIALS AND METHODS Radiographic assessment of arthritis. At the end of the experiment (day 40), the mice were killed using ether Materials and animals. The WP9QY peptide, which anesthesia, and the hind paws were removed and fixed in was known to be a TNF␣ antagonist (10), was synthesized phosphate buffered glutaraldehyde (2.5%)–formalin (4%) fix- (American Peptide Company, Sunnyvale, CA). Anti-TNF ative (pH 7.4) for 2 days, washed with water for 1 day, and then monoclonal antibody (infliximab) was kindly provided by used for the radiographic analyses. Three-dimensional recon- Tanabe Seiyaku (Osaka, Japan). Bovine type II collagen was struction images and sagittal images of the knee joints were obtained from the Collagen Research Center (Tokyo, Japan), obtained by microfocal computed tomography (micro-CT) and Freund’s complete adjuvant (CFA) was from Difco (De- (SMX-90CT; Shimadzu, Kyoto, Japan). Using the recon- troit, MI). Six-week-old male DBA/1J mice were purchased structed sagittal image from micro-CT scanning data, histo- from Charles River Laboratories Japan (Kanagawa, Japan) morphometric analysis of the tibial and femoral epiphysis was and were allowed food (MF; Oriental Yeast Company, Tokyo, performed in the measurement area, which was 0.25 mm from Japan) and distilled water ad libitum. Mice were maintained the surface of the femoral or tibial epiphysis facing along the under the conditions of a 12-hour light/dark cycle. The exper- articular cavity at the knee joints. Bone area and bone imental procedures were reviewed and approved by the Ani- perimeter (BPm) were measured using an image analyzing mal Care and Use Committee of Tokyo Medical and Dental system (KS400; Carl Zeiss, Jena, Germany), and the mean University (Tokyo, Japan). bone thickness was calculated as (bone area/BPm) ϫ 2. The Induction of CIA. The induction and assessment of bone area at the tibial secondary spongiosa was measured CIA were performed as previously described (13). Briefly, starting 0.3 mm distal from the growth plate to exclude the male DBA/1J mice (age 7 weeks, 6–7 per group) were intra- primary spongiosa. 1166 SAITO ET AL

The bone mineral density (BMD) of the knee joints cant difference post hoc test. Tests were carried out using was measured by dual x-ray absorptiometry (DXA) (DCS- Apple software, StatView 4.1 (SAS Institute, Cary, NC). P 600R; Aloka, Tokyo, Japan) using the high-resolution scanning values less than 0.05 were considered significant. mode. The region of interest (ROI), 2.1 mm ϫ 2.1 mm, for measuring BMD at knee joints was determined to exclude the secondary spongiosa of the tibial and femoral metaphysis. RESULTS Histologic assessment of arthritis. The removed hind paws were embedded in a mixture composed of 40 ml methyl- Weak inhibitory effect of WP9QY peptide on methacrylate (Wako Pure Chemical Industries, Osaka, Japan), inflammation compared with that of anti-TNF antibody 60 ml 2-hydroxyethyl methacrylate (Wako Pure Chemical in mice with CIA. To clarify the effects of the WP9QY Industries), 10 ml 2-hydroxyethyl acrylate (Wako Pure Chem- peptide on inflammation in mice with CIA, we assessed ical Industries), 10 ml 2-propanol (Wako Pure Chemical In- the development of inflammation by scoring the clinical dustries), 0.3 ml N,N-dimethyl aniline (Wako Pure Chemical Industries), and 0.4 gm benzoyl peroxide (Nakarai Tesque, disease activity daily from day 21 after the first immu- Kyoto, Japan) as described elsewhere (15), with some modifi- nization. The disease activity in vehicle-treated mice cations. Polymerization was performed at 4°C. Standard unde- with CIA first appeared on day 26, which was 5 days ␮ calcified sections (3 m) were prepared using a Reichert-Jung after the second immunization (Figure 1A). The severity microtome 2050 Supercut (Cambridge Instrument, Heidel- berger, Switzerland). Inflammation at the knee joints was of the disease gradually increased, and the mean arthri- scored as the histologic score on a scale ranging from 0 (no tis score reached 8.5 in the vehicle-treated mice with inflammation) to 5 (severely inflamed joint), using hematoxylin CIA on day 40. The severity of disease in mice with CIA and eosin (H&E)–stained sections as previously described (16). treated with WP9QY or anti-TNF antibody was less than Using toluidine blue–stained sections, cartilage destruction was that in vehicle-treated mice with CIA (Figure 1A). scored as the cartilage score on a scale ranging from 0 (normal) to 3 (completely destroyed) as previously described (14). Significant reductions of the arthritis scores in the 3 Histomorphometric evaluation of arthritis. To obtain treated groups compared with scores in vehicle-treated a quantitative assessment of arthritis, we calculated the rate of mice with CIA appeared starting on day 29. On day 40, infiltration of synovial pannus in the articular cartilage surface a significant reduction in the arthritis score was still at the proximal end of the tibia, using undecalcified H&E- stained sections. The calculation formula was as follows: observed in 2 mg and 4 mg WP9QY–treated mice with infiltration rate of pannus (%) ϭ (length of the synovial CIA (mean scores of 5.8 and 4.6, respectively) and in 4 pannus facing the articular cavity along the proximal end of the mg anti-TNF–treated mice with CIA (mean score 3.3) ϫ tibia/total length of the proximal end of the tibia) 100. To compared with vehicle-treated mice with CIA (P Ͻ 0.05) evaluate bone destruction in the knee joints and to exclude the cortical bone facing the epiphyseal growth plate from the (Figure 1A). When we compared the severity of disease measurement area, an ϳ0.65-mm2 area of the epiphysis start- in the anti-TNF–treated group with that in both ing 0.2 mm proximal from the growth plate–epiphyseal junc- WP9QY-treated groups, the arthritis score in anti-TNF– tion was determined, using undecalcified sections that were treated mice was significantly lower than that in both stained with tartrate-resistant acid phosphatase (TRAP) and WP9QY–treated groups from day 29 to the day the mice counterstained with toluidine blue. The bone volume/total volume (BV/TV) and osteoclast number/bone surface (NOc/ were killed. BS) in the epiphysis of the proximal tibia were measured using Arthritis onset was delayed for 3 days by anti- the KS400 image analyzing system as previously described (17). TNF antibody treatment, while arthritis in vehicle- TRAP-positive cells that formed resorption lacunae at the treated mice with CIA appeared on day 26 (Figure 1B). Ն surface of the trabeculae and contained 2 nuclei were In contrast, both WP9QY–treated groups of mice had designated osteoclasts. Bone histomorphometry of the tibial metaphysis. To no delay in clinical onset of disease compared with investigate the secondary effect of CIA on bone resorption in vehicle-treated mice with CIA (Figure 1B). Moreover, addition to resorption of periarticular bone, a standard histo- all mice treated with anti-TNF antibody had developed morphometric analysis in the tibial metaphysis was performed swelling and/or erythema (100% incidence) by day 34, (18), using the image analyzing system as described above. The while 100% incidence was attained 1 day earlier in mice measurements were performed in a 0.76-mm2 area starting 0.15 mm from the proximal growth plate, using the KS400 treated with 4 mg/kg/day WP9QY. These data indicate image analyzing system as previously described (19), with some that the WP9QY peptide inhibited the severity of arthri- modifications. tis in a dose-dependent manner, but its efficacy in Statistical analysis. The Kruskal-Wallis test was per- inhibiting disease activity was lower compared with the formed for analysis of the arthritis score. The Mann-Whitney same dose of anti-TNF antibody in mice with CIA. U test was performed for analyses of the histologic score and cartilage score. The other data were analyzed by analysis of Similar effects of WP9QY peptide and anti-TNF variance. When an F test yielded significant results (P Ͻ 0.05), antibody on inhibition of articular bone destruction. groups were compared using Fisher’s protected least signifi- Next, the effect of the WP9QY peptide on bone erosion PROPERTIES OF ANTI-TNF␣ PEPTIDE IN MURINE CIA 1167

surface of the epiphysis of femur facing along the articular cavity at the knee joints (Figures 2A–E). Severe bone erosion was observed in vehicle-treated mice with CIA (Figure 2B). Almost no erosion was observed in the WP9QY- and anti-TNF–treated groups (Figures 2C–E), the same as in the vehicle-treated normal mice (Figure 2A). To compare the efficacy of the treatments in

Figure 1. Weak inhibitory effect of WP9QY peptide on inflammation compared with that of anti–tumor necrosis factor antibody (anti-TNF Ab) in mice with collagen-induced arthritis (CIA). CIA was induced by primary (day 0) and secondary (day 21) immunizations with bovine type II collagen in Freund’s complete adjuvant. Infusion pumps were implanted subcutaneously in mice with CIA on day 21. Shown are Figure 2. Inhibitory effects of WP9QY peptide on CIA-induced results in vehicle-treated mice with CIA (diamonds), mice with CIA periarticular bone erosion illustrated by microfocal computed tomo- treated with 2 mg/kg/day WP9QY peptide (circles), mice with CIA graphy (micro-CT) reconstruction images of knee joints. A–E, Three- treated with 4 mg/kg/day WP9QY peptide (squares), and mice with dimensional (3-D) micro-CT images of knee joints of femurs from CIA treated with 4 mg/kg/day anti-TNF␣ monoclonal antibody (trian- vehicle-treated normal (nonimmunized) mice (A), vehicle-treated mice gles). A, Arthritis score (clinical severity of arthritis). The maximum with CIA (B), mice with CIA treated with 2 mg/kg/day WP9QY possible score is 12, as described in Materials and Methods. Values are peptide (C), mice with CIA treated with 4 mg/kg/day WP9QY peptide ϭ P Ͻ 0.05 versus (D), and mice with CIA treated with 4 mg/kg/day anti-TNF␣ antibody ء .(the mean Ϯ SD (n ϭ 6–7 mice per group vehicle-treated mice with CIA; † ϭ P Ͻ 0.05 versus mice with CIA (E). Representative 3-D images from each group are shown. Bar ϭ 1 treated with anti-TNF antibody. B, Incidence of arthritis (percentage mm. F, Measurement area of the distal femoral epiphysis facing the of mice that developed CIA). articular cavity in vehicle-treated normal mice (filled white; the width of the measurement area is 0.25 mm). Bar ϭ 0.5 mm. G, The femoral bone area facing the articular cavity was measured using the sagittal ϭ P Ͻ ء .was investigated by micro-CT. Three-dimensional recon- micro-CT image of the femur. Values are the mean and SD struction images from the micro-CT scanning data re- 0.05 versus vehicle-treated normal mice; † ϭ P Ͻ 0.05 versus vehicle- vealed the degree of articular bone erosions on the treated mice with CIA. See Figure 1 for other definitions. 1168 SAITO ET AL

Figure 3. Periarticular bone erosion at knee joints inhibited equally by WP9QY peptide and anti-TNF antibody. A–E, Representative sagittal reconstructed microfocal computed tomography (micro-CT) images of knee joints from vehicle-treated normal (nonimmunized) mice (A), vehicle-treated mice with CIA (B), mice with CIA treated with 2 mg/kg/day WP9QY peptide (C), mice with CIA treated with 4 mg/kg/day WP9QY peptide (D), and mice with CIA treated with 4 mg/kg/day anti-TNF␣ antibody (E). Representative images from each group are shown. Bar ϭ 1 mm. F, Measurement area of the proximal end of the tibia facing the articular cavity in vehicle-treated normal mice (filled white; the width of the measurement area is 0.25 mm [arrows]). Bar ϭ 0.5 mm. Fem ϭ femur; Tib ϭ tibia. G, Tibial bone area was measured in the area shown in F using sagittal micro-CT images of the tibia. H, Mean bone thickness was calculated as described in Materials and Methods. I, The region of interest, 2.1 mm ϫ 2.1 mm (boxed area), at the knee joint for measuring bone mineral density (BMD) by dual x-ray absorptiometry was determined to exclude the secondary spongiosa of the tibial and femoral metaphysis (in vehicle-treated normal mice). Bar ϭ 1 mm. J, BMD at the knee joints. Values are ϭ P Ͻ 0.05 versus vehicle-treated normal mice; † ϭ P Ͻ 0.05 and †† ϭ P Ͻ 0.005 versus vehicle-treated mice with CIA. See ء .the mean and SD Figure 1 for other definitions. inhibition of bone destruction, quantitative analyses of scanning data at the distal end of the femoral epiphysis the bone area facing along the articular cavity were (Figure 2F). Significant reduction of bone area in performed using the sagittal images from micro-CT vehicle-treated mice with CIA was detected compared PROPERTIES OF ANTI-TNF␣ PEPTIDE IN MURINE CIA 1169

with vehicle-treated normal mice (Figure 2G). WP9QY treatment inhibited this reduction of bone area significantly and to the same extent as anti-TNF antibody treatment (Figure 2G). In a similar manner, the bone area facing along the articular cavity at the proximal end of the tibial epiphysis, which is shown in Figure 3F, was measured using a reconstructed micro-CT image, and the bone thickness was calculated. The quantitative analyses of bone area at the tibial epiphysis yielded results similar to those shown at the femoral epiphysis (Figures 2G and 3G). Measurement of bone thickness, which reflects the average thickness of the tibial epiphysis in the measurement area, also showed that WP9QY treatment blocked bone destruction at the tibial epiphysis to the same extent as did anti-TNF antibody (Figure 3H). To examine the overall changes in the periarticular bone at the knee joints, the BMD measurements were performed using the restricted ROI as shown in Figure 3I. BMD measurement by DXA also showed that the WP9QY treatments prevented bone resorption induced by CIA to the same extent as did anti-TNF treatment (Figure 3J). Inhibitory effects of WP9QY peptide on invasion of inflammatory cells, pannus formation, and cartilage destruction in the knee joints of mice with CIA. In order to confirm the results of micro-CT analyses showing the inhibitory effects of WP9QY treatment on bone destruction, the undecalcified sections were used for histologic analyses. TRAP staining with toluidine blue counterstaining was performed using sagittal sections from the Figure 4. Inhibition by WP9QY peptide of CIA-induced cartilage knee joints. Many TRAP-positive multinucleated cells destruction, pannus formation, bone destruction, and increase of osteoclast number/bone surface (NOc/BS) in the proximal end of the were observed on the bone surface close to the pannus in tibial epiphysis. Undecalcified sections were prepared as described in vehicle-treated mice with CIA compared with vehicle- Materials and Methods. A–C, Representative photomicrographs of treated normal mice (Figures 4A and B). In contrast, tibial epiphysis from vehicle-treated normal (nonimmunized) mice (A), there were fewer TRAP-positive multinucleated cells in vehicle-treated mice with CIA (B), and mice with CIA treated with 2 2 mg WP9QY–treated mice compared with vehicle- mg/kg/day WP9QY peptide (C). Tartrate-resistant acid phosphatase (TRAP) staining with toluidine blue counterstaining was used. Severe treated mice with CIA. The intensity of toluidine blue, bone erosion (arrows) and many TRAP-positive cells (arrowheads) which stains mucopolysaccharide containing proteogly- were observed in vehicle-treated mice with CIA. Bar ϭ 200 ␮m. D and can, the major matrix protein of cartilage, was lower at E, Histologic score (D) and cartilage score (E) were evaluated as the growth plate of the proximal tibiae in vehicle-treated described in Materials and Methods. F–H, Histomorphometric analy- mice with CIA compared with vehicle-treated normal ses were performed to calculate the rate of synovial pannus infiltration (the distance between arrows in B was considered as the length of the mice. WP9QY treatment reversed these changes in synovial pannus) (F), bone volume/total volume (BV/TV) (G), and staining intensity (Figures 4A–C), suggesting that carti- NOc/BS (H) in the proximal end of the tibial epiphysis as described in lage destruction was prevented by this treatment. Materials and Methods. Values are the mean and SD. # ϭ P Ͻ 0.05 ϭ P Ͻ 0.01 ءء ϭ P Ͻ 0.05 and ء ;To investigate the inhibitory effects of the versus vehicle-treated normal mice ؍ WP9QY peptide on disease activity in mice with CIA, versus vehicle-treated mice with CIA. W9 WP9QY peptide (see Figure 1 for other definitions). histologic assessment of the knee joints was performed. Analysis of H&E-stained sections of knee joints revealed that the histologic score, which reflects the abnormalities pared with vehicle-treated mice with CIA (Figure 4D). in the invasion of inflammatory cells, was reduced in a To examine the effects of the WP9QY peptide on knee dose-dependent manner in WP9QY-treated mice com- joint cartilage, histopathologic assessment of the knee 1170 SAITO ET AL

manner (Figure 4F). The effects of anti-TNF antibody were almost the same as those of 4 mg/kg/day WP9QY treatment. These results suggest that the WP9QY peptide inhibited inflammatory articular destruction at the knee joints in mice with CIA to the same extent as did the anti-TNF antibody at the same dose. To further investigate the effects of the WP9QY peptide on bone erosion accompanying CIA of the knee joints, histologic and histomorphometric assessments of the epiphysis of the proximal tibia were performed as described in Materials and Methods. The histomorphometric measurement showed that the BV/TV at the tibial epiphysis in vehicle-treated mice with CIA was significantly reduced compared with that in vehicle- treated normal mice, and this reduction in BV/TV was inhibited in a dose-dependent manner by WP9QY treatment (Figure 4G). NOc/BS at the tibial epiphysis was significantly increased in vehicle-treated mice with CIA compared with that in vehicle-treated normal mice. This increase in NOc/BS was also inhibited in a dose- dependent manner by WP9QY treatment (Figure 4H). Again, the effects of anti-TNF antibody treatment on bone volume and osteoclast parameters were almost the same as those of WP9QY peptide at the same dose. These results suggest that the WP9QY peptide prevented bone destruction in the knee joints of mice with Figure 5. Complete inhibition of CIA-induced systemic bone loss in CIA by inhibiting osteoclast formation to the same WP9QY-treated mice, but not in anti-TNF antibody–treated mice. extent as was observed with anti-TNF antibody treat- A–E, Representative sagittal images, obtained by microfocal computed tomography, of the tibial metaphysis from vehicle-treated normal ment. (nonimmunized) mice (A), vehicle-treated mice with CIA (B), mice Systemic bone loss in mice with CIA inhibited by with CIA treated with 2 mg/kg/day WP9QY peptide (C), mice with WP9QY peptide, but not apparently by anti-TNF anti- CIA treated with 4 mg/kg/day WP9QY peptide (D), and mice with CIA body. Since it has been reported that the disease activity ␣ ϭ treated with 4 mg/kg/day anti-TNF antibody (E). Bar 0.5 mm. F, of CIA causes marginal (systemic) bone loss as well as Measurement area at the tibial metaphysis in vehicle-treated normal mice (framed by solid line to exclude the primary spongiosa). a ϭ 0.3 periarticular bone destruction (20), the secondary spon- mm; b ϭ 1.1 mm; c ϭ 0.2 mm. G, Bone area in the secondary spongiosa giosa in the tibial metaphysis was analyzed using of the proximal tibia was assessed by using the measurement area micro-CT reconstruction images (Figures 5A–E). The ϭ P Ͻ 0.001 versus bone area at the secondary spongiosa was significantly ءء .shown in F. Values are the mean and SD ϭ Ͻ ϭ Ͻ vehicle-treated normal mice; † P 0.05 and †† P 0.005 versus reduced in vehicle-treated mice with CIA (Figure 5B). vehicle-treated mice with CIA. See Figure 1 for definitions. The WP9QY peptide inhibited the CIA-induced systemic bone loss in a dose-dependent manner (Figures joints was performed, using undecalcified sections 5C and D). The inhibition of this systemic bone loss by stained with toluidine blue. Cartilage destruction was WP9QY was much more apparent than that observed scored, and a significant reduction of cartilage damage with anti-TNF antibody treatment (Figure 5E). was observed in both WP9QY-treated groups compared To investigate whether the inhibition of this with vehicle-treated mice with CIA (Figure 4E). In order systemic bone loss by WP9QY peptide was accompanied to evaluate the pannus formation quantitatively, we by structural changes and the reduction of osteoclasts, calculated the rate of infiltration of synovial pannus standard histomorphometric analysis of the tibial me- along the proximal end of the tibial epiphysis. This taphysis was performed (Figure 6). The bone structure histomorphometric assessment revealed that the parameters (bone volume/total volume, trabecular thick- WP9QY peptide significantly inhibited CIA-induced ness, and trabecular number) were significantly de- pannus formation and infiltration in a dose-dependent creased in vehicle-treated mice with CIA compared with PROPERTIES OF ANTI-TNF␣ PEPTIDE IN MURINE CIA 1171

4 mg/kg/day anti-TNF antibody treatment were much weaker compared with those of WP9QY peptide at the same dose (Figures 6A–C). Another structure parameter, trabecular separation (TbSp), was significantly increased in vehicle-treated mice with CIA compared with vehicle-treated normal mice, but TbSp in both groups of WP9QY-treated mice was decreased in a dose-dependent manner compared with that in vehicle- treated mice with CIA. Anti-TNF antibody treatment significantly prevented the CIA-induced increase of TbSp, but the effect of anti-TNF antibody was not as great as that of WP9QY treatment (Figure 6D). The bone resorption parameters (NOc/BS, osteoclast surface/bone surface) were significantly increased in vehicle-treated mice with CIA compared with vehicle- treated normal mice. These increases in bone resorption parameters were significantly reduced in both groups of WP9QY-treated mice. Anti-TNF antibody treatment reduced bone resorption parameters as well, but to a lesser extent than was observed with WP9QY treatment (Figures 6E and F). These results suggest that the WP9QY peptide can inhibit cancellous bone loss in the tibial metaphysis by inhibiting osteoclast differentiation, and that its effects on systemic bone loss are much stronger than those of anti-TNF antibody when administered at the same dose.

DISCUSSION Blocking enhancement of immunologic responses and inhibiting inflammatory cytokines have become standard therapeutic strategies for rheumatoid arthritis (6). TNF␣ stimulates production of inflammatory cytokines such as IL-1, IL-6, and granulocyte– Figure 6. Prevention of cancellous bone loss by WP9QY peptide macrophage colony-stimulating factor in synovial cells, through inhibition of osteoclast differentiation at the marginal site and it enhances the production of RANKL, the oste- apart from the site of inflammation. Histomorphometric analysis of oclast differentiation factor, contributing to the aggra- bone structure parameters (A–D) and bone resorption parameters (E vating lesion of rheumatoid arthritis (21,22). Several and F) in the tibial metaphysis. Parameters were calculated as de- ␣ scribed in Materials and Methods. Shown are bone volume/total studies have shown that TNF -neutralizing therapy us- volume (BV/TV) (A), trabecular thickness (TbTh) (B), trabecular ing anti-TNF␣ monoclonal antibody and soluble TNFR number (TbN) (C), trabecular separation (TbSp) (D), osteoclast inhibits or even reverses inflammatory bone destruction surface/bone surface (OcS/BS) (E), and osteoclast number/bone sur- in rheumatoid arthritis (6,23,24). In the present study, face (NOc/BS) (F). WP9QY peptide treatment blocked the marginal we showed that the inhibitory effects of WP9QY pep- bone loss induced by CIA in a dose-dependent manner. Values are the ␣ ϭ tide, a TNF antagonist, on inflammation are weak ء ;mean and SD. # ϭ P Ͻ 0.05 versus vehicle-treated normal mice ,(ϭ P Ͻ 0.01 versus vehicle-treated mice with CIA. W9 compared with those of anti-TNF antibody (Figure 1 ءء P Ͻ 0.05 and ϭ WP9QY peptide (see Figure 1 for other definitions). but the effects of WP9QY peptide on bone destruction are similar to those of anti-TNF antibody (Figures 2–4), or even stronger in terms of inhibition of systemic bone vehicle-treated normal mice. The bone loss reflected by loss (Figures 5 and 6). these parameters was significantly blocked in both In a previous study, 10 nM anti-TNF antibody groups of WP9QY-treated mice, while the effects of showed effects similar to those of 25 mM WP9QY in 1172 SAITO ET AL

interfering with binding between TNF␣ and TNFR, WP9QY peptide works as a RANKL inhibitor as well as suggesting that a 2,500-fold greater concentration in a a TNF␣ antagonist. We also performed the RANKL- molar base is necessary for WP9QY peptide to achieve induced osteoclastogenesis assay, and the anti-TNF an- the same inhibitory effect as anti-TNF antibody (10). tibody infliximab did not inhibit osteoclastogenesis in Since the molecular weight of WP9QY peptide is ϳ1/ the dose range from 0.75 ␮M to 15 ␮M, while the 100 that of anti-TNF antibody, a 25-fold greater dose WP9QY peptide at 15 ␮M inhibited osteoclastogenesis seems to be necessary for WP9QY to generate the completely (data not shown). We have reported recently same neutralizing effect as anti-TNF antibody. Never- that the WP9QY peptide inhibits RANKL-induced sig- theless, WP9QY treatment using the same dose as that naling, osteoclastogenesis, and bone resorption activity. of anti-TNF antibody showed the same inhibitory effect Furthermore, bone resorption is inhibited by the on destruction of bone facing the articular cavities WP9QY peptide even under TNF-independent condi- (Figures 2 and 3), suggesting that the WP9QY peptide tions in studies using TNFR-deficient mice (34), suggest- could inhibit bone erosion by mechanisms other than ing that the TNF␣ antagonist WP9QY also works as a working as a TNF␣ antagonist. RANKL antagonist in this CIA model. It has been demonstrated that RANKL, the We have previously reported that the WP9QY osteoclast differentiation factor, has a pivotal role in peptide injected subcutaneously on days 21, 23, and 25 joint destruction in rheumatoid arthritis (25–27). The inhibits CIA-induced bone resorption, although the dose importance of RANKL is clearly highlighted by 2 stud- of the peptide was much higher than the doses used in ies. One study showed that osteoprotegerin (OPG), a the present study (35). A very high dose was used in the decoy receptor of RANKL, suppressed only CIA- previous study because of the short half-life of the induced bone destruction but not inflammation (28). peptide, and this was also the reason infusion pumps The other study showed that inflammation, but not bone were used to compare this peptide with anti-TNF anti- destruction, occurred when arthritis was induced in body at an appropriate dose level. Although the WP9QY RANKL-deficient mice by transplanting serum from peptide is only the template for development of small K/BxN mice, in which arthritis develops spontaneously molecular inhibitors and is not ready for clinical use, (29). TNF␣ also induces osteoclastogenesis (30,31); compared with investigations using a reagent that simply therefore, blocking the action of TNF␣ leads to the neutralizes TNF action it can be a useful tool for prevention of bone resorption, showing the clear inhibi- investigating the involvement of RANKL, since this tion of periarticular bone destruction in rheumatoid peptide blocks both TNF and RANKL. In the present arthritis. In the present study, however, the WP9QY study, the WP9QY peptide inhibited marginal bone loss peptide inhibited bone destruction to the same extent as while anti-TNF antibody did not, whereas anti-TNF anti-TNF antibody in spite of weak inhibition of inflam- antibody and this peptide almost equally blocked bone mation compared with anti-TNF antibody. Therefore, loss at the site of inflammation, suggesting that bone loss the possibility that the WP9QY peptide might inhibit at the marginal site occurs via a mechanism different bone resorption by working as a RANKL antagonist as from that at the site of inflammation. Our data indicate well as by blocking TNF␣ cannot be excluded in this CIA that the involvement of RANKL in bone resorption model. might be higher at the marginal site than at the site of Recently, it was shown that the simultaneous inflammation, which is not apparent from our previous administration of anti-TNF antibody and OPG, neutral- findings (35). izing both TNF␣ and RANKL, in arthritis models using A related study shows that the soluble protein TNF-transgenic mice or mice with CIA prevented the designed from the pre–ligand assembly domain (PLAD) systemic bone loss that could not be inhibited effectively of TNFRI ameliorates disease activity in the mouse by anti-TNF antibody alone. These results suggest that model of CIA (36). The half-life of our WP9QY peptide systemic bone loss (so-called “marginal osteoporosis”) would be shorter than that of this soluble PLAD protein accompanied by rheumatoid arthritis, at least in these (although investigators in that study claim that soluble arthritis models, cannot be prevented without blocking PLAD has a short half-life), since injection of WP9QY RANKL (32,33). In our study, micro-CT and histomor- peptide every other day does not work (data not shown), phometric analysis revealed that inhibition of CIA- while injection of soluble PLAD every other day is induced systemic bone loss by WP9QY was much more effective. The WP9QY peptide, however, is lower in apparent than that by anti-TNF antibody treatment molecular weight (ϳ1/30 that of soluble PLAD) and (Figures 5 and 6). These data strongly suggest that the blocks actions of both RANKL and TNF, while soluble PROPERTIES OF ANTI-TNF␣ PEPTIDE IN MURINE CIA 1173

PLAD inhibits only TNF action, although both of the 2. Pincus T. Long-term outcomes in rheumatoid arthritis. Br J reagents seem to be necessary to develop some suitable Rheumatol 1995;34 Suppl 2:59–73. 3. Choy EH, Panayi GS. Cytokine pathways and joint inflammation scaffold for clinical use in the future. in rheumatoid arthritis. N Engl J Med 2001;344:907–16. The anti-TNF antibody infliximab, used as a 4. Brennan FM, Chantry D, Jackson A, Maini R, Feldmann M. Inhibitory effect of TNF ␣ antibodies on synovial cell interleukin-1 positive control in the present study, is a chimeric production in rheumatoid arthritis. Lancet 1989;2:244–7. antibody of human and mouse, and its effects on mouse 5. Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell TNF␣ have not been clarified. Infliximab inhibited both 1996;85:307–10. 6. Lipsky PE, van der Heijde DM, St. Clair EW, Furst DE, Breedveld inflammation and bone destruction in the knee joints of FC, Kalden JR, et al, for the Anti–Tumor Necrosis Factor Trial in mice with CIA, indicating that it also has mouse TNF␣– Rheumatoid Arthritis with Concomitant Therapy Study Group. neutralizing activity. This may be caused by the struc- Infliximab and methotrexate in the treatment of rheumatoid ␣ arthritis. N Engl J Med 2000;343:1594–602. tural similarity between mouse TNF and human 7. Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleisch- TNF␣. mann RM, Fix RI, et al. A trial of etanercept, a recombinant In conclusion, WP9QY peptide was weak in tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. N Engl J Med 1999; inhibiting inflammation compared with the same dose of 340:253–9. anti-TNF antibody, but both agents were similar in their 8. Zhang X, Piatier-Tonneau D, Auffray C, Murali R, Mahapatra A, effect on bone destruction, suggesting that WP9QY Zhang F, et al. Synthetic CD4 exocyclic peptides antagonize CD4 ␣ holoreceptor binding and T cell activation. Nat Biotechnol 1996; peptide might have effects in addition to TNF inhibi- 14:472–5. tion. Furthermore, the inhibition of CIA-induced sys- 9. Zhang X, Gaubin M, Briant L, Srikantan V, Murali R, Saragovi U, temic bone loss by WP9QY was more apparent than that et al. Synthetic CD4 exocyclics inhibit binding of human immuno- ␣ deficiency virus type 1 envelope to CD4 and virus replication in T achieved with anti-TNF antibody. Since the TNF an- lymphocytes. Nat Biotechnol 1997;15:150–4. tagonist WP9QY has been proven to also be a RANKL 10. Takasaki W, Kajino Y, Kajino K, Murali R, Greene MI. Structure- antagonist (34), it could be more useful than reagents based design and characterization of exocyclic peptidomimetics ␣ that inhibit TNF ␣ binding to its receptor. Nat Biotechnol 1997; that simply block TNF , as a tool for understanding the 15:1266–70. role of TNF␣ in bone resorption in CIA. This peptide is 11. Banner DW, D’Arcy A, Janes W, Gentz R, Schoenfeld HJ, Broger a useful template for the development of small molecu- C, et al. Crystal structure of the soluble human 55 kd TNF receptor-human TNF ␤ complex: implications for TNF receptor lar inhibitors to prevent bone resorption both at the site activation. Cell 1993;73:431–45. of inflammation and at the marginal site in rheumatoid 12. Eck MJ, Sprang SR. The structure of tumor necrosis factor-␣ at arthritis by blocking both RANKL and TNF␣. 2.6 Å resolution: implications for receptor binding. J Biol Chem 1989;264:17595–605. 13. Iwai H, Kozono Y, Hirose S, Akiba H, Yagita H, Okumura K, et al. Amelioration of collagen-induced arthritis by blockade of ACKNOWLEDGMENTS inducible costimulator-B7 homologous protein costimulation. J Immunol 2002;169:4332–9. We thank Dr. Miyuki Azuma (Tokyo Medical and 14. Williams RO, Feldmann M, Maini RN. Anti-tumor necrosis factor Dental University, Tokyo, Japan) for helpful suggestions. We ameliorates joint disease in murine collagen-induced arthritis. also thank Drs. Hideyuki Iwai (Tokyo Medical and Dental Proc Natl Acad SciUSA1992;89:9784–8. University, Tokyo, Japan) and Hisashi Murayama (Kureha 15. Tsurukami H, Nakamura T, Suzuki K, Sato K, Higuchi Y, Nishii Y. Biomedical Research Laboratories, Tokyo, Japan) for techni- A novel synthetic vitamin D analogue, 2 ␤-(3-hydroxypropoxy)1 ␣, cal suggestions. 25-dihydroxyvitamin D3 (ED-71), increases bone mass by stimulating the bone formation in normal and ovariectomized rats. Calcif Tissue Int 1994;54:142–9. AUTHOR CONTRIBUTIONS 16. Bendele A, McAbee T, Sennello G, Frazier J, Chlipala E, McCabe D. Efficacy of sustained blood levels of interleukin-1 receptor Dr. Aoki had full access to all of the data in the study and antagonist in animal models of arthritis: comparison of efficacy in takes responsibility for the integrity of the data and the accuracy of the animal models with human clinical data. Arthritis Rheum 1999; data analysis. 42:498–506. Study design. Saito, Horne, Baron, Ohya, Aoki. 17. Seto H, Aoki K, Kasugai S, Ohya K. Trabecular bone turnover, Acquisition of data. Saito, Kojima, Takahashi, Ohya, Aoki. bone marrow cell development, and gene expression of bone Analysis and interpretation of data. Saito, Kojima, Horne, Baron, matrix proteins after low calcium feeding in rats. Bone 1999;25: Ohya, Aoki. 687–95. Manuscript preparation. Saito, Baron, Amagasa, Ohya, Aoki. 18. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Statistical analysis. Saito, Aoki. Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histo- morphometry Nomenclature Committee. J Bone Miner Res 1987; REFERENCES 2:595–610. 19. Aoki K, Didomenico E, Sims NA, Mukhopadhyay K, Neff L, 1. Chen E, Keystone EC, Fish EN. Restricted cytokine expression in Houghton A, et al. The tyrosine phosphatase SHP-1 is a negative rheumatoid arthritis. Arthritis Rheum 1993;36:901–10. regulator of osteoclastogenesis and osteoclast resorbing activity: 1174 SAITO ET AL

increased resorption and osteopenia in mev/mev mutant mice. bone erosion in collagen-induced arthritis. Am J Pathol 2002;161: Bone 1999;25:261–7. 1419–27. 20. Nishida S, Tsurukami H, Sakai A, Sakata T, Ikeda S, Tanaka M, et 29. Pettit AR, Ji H, von Stechow D, Muller R, Goldring SR, Choi Y, al. Stage-dependent changes in trabecular bone turnover and et al. TRANCE/RANKL knockout mice are protected from bone osteogenic capacity of marrow cells during development of type II erosion in a serum transfer model of arthritis. Am J Pathol collagen-induced arthritis in mice. Bone 2002;30:872–9. 2001;159:1689–99. 21. Tanaka S, Nakamura K, Takahasi N, Suda T. Role of RANKL in 30. Azuma Y, Kaji K, Katogi R, Takeshita S, Kudo A. Tumor necrosis physiological and pathological bone resorption and therapeutics factor-␣ induces differentiation of and bone resorption by oste- targeting the RANKL-RANK signaling system. Immunol Rev oclasts. J Biol Chem 2000;275:4858–64. 2005;208:30–49. 31. Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, 22. Feldmann M. The cytokine network in rheumatoid arthritis: Kotake S, et al. Tumor necrosis factor ␣ stimulates osteoclast ␣ definition of TNF as a therapeutic target. J R Coll Physicians differentiation by a mechanism independent of the ODF/RANKL- Lond 1996;30:560–70. RANK interaction. J Exp Med 2000;191:275–86. 23. Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff MH, 32. Redlich K, Gortz B, Hayer S, Zwerina J, Doerr N, Kostenuik P, et Keystone EC, et al. A comparison of etanercept and methotrexate al. Repair of local bone erosions and reversal of systemic bone loss in patients with early rheumatoid arthritis. N Engl J Med 2000; upon therapy with anti-tumor necrosis factor in combination with 343:1586–93. osteoprotegerin or parathyroid hormone in tumor necrosis factor- 24. Jones G, Halbert J, Crotty M, Shanahan EM, Batterham M, Ahern mediated arthritis. Am J Pathol 2004;164:543–55. M. The effect of treatment on radiological progression in rheu- 33. Saidenberg-Kermanac’h N, Corrado A, Lemeiter D, deVernejoul matoid arthritis: a systematic review of randomized placebo- ␣ controlled trials. Rheumatology (Oxford) 2003;42:6–13. MC, Boissier MC, Cohen-Solal ME. TNF- antibodies and osteo- 25. Fujikawa Y, Shingu M, Torisu T, Itonaga I, Masumi S. Bone protegerin decrease systemic bone loss associated with inflamma- resorption by tartrate-resistant acid phosphatase-positive multinu- tion through distinct mechanisms in collagen-induced arthritis. clear cells isolated from rheumatoid synovium. Br J Rheumatol Bone 2004;35:1200–7. 1996;35:213–7. 34. Aoki K, Saito H, Itzstein C, Ishiguro M, Shibata T, Blanque R, et ␣ 26. Kotake S, Sato K, Kim KJ, Takahashi N, Udagawa N, Nakamura al. TNF- receptor loop peptide mimic blocks RANK ligand- I, et al. Interleukin-6 and soluble interleukin-6 receptors in the induced signaling, bone resorption, and bone loss. J Clin Invest synovial fluids from rheumatoid arthritis patients are responsible 2006;116:1525–34. for osteoclast-like cell formation. J Bone Miner Res 1996;11: 35. Kojima T, Aoki K, Nonaka K, Saito H, Azuma M, Iwai H, et al. 88–95. Subcutaneous injections of a TNF-␣ antagonistic peptide inhibit 27. Takayanagi H, Oda H, Yamamoto S, Kawaguchi H, Tanaka S, both inflammation and bone resorption in collagen-induced mu- Nishikawa T, et al. A new mechanism of bone destruction in rine arthritis. J Med Dent Sci 2005;52:91–9. rheumatoid arthritis: synovial fibroblasts induce osteoclastogen- 36. Deng GM, Zheng L, Chan FK, Leonardo M. Amelioration of esis. Biochem Biophys Res Commun 1997;240:279–86. inflammatory arthritis by targeting the pre-ligand assembly do- 28. Romas E, Sims NA, Hards DK, Lindsay M, Quinn JW, Ryan PF, main of tumor necrosis factor receptors. Nat Med 2005;10: et al. Osteoprotegerin reduces osteoclast numbers and prevents 1066–72. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1175–1186 DOI 10.1002/art.22511 © 2007, American College of Rheumatology

Cell Therapy Using Allogeneic Bone Marrow Mesenchymal Stem Cells Prevents Tissue Damage in Collagen-Induced Arthritis

Andrea Augello,1 Roberta Tasso,2 Simone Maria Negrini,2 Ranieri Cancedda,3 and Giuseppina Pennesi2

Objective. Mesenchymal stem cells (MSCs) are nism of autoimmune arthritis using allogeneic MSCs. precursors of tissue of mesenchymal origin, but they However, further studies are required before these also have the capacity to regulate the immune response results can be translated to clinical settings. by suppressing T and B lymphocyte proliferation in a non–major histocompatibility complex–restricted man- Although mesenchymal tissue and the immune ner. Use of MSCs as immunosuppressant agents in system have different functions (skeletal support and autoimmune diseases has been proposed and success- defense of the organism, respectively), they share some fully tested in animal models. We explored the feasibil- characteristics, such as heterogeneity of their cellular ity of using allogeneic MSCs as therapy for collagen- components and the molecular mechanisms regulating induced arthritis, a mouse model for human their interactions (1). Experimental evidence suggests rheumatoid arthritis. that bone development and hematopoiesis are strictly Methods. DBA/1 mice were immunized with type intertwined processes; not only do hematopoietic pre- II collagen in Freund’s complete adjuvant, and some of cursor cells reside close to endosteal surfaces, but osteo- the animals received an intraperitoneal injection of progenitor cells produce many factors essential for the allogeneic MSCs. survival, renewal, and maturation of hematopoietic stem Results. A single injection of MSCs prevented the cells (HSCs), which are precursors of the cell compo- occurrence of severe, irreversible damage to bone and nents of the immune system (1,2). Osteoprogenitors are cartilage. MSCs induced hyporesponsiveness of T lym- part of the very heterogeneous population of mesenchy- phocytes as evidenced by a reduction in active prolifer- mal stem cells (MSCs), which reside primarily in the ation, and modulated the expression of inflammatory bone marrow but can be found in other tissue (e.g., fat) cytokines. In particular, the serum concentration of tumor necrosis factor ␣ was significantly decreased. and are capable of self-renewal and multilineage differ- MSCs exerted their immunomodulatory function by ed- entiation (3,4). Under appropriate stimulation, MSCs ucating antigen-specific Tregs. undergo orthodox differentiation into the 3 mesenchy- Conclusion. Our results suggest an effective new mal lineages: adipocytes, chondrocytes, and osteoblasts therapeutic approach to target the pathogenic mecha- (3,4). MSCs may also be induced experimentally to undergo unorthodox differentiation, i.e., neural and Supported by the Italian Ministry of University and Research myogenic cells (5–7). (grant FIRB RBNE01YANS). The heterogeneity of the MSC population is 1 Andrea Augello, PhD: National Institute for Cancer Re- reflected by the absence of a unique, specific molecular search, Genoa, Italy; 2Roberta Tasso, BS, Simone Maria Negrini, MD, PhD, Giuseppina Pennesi, MD, PhD: University of Genoa, Genoa, marker. MSCs derived from different tissues express Italy; 3Ranieri Cancedda, MD: National Institute for Cancer Research, developmental markers of mesenchymal, endothelial, and University of Genoa, Genoa, Italy. Address correspondence and reprint requests to Giuseppina and hematopoietic tissues (8–10), but they also produce Pennesi, MD, PhD, Department of Oncology, Biology, and Genetics, molecules directly involved in regulation of the immune University of Genoa, Largo Rosanna Benzi 10, 16132 Genoa, Italy. response, such as the costimulatory molecule CD28, E-mail: [email protected]. Submitted for publication March 17, 2006; accepted in revised the inhibitory molecules programmed death ligand 1 form January 4, 2007. (PDL-1) and PDL-2, and an array of different cyto-

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kines (11,12). Through these molecules, MSCs can reg- MATERIALS AND METHODS

ulate the immune response. It has been shown that q b Mice. DBA/1 (H-2 haplotype) and C57Bl/10 (H-2 MSCs suppress in vitro proliferation of T and B lympho- haplotype) mice, 6–8 weeks old, were purchased from Charles cytes, triggered by cellular stimuli, nonspecific mito- River (Calco, Italy). Green-fluorescent protein (GFP)– genic stimuli, or antigenic peptides. Immunologic re- transgenic mice of the C57Bl/6 background (H-2b haplotype) striction appears not to be involved, indicating that were kindly donated by Dr. Giuliana Ferrari (Telethon In- stitute for Gene Therapy, San Raffaele Hospital, Milan, Italy) third-party nonhistocompatible cells can be used to (for more information, see the following Web site: http:// suppress lymphocyte activation (11,13,14). In vitro jaxmice.jax.org). MSC-mediated immunoregulation also involves differ- Mice were bred and maintained at the institution’s entiation of alloantigen-induced dendritic cells activat- animal facility of the National Institute for Cancer Research, ing CD4ϩ, CD25ϩ T cell subsets (15). The immuno- Genoa, Italy. The care and use of the animals were in compliance with laws of the Italian Ministry of Health and the regulatory function of MSCs has also been observed in guidelines of the European Community. in vivo settings. In humans, treatment with MSCs im- Cells. MSCs were isolated from male and female proved the outcome of allogeneic transplantation by C57Bl/10 and GFP-transgenic mice. Bone marrow cells were promoting hematopoietic engraftment and limiting collected by flushing the cells out of femurs and tibiae with cold phosphate buffered saline (PBS). Cells were cultured at a graft-versus-host disease (GVHD) (16,17), while in an- concentration of 10 ϫ 106 nucleated cells per 10-cm Petri dish imal models, MSCs were successfully used to ameliorate in Coon’s modified Ham’s F-12 medium (Biochrom, Berlin, experimental autoimmune encephalomyelitis (18) and Germany) supplemented with 10% fetal calf serum (Gibco, prevent the recurrence of autoimmunity in lupus-prone Milan, Italy), 1% glutamine, and 1% penicillin–streptomycin (standard medium). No cytokines were added at any stage. mice (19). Cultures (stage P0) were incubated at 37°C in a 5% CO2 The activation and ending of the immune re- atmosphere. After 3 days, nonadherent cells were removed. sponse are tightly regulated by a set of connections When they reached 80% confluency in the dish, adherent cells involving regulatory and suppressive cytokines and cells were trypsinized (0.05% trypsin–EDTA at 37°C for 15 minutes) and expanded (stage P1). (20–22). When this network is not well balanced and the Randomly chosen MSCs from individual male or fe- immune response remains abnormally induced, a patho- male animals were pooled and tested for their capacity to form logic autoimmune reaction occurs. Autoimmune dis- colonies and for their ability to undergo differentiation into eases are the result of interactions between genes and chondrocytes, osteocytes, and adipocytes, as well as the MSC immunophenotype, as previously described (11). Only pools of environmental factors and can be experimentally in- MSCs showing the potential for full differentiation into mes- duced in susceptible animals. Collagen-induced arthritis enchymal lineages were selected and used for the experiments. (CIA) is an experimental autoimmune disease that can More than 20 different cell pools were used in the in vitro and be elicited in susceptible strains of rodents (rats and in vivo tests. After GFP-transgenic mice were killed, spleen cells mice) and nonhuman primates by immunization with were obtained by mechanical shredding and collected in RPMI type II collagen (CII), the major constituent protein of 1640 medium (Sigma, Milan, Italy) supplemented with articular cartilage. Following immunization in the ani- 2-mercaptoethanol, glutamine, nonessential amino acids, so- mals, an autoimmune polyarthritis develops that shares dium pyruvate, antibiotics (Sigma), and 1% normal mouse serum. several clinical and histologic features with rheumatoid CIA induction and clinical scoring. We immunized arthritis (RA). Susceptibility to CIA in rodents, and to male and female DBA/1 mice by injecting an emulsion of 100 RA in humans, is linked to the class II molecules of the ␮g of murine acid-soluble CII (Sigma) dissolved in 0.1N acetic ␮ major histocompatibility complex (MHC), and the im- acid and mixed with 50 l Freund’s complete adjuvant (CFA) (Sigma) at the base of the tail. An immunization boost was mune response to CII is characterized by both the given on day 21, by injecting 50 ␮g of murine acid-soluble CII stimulation of collagen-specific T cells and the produc- dissolved in 0.1N acetic acid and mixed with 25 ␮l CFA at the tion of high titers of specific antibodies (23). base of the tail. Given the success of cell therapy with MSCs for A first regimen of immunosuppressive cell therapy with MSCs was tested by cotreating, at the beginning of the the treatment of GVHD in humans (17), and for some experiment (day 0), DBA/1 mice with CII as described above autoimmune conditions in animal models (18,19), we and with an intraperitoneal injection of 100 ␮l of a cell 6 explored the feasibility of using MSCs as immunosup- suspension containing 5 ϫ 10 allogeneic MSCs from C57Bl/10 pressant agents in therapy for CIA, and also examined or GFP-transgenic mice. As a control, another group of CII-immunized mice was cotreated with a cell suspension of the mechanisms by which MSC-mediated immunosup- 5 ϫ 106 allogeneic splenocytes from GFP-transgenic mice in a pression occurs in in vivo settings. volume of 100 ␮l, injected intraperitoneally. ALLOGENEIC MSC THERAPY FOR CIA 1177

A second regimen of immunosuppressive cell therapy cytometry to evaluate the expression of CD4, CD25, and with MSCs was tested by treating CII-immunized DBA/1 mice CD27. with an intraperitoneal injection of 100 ␮l of a cell suspension To test the antigen specificity of CD4ϩ,CD25ϩ Tregs, containing 5 ϫ 106 allogeneic MSCs from C57Bl/10 or GFP- another set of DBA/1 mice was immunized with 100 ␮gof transgenic mice at the moment of the boost (day 21). At the bovine serum albumin (BSA) (Sigma) dissolved in 0.1N acetic end of the experiments (day 42), we killed the animals and acid and mixed with 50 ␮l CFA (Sigma). Some of the animals collected lymph nodes, splenocytes, peritoneum specimens, were treated with 5 ϫ 106 allogeneic MSCs from GFP- and limbs for further studies. transgenic mice. Tregs were isolated using the CD4ϩ,CD25ϩ Clinical scores were assessed on a 4-point scale com- Regulatory T Cell Isolation Kit (Miltenyi Biotec), as described bining clinical signs, such as limb redness, swelling, and above. deformities, and histologic characteristics of joint tissues, FoxP3 expression analysis. Total RNA was isolated particularly synovial infiltration and cartilage or bone destruc- from CD4ϩ,CD25ϩ T lymphocytes, pooled within each exper- tion. The scoring system was as follows: 0 ϭ no damage, 1 ϭ imental group, using an RNeasy Mini Kit (Qiagen, Milan, limb redness without histologic lesions, 2 ϭ limb swelling Italy). RNA was treated with DNase (RNase-Free DNase Set; without histologic lesions, 3 ϭ limb deformities with reversible Qiagen) to avoid contamination of genomic DNA. We used a histologic lesions, and 4 ϭ limb deformities accompanied by SuperScript II First-Strand Synthesis System (Invitrogen, San permanent histologic lesions such as bone or cartilage ero- Diego, CA) to synthesize complementary DNA. Real-time sions. Each limb of a mouse was scored separately, and the polymerase chain reaction (PCR) was performed using FoxP3- specific primers (forward 5Ј-CTCACCCCACCTACAGGCC- averaged score for each animal was calculated, using a modi- Ј Ј Ј fication of the method described by Thornton et al (24). 3 , reverse 5 -GGCATCCACAGTGGAGAGCT-3 ) and probe (5Ј-6-FAM-TCTCCAGGACAGACCACACTTCA Delayed-type hypersensitivity (DTH) response. Seven Ј days after the boost (day 28), the DTH response of immunized TGCAT-XTP-3 ). Gene expression levels were measured as the ratio of expression values and internal GAPDH (Rodent animals was tested by treating mice with a suspension of 10 ␮l GAPDH Control Reagents [VIC-labeled]; Applied Biosys- of CII administered intradermally into the pinna of one ear. tems, Monza, Italy). The other ear was injected similarly, but with medium (0.1N Proliferation assay. T lymphocytes were isolated from acetic acid diluted in PBS). Ear swelling was measured 48 the spleens of untreated or MSC-treated immunized DBA/1 hours later with a spring-loaded micrometer. mice, by negative selection using the Pan T Cell Isolation Kit Histologic and immunohistochemical analyses. (Miltenyi Biotec). In proliferation and blocking assays, re- Formalin-fixed limbs were decalcified and paraffin-embedded ϫ 5 ␮ sponder T lymphocytes (5 10 cells/well) were plated in a using standard histologic techniques. Serial 4- m sections were round-bottomed 96-well plate (Corning Life Sciences, Pero, cut and stained with hematoxylin and eosin to examine mor- Italy). We examined the proliferation of DBA/1 T cells in phologic features and assess the histologic arthritis score. response to stimulation with 0.2 ␮M phytohemagglutinin To detect the presence of allogeneic MSCs from (Sigma), 10 ␮g murine CII (Sigma), or 10 ␮g BSA (Sigma) in C57Bl/10 or GFP-transgenic mice, immunohistochemical ana- a final volume of 200 ␮l/well. Cell proliferation was tested in b lysis using an anti–class I H-2 monoclonal antibody (clone the presence or absence of 5 ϫ 105 ␥-irradiated allogeneic AF6-88.5; BD PharMingen, Milan, Italy) or rabbit polyclonal MSCs/well or 5 ϫ 105 Tregs/well, derived from CII- or anti-GFP antibody (Molecular Probes, Leiden, The Nether- BSA-immunized mice that were untreated or treated with lands) was performed on limbs, peritoneum sections, and allogeneic MSCs. spleens collected at different time points during the experi- Proliferation of Tregs in response to stimulation with ment (days 3, 7, 11, and 42). CII in the presence or absence of interleukin-2 (IL-2) was Primary antibody staining was followed by treatment tested by plating 5 ϫ 105 Tregs/well in a round-bottomed with biotinylated goat anti-mouse or anti-rabbit secondary 96-well plate (Corning) and stimulating them with 10 ␮gof antibody (Vector, Burlingame, CA) and peroxidase- murine CII (Sigma), 33 units of IL-2 (PeproTech, Rocky Hill, conjugated streptavidin (BD PharMingen) using 3-amino-9- NJ), or both in a final volume of 200 ␮l/well. The cultures were ethylcarbazole substrate (Sigma), generating a red precipitate. incubated for 60 hours and pulsed with 3H-thymidine (1.0 Images were captured by transmitted-light microscopy using a ␮Ci/well) (Amersham Biosciences, Cologno Monzese, Italy) Zeiss Axiovert 200M microscope equipped with a Zeiss Axio- for the last 18 hours. The cells were harvested, and cell Cam MRc color chilled 3CCD camera (Zeiss, Wetzlar, Ger- proliferation was evaluated by counting thymidine uptake, many). measured as counts per minute. Separation of Tregs. At the end of the experiment (day Cytokine profile. We tested IL-2, IL-4, IL-10, and 42), we collected spleens from CII-immunized DBA/1 mice interferon-␥ (IFN␥) concentrations in sera from blood of that were treated or untreated with allogeneic MSCs. Spleen MSC-treated and untreated immunized mice, using a mouse cells were obtained by mechanical shredding, collected in Th1/Th2 ELISA Ready-SET-Go! kit (eBioscience, San Diego, complete RPMI medium, and pooled within each experimen- CA) according to the manufacturer’s instructions. Serum con- tal group. We first isolated T lymphocytes using a Pan T Cell centrations of TNF␣ were also measured, using a mouse TNF␣ Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) ELISA Ready-SET-Go! kit (eBioscience) according to the by depletion of non–T cells, then we isolated CD4ϩ,CD25ϩ T manufacturer’s instructions. lymphocytes (Tregs) from this CD4ϩ cell–enriched population Concentrations of IL-2, IL-4, IL-10, IFN␥, and TNF␣ using a CD4ϩ,CD25ϩ Regulatory T Cell Isolation Kit (Milte- in supernatants of CII-primed T lymphocytes in vitro rechal- nyi Biotec). Cells were then counted and examined by flow lenged with CII were also assessed. Responder T lymphocytes 1178 AUGELLO ET AL

Figure 1. Disease severity in mice with collagen-induced arthritis. A, Disease scores in individual male DBA/1 mice after immunization with type II collagen (CII) and treatment with allogeneic mesenchymal stem cells (MSCs) injected on day 0 or day 21 after immunization or with an equal amount of allogeneic spleen cells. CII-immunized MSC-treated or untreated female DBA/1 mice were used as controls. Bars show the mean. B, Disease scores were assigned according to clinical and histologic evidence, as follows: 0 ϭ no damage, 1 ϭ limb redness without histologic lesions, 2 ϭ limb swelling without histologic lesions, 3 ϭ limb deformities with reversible histologic lesions, and 4 ϭ limb deformities accompanied by permanent histologic lesions such as bone or cartilage erosions. Each limb of a mouse was scored separately, and the average score for each animal was calculated. C, Delayed-type hypersensitivity (DTH) responses in MSC-treated or untreated CII- immunized mice. Values are the mean and SD.

(5 ϫ 105) were plated in each well of a round-bottomed 96-well response values (calculated as ⌬mm), the frequency of plate and stimulated with 10 ␮g of murine CII (Sigma) in the CD4ϩ,CD25ϩ,CD27ϩ T lymphocytes (calculated by flow presence or absence of 5 ϫ 105 ␥-irradiated allogeneic MSCs/ cytometric analysis of pooled spleen cell populations), FoxP3 well in a final volume of 200 ␮l. Supernatants were collected expression data (obtained using real-time PCR after calcula- and tested using mouse Th1/Th2 ELISA Ready-SET-Go! and tion of the averaged gene:internal GAPDH ratio), serum or ␣ mouse TNF ELISA Ready-SET-Go! kits (eBioscience) ac- supernatant concentrations of IL-2, IFN␥, IL-4, IL-10, and cording to the manufacturer’s instructions. TNF␣ (measured as pg/100 ␮l in 4 independent experiments), ␣ Concentrations of TNF were also measured in super- and cell proliferation rates (measured as counts per minute). natants of MSCs, cultured at a concentration of 5 ϫ 105/well and treated or untreated with 10 ␮g of murine CII (Sigma) in a final volume of 200 ␮l/well, using a mouse Th1/Th2 ELISA RESULTS Ready-SET-Go! kit and a mouse TNF␣ ELISA Ready-SET- Go! kit (eBioscience) according to the manufacturer’s instruc- Prevention of severe tissue damage in CII- tions. Reactions were read at 450 nm, obtained values were immunized mice by MSCs. Male and female DBA/1 normalized with given standard solutions, and concentrations q (pg/100 ␮l) were calculated. mice bearing the mouse MHC H-2 haplotype were Statistical analysis. A disease score was assessed for immunized with CII emulsified in CFA, to generate each treated animal, as previously described. The results of 5 CIA. In this model, male DBA/1 mice are described as independent immunization experiments were compiled, and being susceptible to disease induction, while female mice the statistical significance of observed differences among experimental groups was calculated by nonparametric Mann- fail to develop severe arthritis but show some of the Whitney U test. A 2-tailed t-test was used to evaluate the immunopathogenic features of disease (25) and thus statistical significance of observed differences between DTH served as a negative disease control. In our experiments, ALLOGENEIC MSC THERAPY FOR CIA 1179

all 27 immunized male DBA/1 mice (100%) showed signs of CIA, with a mean Ϯ SD disease score of 2.64 Ϯ 1.17, while 10 (71.42%) of 14 female mice showed significantly milder signs of disease (mean Ϯ SD disease score 1.21 Ϯ 1.13; P ϭ 0.0006) (Figure 1A), indicating that the vast majority of immunized male animals developed irreversible bone or cartilage erosions in at least 1 joint, while female mice showed only reversible signs of synovial inflammation without permanent tissue damage. Because data from the literature have shown that the immunosuppressant function of MSCs is exerted in a non–MHC-restricted manner (11,13,14,17), and that allogeneic MSCs might be more exploitable in clinical settings (17,26), we used allogeneic MSCs from C57Bl/10 mice (MHC H-2b haplotype) or GFP- transgenic mice (MHC H-2b haplotype) as immunosuppressant agents for CIA. We first intraperitoneally injected MSCs in both male and female mice at the time of immunization (day 0). The incidence (17 [70.83%] of 24 mice) and severity of disease were significantly reduced in MSC-treated male mice (mean Ϯ SD disease score 0.59 Ϯ 0.59) compared with the incidence and severity in mice that were immunized but not treated with MSCs Figure 2. Proliferation rate of T lymphocytes from mesenchymal stem (P Ͻ 0.0001), while MSC treatment did not cause any cell (MSC)–treated and untreated immunized mice upon mitogenic or antigen-specific stimulation. Results are representative of 3 indepen- significant change in disease severity in 6 (85.71%) of 7 ϭ Ϯ Ϯ dent experiments. Values are the mean and SD. PHA phytohemag- .ϭ P Ͻ 0.01 versus medium ء .female mice (mean SD disease score 1.15 0.87) glutinin; CII ϭ type II collagen compared with female mice that did not receive treatment with MSCs (P ϭ 0.9109) (Figure 1A). Immunosuppression appeared to be a specific property of MSCs, because when allogeneic spleen cells Effect of MSCs on lymphocyte priming. The were used for treatment of CIA, disease was exacerbated results of these experiments suggested that MSCs did in all 6 treated animals (mean Ϯ SD disease score 3.32 Ϯ not affect priming of antigen-specific T lymphocytes, 0.47; P Ͻ 0.0001) (Figure 1A). because the DTH response was positively recalled using To establish whether cell therapy with osteopro- murine CII in mice in all experimental groups (⌬mm genitor cells was also efficient when the pathogenic 0.03–0.13), and no statistically significant differences mechanisms of the disease were established, we intra- between groups were observed, albeit the response peritoneally injected MSCs in CII-immunized male tended to be less vigorous in MSC-treated mice (Fig- DBA/1 mice at the moment of the boost (day 21). At ure 1C). that time, the mice already showed clinical signs of MSC-induced hyporesponsiveness of T lympho- disease, such as redness and/or swelling of the joints, cytes. Lymphocyte proliferation experiments showed corresponding to a clinical score of 1–2 (Figure 1B). that treatment with allogeneic MSCs induced hypore- Treatment with MSCs prevented exacerbation of the sponsiveness of T lymphocytes from immunized mice. T disease; at the end of the experiment, only 7 (70%) of 10 lymphocytes from mice that were not treated with MSCs animals treated with MSCs at the moment of the boost actively proliferated when stimulated with PHA (P Ͻ showed signs of disease, with a mean Ϯ SD disease score 0.01 versus medium) and CII (P Ͻ 0.01 versus medium). of 1.05 Ϯ 1.29; the incidence and disease score were T cells from immunized mice that were treated with significantly lower than the incidence and disease score MSCs showed basal in vitro proliferation, mitogen- of mice that were immunized but did not receive treat- induced proliferation, and CII-recalled proliferation, all ment with MSCs (P ϭ 0.001) and were comparable with of which were significantly reduced compared with pro- the incidence and disease score of animals that received liferation of T lymphocytes from immunized mice that MSC therapy at the time of immunization (P ϭ 0.1590). did not receive MSCs and were cultured under the same 1180 AUGELLO ET AL

Figure 3. Cytokine profiles. A, Serum concentrations of interleukin-2 (IL-2), interferon-␥ (IFN␥), IL-4, IL-10, and tumor necrosis factor ␣ (TNF␣) at the end of the experiment, analyzed by enzyme-linked immunosorbent assay. Results are representative of 3 independent experiments. B, Supernatant concentrations of IL-2, IFN␥, IL-4, and IL-10 produced by type II collagen (CII)–primed T lymphocytes stimulated in vitro with CII in the presence or absence of mesenchymal stem cells (MSCs). The TNF␣ concentration was measured in supernatants of cultures of CII-primed T lymphocytes challenged in vitro with the immunizing antigen and cultured in the presence or absence of allogeneic MSCs. The expression profile of TNF␣ secreted by MSCs cultured in the presence or absence of CII is also shown. Results are representative of 3 independent experiments. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes .ϭ P Ͻ 0.03 ء .represent the 10th and 90th percentiles ALLOGENEIC MSC THERAPY FOR CIA 1181

Figure 4. Immunohistochemical analysis of peritoneum and spleens from DBA/1 mice treated with intraperitoneally injected mesenchymal stem cells from green fluorescent protein (GFP)– transgenic mice and collected on days 3, 7, and 11 after treatment. An anti-GFP monoclonal antibody was used for cell detection. Peritoneum and spleens from GFP-transgenic (GFP-Tg) mice were used as positive controls, and preimmune peritoneum and spleens from DBA/1 mice were used as negative controls. conditions (P ϭ 0.0005, P ϭ 0.0219, P ϭ 0.0019, presence of MSCs did not affect IL-2 concentrations, respectively) (Figure 2). tended to reduce IFN␥ and IL-4 concentrations, and Effect of MSC treatment on cytokine production significantly lowered concentrations of IL-10 (P ϭ in immunized mice. At the end of the experiment, we 0.0084) (Figure 3B). assessed serum concentrations of IL-2, IL-4, IL-10, TNF␣ is a pivotal cytokine in the pathogenesis of IFN␥, and TNF␣. In parallel, we checked the cytokine RA (27), and the effect of MSCs on TNF␣ production expression profiles in supernatants of CII-primed T during CIA appeared to be an extremely complex phe- lymphocytes that were rechallenged in vitro with the nomenon. The serum concentration of TNF␣ was sig- immunizing antigen, in the presence or absence of nificantly decreased in immunized mice treated with allogeneic MSCs. allogeneic MSCs (mean Ϯ SD TNF␣ concentration in As a result, we observed that serum concentra- MSC-treated mice 37 Ϯ 45 pg/100 ␮l versus 145 Ϯ 21 tions of IL-2 were not modified by the effect of MSCs pg/100 ␮l in untreated mice; P ϭ 0.0197) (Figure 3A). In (Figure 3A), while MSCs tended to decrease serum contrast, secretion of TNF␣ by in vitro–stimulated CII- concentrations of IFN␥, IL-4, and IL-10 (Figure 3A). primed T lymphocytes was significantly increased when These data paralleled the results obtained in an analysis they were cocultured with allogeneic MSCs (mean Ϯ SD of supernatants of lymphocyte cultures, in which the concentration 686 Ϯ 109 pg/100 ␮l) compared with 1182 AUGELLO ET AL

TNF␣ production in the absence of MSCs (mean Ϯ SD concentration 414 Ϯ 156 pg/100 ␮l; P ϭ 0.029) (Figure 3B). When we tested whether MSCs would produce TNF␣ if they were cultured in the presence of CII, we surprisingly observed that adding CII to the culture medium induced the secretion of significantly higher amounts of TNF␣ (mean Ϯ SD concentration 627 Ϯ 28 pg/100 ␮l) compared with baseline levels (mean Ϯ SD concentration 0.14 Ϯ 0.11 pg/100 ␮l; P Ͻ 0.0001) (Fig- ure 3B). Role of MSC viability in immunosuppression of CIA. We tracked the fate of MSCs in the recipient organism by the detection of H-2b–positive or fluorescence-labeled MSCs isolated from GFP- transgenic mice and injected in CII-immunized mice. At the end of the experiment, H-2b–positive, green- fluorescent cells were not detectable by immunohistochemistry in the joints of MSC-treated mice, suggesting that injected MSCs did not restore tissue integrity by mechanisms of tissue repair (data not shown [28,29]). At the end of the experiment, H-2b–positive, green-fluorescent cells were not evident at the site of injection, in the peritoneum, or in secondary lymphoid organs, such as the spleen (data not shown); however, we were able to detect these cells at intermediate time points during the course of the disease (Figure 4). Briefly, MSCs were found to colonize the peritoneum throughout the first week after the initiation of treatment (Figure 4). At the same time, they possibly started to circulate through the bloodstream, ending in the Figure 5. Immunophenotype of mesenchymal stem cell (MSC)– educated Tregs. Top, Frequency of CD4ϩ,CD25ϩ,CD27ϩ Tregs spleen, where they were found as cell ghosts 7 day after isolated from MSC-treated or untreated immunized mice. Results are treatment (Figure 4). At day 11, both the peritoneum representative of 1 experiment. Bottom, FoxP3 expression levels of and spleen were negative (Figure 4). CD4ϩ,CD25Ϫ or CD4ϩ,CD25ϩ T lymphocytes isolated from MSC- Mediation of MSC action by antigen-specific treated or untreated type II collagen (CII)–immunized mice. Expres- Tregs. To explore the possibility that the in vivo immu- sion levels were analyzed by real-time polymerase chain reaction. ϭ ء .Values are the mean and SD. CFA ϭ Freund’s complete adjuvant nosuppressant action of MSCs was mediated by activa- P Ͻ 0.0001. tion of a cascade of different cell types, i.e., Tregs, we compared the frequency of peripheral Tregs characterized by the CD4ϩ,CD25ϩ,CD27ϩ,FoxP3ϩ phenotype forkhead transcription factor FoxP3, a molecular marker (30) within spleen cell populations from mice immu- characterizing cells with an immunoregulatory function nized with CII with the frequency in spleen cells from (31). FoxP3 was expressed at significantly higher levels mice immunized with CII and treated with MSCs at the in CD4ϩ,CD25ϩ T lymphocytes than in CD4ϩ,CD25Ϫ beginning of the experiment. We observed that cells isolated from splenocytes of both MSC-untreated CD4ϩ,CD25ϩ,CD27ϩ T lymphocytes represented a and MSC-treated immunized mice (mean Ϯ SD relative mean Ϯ SD of 5.39 Ϯ 4.01% (range 1.19–9.57%) of the expression rates 6.8 Ϯ 0.3 versus 1.1 Ϯ 0.2 and 8 Ϯ 0.4 spleen cell populations of immunized and MSC-treated versus 0.7 Ϯ 0.1, respectively; P Ͻ 0.0001 for both mice, while they were virtually absent in the spleens of comparisons) (Figure 5). Moreover, Tregs from immu- mice receiving only immunization with CII (mean Ϯ SD nized mice treated with MSCs expressed significantly frequency 0.19 Ϯ 0.21% [range 0–0.37%]; P ϭ 0.0412) higher levels of messenger RNA for FoxP3 (mean Ϯ SD (Figure 5). relative expression rate 8 Ϯ 0.4) than immunized but We also assessed the expression rate of the untreated mice (relative expression rate 6.8 Ϯ 0.3; P ϭ ALLOGENEIC MSC THERAPY FOR CIA 1183

ited by the presence of Tregs from MSC-treated immunized mice (P ϭ 0.0053) (Figure 6). In order to countercheck the antigen specificity of MSC-educated Tregs, we immunized DBA/1 mice with BSA, treating some of the animals with allogeneic MSCs. We isolated Tregs from MSC-treated animals and performed proliferation assays, using CD4ϩ lymphocytes from BSA-treated animals as responder cells. These cells did not proliferate when stimulated with CII (P ϭ 0.4222 versus basal proliferation), but they proliferated when stimulated with BSA (P ϭ 0.0113 versus basal proliferation). Cell proliferation was significantly reduced when Tregs from BSA-immunized and MSC- treated animals were added (P ϭ 0.0366) but not when Tregs from CII-immunized and MSC-treated animals were added (P ϭ 0.3338) (Figure 6). CD4ϩ,CD25ϩ,CD27ϩ,FoxP3ϩ Tregs from immunized and MSC-treated mice did not proliferate when cultured in the presence of CII, but they actively proliferated in the presence of IL-2 (P Ͻ 0.0001), and proliferation tended to increase in the presence of IL-2 plus CII (P Ͻ 0.0001) (Figure 6). Figure 6. Antigen specificity of Tregs isolated from mesenchymal stem cell (MSC)–treated mice. A, Proliferation rates of type II collagen (CII)–primed T lymphocytes stimulated with the immunizing antigen DISCUSSION alone or in the presence of Tregs isolated from MSC-treated or untreated immunized mice. B, Proliferation rates of bovine serum Cell therapy for autoimmune disease began sev- albumin (BSA)–primed T lymphocytes challenged in vitro with CII or eral years ago when a consensus statement on use of BSA alone or cultured in the presence of Tregs from MSC-treated CII- HSC transplantation in the treatment of severe auto- or BSA-immunized mice. C, Proliferation rates of Tregs from CII- immunized MSC-treated mice challenged in vitro with CII or stimu- immune disease was published (32,33). However, a lated with interleukin-2 (IL-2) or cultured in the presence of CII plus broader application of stem cell therapy requires better ϭ P Ͻ 0.04 versus medium. understanding of how the use of adult stem cells can ء .IL-2. Values are the mean and SD modify the autoimmune process, interfering with hyper- activation of the host’s immune responses. Recent characterization of MSCs and their role 0.002). In contrast, CD4ϩ,CD25Ϫ T lymphocytes from in hematopoiesis (2) and immune modulation (34) sug- mice that were not treated with MSCs expressed higher gests their potential use for cell therapy (35,36). In levels of FoxP3 (mean Ϯ SD relative expression rate animal models, autologous MSCs have been successfully 1.1 Ϯ 0.2) than CD4ϩ,CD25Ϫ T lymphocytes from used to ameliorate experimental autoimmune encepha- MSC-treated mice (relative expression rate 0.7 Ϯ 0.1; litis (18), and therapy with allogeneic osteoprogenitor P ϭ 0.0124). cells prevented recurrence of autoimmunity in lupus- Proliferation and blocking assays showed that prone mice (19). Data in the literature showed that the Tregs isolated from the splenocytes of immunized and immunosuppressant function of MSCs is not MHC MSC-treated mice were antigen specific, being able to restricted (11,13,14,17), and that allogeneic MSCs might suppress the proliferation of primed lymphocytes in be more functional in clinical settings (17,26). There- response to stimulation with CII (Figure 6). In these fore, we used allogeneic MSCs from completely MHC- experiments, T lymphocytes from immunized mice were mismatched mice for the treatment of CIA. rechallenged in vitro with the specific antigen CII that A single intraperitoneal injection of allogeneic recalled a significant cell proliferation (P ϭ 0.0044). MSCs given at the moment of immunization with CII CII-induced lymphocyte proliferation was not blocked was sufficient to prevent the occurrence of bone and by Tregs from immunized mice that were not treated cartilage erosions in the joints of immunized mice. These with MSCs (P ϭ 0.1196), but it was significantly inhib- are permanent, irreversible lesions corresponding to the 1184 AUGELLO ET AL

most severe grade of disease and are never observed in cells or factors. Therefore, it is possible that MSCs lose MSC-treated mice. The observation that the DTH re- the ability to “educate” immunosuppressant effectors sponse to the immunizing antigen was preserved, albeit during the immortalization process, preserving their in reduced, in MSC-treated mice indicates that priming of vitro inhibitory activity, or that the TNF␣ produced by T lymphocytes occurred. the host as an antitumor response to the immortalized Together, the absence of severe tissue injuries cell line (39) could block the antiproliferative cascade and the presence of a positive DTH response suggest initiated in vivo by cells of mesenchymal origin. that MSCs might act by inhibiting the activation and The use of MSCs for cell therapy greatly relies on proliferation of tissue-specific autoreactive T cell clones. their capacity to engraft and survive long-term in the Furthermore, we determined whether the immunosup- appropriate target organs (40), contributing to the repair pressant function of MSCs could also be in effect when of injured tissues (28,29). Their plasticity and capacity to the mechanisms of disease are already established, by undergo orthodox and unorthodox differentiation pro- injecting allogeneic MSCs on day 21, together with the cesses allowed the use of MSCs, alone or combined with immunization boost, when the mice already showed biomaterials, for repair of tissues, such as bone (41), signs of disease. In this setting, MSCs were able to heart (42,43), kidney (44), lung (45,46), and brain (47). prevent the occurrence of severe lesions in the joints of However, in our experiments we found that MSC viabil- immunized mice. ity was not required for their long-term immunosuppres- A first report of cell therapy for CIA using sant action; MSCs were, in fact, detectable in the mesenchymal cell lines was not encouraging, showing recipient for no more than 10 days after treatment. that the use of these cells was not beneficial in curing During this time lag, they were able to “educate” other arthritis, and that the inflammatory milieu in the injured cells to inhibit the pathogenic immune reaction. tissues might reverse their immunomodulatory effect We observed that the in vitro proliferation rate of through activation of the TNF␣ inflammatory pathway T lymphocytes isolated from MSC-treated mice was (37). The discrepancy between those results and ours significantly lower than the proliferation rate of T cells could be attributed to several factors. The main differ- from immunized mice that did not receive MSCs. This ence is that Djouad et al used an immortalized mesen- result was evident either under basal conditions or when chymal cell line (37), while we used primary cultures of the proliferation was recalled by a mitogenic stimulus or mouse MSCs obtained after the first in vitro passage by challenge with the immunizing antigen. The observa- (11). The mesenchymal cell line used by Djouad et al tion that serum concentrations of the different cytokines was not efficient in blocking the antigen-specific immune were lower in MSC-treated animals strengthened the response in in vivo settings, despite the fact that its idea that MSCs down-regulated activation of the patho- inhibitory activity was preserved in vitro (37). Moreover, genic immune mechanism leading to tissue damage. when those investigators added TNF␣ to cocultures of Tregs play an important role in the prevention of splenocytes and immortalized mesenchymal cells, in autoimmunity, and it has been demonstrated that they vitro mimicking the inflammatory milieu occurring dur- modulate the severity of CIA (48,49). We found that ing CIA, they reverted the immunosuppressant activity MSC treatment in immunized mice induced prolifera- of MSCs. tion of antigen-specific clones of Tregs with a Our data, however, depicted a much more com- CD4ϩ,CD25ϩ,CD27ϩ,FoxP3ϩ phenotype, suggesting plex scenario, in which MSCs themselves produced high that the immunosuppressive activity of MSCs could be levels of TNF␣ when cultured in the presence of CII, in prolonged by the action of Treg clones that can be vitro mimicking the antigen-specific immunizing condi- activated by an antigen-specific stimulus. tions; even if MSCs produced the same high amounts of Current therapy for RA is directed toward dimin- TNF␣ also in the in vivo setting, the final effect of their ishing the inflammatory response and treating the se- action was an overall significant decrease in the TNF␣ quelae of uncontrolled inflammation. To date, it has not concentration in sera from cured animals. This might been possible to prevent the disease or to completely suggest that TNF␣ specifically secreted by MSCs tar- arrest the disease process through medical therapy (50). geted a particular type of cells bearing TNF receptor I, Our results represent an effective new therapeutic ap- leading to a paradoxical antiinflammatory action (38), or proach to target the pathogenic mechanism of auto- that the activity of TNF␣ eventually produced by MSCs immune arthritis using adult stem cells, although further would be, in the long run, counterbalanced by the studies are required before these results can be trans- function of other MSC-educated immunomodulatory lated to the clinical setting. ALLOGENEIC MSC THERAPY FOR CIA 1185

AUTHOR CONTRIBUTIONS hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004;33:597–604. Dr. Pennesi had full access to all of the data in the study and 17. Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, takes responsibility for the integrity of the data and the accuracy of the Uzunel M, et al. Treatment of severe acute graft-versus-host data analysis. disease with third party haploidentical mesenchymal stem cells. Study design. Augello, Pennesi. Lancet 2004;363:1439–41. Acquisition of data. Augello, Tasso, Negrini. 18. Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Analysis and interpretation of data. Augello, Tasso, Cancedda, Pen- Gerdoni E, et al. Mesenchymal stem cells ameliorate experimental nesi. autoimmune encephalomyelitis inducing T cell anergy. Blood Manuscript preparation. Cancedda, Pennesi. 2005;106:1755–61. Statistical analysis. Augello, Tasso, Pennesi. 19. Ishida T, Inaba M, Hisha H, Sugiura K, Adachi Y, Nagata N, et al. Requirement of donor-derived stromal cells in the bone marrow REFERENCES for successful allogeneic bone marrow transplantation: complete prevention of recurrence of autoimmune diseases in MRL/MP-lpr/ 1. Wein M, Jones D, Glimcher L. Turning down the system: coun- lpr mice by transplantation of bone marrow plus bones (stromal terregulatory mechanisms in bone and adaptive immunity. Immu- cells) from the same donor. J Immunol 1994;152:3119–27. nol Rev 2005;208:66–79. 20. Ciubotariu R, Colovai AI, Pennesi G, Liu Z, Smith D, Berlocco P, ϩ 2. Taichman RS. Blood and bone: two tissues whose fates are et al. Specific suppression of human CD4 Th cell responses to ϩ Ϫ intertwined to create the hematopoietic stem-cell niche. Blood pig MHC antigens by CD8 CD28 regulatory T cells. J Immunol 2005;105:2631–9. 1998;161:5193–202. ϩ ϩ 3. Bianco P, Riminucci M, Gronthos S, Robey P. Bone marrow 21. Shevach E. CD4 CD25 suppressor T cells: more questions than stromal stem cells: nature, biology, and potential applications. answers. Nat Rev Immunol 2002;2:389–400. Stem Cells 2001;19:180–92. 22. Khaled A, Durum S. Lymphocide: cytokines and the control of 4. Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow- lymphoid homeostasis. Nat Rev Immunol 2002;2:817–30. derived stem cells. Stem Cells 2004;22:487–500. 23. Seki N, Sudo Y, Mizuhara H, Orito K, Imasaki A, Ono S, et al. 5. Woodbury D, Schwarz E, Prockop D, Black I. Adult rat and Type II collagen-induced murine arthritis: induction of arthritis human bone marrow stromal cells differentiate into neurons. depends on antigen-presenting cell function as well as susceptibil- J Neurosci Res 2000;61:364–70. ity of host to an anticollagen immune response. J Immunol 6. Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford 1992;148:3093–9. T, Willing A, et al. Adult bone marrow stromal cells differentiate 24. Thornton S, Boivin G, Kim K, Finkelman F, Hirsch R. Heteroge- into neural cells in vitro. Exp Neurol 2000;164:247–56. neous effects of IL-2 on collagen-induced arthritis. J Immunol 7. Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H. Sciatic 2000;165:1557–63. nerve regeneration in rats induced by transplantation of in vitro 25. Adarichev VA, Nesterovitch AB, Bardos T, Biesczat D, Chan- differentiated bone-marrow stromal cells. Eur J Neurosci 2001;14: drasekaran R, Vermes C, et al. Sex effect on clinical and immu- 1771–6. nologic quantitative trait loci in a murine model of rheumatoid 8. Kuznetsov S, Mankani M, Gronthos S, Satomura K, Bianco P, arthritis. Arthritis Rheum 2003;48:1708–20. Robey P. Circulating skeletal stem cells. J Cell Biol 2001;153: 26. Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo 1133–9. S, et al. Cardiac repair with intramyocardial injection of allogeneic 9. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keenek CD, mesenchymal stem cells after myocardial infarction. Proc Natl Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells Acad SciUSA2005;102:11474–9. derived from adult marrow. Nature 2002;418:41–9. 27. Firestein GS. Evolving concepts of rheumatoid arthritis [review]. 10. Potian JA, Aviv H, Ponzio NM, Harrison JS, Rameshwar P. Nature 2003;423:356–61. Veto-like activity of mesenchymal stem cells: functional discrimi- 28. Gimeno MJ, Maneiro E, Rendal E, Ramallal M, Sanjurjo L, nation between cellular responses to alloantigens and recall anti- Blanco FJ. Cell therapy: a therapeutic alternative to treat focal gens. J Immunol 2003;171:3426–34. cartilage lesions. Transplant Proc 2005;37:4080–3. 11. Augello A, Tasso R, Negrini S, Amateis A, Indiveri F, Cancedda 29. Caplan AI. Review: mesenchymal stem cells, cell-based recon- R, et al. Bone marrow mesenchymal progenitor cells inhibit structive therapy in orthopedics [review]. Tissue Eng 2005;11: lymphocyte proliferation by activation of the programmed death 1 1198–211. pathway. Eur J Immunol 2005;35:1482–90. 30. Ruprecht CR, Gattorno M, Ferlito F, Gregorio A, Martini A, 12. Liu CH, Hwang SM. Cytokine interactions in mesenchymal stem Lanzavecchia A, et al. Coexpression of CD25 and CD27 identifies ϩ cells from cord blood. Cytokine 2005;32:270–9. FoxP3 regulatory T cells in inflamed synovia. J Exp Med 13. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni P, 2005;201:1793–803. Matteucci P, et al. Human bone marrow stromal cells suppress 31. Muthukumar T, Dadhania D, Ding R, Snopkowski C, Naqvi R, T-lymphocyte proliferation induced by cellular or nonspecific Lee JB, et al. Messenger RNA for FOXP3 in the urine of mitogenic stimuli. Blood 2002;99:3838–43. renal-allograft recipients. N Engl J Med 2005;353:2342–51. 14. Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E, 32. Tyndall A, Daikeler T. Autologous hematopoietic stem cell trans- et al. Bone marrow mesenchymal stem cells inhibit the response of plantation for autoimmune diseases. Acta Haematol 2005;114: naive and memory antigen-specific T cells to their cognate pep- 239–47. tide. Blood 2003;101:3722–9. 33. Hough RE, Snowden JA, Wulffraat NM. Haemopoietic stem cell 15. Maccario R, Podesta M, Moretta A, Cometa A, Comoli P, transplantation in autoimmune diseases: a European perspective Montagna D, et al. Interaction of human mesenchymal stem cells [review]. Br J Haematol 2005;128:432–59. with cells involved in alloantigen-specific immune response favors 34. Frank M, Sayegh M. Immunomodulatory functions of mesenchy- the differentiation of CD4ϩ T-cell subsets expressing a regulatory/ mal stem cells. Lancet 2004;363:1411–2. suppressive phenotype. Haematologica 2005;90:516–25. 35. Ikehara S. Successful allogeneic bone marrow transplantation: 16. Maitra B, Szekely E, Gjini K, Laughlin M, Dennis J, Haynesworth crucial roles of stromal cells in prevention of graft rejection. Acta S, et al. Human mesenchymal stem cells support unrelated donor Haematol 2001;105:172–8. 1186 AUGELLO ET AL

36. El-Badri NS, Maheshwari A, Sanberg PR. Mesenchymal stem cells Bone marrow stem cells contribute to repair of the ischemically in autoimmune disease [review]. Stem Cells Dev 2004;13:463–72. injured renal tubule. J Clin Invest 2003;112:42–9. 37. Djouad F, Fritz V, Apparailly F, Louis-Plence P, Bony C, Sany J, 45. Albera C, Polak JM, Janes S, Griffiths MJ, Alison MR, Wright et al. Reversal of the immunosuppressive properties of mesenchy- NA, et al. Repopulation of human pulmonary epithelium by bone mal stem cells by tumor necrosis factor ␣ in collagen-induced marrow cells: a potential means to promote repair. Tissue Eng arthritis. Arthritis Rheum 2005;52:1595–603. 2005;11:1115–21. 38. Zakharova M, Ziegler HK. Paradoxical anti-inflammatory actions 46. Wang G, Bunnell BA, Painter RG, Quiniones BC, Tom S, Lanson ␣ of TNF- : inhibition of IL-12 and IL-23 via TNF receptor 1 in NA Jr, et al. Adult stem cells from bone marrow stroma differen- macrophages and dendritic cells. J Immunol 2005;175:5024–33. tiate into airway epithelial cells: potential therapy for cystic 39. Parmiani G. Tumor-infiltrating T cells: friend or foe of neoplastic fibrosis. Proc Natl Acad SciUSA2005;102:186–91. cells? N Engl J Med 2005;353:2640–1. 40. Allers C, Sierralta WD, Neubauer S, Rivera F, Minguell JJ, 47. Lu D, Mahmood A, Wang L, Li Y, Lu M, Chopp M. Adult bone Conget PA. Dynamic of distribution of human bone marrow- marrow stromal cells administered intravenously to rats after derived mesenchymal stem cells after transplantation into adult traumatic brain injury migrate into brain and improve neurological unconditioned mice. Transplantation 2004;78:503–8. outcome. Neuroreport 2001;12:559–63. 41. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov S, Mukh- 48. Morgan ME, Flierman R, van Duivenvoorde LM, Witteveen HJ, achev V, Lavroukov A, et al. Repair of large bone defects with the van Ewijk W, van Laar JM, et al. Effective treatment of collagen- ϩ use of autologous bone marrow stromal cells. N Engl J Med induced arthritis by adoptive transfer of CD25 regulatory T cells. 2001;344:385–6. Arthritis Rheum 2005;52:2212–21. 42. Natsu K, Ochi M, Mochizuki Y, Hachisuka H, Yanada S, Yasu- 49. Kelchtermans H, De Klerck B, Mitera T, Van Balen M, Bullens D, naga Y. Allogeneic bone marrow-derived mesenchymal stromal Billiau A, et al. Defective CD4ϩCD25ϩ regulatory T cell func- cells promote the regeneration of injured skeletal muscle without tioning in collagen-induced arthritis: an important factor in patho- differentiation into myofibers. Tissue Eng 2004;10:1093–112. genesis, counter-regulated by endogenous IFN-␥. Arthritis Res 43. Peschle C, Condorelli G. Stem cells for cardiomyocyte regenera- Ther 2005;7:R402–15. tion: state of the art [review]. Ann N Y Acad Sci 2005;1047: 50. Choo-Kang BS, Hutchison S, Nickdel MB, Bundick RV, Leishman 376–85. AJ, Brewer JM, et al. TNF-blocking therapies: an alternative mode 44. Kale S, Karihaloo A, Clark P, Kashgarian M, Krause D, Cantley L. of action? Trends Immunol 2005;26:518–22. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1187–1197 DOI 10.1002/art.22492 © 2007, American College of Rheumatology

Selective Therapeutic Control of C5a and the Terminal Complement Complex by Anti-C5 Single-Chain Fv in an Experimental Model of Antigen-Induced Arthritis in Rats

Fabio Fischetti, Paolo Durigutto, Paolo Macor, Roberto Marzari, Renzo Carretta, and Francesco Tedesco

Objective. To determine the role of the terminal Conclusion. These 2 human anti-C5 scFv could complement complex (TCC) in the development of ex- represent potential therapeutic reagents to be used in perimental antigen-induced arthritis (AIA) and the patients with rheumatoid arthritis. In addition, the therapeutic effects of human anti-C5 single-chain Fv finding that TS-A 8 was as effective as TS-A 12/22 in (scFv). reducing disease severity suggests that the TCC is Methods. Two different anti-C5 scFv, one that mainly responsible for the joint inflammation and dam- inhibits both release of C5a and assembly of the TCC age observed in AIA. (TS-A 12/22) and another that selectively blocks formation of the TCC (TS-A 8), were injected at the onset of The complement system has been implicated in AIA. The effects of these scFv on disease severity were the development and amplification of the inflammatory evaluated for up to 21 days and compared with the process at the tissue level in various pathologic condi- effects of injection of an unrelated scFv. AIA was also tions, including rheumatoid arthritis (RA). Although the established in C6-deficient and C6-sufficient PVG rats pathogenesis of RA is multifactorial and involves the to obtain further information on the role of the TCC in contribution of both cell-mediated and antibody- this model. dependent tissue damage, complement activation has Results. TS-A 12/22 and TS-A 8 proved to be long been recognized as one of the initial events that equally effective in reducing joint swelling, cell counts occur in the acute phase of this disease and may also be and tumor necrosis factor ␣ levels in synovial lavage responsible for amplification of the inflammatory pro- fluids, and the degree of histomorphologic changes cess during progression of RA (1–3). Complement acti- compared with the effects of the unrelated scFv. TS-A 12/22 and TS-A 8 prevented the deposition of C9 but not vation products are detected in the circulation of pa- that of C3, confirming the ability of the 2 scFv to tients with RA, and the levels of these products have neutralize C5. Administration of the 2 anti-C5 scFv been shown to correlate with disease activity (4–8). The after AIA onset also reduced disease severity. In C6- contribution of the complement system to inflammation- deficient rats with AIA, disease activity was reduced related damage of the synovial membrane in these markedly compared with that in C6-sufficient rats. patients is further supported by the finding of complement activation products in synovial fluid and also by the detection of complement deposits in synovial tissue Supported by the Italian Ministry of University and Research during the chronic phase of RA (3,9–19). (grant COFIN-2005 060371-005). Recognition that the complement system plays an Fabio Fischetti, MD, Paolo Durigutto, BSc, Paolo Macor, BSc, Roberto Marzari, PhD, Renzo Carretta, MD, Francesco Tedesco, important role in the development of synovitis in pa- MD: University of Trieste, Trieste, Italy. tients with RA has therapeutic implications, because Drs. Marzari and Tedesco hold a patent (no. MI2002 A001527) for TS-A 12/22. antibodies or synthetic compounds that neutralize com- Address correspondence and reprint requests to Francesco plement components may be added to the growing list of Tedesco, MD, Department of Physiology and Pathology, University of biologic response modifiers that inhibit the effect of Trieste, via Valerio 28, 34127 Trieste, Italy. E-mail: [email protected]. Submitted for publication November 20, 2006; accepted in tumor necrosis factors (TNFs) and other cytokines (20– revised form December 31, 2006. 29). In this regard, animal models of arthritis, which

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resemble RA in many ways, have been a valuable tool to antagonist had a beneficial effect on the development of elucidate the beneficial effect of anticomplement ther- AIA, reducing the severity of arthritis. Similar results apy. Collagen-induced arthritis (CIA) and antigen- were obtained in a model of CIA triggered by antibodies induced arthritis (AIA) are 2 such models, which share to type II collagen, in which the role of C5a in the with RA the features of an inflammatory synovitis development of arthritis was confirmed by the observa- characterized by synovial hyperplasia and leukocyte in- tion that the disease course was milder in C5aR- filtration and, as in the chronic phase of RA, pannus deficient mice compared with wild-type mice (36). formation and cartilage erosions (23,30–32). These conflicting results leave open the question Several lines of evidence indicate that comple- of the relative contributions of C5a and the membrane ment plays a role in the pathophysiology of CIA. Thus, attack complex to joint injury. This is an important point the arthritis induced following intravenous injection of to consider when designing a therapeutic strategy to antibodies directed against type II collagen was found to control the proinflammatory effects of the biologically be milder in C3-deficient and factor B–deficient DBA/1J active products of the terminal complement compo- mice compared with control mice. These findings sug- nents. We addressed this issue using 2 anti-human C5 gest that both classical and alternative pathways of single-chain Fv (scFv) to prevent the development of complement activation contribute to development of the acute synovitis or reduce its progression to a chronic inflammatory process (2). The observation that C5 phase in a rat model of AIA. One of these scFv deficiency partially protects susceptible DBA/1 mice selectively blocks assembly of the membrane attack against CIA is an indication that proinflammatory bio- complex, thus providing useful information on the role logically active products released from the terminal played by the complex. Our aim was to compare the complement components may be involved in develop- effect of this scFv with that of another anti-C5 scFv that, ment of the inflammatory process (33). This view is also conversely, inhibits both the release of C5a and the supported by the finding that a monoclonal antibody formation of the membrane attack complex, and with (mAb) to murine C5 prevented the onset of CIA and the clinical manifestations of AIA in C6-deficient rats, greatly reduced the clinical severity of established arthri- which are unable to form the membrane attack complex. tis in a susceptible DBA/1LacJ mouse strain (20,34). An additional aim was to investigate the effect of the 2 Additional information on the role played by late scFv on rats with established arthritis, to mimic the complement components in promoting joint inflamma- situation that can be encountered in patients with RA. tion was obtained in a rat model of acute arthritis triggered by an intraarticular injection of neutralizing MATERIALS AND METHODS antibody against rat CD59 (22). The clinical and histologic findings of a marked inflammatory process induced Animals. Wistar rats were obtained from a colony kept in the animal house at our university. C6ϩ/ϩ PVG rats were by this treatment in the rat joint, associated with local Ϫ Ϫ purchased from Harlan Italy (Corezzano, Italy), and C6 / deposition of the membrane attack complex on the PVG rats were from a previously described rat colony (37) that synovial surface, led those investigators to suspect that was established in our animal house. Male animals weighing the terminal complement complex (TCC) was responsi- 230–260 gm were used in this study. ble for the synovial injury. This assumption was further The in vivo experiments were performed in compliance strengthened by the observation that AIA was more with the guidelines of European (86/609/EEC) and Italian Ϫ/Ϫ ϩ/ϩ (D.L.116/92) laws and were approved by the Italian Ministry of severe in CD59a mice than in CD59a control University and Research and by the University Institutional mice, and that local reconstitution of CD59 with Committee. All treatments were performed under total anes- membrane-targeted recombinant rat CD59 markedly thesia induced with 25 mg/kg bromoethanol (Avertin; Sigma, reduced disease severity in CD59aϪ/Ϫ mice (35). More- St. Louis, MO). over, the failure of a peptide antagonist of the C5a Preparation and functional characterization of human scFv antibodies. Human scFv antibodies against C5 were receptor (C5aR) to suppress the arthritis induced by a obtained from a human antibody library and tested for their neutralizing antibody to rat CD59 tends to exclude a reactivity against C5 and for their neutralizing activity, follow- major role of C5a in promoting inflammation and ing a procedure previously described in detail (28). Briefly, instead points to the membrane attack complex as the purified scFv were obtained by affinity chromatography of the critical mediator of synovitis (24). bacterial extract on nickel–nitrilotriacetic acid resin (Qiagen, Milan, Italy), which selectively binds the 6 histidines at the Using a different peptide antagonist of the C5aR C-terminus of the scFv fragment. Hemolytic assay of C5 was in a rat model of AIA, Woodruff and colleagues (29) performed using sheep red blood cells sensitized with a reached a different conclusion, showing that the C5aR subagglutinating amount of rabbit IgM (EA) and then incu- HUMAN ANTI-C5 scFv AS THERAPEUTIC REAGENTS IN RA 1189

bated with a 1:10 dilution of C5-deficient serum in glucose– lytic activity of C5 present in 2 ml of rat serum. This amount of veronal buffered saline (GVBS). Fifty microliters of 1% EA the scFv was expected to be well above the level of C5 in the bearing classic C5 convertase (EAC1-3b) was incubated in the synovial lavage fluid. presence of 100 ␮l of C5-deficient serum (1:100 dilution) and In a second set of experiments, the same intraarticular 50 ng of C5 to a final volume of 250 ␮l, using GVBS for 30 injections of the 2 anti-C5 scFv were administered after the minutes at 37°C. Erythrocytes were then removed by centrif- development of arthritis, and the effects of these injections ugation, and hemoglobin release was measured at 405 nm to were compared with those induced by Un-scFv or saline. Five calculate the percentage of lysis. The levels of C5a in the days after arthritis induction, in 3 groups of 6 rats each, TS-A8 supernatant were measured by enzyme-linked immunosorbent scFv, TS-A12/22 scFv, or Un-scFv, respectively, was injected assay (ELISA) using mAb C17/5 and biotin-labeled G25/2, as intraarticularly, while another group of 3 arthritic rats was described by Oppermann et al (38). The amount of TCC was treated with saline and served as a positive control. The 3 other evaluated by ELISA using mAb aE11 (kindly provided by Prof. preimmunized rats received sequential intraarticular injections S. Meri, Helsinki University, Helsinki, Finland) and biotin- of only saline, thus serving as negative controls. labeled anti-C5 IgG. Alkaline phosphatase–conjugated Measurement of TNF␣ concentrations in synovial streptavidin (Sigma-Aldrich, Milan, Italy) was then used, ac- lavage fluids. Undiluted samples of lavage fluids were assayed cording to published procedures (39). for TNF␣ levels, using an ELISA kit (Euroclone, Milan, Italy). Induction of AIA. The animals received 2 intradermal Concentrations of TNF␣ in the samples were determined by injections of an emulsion containing an equal volume of 100 ␮g linear regression analysis of the standard curve. methylated bovine serum albumin (mBSA) in 200 ␮l of sterile Immunohistochemical analysis. Tissue deposition of saline and Freund’s complete adjuvant (CFA) (both from C3 was assessed on frozen sections incubated with goat anti-rat Sigma-Aldrich), at the base of the tail. Fourteen days after the C3 IgG at a dilution of 1:200 (Cappel, ICN Biomedicals, second injection, arthritis was induced by intraarticular admin- Milan, Italy) for 60 minutes at room temperature and further istration of mBSA (100 ␮gin100␮l of saline) into the right exposed to fluorescein isothiocyanate (FITC)–labeled rabbit Ј knee, while saline was injected into the left knee, which served F(ab )2 anti-goat IgG at a dilution of 1:200 (Southern Biotech- as a control. nology, Birmingham, AL) for an additional 60 minutes at room Animals were examined for the development of arthri- temperature. A similar approach was followed to examine tis by measuring swelling of the right and left knees with a synovial tissue for the presence of C9, using rabbit anti-rat C9 plethysmography device (Ugo Basile, Varese, Italy). At differ- IgG (a kind gift from Prof. P. Morgan, Cardiff, UK) at a ent time points after arthritis induction (1, 3, 7, 14, and 21 1:1,000 dilution, followed by biotin-labeled goat anti-rabbit days), the animals were killed, and the periarticular tissue of IgG (Sigma-Aldrich) at a 1:400 dilution, and FITC-labeled the knee joints was gently removed to expose the intraarticular streptavidin (Dako, Milan, Italy) at a 1:50 dilution. cavity for lavage with 2 ml of saline. A ZB1 Coulter Counter Histologic evaluation. Serial sections (6–8 ␮m) of (Beckman Coulter, Luton, UK) was used to measure the total paraffin-embedded specimens were cut and stained with hema- cell count in the synovial lavage fluid, and the number of toxylin and eosin. Two observers (FF, PD) examined the slides polymorphonuclear neutrophils (PMNs) was assessed by mea- for signs of tissue damage, and a cumulative score was attrib- suring their myeloperoxidase content, as previously reported uted after evaluation of the following 3 parameters: thickness (28). The same fluids were centrifuged to remove the cells and and hyperplasia of synovial membrane, scored as 0 (thin layer used to evaluate TNF␣ levels. of 1–2 lines of cells on adipose-rich tissue), 1 (focal hyperpla- Part of the anterior capsule of the joint was removed, sia, revealed by the detection of 2–3 lines of flat or round cells embedded in OCT compound (Miles, Milan, Italy), snap- randomly distributed over thickened fibroadipose tissue), 2 frozen in liquid nitrogen, and kept at Ϫ80°C until used for (diffuse hyperplasia, assessed by the appearance of a continu- immunofluorescence analysis. The knee joints were fixed for 6 ous layer of 2–5 lines of cells over thickened connective tissue days in 10% buffered formalin, decalcified for 5 days in associated with a Ͼ50% reduction in the amount of adipose Decalcifier I (SurgiPath, Richmond IL [distributed by Bio- tissue), or 3 (severe hyperplasia, characterized by the presence Optica, Milan, Italy]), and embedded in paraffin. of Ն5 lines of synovial cells covering areas of completely Study design and treatment groups. The therapeutic deranged tissue, where adipose tissue was substituted almost activity of the anti-C5 scFv was evaluated in 2 sets of experi- completely by fibrous, dense connective tissue); leukocyte ments. In the first set of experiments, either TS-A 12/22 or infiltration in synovial tissue, scored as 0 (no infiltration), 1 TS-A 8 (each at a dose of 400 ␮g/50 ␮l saline) was added to (scattered cells), 2 (focal infiltration in up to 50% of the mBSA (100 ␮g/50 ␮l saline) and injected intraarticularly (at synovial tissue), or 3 (diffuse infiltration, with or without the time of arthritis onset) into 2 groups of 30 rats each. follicular aspects); and cartilage erosions, scored as 0 (no Following arthritis onset, 6 rats per group were killed in order erosions), 1 (scanty erosions, present in Ͻ25% of the cartilage to evaluate disease at each of the considered time points. Two surface), or 2 (erosions detected on Ͼ25% of the visible additional groups of rats received an intraarticular injection of cartilage surface). mBSA (100 ␮g/100 ␮l saline) (n ϭ 20 rats) or mBSA (100 Statistical analysis. Results are expressed as the ␮g/50 ␮l saline) mixed with an unrelated human anti-gliadin mean Ϯ SD. Data were compared by analysis of variance using scFv (Un-scFv) at a dose of 400 ␮g/50 ␮l saline (n ϭ 30 rats). post hoc analysis for paired multiple comparisons, with Fish- Another group of preimmunized rats (n ϭ 20) was treated er’s corrected t-test. A nonparametric Mann-Whitney test was intraarticularly with saline alone and served as negative con- used to determine the significance of differences between trols. The scFv were injected at a concentration that, in tissue damage scores in the tested groups. P values less than or preliminary experiments, was shown to inhibit completely the equal to 0.05 were considered significant. 1190 FISCHETTI ET AL

Figure 1. Inhibition of C5a release, terminal complement complex (TCC) formation, and complement activity by the anti-C5 single-chain Fv (scFv). Purified human C5 was activated by the C5 convertase expressed on EAC1-3b in the presence of either TS-A 8 or TS-A 12/22 or an unrelated anti-gliadin scFv (Un-scFv) and C5-deficient serum. Formation of C5a and the TCC was measured by enzyme-linked immunosorbent assay (A), and complement hemolytic activity was expressed as the percentage of red cell lysis (B). Details of the experimental procedure are provided in Materials and Methods. Values are the mean and SD. OD ϭ optical density.

RESULTS intraarticular injections of TS-A 8, either at the onset of arthritis or after its induction, were evaluated and com- Inhibition of functional activity of C5 by scFv pared with those induced by TS-A 12/22 as well as those TS-A 12/22 and TS-A 8. TS-A 12/22 was identified in a obtained in animals treated with mBSA alone or mBSA previous study (28) and was characterized as an scFv that reacts with C5 at the cleavage site of the C5 plus Un-scFv. convertases of both the classical and alternative path- In the group of mBSA-treated animals, the mod- ways. This was shown by the total loss of C5 lytic activity ifications observed in knee joint swelling, total cell ␣ and also by the ability of the scFv to inhibit generation of counts and TNF concentrations in synovial lavage C5a and the TCC. The same human library was screened fluids, as well as the histology damage score appeared to for the selection of TS-A 8, which showed strong reac- be comparable with those observed in rats treated with tivity for C5 when examined by ELISA. TS-A 8 and mBSA plus Un-scFv (data not shown). Therefore, only TS-A 12/22 shared the ability to inhibit the lytic activity data from the group of animals treated with mBSA plus of C5. However, the 2 scFv differed in terms of the Un-scFv were chosen as reference values for further functional consequences of C5 inactivation, as assessed statistical comparisons. by the quantitation of C5a and the TCC released from In rats treated with mBSA plus Un-scFv, joint activated C5. TS-A 8, when added to the incubation swelling was already apparent 1 day after the intraartic- mixture containing C5 and the source of C5 convertase, ular injection and peaked on day 3 (Figure 2, top), with prevented the formation of the TCC but did not affect a gradual decrease over the next 3 weeks. The 2 intraar- the release of C5a (Figure 1A). Conversely, C5a and the ticularly injected anti-C5 scFv were equally effective in TCC were almost undetectable when C5 was activated reducing joint swelling to levels slightly higher than by the C5 convertase in the presence of TS-A 12/22. As those observed in rats receiving saline. The maximal previously demonstrated for TS-A 12/22 (28), TS-A 8 increase in knee diameters was observed on day 1 was also able to neutralize rat C5 inhibiting the postinjection, with a decline to control values on day 6. complement-dependent hemolytic activity of rat serum Analysis of the cell content in lavage fluid from the rat (Figure 1B). knee injected with Un-scFv plus mBSA revealed a sharp Effect of scFv TS-A 12/22 and TS-A 8 on AIA. increase in cell counts (Figure 2, middle). The highest Having shown that TS-A 8 selectively inhibited assembly value was reached on day 1 postinjection, with a proof the TCC and did not interfere with the release of C5a gressive decline to a low level on day 6. The majority of from activated C5, we considered that this scFv was a cells collected in lavage fluids from the joint were PMNs. suitable reagent to evaluate the contribution of the Administration of TS-A 8 led to a significant decrease in complement complex to the development of joint in- the PMN count, varying from 45% to 65% in the first 3 flammation in AIA. For this purpose, the effects of days postinjection. The inhibitory effect of TS-A 12/22 HUMAN ANTI-C5 scFv AS THERAPEUTIC REAGENTS IN RA 1191

Figure 2. Effect of TS-A 8 and TS-A 12/22 on the development of antigen-induced arthritis in rats. Joint swelling and total cell counts and levels of tumor necrosis factor ␣ (TNF␣) in synovial lavage fluids were assessed at different time points following the intraarticular injection of methylated bovine serum albumin (mBSA) alone (100 ␮gin100␮l of sterile saline) or mBSA mixed with either TS-A 8, TS-A 12/22, or an unrelated human anti-gliadin single-chain Fv (Un-scFv), to a final concentration of 400 ␮g/100 ␮l. A fourth group of rats that were preimmunized with mBSA and received an intraarticular injection of 100 ␮l of saline alone served as a negative control. In the groups treated with TS-A 12/22, TS-A 8, and Un-scFv, 6 rats were studied at each time point; in the saline-treated group, 4 rats were examined at each time point. In another group of 20 animals, in which mBSA alone was intraarticularly injected (results not shown), the modifications observed in knee joint swelling and total cell counts and TNF␣ concentrations in the synovial lavage fluids were comparable with those observed in the rats treated with mBSA plus Un-scFv. Values ;ϭ P Ͻ 0.05 ء .are the mean and SD. Arrow indicates the time of arthritis induction .ϭ P Ͻ 0.01 versus mBSA plus Un-scFv; § ϭ P Ͻ 0.05 versus saline ءء

was slightly higher on day 1 postinjection but did not Histologic and immunofluorescence findings. differ from that of TS-A 8 on day 3. The inflammatory Histologic analysis of the joints obtained from Un-scFv– process was also characterized by a slow increase in the treated rats revealed the presence of hyperplasia of TNF␣ concentration, which reached the highest level on synovial lining cells and marked leukocyte infiltration in day 3 and remained elevated up to day 21 (Figure 2, the synovial tissue (Figure 3). With disease progression, bottom). Rats treated with TS-A 8 had a significantly the accumulation of leukocytes was accompanied by lower level of this cytokine, although the level did not development of fibrosis, and major tissue changes with return to the control value observed in saline-treated cartilage erosions were observed at a later phase. The rats. TS-A 12/22 had a similar inhibitory effect on the overall score for joint inflammation, based on the eval- TNF␣ level, and no significant difference was observed uation of synovial thickness, cell infiltrates, and cartilage between the 2 scFv. erosions, reached the highest value 1 week after injec- 1192 FISCHETTI ET AL

Figure 3. A–D, Photomicrographs of knee joints obtained from rats preimmunized with mBSA, 7 days after the intraarticular injection of saline (A), mBSA plus Un-scFv as a positive control (B), mBSA plus TS-A 8 (C), or mBSA plus TS-A 12/22 (D). Note the morphologic feature of an apparently normal synovium in A (score ϭ 0) as opposed to the synovial hyperplasia and leukocyte infiltration seen in B (score ϭ 4) and the marked reduction in the inflammatory process shown in C (score ϭ 2) and D (score ϭ 2). (Original magnification ϫ 10.) Bottom, The 4 groups of animals treated as described above were examined for histomorphologic modifications at different time points after arthritis induction. Sections of paraffin-embedded specimens of the knee joints were stained with hematoxylin and eosin and analyzed for tissue alterations (damage score) by 2 observers (FF, PD). Results for rats treated with mBSA alone are not shown, because they were absolutely comparable with those observed in the animals treated with mBSA plus Un-scFv. Values ϭ P Ͻ 0.01 versus mBSA plus Un-scFv; § ϭ P Ͻ 0.01 versus ءء ;ϭ P Ͻ 0.05 ء .are the mean and SD saline. See Figure 2 for other definitions. tion of mBSA and leveled off thereafter. Treatment of to that observed in Un-scFv–treated rats. Conversely, rats with the 2 scFv markedly reduced the synovial TS-A 8 was effective in inhibiting the deposition of C9, hyperplasia and leukocyte infiltration. The scores ob- confirming similar data obtained with TS-A 12/22 (28). served with TS-A 8 were not significantly different from Treatment of established arthritis with TS-A 8 those obtained with TS-A 12/22 at each time point of and TS-A 12/22. To evaluate the beneficial effect of the observation. 2 anti-C5 scFv on established arthritis, scFv were in- Immunofluorescence analysis of synovial tissue jected intraarticularly 5 days after the initiation of AIA from rats receiving Un-scFv revealed linear deposits of by mBSA. As shown in Figure 5, the total cell counts C3 and C9 on cells along the synovial lining layer, as well (top panel) and TNF␣ levels (middle panel) in synovial as deposition of these complement components in the lavage fluids were markedly decreased in rats treated subsynovial connective tissue (Figure 4). No staining was with the 2 anti-C5 scFv, as compared with the values observed in tissue obtained from the control rats treated obtained in Un-scFv–treated rats. The inhibitory effect with saline. The injection of TS-A 8 plus mBSA into the of TS-A 12/22 was slightly higher than that of TS-A 8, rat knees did not prevent the binding of C3, which but the values were not significantly different. The exhibited a distribution pattern and an intensity similar histomorphologic changes observed in the arthritic joints HUMAN ANTI-C5 scFv AS THERAPEUTIC REAGENTS IN RA 1193

kinetics of the changes were essentially similar. The arthritis induced in C6Ϫ/Ϫ rats was much milder, with joint swelling and values of the total cell count and TNF␣ level slightly increased but not significantly different from the values observed in a control group of 20 C6ϩ/ϩ rats that received only saline. Similarly, the arthritic joints of mBSA-treated C6Ϫ/Ϫ rats showed histomorphologic changes that were clearly different from those observed in C6ϩ/ϩ rats. These differences were particularly evident in the first 2 weeks after the initiation of AIA and were no longer seen at a later

Figure 4. Immunofluorescence analysis of rat knee joints for the deposition of C3 and C9. The animals were killed 3 days after injection of saline (A), methylated bovine serum albumin (mBSA) plus an unrelated anti-gliadin single-chain Fv (Un-scFv) (B), mBSA plus TS-A 8(C), or mBSA plus TS-A 12/22 (D). Note that both anti-C5 scFv prevented deposition of C9 but failed to inhibit deposition of C3. (Original magnification ϫ 20.)

treated with Un-scFv and characterized by synovial hyperplasia, leukocyte infiltration, and cartilage erosions were also reduced in rats treated with TS-A 8 and TS-A 12/22 (Figure 5, bottom panel). Again, the effects induced by the 2 anti-C5 scFv were not significantly Figure 5. Effect of the anti-C5 scFv on established arthritis in rats. Antigen-induced arthritis (AIA) was initiated in rats by the standard different. procedure described in Materials and Methods. Five days after the Initiation of AIA in C6-deficient animals. To intraarticular injection of mBSA, the animals were divided into 3 further prove that the TCC plays an important role in groups of 6 rats each that, respectively, received intraarticular injec- promoting arthritis in our model of AIA, as suggested by tions of 400 ␮g of the following scFv: TS-A 8 (shaded bars), TS-A 12/22 ␮ the beneficial results obtained with TS-A 8, we decided (right-hatched bars), or Un-scFv (solid bars) diluted in 100 l of saline. Ϫ/Ϫ Two additional groups of preimmunized rats (n ϭ 3 each) with either to establish this model in 25 PVG C6 rats and, for an arthritic condition (open bars) or with no induced damage (left- ϩ/ϩ comparison, in 20 PVG C6 rats. Joint swelling, the hatched bars) received an intraarticular injection of 100 ␮l of saline total cell count and the TNF␣ level in synovial lavage alone, thus serving as positive and negative controls, respectively. The fluids, and the histomorphology damage score measured animals were killed 21 days after the initiation of AIA. Values are the ␣ ϭ ␣ in complement-sufficient animals following the intraar- mean and SD. TNF tumor necrosis factor (see Figure 4 for other ϭ P Ͻ 0.05 versus arthritic rats treated with mBSA ء .(definitions ticular injection of mBSA were all increased (Figure 6). (positive controls) and rats treated with mBSA plus Un-scFv; § ϭ P Ͻ The values of these parameters were slightly lower than 0.01 versus preimmunized rats treated with saline alone (negative those observed in mBSA-treated Wistar rats, even if the controls). 1194 FISCHETTI ET AL

Figure 6. Antigen-induced arthritis (AIA) in C6Ϫ/Ϫ PVG rats. AIA was induced in 25 C6Ϫ/Ϫ PVG rats that were killed at different time points after the induction of arthritis. Animals were examined for the degree of joint swelling, total cell counts and tumor necrosis factor ␣ (TNF␣) levels in synovial lavage fluids, and the histomorphology of joint sections. The results were compared with those observed in a group of 20 comparably treated C6ϩ/ϩ PVG rats. A third group of 20 C6ϩ/ϩ animals preimmunized with methylated bovine serum albumin (mBSA) received an intraarticular injection of saline and served as negative controls. Three rats in each ϭ P Ͻ 0.01 versus C6ϩ/ϩ rats treated with ءء ;ϭ P Ͻ 0.05 ء .group were examined. Values are the mean and SD mBSA; § ϭ P Ͻ 0.05 versus C6ϩ/ϩ rats treated with saline. phase. Of note is the finding that, unlike the joint These data extended similar findings previously ob- swelling and the total cell count, the TNF␣ levels and the tained in mice using cobra venom factor to deplete the Ϫ Ϫ scores for tissue damage in the C6 / AIA rats were complement system (40). We provided further evidence slightly higher than the values observed in saline-treated for the contribution of complement to the onset of rats, even after 21 days of observation. synovitis in this model, showing that C3 and TCC are deposited on the synovial membranes of mBSA-treated DISCUSSION rats (28). However, the finding that treatment with sCR1 and cobra venom factor suppressed synovial inflamma- Our results demonstrate that TCC plays a major role in the development of immune complex–mediated tion, although highly suggestive for the contribution of arthritis induced in rats by immunization with mBSA. complement to the development of AIA, did not clarify This model was selected for the present study because it the biologically active product of the complement system resembles RA in many respects, including hyperplasia of responsible for the promotion of inflammation. synovium, leukocyte infiltration of synovial tissue, and Attempts have been made to identify the comple- cartilage erosions. Complement has been implicated in ment activation products that may act as effectors of the onset and progression of AIA in rats, following the synovial changes in AIA, and attention has focused on observation by Goodfellow et al (21) that soluble CR1 C5a and TCC as the most likely mediators of tissue (sCR1), a potent inhibitor of complement activation damage. These biologically active products are released through the classical and alternative pathways, injected at the tissue level as a result of complement activation. intraarticularly prevented the development of arthritis. C5a and C5aR have been implicated in the development HUMAN ANTI-C5 scFv AS THERAPEUTIC REAGENTS IN RA 1195

of arthritis in mice, using C5aR-deficient animals (36,41) also can be induced by the cytolytically inactive SC5b–9 or neutralizing antibodies against C5aR (42). However, complex, the type of complex that is most abundantly data are also available suggesting a role of the mem- present in the circulation and at the tissue level under brane attack complex in a murine model of AIA, as conditions of complement activation (39). In vivo mod- revealed by the increased disease severity observed in els have shown that this complex can sup- CD59aϪ/Ϫ mice as compared with CD59aϩ/ϩ mice (35) port transendothelial migration of PMNs through the and by the finding that the clinical and histologic man- mesenteric vessels (47) and promotes a marked inflam- ifestations of AIA improved following intraarticular matory response when injected into the lateral ventricle injection of recombinant CD59. Furthermore, Tramon- of rats (48). tini et al (43) showed, in a model of AIA established in TS-A 8 prevented the development of joint in- rabbits, that leukocyte counts and interleukin-8 (IL-8) flammation even when administered 1 week after the levels in synovial lavage fluids were significantly de- induction of arthritis, and, again, no significant differ- creased in C6-deficient animals compared with C6- ence between the effect obtained with TS-A 12/22 was sufficient animals. observed. This is an important point to consider when The contribution of C5a and the membrane evaluating the efficacy of this scFv as a therapeutic attack complex to the development of AIA in rats agent. It must be pointed out that the tissue damage remains a controversial issue. The marked reduction in characterized by synovial hyperplasia, leukocyte infiltra- knee swelling, histologic abnormalities of the synovial tion, and cartilage erosions was suppressed only partially membrane, and levels of IL-6 and TNF␣ observed in rats by TS-A 8, whether it was injected simultaneously with treated with a cyclic peptide antagonist of C5aR led mBSA or 5 days later. This suggests that this process Woodruff and colleagues (29) to suggest that C5a plays may be, at least in part, complement independent or an important role in causing inflammation in this model, dependent on the activation of early complement com- although those investigators did not investigate the ponents. contribution of the membrane attack complex in their Proinflammatory cytokines have been implicated model. We took a different approach based on analysis in the induction of arthritis, as shown by the increased of the beneficial effect of 2 different neutralizing anti- levels of TNF␣, IL-1␤, and IL-6 in synovial fluids from bodies to C5 on the development of synovitis in AIA. patients with RA (49–52) and in joint lavage fluids from Our finding that TS-A 8 was as effective as TS-A 12/22 rats undergoing AIA, as observed in this and other in inhibiting the inflammatory reaction in the rat knee studies (29). Based on the satisfactory results of anti- suggests that TCC is the major effector of this process. TNF␣ mAb in suppressing joint inflammation in exper- This is also supported by the observation that the joint imental models of arthritis, these antibodies and a inflammation observed in C6-deficient rats was mark- recombinant human soluble TNF receptor fusion pro- edly reduced, as compared with the arthritis induced in tein are now extensively used in the treatment of pa- C6-sufficient animals. One possible explanation for the tients with RA, in association with more conventional different results obtained in the study by Woodruff et al therapy (53). However, 25–45% of patients do not seem (29) might be related to the concentration of mBSA to benefit from this therapy (54–56). We believe that injected intraarticularly, because those investigators inhibitors of complement activation, and in particular used a dose that was 5-fold higher than that used in our reagents that prevent activation of the terminal compo- model, thus possibly leading to more increased levels of nents and assembly of the TCC, may provide an addi- immune complexes, enhanced complement activation, tional, and not necessarily alternative, therapeutic ap- and release of a higher amount of C5a. proach to controlling arthritis. The advantage of the Our finding that the TCC was mainly responsible scFv used in this study is that, although they were for the development of arthritis was not surprising in selected from a human phage display library, they view of the wealth of data accumulated in recent years neutralize C5 from human and other animal species, supporting the proinflammatory activity of the complex. including rat and mouse. The obvious advantage is that The membrane attack complex has been localized in the once they have been tested for their efficacy in animal synovial tissue of patients with RA (16), and a sublytic models, they can be used in humans. form of this complex has been shown to trigger synovial In conclusion, we have provided data suggesting and endothelial cells to release inflammatory cytokines that a human anti-C5 scFv that prevents the assembly of (44–46). The proinflammatory effect not only is re- TCC suppresses the development of AIA in rats and is stricted to the sublytic membrane attack complex but also effective in rats with established arthritis. 1196 FISCHETTI ET AL

AUTHOR CONTRIBUTIONS levels of complement SC5b–9 and fragment Bb are elevated in patients with rheumatoid arthritis. Arthritis Rheum 1991;34: Dr. Tedesco had full access to all of the data in the study and 1531–7. takes responsibility for the integrity of the data and the accuracy of the 16. Corvetta A, Pomponio G, Rinaldi N, Luchetti MM, Di Loreto C, data analysis. Stramazzotti D. Terminal complement complex in synovial tissue Study design. Fischetti, Marzari, Carretta, Tedesco. from patients affected by rheumatoid arthritis, osteoarthritis and Acquisition of data. Fischetti, Durigutto, Macor. acute joint trauma. Clin Exp Rheumatol 1992;10:433–8. Analysis and interpretation of data. Fischetti, Carretta. 17. Shingu M, Watanabe Y, Tomooka K, Yoshioka K, Ohtsuka E, Manuscript preparation. Fischetti, Tedesco. Nobunaga M. Complement degradation products in rheumatoid Statistical analysis. Fischetti. arthritis synovial fluid. Br J Rheumatol 1994;33:299–300. 18. Hogasen K, Mollnes TE, Harboe M, Gotze O, Hammer HB, Oppermann M. Terminal complement pathway activation and low REFERENCES lysis inhibitors in rheumatoid arthritis synovial fluid. J Rheumatol 1. Morgan BP. The complement system: an overview [review]. Meth- 1995;22:24–8. ods Mol Biol 2000;150:1–13. 19. Konttinen YT, Ceponis A, Meri S, Vuorikoski A, Kortekangas P, 2. Hietala MA, Nandakumar KS, Persson L, Fahlen S, Holmdahl R, Sorsa T, et al. Complement in acute and chronic arthritides: Pekna M. Complement activation by both classical and alternative assessment of C3c, C9, and protectin (CD59) in synovial mem- pathways is critical for the effector phase of arthritis. Eur J Im- brane. Ann Rheum Dis 1996;55:888–94. munol 2004;34:1208–16. 20. Wang Y, Rollins SA, Madri JA, Matis LA. Anti-C5 monoclonal 3. Neumann E, Barnum SR, Tarner IH, Echols J, Fleck M, Judex M, antibody therapy prevents collagen-induced arthritis and amelio- et al. Local production of complement proteins in rheumatoid rates established disease. Proc Natl Acad SciUSA1995;92: arthritis synovium. Arthritis Rheum 2002;46:934–45. 8955–9. 4. Takemura S, Ueda M, Tagami H, Kato H, Yoshikawa T, 21. Goodfellow RM, Williams AS, Levin JL, Williams BD, Morgan Kawakami N, et al. Relation of clinical activity of rheumatoid BP. Local therapy with soluble complement receptor 1 (sCR1) arthritis to immune complexes, complement components and suppresses inflammation in rat mono-articular arthritis. Clin Exp anti-immunoglobulins. Rheumatol Int 1984;4:159–63. Immunol 1997;110:45–52. 5. Makinde VA, Senaldi G, Jawad AS, Berry H, Vergani D. Reflec- 22. Mizuno M, Nishikawa K, Goodfellow RM, Piddlesden SJ, Morgan tion of disease activity in rheumatoid arthritis by indices of BP, Matsuo S. The effects of functional suppression of a mem- activation of the classical complement pathway. Ann Rheum Dis brane-bound complement regulatory protein, CD59, in the syno- 1989;48:302–6. vial tissue in rats. Arthritis Rheum 1997;40:527–33. 6. Petersen NE, Elmgreen J, Teisner B, Svehag SE. Activation of 23. Linton SM, Morgan BP. Complement activation and inhibition in classical pathway complement in chronic inflammation: elevated experimental models of arthritis. Mol Immunol 1999;36:905–14. levels of circulating C3d and C4d split products in rheumatoid 24. Mizuno M, Nishikawa K, Morgan BP, Matsuo S. Comparison of arthritis and Crohn’s disease. Acta Med Scand 1988;223:557–60. the suppressive effects of soluble CR1 and C5a receptor antagonist 7. Molenaar ET, Voskuyl AE, Familian A, van Mierlo GJ, Dijkmans in acute arthritis induced in rats by blocking of CD59. Clin Exp BA, Hack CE. Complement activation in patients with rheumatoid Immunol 2000;119:368–75. arthritis mediated in part by C-reactive protein. Arthritis Rheum 25. Linton SM, Williams AS, Dodd I, Smith R, Williams BD, Morgan 2001;44:997–1002. BP. Therapeutic efficacy of a novel membrane-targeted comple- 8. Wouters D, Voskuyl AE, Molenaar ET, Dijkmans BA, Hack CE. ment regulator in antigen-induced arthritis in the rat. Arthritis Evaluation of classical complement pathway activation in rheuma- Rheum 2000;43:2590–7. toid arthritis: measurement of C1q–C4 complexes as novel activa- 26. Goodfellow RM, Williams AS, Levin JL, Williams BD, Morgan tion products. Arthritis Rheum 2006;54:1143–50. BP. Soluble complement receptor one (sCR1) inhibits the devel- 9. Ruddy S, Colten HR. Rheumatoid arthritis: biosynthesis of com- opment and progression of rat collagen-induced arthritis. Clin Exp plement proteins by synovial tissues. N Engl J Med 1974;290: Immunol 2000;119:210–6. 1284–8. 27. Mizuno M, Nishikawa K, Spiller OB, Morgan BP, Okada N, 10. Mollnes TE, Lea T, Mellbye OJ, Pahle J, Grand O, Harboe M. Okada H, et al. Membrane complement regulators protect against Complement activation in rheumatoid arthritis evaluated by C3dg the development of type II collagen–induced arthritis in rats. and the terminal complement complex. Arthritis Rheum 1986;29: Arthritis Rheum 2001;44:2425–34. 715–21. 28. Marzari R, Sblattero D, Macor P, Fischetti F, Gennaro R, Marks 11. Swaak AJ, van Rooyen A, Planten O, Han H, Hattink O, Hack E. JD, et al. The cleavage site of C5 from man and animals as a An analysis of the levels of complement components in the common target for neutralizing human monoclonal antibodies: in synovial fluid in rheumatic diseases. Clin Rheumatol 1987;6:350–7. vitro and in vivo studies. Eur J Immunol 2002;32:2773–82. 12. Doherty M, Richards N, Hornby J, Powell R. Relation between 29. Woodruff TM, Strachan AJ, Dryburgh N, Shiels IA, Reid RC, synovial fluid C3 degradation products and local joint inflamma- Fairlie DP, et al. Antiarthritic activity of an orally active C5a tion in rheumatoid arthritis, osteoarthritis, and crystal associated receptor antagonist against antigen-induced monarticular arthritis arthropathy. Ann Rheum Dis 1988;47:190–7. in the rat. Arthritis Rheum 2002;46:2476–85. 13. Olmez U, Garred P, Mollnes TE, Harboe M, Berntzen HB, 30. Andersson SE, Lexmuller K, Ekstrom GM. Physiological charac- Munthe E. C3 activation products, C3 containing immune com- terization of mBSA antigen induced arthritis in the rat. I. Vascular plexes, the terminal complement complex and native C9 in pa- leakiness and pannus growth. J Rheumatol 1998;25:1772–7. tients with rheumatoid arthritis. Scand J Rheumatol 1991;20: 31. Andersson SE, Johansson A, Lexmuller K, Ekstrom GM. Physio- 183–9. logical characterization of mBSA antigen induced arthritis in the 14. Firestein GS, Paine MM, Littman BH. Gene expression (collage- rat. II. Joint blood flow, glucose metabolism, and cell proliferation. nase, tissue inhibitor of metalloproteinases, complement, and J Rheumatol 1998;25:1778–84. HLA–DR) in rheumatoid arthritis and osteoarthritis synovium: 32. Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala quantitative analysis and effect of intraarticular corticosteroids. E, et al. Animal models of arthritis: relevance to human disease. Arthritis Rheum 1991;34:1094–105. Toxicol Pathol 1999;27:134–42. 15. Brodeur JP, Ruddy S, Schwartz LB, Moxley G. Synovial fluid 33. Wang Y, Kristan J, Hao L, Lenkoski CS, Shen Y, Matis LA. A role HUMAN ANTI-C5 scFv AS THERAPEUTIC REAGENTS IN RA 1197

for complement in antibody-mediated inflammation: C5-deficient of human synovial fibroblast-like cells. Int Arch Allergy Appl DBA/1 mice are resistant to collagen-induced arthritis. J Immunol Immunol 1989;90:248–55. 2000;164:4340–7. 45. Daniels RH, Houston WA, Petersen MM, Williams JD, Williams 34. Banda NK, Kraus D, Vondracek A, Huynh LH, Bendele A, Holers BD, Morgan BP. Stimulation of human rheumatoid synovial cells VM, et al. Mechanisms of effects of complement inhibition in by non-lethal complement membrane attack. Immunology 1990; murine collagen-induced arthritis. Arthritis Rheum 2002;46: 69:237–42. 3065–75. 46. Niculescu F, Rus H. Mechanisms of signal transduction activated 35. Williams AS, Mizuno M, Richards PJ, Holt DS, Morgan BP. by sublytic assembly of terminal complement complexes on nucle- Deletion of the gene encoding CD59a in mice increases disease ated cells. Immunol Res 2001;24:191–9. severity in a murine model of rheumatoid arthritis. Arthritis 47. Dobrina A, Pausa M, Fischetti F, Bulla R, Vecile E, Ferrero E, et Rheum 2004;50:3035–44. al. Cytolytically inactive terminal complement complex causes 36. Grant EP, Picarella D, Burwell T, Delaney T, Croci A, Avitahl N, transendothelial migration of polymorphonuclear leukocytes in et al. Essential role for the C5a receptor in regulating the effector vitro and in vivo. Blood 2002;99:185–92. phase of synovial infiltration and joint destruction in experimental 48. Casarsa C, De Luigi A, Pausa M, De Simoni MG, Tedesco F. arthritis. J Exp Med 2002;196:1461–71. Intracerebroventricular injection of the terminal complement 37. Leenaerts PL, Stad RK, Hall BM, van Damme BJ, Vanrenterghem complex causes inflammatory reaction in the rat brain. Eur Y, Daha MR. Hereditary C6 deficiency in a strain of PVG/c rats. J Immunol 2003;33:1260–70. Clin Exp Immunol 1994;97:478–82. 49. De Benedetti F, Pignatti P, Gerloni V, Massa M, Sartirana P, 38. Oppermann M, Schulze M, Gotze O. A sensitive enzyme immu- Caporali R, et al. Differences in synovial fluid cytokine levels noassay for the quantitation of human C5a/C5a(desArg) anaphy- between juvenile and adult rheumatoid arthritis. J Rheumatol latoxin using a monoclonal antibody with specificity for a neo- 1997;24:1403–9. epitope. Complement Inflamm 1991;8:13–24. 50. Lettesjo H, Nordstrom E, Strom H, Nilsson B, Glinghammar B, 39. Tedesco F, Pausa M, Nardon E, Introna M, Mantovani A, Dobrina Dahlstedt L, et al. Synovial fluid cytokines in patients with A. The cytolytically inactive terminal complement complex acti- rheumatoid arthritis or other arthritic lesions. Scand J Immunol vates endothelial cells to express adhesion molecules and tissue 1998;48:286–92. factor procoagulant activity. J Exp Med 1997;185:1619–27. 51. Manicourt DH, Poilvache P, van Egeren A, Devogelaer JP, Lenz 40. Van Lent PL, van den Bersselaar LA, van den Hoek AE, van de ME, Thonar EJ. Synovial fluid levels of tumor necrosis factor ␣ Loo AA, van den Berg WB. Cationic immune complex arthritis in and oncostatin M correlate with levels of markers of the degrada- mice—a new model: synergistic effect of complement and inter- tion of crosslinked collagen and cartilage aggrecan in rheumatoid leukin-1. Am J Pathol 1992;140:1451–61. arthritis but not in osteoarthritis. Arthritis Rheum 2000;43:281–8. 41. Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA, 52. Saxena N, Aggarwal A, Misra R. Elevated concentrations of et al. Arthritis critically dependent on innate immune system monocyte derived cytokines in synovial fluid of children with players. Immunity 2002;16:157–68. enthesitis related arthritis and polyarticular types of juvenile 42. Lee H, Zahra D, Vogelzang A, Newton R, Thatcher J, Quan A, et idiopathic arthritis. J Rheumatol 2005;32:1349–53. al. Human C5aR knock-in mice facilitate the production and 53. Moreland LW. Drugs that block tumour necrosis factor: experi- assessment of anti-inflammatory monoclonal antibodies. Nat Bio- ence in patients with rheumatoid arthritis. Pharmacoeconomics technol 2006;24:1279–84. 2004;22:39–53. 43. Tramontini NL, Kuipers PJ, Huber CM, Murphy K, Naylor KB, 54. Schwartzman S, Fleischmann R, Morgan GJ Jr. Do anti-TNF Broady AJ, et al. Modulation of leukocyte recruitment and IL-8 agents have equal efficacy in patients with rheumatoid arthritis? expression by the membrane attack complex of complement Arthritis Res Ther 2004;6 Suppl 2:S3–11. (C5b-9) in a rabbit model of antigen-induced arthritis. Inflamma- 55. Ranganathan P. Pharmacogenomics of tumor necrosis factor tion 2002;26:311–9. antagonists in rheumatoid arthritis [review]. Pharmacogenomics 44. Von Kempis J, Torbohm I, Schonermark M, Jahn B, Seitz M, 2005;6:481–90. Hansch GM. Effect of the late complement components C5b-9 56. Symmons DP, Silman AJ. The world of biologics [review]. Lupus and of platelet-derived growth factor on the prostaglandin release 2006;15:122–6. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1198–1203 DOI 10.1002/art.22516 © 2007, American College of Rheumatology

A Randomized Crossover Trial of a Wedged Insole for Treatment of Knee Osteoarthritis

Kristin Baker,1 Joyce Goggins,1 Hui Xie,2 Karen Szumowski,2 Michael LaValley,2 David J. Hunter,2 and David T. Felson1

Objective. In uncontrolled studies, a lateral- wedge insole for knee OA was neither statistically wedge insole has reduced knee pain in patients with significant nor clinically important. medial knee osteoarthritis (OA). The aim of this study was to test the efficacy of this simple, low-cost interven- Knee osteoarthritis (OA) is a disabling disorder tion for pain in patients with medial knee OA. affecting ϳ13% of individuals ages 65 years and older Methods. We conducted a double-blind, random- (1). According to a recent meta-analysis, 60% of OA ized, crossover trial designed to detect a small effect of trials assess drug treatments, and 26% assess surgical treatment. Participants were at least 50 years of age and procedures (2). The remarkable lack of studies evaluat- had medial joint space narrowing on posteroanterior ing rehabilitation and physical therapy techniques is a semiflexed radiographs and scores indicating moderate consequence of lucrative opportunities for the develop- pain for 2 of the 5 items on the Western Ontario and ment of drug therapy (2). The toxicity/adverse event McMaster Universities Osteoarthritis Index (WOMAC) profile of the most commonly used existing therapies, pain scale. Participants were randomized to receive a 5° such as nonsteroidal antiinflammatory drugs (NSAIDs), lateral-wedge insole or a neutral insole for 6 weeks. is very unfavorable when compared with conservative Following a 4-week washout period, participants crossed interventions such as braces and orthotics (3), suggesting over to the other treatment for 6 weeks. Knee pain, the primary outcome, was assessed by the WOMAC pain that such therapies, if efficacious, might be safe options scale (visual analog scale version). Secondary outcomes for treatment of this common disabling disorder. included the WOMAC disability subscale, overall knee Most knee OA affects the medial compartment, Ն pain, 50-feet walk time, chair-stand time, and use of which bears 60% of the load during weight-bearing medications for knee pain. (4,5). Degeneration of the medial compartment may Results. Ninety patients were randomized. The increase loading there and be a source of pain. Japanese mean difference in pain between the 2 treatments was investigators have developed a simple lateral-wedge 13.8 points on the WOMAC pain scale (95% confidence insole for treatment of medial knee OA that shifts the We observed similar distribution of load in the foot laterally, thereby inducing .([0.13 ؍ interval ؊3.9, 31.4 [P small effects for the secondary outcomes. a lateral shift in the load across the knee, unloading the Conclusion. The effect of treatment with a lateral- medial compartment (4). Uncontrolled Japanese studies using lateral wedges have shown a benefit on pain (6,7). In a pilot study, we observed that use of a lateral-wedge ClinicalTrials.gov identifier: NCT00032240. shoe insert led to a significant reduction in the adduction Supported by the NIH (grant P60-AR-47785). moment, which is the measure of dynamic medial load 1Kristin Baker, PhD, Joyce Goggins, MPH, David T. Felson, MD, MPH: Veterans Affairs Boston Health Care System, Boston, across the knee (8). There has been one parallel-design Massachusetts; 2Hui Xie, PhD, Karen Szumowski, BA, Michael La- trial comparing the effects of a neutral, nonwedged Valley, PhD, David J. Hunter, MBBS, PhD: Boston University, and Veterans Affairs Boston Health Care System, Boston, Massachusetts. insole with lateral-wedge insoles, which demonstrated a Address correspondence and reprint requests to Kristin null effect on pain but a decrease in the use of pain Baker, PhD, Boston University Medical School, 715 Albany Street, medication in individuals wearing the lateral-wedge in- A203, Boston, MA 02118. E-mail: [email protected]. Submitted for publication September 19, 2006; accepted in sole compared with the neutral insole (9). Several fea- revised form January 16, 2007. tures of that trial may have influenced its null result,

1198 LATERAL-WEDGE INSOLE FOR KNEE OA 1199

including compression of the insert during wear and inadequate power to detect a small effect of treatment. Because use of a standard lateral-wedge insert is a simple, low-cost, and likely safe intervention, even a small improvement in pain with lateral-wedge insoles could translate to large public health benefits and probably widespread use. We conducted a double-blind crossover trial to test for a small effect of a 5° lateral- wedge insole, the same insole we tested previously (8). Compared with a parallel-design trial, a crossover trial Figure 1. Trial timeline. usually provides greater statistical power to detect a small therapeutic effect.

PATIENTS AND METHODS likely followup attendance. It also allowed us to assess pain status twice, in order to obtain an average of knee pain scores Participants. Participants were recruited from the prior to the trial, reducing the variability in pain measurement. following 3 sources: a previous natural history study (10), lists Participants were randomized to receive initially either of individuals seeking care at a local facility who said they were a 5° lateral-wedge insole or a neutral insole for 6 weeks (phase interested in participating in research, and advertisements in 1). Inserts were removed, and, following a washout period of 4 local newspapers. weeks, the patients were crossed over to the other treatment The eligibility criteria for participation in the study for 6 weeks (phase 2) (Figure 1). Randomization codes were were as follows: age Ն50 years, medial but not lateral tib- generated and held at the Data Coordinating Center at Boston iofemoral narrowing (Ն1 on a 0–3-point scale) on posteroan- University. We stratified randomization by 2 factors that were terior semiflexed radiographs (11), and scores reflecting at potentially predictive of treatment response: whether the knee least moderate pain for 2 of the 5 items of the Western Ontario to be studied had a Kellgren/Lawrence (K/L) (13) grade of 4 and McMaster Universities Osteoarthritis Index (WOMAC) versus a K/L grade of Ͻ4, and whether the patient had pain subscale (5 questions regarding the level of pain during unilateral or bilateral disease. Participants had to fulfill eligi- different activities) (12). bility requirements (as described above) in both knees to be Patients were excluded if they were bed- or chair- defined as having bilateral disease. Data collection occurred at bound or usually used an ambulation aid to walk; had limited the General Clinical Research Center at Boston University ability to wear shoes (Ͻ8 hours/day); had undergone amputa- School of Medicine. tion of or previous major trauma to a foot, raising concerns Interventions. Treatment A consisted of use of a flat, that using an insert might worsen foot pain; had foot sores or 1⁄8-inch–thick shoe insert on the side of the affected knee. ulcers; had neuropathy attributable to diabetes or other causes; Treatment B consisted of use of a 5° lateral-wedge insole on were not fluent in English; experienced pain emanating more the side of the affected knee; this insole was made of the same from the back or hip than from the knee; planned to move material as that for the treatment A inserts (the mean medial– from the area within 7 months of screening; had symptomatic lateral thickness of this wedge was 1⁄8 inch). Both the neutral comorbid disease that limited walking more than knee pain and the lateral-wedge insole were made of NickelPlast (AliMed, limited walking; had received a corticosteriod injection in the Dedham, MA). In a 3-month pilot study, we observed that this knee in the month before screening; had bilateral total knee material does not compress over time. replacements (TKRs) or plans for TKR surgery during the trial Because shock-absorbing insole material may itself period; had known inflammatory arthritis; failed to pass the lessen heel-strike impulse load, we used the same material for run-in test (see below); had undergone initiation of glu- active and control inserts. We provided 2 sets of inserts (2 cosamine and/or chondroitin and/or NSAID treatment 2 pairs) to each patient. Participants returned twice during each months prior to screening; or were unwilling to forego starting treatment, at 3 weeks and again at 6 weeks, with a 4-week any new medication during the trial period. washout period between treatments. Screening was performed via telephone. Eligible par- Patients with unilateral medial knee OA were treated ticipants were invited to a prerandomization visit where in- with a lateral-wedge shoe insole on the side of the diseased formed consent was obtained, radiographs were obtained, and knee and a neutral shoe insert on the side of the unaffected the WOMAC questionnaire was administered. At this visit, we knee, whereas those with bilateral medial knee OA were also conducted the clinical knee examination (described be- treated with lateral-wedge shoe insoles on both sides. We low) and excluded anyone in whom the main source of knee chose this approach in patients with unilateral disease to pain was pes anserine tenderness. The run-in for this study eliminate any leg-length discrepancy caused by the insert and started at the prerandomization visit and lasted 2 weeks, at potential gait alterations. which time participants attended the baseline visit and were For patients with bilateral medial knee OA, we chose randomized. In addition to determining whether patients were the more symptomatic knee as the study knee. If both knees likely to return for followup visits, the run-in allowed us to were equally symptomatic, we selected the knee with the most review radiographs prior to randomization and to determine medial joint space narrowing on radiography. If both knees 1200 BAKER ET AL

were equally symptomatic and equally affected, we randomly chose a knee as the study knee. Measurements and procedures. Data collection occurred at the prerandomization visit, the randomization visit, 3 weeks, 6 weeks, 10 weeks (baseline after washout), 13 weeks, and 16 weeks. Staff performing the clinical examination on the knees were blinded to the treatment assignment of the patients. Primary outcome. The primary outcome was the WOMAC pain score on a visual analog scale (VAS); the WOMAC pain subscale consists of 5 questions regarding the level of pain experienced during a particular activity over the past 48 hours. We used a version that queries patients about knee-specific pain. The primary outcome was assessed in the study knee. The WOMAC pain subscale has been used as a principal outcome measure in several randomized trials in OA and has been shown to be more sensitive to change than the other subscales of the WOMAC (14). Figure 2. Flow chart of participants. OA ϭ osteoarthritis. Secondary outcomes and other measurements. Second- ary outcomes included the WOMAC disability subscale, overall knee pain (on a VAS), physical performance measures knees without patellofemoral tenderness on clinical examina- including 50-feet walk time and 5 chair-stand time, and use of tion (all knees with scores for at least moderate pain on analgesic and antiinflammatory medications for knee pain (as palpation of the patella were eliminated), because patel- recorded in medication diaries). At each visit, we also collected lofemoral pain would not be improved by the inserts. These information on other musculoskeletal pain and performed a subgroup analyses used the same statistical model described clinical knee examination that assessed pain with palpation of above. the joint, pain with passive motion, and pain at rest. In a subset We powered the study to detect a difference of 22 analysis, we used the examination focusing on pain with points (0–500-point scale) between treatment groups. Given palpation of the medial and lateral patellae to exclude patients that the mean WOMAC score of patients with knee OA is with patellofemoral tenderness. ϳ230 (15), this would correspond, for example, to a response Foot inserts have been used to treat back pain, but the of 30% with treatment versus slightly more than 20% with unique effects of lateral-wedge insoles on preexisting or devel- placebo, a treatment effect of slightly less than 10%. Because oping back pain or foot pain were assessed as part of this trial. we were interested in detecting minimal clinical effects of We also collected information about falls that occurred during treatment, we focused in secondary analyses on the number of the study period. To track adherence to treatment, patients patients achieving minimal clinical improvement as defined by were asked to record in a diary the number of hours per day Ehrich et al (16) and compared the proportion in each that they wore the insoles. treatment group. To compare the 2 treatments in terms of the Data analysis. Analysis of the results of this trial number of patients achieving minimal clinical improvement, focused on the primary outcome measure of pain during we dichotomized the change in pain into an indicator for a treatment. The knee-specific WOMAC pain subscale (0– Ն50-point improvement and compared the rates using McNe- 500-mm VAS) was administered at baseline, week 3, and week mar’s test (17). The study protocol was approved by the Boston 6 of each treatment period. The primary outcome measure was University Medical Campus institutional review board. the average pain score from these 2 time points in each period. Treatment effects were measured by regression analysis, with generalized estimating equation (GEE) adjustment for re- RESULTS peated measures within subjects. We used the GEE method to fit 2 models: treatment effect, treatment-period effect, and Participants were recruited from February 2002 differential carryover effect adjusting for the baseline pain, and to November 2004, with followup data collected until treatment effect and treatment-period effect with exclusion of March 2005. Of the 526 potential participants who were the differential carryover effect. screened but not randomized, 177 were ineligible be- We defined 2 subgroups of a priori interest. The first cause their knee pain was not severe enough (Figure 2). subgroup comprised patients with severe OA (K/L grade 4) versus those with nonsevere OA (K/L grade Ͻ4) and was based Other common reasons for ineligibility included pain at on previous literature (6). The second subgroup comprised other sites, such as the lower back or foot, and other patients who were obese (body mass index [BMI] Ն30 kg/m2) rheumatic diseases. Of the 90 patients who were ran- versus those who were not obese, because obesity is such a domized, 86 who did not violate protocol and had at strong risk factor for knee OA, including symptomatic knee least 1 visit while receiving treatment were analyzed. OA, that it may negate any small effect of the inserts. It should be noted that our radiographs were obtained with the knee in Three patients withdrew consent (2 due to travel and a semiflexed position, and the K/L grades utilized were based time commitment and 1 on the advice of his physician). on a fully extended knee position. We also analyzed separately In 1 patient, painful patellofemoral OA rather than LATERAL-WEDGE INSOLE FOR KNEE OA 1201

Table 1. Characteristics of the participants at baseline* Table 3. Adherence to and side effects of treatment Neutral to Wedged to Neutral Wedged wedged neutral insole insole Characteristic (n ϭ 41) (n ϭ 45) Adherence, mean Ϯ SD Age, mean Ϯ SD years 67.8 Ϯ 9.9 68.2 Ϯ 8.7 hours of wear/day Male sex, % 34 47 Period 1 7.7 Ϯ 1.6 7.3 Ϯ 2.0 Body mass index, mean Ϯ SD kg/m2 32.9 Ϯ 6.4 33.0 Ϯ 4.8 Period 2 7.5 Ϯ 2.5 7.2 Ϯ 1.9 Kellgren/Lawrence grade Ն3, % 93 93 Side effect, no. of patients WOMAC pain score, mean Ϯ SD 263 Ϯ 95 268 Ϯ 115 Musculoskeletal symptoms 10 6 (0–500 scale) Blisters 5 1 Unilateral/bilateral osteoarthritis, % 63/37 69/31 Falls 3 4 * WOMAC ϭ Western Ontario and McMaster Universities Osteoar- thritis Index. was 13.8 points (95% confidence interval [95% CI] Ϫ3.9, 31.4) on the 500-point WOMAC pain scale. We found similar small and nonsignificant effects of treatment for tibiofemoral OA was diagnosed just after randomiza- secondary outcomes, the WOMAC disability subscale, tion. There were no differences between the randomiza- overall pain on a VAS, and physical performance mea- tion groups in terms of age, BMI, K/L grade, and the sures (data not shown). presence of unilateral versus bilateral disease (Table 1). We observed that 11 of 86 subjects experienced When analyzing the crossover trial findings, we minimal clinical improvement in the WOMAC pain first tested for treatment period and differential car- score (Ն50 points) after both treatments. Twenty-one ryover effects (Table 2). The differential carryover was a patients achieved this level of improvement only with the 1.5-point difference in the WOMAC pain score (P ϭ wedged insole, but 19 patients achieved it only with the 0.96), showing that the comparison of treatment efficacy neutral insole (P ϭ 0.75). was not different if the patients were randomized to the The lateral-wedge insole improved pain in pa- neutral insert or the wedged insert first. Therefore, we tients with a K/L grade Ͻ4 by 21 points, compared with removed the carryover term from the model. The a 2-point improvement in those with a K/L grade of 4. treatment-period effect was a 6-point difference (P ϭ Those with a BMI of Ͻ30 kg/m2 had a 29-point improve- 0.50) between WOMAC pain scores. The mean differ- ment in pain, compared with a 6-point improvement in ence between the 2 treatments across the time periods those with a BMI Ն30 kg/m2 (P ϭ 0.06 for both). No effects on the WOMAC pain score with treatment were Table 2. Predictors of WOMAC pain scores during the crossover observed in the subset of participants who did not have trial* patellofemoral tenderness (data not shown). There also Predictor Model 1 Model 2 was no treatment effect for the reported number of days receiving rescue medication (data not shown). Baseline WOMAC pain score 0.67 0.67 95% confidence interval 0.53, 0.82 0.53, 0.82 On average, patients reported wearing the inserts P Ͻ0.0001 Ͻ0.0001 7 hours per day, with no difference observed between Treatment 14.5 13.8 groups or time periods (Table 3). This level of adher- 95% confidence interval Ϫ23.1, 52.2 Ϫ3.9, 31.4 P 0.45 0.13 ence was close to the 8 hours or more per day prescribed Treatment, period 1 vs. period 2 6.8 6.1 after an initial 1-week buildup period. There was no 95% confidence interval Ϫ31.1, 44.7 Ϫ11.6, 23.7 increase in musculoskeletal symptoms among patients P 0.72 0.50 Period by treatment interaction† Ϫ1.5 wearing the lateral-wedge insole, and no falls were 95% confidence interval Ϫ64.7, 61.6 attributable to the insert (Table 3). The most common P 0.96 side effects were blisters on the tops of the toes when the * Values for model predictors are beta coefficients. For treatment as a wedge pushed feet up to the tops of the shoes. These predictor, a value of 13.8 means that active treatment was associated resolved with temporary discontinuation of the wedge with a 13.8 lower score on the Western Ontario and McMaster and/or by refitting the wedges. Universities Osteoarthritis Index (WOMAC) pain scale compared with control treatment. An unstructured correlation matrix for observations within subjects was used in generalized estimating equation fitting of the marginal model (1). Model 2 was conducted with DISCUSSION exclusion of the differential carryover effect. † Tests whether treatment effects differed according to use in period 1 We observed that a 5° lateral-wedge insole, which or period 2, constituting the differential carryover effect. was documented in a previous study to modestly de- 1202 BAKER ET AL

crease the knee adduction moment, had only a 13.8- NSAIDs or acetaminophen with placebo, effect sizes of point effect (95% CI Ϫ3.9, 31.4) on pain in patients with ϳ0.50 and ϳ0.3, respectively, have been reported (19,20). medial knee OA. Is a 13.8-point therapeutic effect Other studies have found that lateral-wedge in- meaningful? Although Ehrich and colleagues focused on soles improve pain, but the study designs and treatment the perceptible effect of treatment in 1 group of patients protocols differed from those used in our study (21,22). (16), those investigators suggested that a minimally Toda et al reported a decrease in pain among patients perceptible improvement in the WOMAC pain score in who used a lateral-wedge insole in a controlled but patients with knee OA is ϳ10 mm (on a 0–100-mm nonrandomized study. However, that treatment in- scale), translating into a 50-point effect (based on a cluded the use of a subtalar elastic strap, and the wedge 500-point scale) in this study. We tested this and found was made of material that may be compressible. The that the number of patients achieving minimal percep- subtalar strapping may limit mobility of the subtalar tible improvement while wearing the wedged insole was joint during walking and, independent of the lateral no greater than the number of patients achieving this wedge, may correct the femorotibial angle in patients level of improvement while wearing the neutral insert. with medial compartment knee OA. The findings by Maillefert et al also reported a null effect on knee Toda et al suggest that it may be necessary to immobilize pain with a lateral-wedge insole, but they did observe the ankle joint in conjunction with use of a lateral-wedge some decrease in concomitant drug therapy in partici- insole. pants using the lateral-wedge insole (9). In our trial, we A lateral-wedge insole can accentuate foot pro- did not observe an effect of the lateral-wedge insole on nation and retard foot supination during gait in a medication use. There are several differences regarding well-balanced foot and ankle and might accentuate the treatments in our trial and the Maillefert et al trial. pronation in an already overpronated foot and ankle. In the trial by Maillefert et al, the lateral wedge was Also, changes in a normal gait may lead to problems in individualized, with the wedge elevation determined by a other joints. In our study, we did not find that the use of static pedometer evaluation, and treatment was always a lateral-wedge insole for 6 weeks created problems in bilateral. It is unclear whether treating bilaterally when the foot or elsewhere in the body. disease is unilateral has a negative effect on gait and Several limitations of our trial, or factors for outcome. It is also not clear to what extent the insole which we did not control, may be important for future material was compressible. We treated all patients with trials, including shoe wear and physical activity levels of the same 5° wedge on the affected side(s) only, and used the participants. The use of nonsupportive, worn-out a wedge that did not compress after prolonged use. Fur- shoes may lessen the effectiveness of the lateral-wedge thermore, in the trial by Maillefert et al, subject eligibility insole, and very sedentary individuals may not utilize the did not include medial knee joint space narrowing, insert enough to make it beneficial. leaving open the possibility that subjects whose medial In conclusion, wedged shoe insoles were not moment was not increased could have been studied. efficacious in patients with medial knee OA. Although Our crossover trial had enough power to detect a our results do not completely rule out a modest effect of small effect of treatment, a difference of 10% between this treatment in some patients, the effects were so small groups (versus 20% seen in the Maillefert et al trial). We that they were not likely to be clinically important. powered this study for a small effect because of the Future studies are needed to examine subgroups of public health implications of even a small clinical im- patients who may benefit, and to examine the approach provement with use of a simple, inexpensive therapy. We of combining wedged insoles with ankle immobilization had adequate power to detect a difference of 22 points or with additional realignment therapies. between groups on the 500-point WOMAC pain subscale (although we observed a difference of only 13.8 points). Our power would have permitted us, for exam- AUTHOR CONTRIBUTIONS ple, to detect a 30% reduction in pain from baseline with Dr. Baker had full access to all of the data in the study and the lateral-wedge insole versus a 20% reduction with the takes responsibility for the integrity of the data and the accuracy of the data analysis. neutral insole. To provide perspective on this treatment Study design. Baker, LaValley, Felson. effect, given that the standard deviations seen in other Acquisition of data. Baker, Goggins, Szumowski, LaValley, Hunter. trials using this outcome measure were ϳ105 (15,18), Analysis and interpretation of data. Baker, Xie, Szumowski, LaValley, Hunter, Felson. this 22-point difference in the WOMAC pain subscale is Manuscript preparation. Baker, Xie, Hunter, Felson. equivalent to an effect size of 0.2. In trials comparing Statistical analysis. Baker, Xie. LATERAL-WEDGE INSOLE FOR KNEE OA 1203

REFERENCES 12. Goggins J, Baker K, Felson D. What WOMAC pain score should make a patient eligible for a trial in knee osteoarthritis? J Rheu- 1. Felson DT, Zhang Y. An update on the epidemiology of knee and matol 2005;32:540–2. hip osteoarthritis with a view to prevention. Arthritis Rheum 13. Kellgren JH, Lawrence JS. Radiological assessment of osteo- 1998;41:1343–55. arthrosis. Ann Rheum Dis 1957;16:494–502. 2. Tallon D, Chard J, Dieppe P. Relation between agendas of the 14. Bellamy N, Buchanan WW, Goldsmith CH, Campbell J, Stitt LW. research community and the research consumer. Lancet 2000; Validation study of WOMAC: a health status instrument for 9220:2037–40. measuring clinically important patient relevant outcomes to anti- 3. Jordan KM, Arden NK, Doherty M, Bannwarth B, Bijlsma JW, rheumatic drug therapy in patients with osteoarthritis of the hip or Dieppe P. EULAR Recommendations 2003: an evidence based knee. J Rheumatol 1988;15:1833–40. approach to the management of knee osteoarthritis: report of a task force of the Standing Committee for International Clinical 15. Bellamy N, Buchanan WW, Chalmers A, Ford PM, Kean WF, Studies Including Therapeutic Trials (ESCISIT) [review]. Ann Kraag GR, et al. A multicenter study of tenoxicam and diclofenac Rheum Dis 2003;62:1145–55. in patients with osteoarthritis of the knee. J Rheumatol 1993;20: 4. Yasuda K, Sasaki T. The mechanics of treatment of the osteoar- 999–1004. thritic knee with a wedged insole. Clin Orthop 1987;215:162–72. 16. Ehrich EW, Davies GM, Watson DJ, Bolognese JA, Seidenberg 5. Andriacchi TP. Dynamics of knee malalignment. Orthop Clin BC, Bellamy N. Minimal perceptible clinical improvement with the North Am 1994;25:395–403. Western Ontario and McMaster Universities osteoarthritis index 6. Sasaki T, Yasuda K. Clinical evaluation of the treatment of questionnaire and global assessments in patients with osteoarthri- osteoarthritic knees using a newly designed wedged insole. Clin tis. J Rheumatol 2000;27:2635–41. Orthop Relat Res 1987;221:181–7. 17. Kirkwood BR, Sterne JA. Essential medical statistics. 2nd ed. 7. Tohyama H, Yasuda K, Kaneda K. Treatment of osteoarthritis of Malden (MA): Blackwell Science; 2003. the knee with heel wedges. Int Orthop 1991;15:31–3. 18. Bellamy N, Carette S, Ford PM, Kean WF, le Riche NG, Lussier 8. Kerrigan DC, Lelas JL, Goggins J, Merriman GJ, Kaplan RJ, A, et al. Osteoarthritis antirheumatic drug trials. II. Tables for Felson D. Effectiveness of a lateral wedge insole on knee varus calculation sample size for clinical trials. J Rheumatol 1992;19: torque in patients with knee osteoarthritis. Arch Phys Med Reha- 444–50. bil 2002;83:889–93. 19. Felson D. The verdict favors nonsteroidal antiinflammatory drugs 9. Maillefert JF, Hudry C, Baron G, Kieffert P, Bourgeois P, for treatment of osteoarthritis and a plea for more evidence on Lechevalier D, et al. Laterally elevated wedged insoles in the other treatments [review]. Arthritis Rheum 2001;44:1477–80. treatment of medial knee osteoarthritis: a prospective randomized controlled study. Osteoarthritis Cartilage 2001;9:738–45. 20. Towheed TE. Published meta-analyses of pharmacological thera- 10. Felson D, McLaughlin S, Goggins J, LaValley MP, Gale ME, pies for osteoarthritis. Osteoarthritis Cartilage 2002;10:836–7. Totterman S, et al. Bone marrow edema and its relation to 21. Toda Y, Segal N. Usefulness of an insole with subtalar strapping progression of knee osteoarthritis. Ann Intern Med 2003;139: for analgesia in patients with medial compartment osteoarthritis of 330–6. the knee. Arthritis Rheum 2002;47:468–73. 11. Altman RD, Hochberg M, Murphy WA Jr, Wolfe F, Lequesne M. 22. Toda Y, Segal N, Kato A, Yamamoto S, Irie M. Effect of a novel Atlas of individual radiographic features in osteoarthritis. Osteo- insole on the subtalar joint of patients with medial compartment arthritis Cartilage 1995;3 Suppl A:3–70. osteoarthritis of the knee. J Rheumatol 2001;28:2705–10. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1204–1211 DOI 10.1002/art.22515 © 2007, American College of Rheumatology

Association Between Valgus and Varus Alignment and the Development and Progression of Radiographic Osteoarthritis of the Knee

G. M. Brouwer, A. W. van Tol, A. P. Bergink, J. N. Belo, R. M. D. Bernsen, M. Reijman, H. A. P. Pols, and S. M. A. Bierma-Zeinstra

Objective. Although knee malalignment is as- increased risk was especially seen in overweight and sumed to correlate with knee osteoarthritis (OA), it is obese individuals but not in non-overweight persons. still unknown whether malalignment precedes the de- The risk of OA progression was also significantly in- velopment of OA or whether it is a result of OA. The aim creased in the group with varus alignment compared of this study was to assess the relationship between with the group with normal alignment (OR 2.90, 95% CI malalignment and the development of knee OA as well 1.07–7.88). as progression of knee OA. Conclusion. An increasing degree of varus align- Methods. A total of 1,501 participants in the ment is associated not only with progression of knee OA Rotterdam study were randomly selected. Knee OA at but also with development of knee OA. However, this baseline and at followup (mean followup 6.6 years) was association seems particularly applicable to overweight scored according to the Kellgren/Lawrence (K/L) grad- and obese persons. ing system. Alignment was measured by the femorotibial angle on radiographs at baseline. Multivariable logistic Knee osteoarthritis (OA) is the most common regression for repeated measurements was used to joint disorder and is characterized by abnormal articular analyze the association of malalignment with the devel- cartilage and subchondral bone of the tibiofemoral joint. opment and progression of OA. In The Netherlands, Ͼ335,000 of the 16 million inhab- Results. Of 2,664 knees, 1,012 (38%) were consid- itants have knee OA (1). The risk of disability attribut- ered to have normal alignment, 693 (26%) had varus able to knee OA alone is as great as that attributable to alignment, and 959 (36%) had valgus alignment. A cardiac disease and greater than that attributable to any comparison of valgus alignment and normal alignment showed that valgus alignment was associated with a other medical condition in elderly persons (2). Knee OA borderline significant increase in development of knee also substantially increases the risk of disability due to OA (odds ratio [OR] 1.54, 95% confidence interval [95% other medical conditions (3). Because of aging of the CI] 0.97–2.44), and varus alignment was associated with population, the prevalence of OA is expected to increase a 2-fold increased risk (OR 2.06, 95% CI 1.28–3.32). substantially in the coming decades (4). Stratification for body mass index showed that this Malalignment (valgus or varus) of the knee is assumed to correlate with unicompartmental OA of the knee. However, it is still unknown whether malalignment G. M. Brouwer, MSc, A. W. van Tol, MSc, A. P. Bergink, precedes the development of radiographic OA, whether MD, J. N. Belo, MD, R. M. D. Bernsen, PhD, M. Reijman, PhD, malalignment is a result of OA, or (even more likely) H. A. P. Pols, MD, PhD, S. M. A. Bierma-Zeinstra, PhD: Erasmus Medical Center, Rotterdam, The Netherlands. whether the relationship between malalignment and OA Mrs. Brouwer and Mrs. van Tol contributed equally to this is bidirectional. A reason for the relatively small number work. Address correspondence and reprint requests to S. M. A. of epidemiologic studies dealing with malalignment Bierma-Zeinstra, PhD, Department of General Practice, Erasmus might be that malalignment is mainly measured by Medical Center, PO Box 2040, 3000 CA, Rotterdam, The Netherlands. means of the hip–knee–ankle (HKA) angle on full-limb E-mail: [email protected]. Submitted for publication March 29, 2006; accepted in revised radiographs, assessing the mechanical axis in the knee. form January 16, 2007. Full-limb radiographs are used specifically to determine

1204 MALALIGNMENT AND DEVELOPMENT OF KNEE OA 1205

the HKA angle and, in most cases, to surgically adjust this angle. However, this method is cumbersome, requires specialized equipment and expertise, and is costly (particularly for large epidemiologic studies) (5). Whereas some studies have shown a positive relationship between malalignment and progression of knee OA (6–9), others found that the presence of a varus or valgus deformity, although it is unclear how this was assessed, did not differ between patients in whom OA progressed and those in whom OA did not progress (10). No study has investigated the relationship between malalignment and development of OA using HKA angle measurements. In clinical practice, obtaining anteroposterior (AP) knee radiographs is the most common way to evaluate knee OA radiographically; these radiographs are also used in most large epidemiologic studies. On AP radiographs, the femorotibial (FT) angle can be measured, which defines the anatomic axis in the knee. It was recently shown that this method correlates moderately (r ϭ 0.75) with measurement of the HKA angle on Figure 1. Flow chart showing subjects from eligibility to inclusion in full-limb radiographs (5). Even more recently, Hinman the present study. et al (11) confirmed in another study that the anatomic axis is a valid alternative to the HKA angle for determining frontal plane knee alignment (r ϭ 0.88). This for a home interview. When these participants were willing and method, however, has not been used to determine the able to visit the research center, radiographic and other influence of alignment on the development of knee OA. medical examinations were performed 2 weeks later. Of the We identified one study in which this method was used 7,983 participants, 6,450 visited the research center for a to determine the influence of the FT angle on annual baseline medical examination, and 3,585 of these revisited the cartilage loss measured on magnetic resonance imaging center after 6.6 years of followup (Figure 1). Compared with the total Rotterdam Study population, the population available in patients with moderate knee OA; that study showed a for followup was significantly younger (70.6 years versus 66.4 significant relationship between medial femoral carti- years) (13). For the present study, a random subset of 1,501 of lage loss and the baseline FT angle (12). the participants available for followup was used, and radio- Therefore, in the present study we investigated graphs of the knees from this group were read. the association between malalignment, based on the FT Baseline measurements were obtained between April 1990 and July 1993, and the followup measurements were angle on AP radiographs, and the development of OA in obtained between 1996 and 1999 (mean Ϯ SD followup 6.6 Ϯ individuals who did not have knee OA at baseline. We 0.5 years). The Medical Ethics Committee of the Erasmus also investigated whether malalignment assessed with Medical Center approved the Rotterdam Study. the FT angle is a prognostic factor for progression of OA Radiographic measurements. To assess radiographic in individuals with knee OA at baseline. OA, AP radiographs of the lower extremity with weight- bearing were obtained at 70 KV, a focus of 1.8 mm2, and a focus-to-film distance of 120 cm, using High Resolution G PATIENTS AND METHODS 35 ϫ 43–cm radiographic film (Fujifilm Medical Systems, Stamford, CT). Radiographs of the extended knee were ob- Participants. The current study population comprised tained with the patella in central position. participants in the Rotterdam Study, which is an open- Radiographic OA was graded using the Kellgren/ population, prospective cohort study on the incidence of and Lawrence (K/L) scale for both tibiofemoral compartments risk factors for chronic disabling diseases. In the Rotterdam together (14). Two trained readers who were blinded to the Study, all 10,275 inhabitants of one district of Rotterdam clinical status of patients independently evaluated the radio- (Ommoord) ages 55 years and older were invited to partici- graphs of the knee. After each set of 150 radiographs was pate. The response rate was 78%, which means that 7,983 men evaluated, the scores of the 2 readers were assessed. Whenever and women participated. All participants gave written in- the K/L grade differed, the 2 readers met to read the radio- formed consent and were visited by a trained research assistant graphs together, and a consensus score was determined. Ra- 1206 BROUWER ET AL

yses were adjusted for age (continuous variable), sex, and body mass index (BMI; continuous variable). The relationship between valgus or varus alignment and the development of knee OA (K/L grade Ն2) was assessed for knees with K/L grades 0 and 1 together and for knees with K/L grade 0 and K/L grade 1 separately. Progression of OA of the knee was defined by subtracting the K/L grade at baseline from the K/L grade at followup (outcome Ն1) and was assessed in knees with K/L grade 2. Knees with K/L grades 3 and 4 were excluded due to very few cases, and knees that were not able to progress any further (K/L grade 4 at baseline) were also excluded. Again, alignment was assessed as normal versus valgus and varus. Furthermore, given the role of being overweight in the development of knee OA (17,18), additional analyses were executed stratified for participants who were obese, those who were overweight, and those who were not overweight. The BMI was used for this stratification, with the following 3 groups: Ͻ25 kg/m2, Ն25 kg/m2 and Ͻ30 kg/m2, and Ն30 kg/m2. Overweight was defined as a BMI of 25–30 kg/m2, and obesity Figure 2. Method used to measure the femorotibial angle. was defined as a BMI of Ն30 kg/m2. All statistical tests were 2-sided and were conducted using an alpha error of 0.05. SPSS version 11.0 software (SPSS, Chicago, IL) and SAS version 8.2 software (SAS Institute, Cary, NC) were used for all analyses. diographic OA was defined as being present when the K/L grade was Ն2. Alignment was measured as the medial angle formed Table 1. Baseline characteristics of the study population (n ϭ by the femur and tibia (FT angle) (Figure 2), using a method 1,501)* based on that described by Moreland et al (15). Lines were drawn through the middle of the femoral shaft and through the Characteristic middle of the tibial shaft. The medial angle subtended at the Female sex 59.4 point at which these 2 lines met in the center of the tibial spines Age, mean Ϯ SD years 66.4 Ϯ 6.7 was characterized as the anatomic angle (16). Body mass index, mean Ϯ SD kg/m2 26.3 Ϯ 3.6 The knees were divided into 3 groups. A distinction Femorotibial angle, mean Ϯ SD was made between normal alignment, valgus alignment, and Left knee 183.9 Ϯ 3.6 varus alignment. Normal alignment was considered to be an Right knee 183.3 Ϯ 3.3 FT angle between 182° and 184°. A knee was defined as valgus Varus alignment when alignment was Ͼ184° and as varus when alignment was Left knee 20.8 Ͻ182°. These cutoffs were based on values for normal, varus, Right knee 25.4 and valgus alignment for the mechanical axis angle from a Valgus alignment Left knee 34.5 full-limb radiograph, as described by Moreland et al (15), with ϩ Right knee 28.8 adjustment ( 4°) for the offset in valgus direction when Normal alignment measured as FT angle on AP extended-knee radiographs, as Left knee 31.7 reported by Kraus et al (5). We did not use decimals as in the Right knee 36.2 original cutoff values or offset values, because our measure- K/L grade 0 ments had only 1° increments. Left knee 73.0 Two independent observers who were blinded to fol- Right knee 69.8 lowup data each measured the FT angle of 1,501 knees. To K/L grade 1 assess interobserver reproducibility, 98 radiographs were mea- Left knee 13.9 Right knee 14.2 sured by 2 observers blinded to each other’s results. K/L grade 2 Statistical analysis. Reproducibility of the FT angle Left knee 12.1 measurements was assessed by calculating intraclass correla- Right knee 14.9 tion coefficients (ICCs) using a two-way mixed-effect model. K/L grade 3 Odds ratios (ORs) were calculated using generalized estimat- Left knee 1.1 ing equations (GEEs). ORs represented the likelihood that Right knee 1.1 OA would develop or progress in knees with malalignment at K/L grade 4 0.0 baseline (compared with knees without malalignment at base- Left knee 0.0 line). This is a logistic regression for repeated measurements, Right knee 0.1 to take into account the correlation between the left and right * Except where indicated otherwise, values are the percent. Informa- knees. These analyses were executed for valgus and varus tion on alignment was missing for 13% of left knees and 9.5% of right alignment, with normal alignment as a reference group. Anal- knees. K/L ϭ Kellgren/Lawrence. MALALIGNMENT AND DEVELOPMENT OF KNEE OA 1207

of the FT angle measurement showed high reproducibility (ICC 0.90). Among the 3,002 knees, information on 338 knees (11%) was missing due to the absence (or illegi- bility) of the radiographs. The remaining 2,664 knees were divided into 3 categories as described above. Of these, 1,012 knees (38%) were considered to have normal alignment, 693 (26%) had varus alignment, and 959 (36%) had valgus alignment. Figure 3 shows the distribution of the FT angle over all knees. Development of knee OA. Among knees without OA (K/L grade 0 or 1) at baseline, OA had developed at followup in 45 (5.5%) of the 819 knees with valgus alignment, in 43 (7.4%) of the 579 knees with varus alignment, and in 35 (3.9%) of the 892 knees with normal alignment. In GEE logistic regression analyses, valgus versus normal alignment at baseline was associated with a borderline significantly increased risk of developing OA for those with K/L grade 0 or 1 at baseline (OR 1.54, 95% confidence interval [95% CI] 0.97–2.44). The knees with varus alignment showed a significant increase in the risk of developing OA for Figure 3. Histogram showing the distribution of alignment of the knees. those with K/L grade 0 or 1 at baseline (OR 2.06, 95% CI 1.28–3.32) (Table 2). Similar estimates were obtained when knees with K/L grade 0 and knees with K/L grade 1 at baseline were analyzed separately, although valgus alignment was no longer associated with a significantly RESULTS increased risk of developing OA (Table 2). Baseline characteristics of the cohort. The study Progression of OA. We also examined the rela- population comprised 1,501 participants. The mean age tionship between baseline alignment and progression of was 66.4 years, and almost 60% of the participants were knee OA (increase in the K/L score of Ն1 at followup in women. Table 1 presents the baseline characteristics of knees with a K/L score at baseline of Ն2). Progression the total study population. The interreader assessment occurred in 8 (6.3%) of the 128 knees with valgus

Table 2. Association between alignment at baseline and development of knee OA after 6.6 years of followup in knees without OA at baseline* No. of knees No. of knees without OA with OA at Alignment at baseline followup OR† 95% CI P K/L grades 1 and 2 Normal 892 35 1 (reference) – – Valgus 819 45 1.54 0.97–2.44 0.065 Varus 579 43 2.06 1.28–3.32 0.003 K/L grade 0 Normal 752 19 1 (reference) – – Valgus 694 27 1.63 0.89–3.00 0.112 Varus 484 23 1.95 1.02–3.73 0.043 K/L grade 1 Normal 140 16 1 (reference) – – Valgus 125 18 1.52 0.77–3.01 0.232 Varus 95 20 2.12 1.00–4.46 0.049 * No osteoarthritis (OA) at baseline was defined as a Kellgren/Lawrence (K/L) grade of 0 or 1. OR ϭ odds ratio; 95% CI ϭ 95% confidence interval. † Adjusted for age, sex, and body mass index. 1208 BROUWER ET AL

Table 3. Association between alignment at baseline and progression of knee OA after 6.6 years of followup in knees with OA at baseline* No. of knees No. of knees with with OA at progression Alignment baseline at followup OR† 95% CI P Normal 125 6 1 (reference) – – Valgus 128 8 1.39 0.48–4.05 0.550 Varus 95 11 2.90 1.07–7.88 0.037 * Osteoarthritis (OA) at baseline was defined as a Kellgren/Lawrence (K/L) grade of Ն2. OR ϭ odds ratio; 95% CI ϭ 95% confidence interval. † Adjusted for age, sex, and body mass index. alignment, 11 (11.6%) of the 95 knees with varus group, the analyses showed a nonsignificant OR of 1.08 alignment, and 6 (4.8%) of the 125 knees with normal (95% CI 0.41–2.80) for the group with valgus align- alignment. In GEE logistic regression analyses, valgus ment compared with the group with normal alignment versus normal alignment was not significantly associated (Table 4). with progression of knee OA (OR 1.39, 95% CI 0.48– When we compared the group with varus align- 4.05). In knees with varus alignment, analyses showed ment with the group with normal alignment, a significant significantly increased odds for progression (OR 2.90, OR of 2.02 (95% CI 1.07–3.84) was observed for persons 95% CI 1.07–7.88) compared with knees with normal with a BMI Ն25 kg/m2 but Ͻ30 kg/m2, and an even alignment (Table 3). stronger OR (5.06 [95% CI 1.71–14.94]) was associated Analyses stratified by BMI. For stratification with a BMI of Ն30 kg/m2. The analyses in the non- according to being overweight, fewer knees were in- overweight group showed a nonsignificant OR (1.24 cluded in the analyses due to the absence of 7 BMI [95% CI 0.50–3.10]) (Table 4). Stratification for the measurements. Among individuals (123 knees) with K/L analyses of progression in knees with OA (K/L grade Ն2 grade 0 or 1 at baseline and in whom OA developed, 30 at baseline) yielded very few cases in each stratum, persons had a BMI of Ͻ25 kg/m2, 62 persons had a BMI especially in the non-overweight group, and was deemed of 25–29.99 kg/m2, and 31 persons had a BMI of Ն30 clinically and statistically meaningless. kg/m2. Comparing the valgus group with the normal alignment group, a nonsignificant OR of 1.42 (95% CI DISCUSSION 0.78–2.60) for the development of OA was observed for individuals with a BMI of Ն25 kg/m2 but Ͻ30 kg/m2, and Until now, only animal model data have sup- a significant OR of 3.25 (95% CI 1.14–9.27) was associ- ported a link between preexisting varus or valgus align- ated with a BMI of Ն30 kg/m2. In the non-overweight ment and development of knee OA (19). Using data

Table 4. Association between alignment at baseline and development of knee OA after 6.6 years of followup in knees without OA at baseline, stratified for BMI (kg/m2)* No. of knees No. of knees without OA with OA at Alignment at baseline followup OR† 95% CI P BMI Ͻ25 Normal 347 11 1 (reference) – – Valgus 289 8 1.08 0.41–2.80 0.881 Varus 287 11 1.24 0.50–3.10 0.640 BMI Ն25 and Ͻ30 Normal 442 19 1 (reference) – – Valgus 432 23 1.42 0.78–2.60 0.252 Varus 240 20 2.02 1.07–3.84 0.031 BMI Ն30 Normal 103 5 1 (reference) – – Valgus 98 14 3.25 1.14–9.27 0.028 Varus 52 12 5.06 1.71–14.94 0.003 *OAϭ osteoarthritis; BMI ϭ body mass index; OR ϭ odds ratio; 95% CI ϭ 95% confidence interval. † Adjusted for age and sex. MALALIGNMENT AND DEVELOPMENT OF KNEE OA 1209

from a large population-based cohort study, our study is differences in the cohorts, for example a higher mean the first to show that malalignment is a risk factor for BMI in the population described by Sharma et al, might development of knee OA. It also further strengthened explain the stronger relationship in that cohort. the concept that malalignment is positively related to Another point of discussion is that the adduction progression of knee OA. moment, rather than alignment, reflects the dynamic A clear limitation of the present study is that we load on the medial compartment of the knee (8). The did not obtain full-limb radiographs for accurate mea- adduction moment of the knee is a major determinant of surement of mechanical alignment in the population medial-to-lateral load distribution; thus, it is responsible studies. Although the anatomic axis of the tibia is for the biomechanical abnormality of medial compart- assumed to be straight (20), the bowing curvature of the ment knee OA. Sharma et al reported that dynamic load tibia might lead to differences between anatomic align- during gait correlated with disease severity in tibiofemo- ment (measured by knee angle) and mechanical align- ral knee OA. They suggested that the magnitude of the ment using the entire tibia. Kraus et al (5) reported a adduction moment possibly influences the structural mean offset of ϳ4° in the valgus direction for anatomic outcome in medial compartment knee OA. Although alignment compared with mechanical alignment. For the FT angle measured in our study might be a proxy for this reason, we used this reported offset in our defini- unfavorable load as a risk factor, this might also be the tions of normal, varus, and valgus alignment. However, case for the HKA angle. Using univariate analyses, Kraus et al and Hinman et al (11) also reported that Miyazaki et al (8) showed that the HKA angle, as well as measurements of mechanical alignment and measure- the adduction moment, were related to radiographic ment of anatomic alignment were strongly but not progression; however, in multivariable analyses only the optimally correlated (r ϭ 0.75 and r ϭ 0.88, respec- adduction moment was significantly related, and the tively). Therefore, the strength of the relationship be- HKA angle lost its relationship and significance. tween alignment and the increase in the K/L score might Because of the arbitrariness of the cutoff point have been stronger if a full-limb assessment of alignment for the absence of radiographic OA, we also assessed the had been used and mechanical alignment had been risk of development of OA in knees with K/L grades 0 measured directly. and 1 at baseline separately. Knees with K/L grade 2 This difference in measurement technique may were used to analyze progression. However, data for explain the stronger relationship (OR 4.1) between knee individuals who had K/L grade 1 at baseline with pro- angle and radiographic progression seen in the study by gression to K/L grade Ն2 that were used for the analysis Sharma et al (7). Another explanation for the difference of development of OA can also be used to assess in ORs between our study and that of Sharma et al might progression. For progression, however, the association be the difference in methods used to assess progression tended to be stronger in persons with K/L grade 2 than of knee OA. Sharma and colleagues used an increase of in those with K/L grade 1. The risk of development of Ͼ1 grade in severity of joint space narrowing, whereas OA was similar for individuals with a baseline K/L grade we used the K/L scale. Both methods are used to assess of 0 or 1, indicating that perhaps the usual definitions for OA, but the sensitivity to measure change is higher for non-OA (K/L grades 0 or 1) and OA (K/L grade Ն2) the methods measuring joint space width than for meth- might be acceptable after all. ods using the K/L score (21). Moreover, the sensitivity to Of the 6,450 participants who visited a research change also depends on the manner in which radio- center for a baseline examination, 3,585 revisited the graphs are obtained. Sharma et al used a semiflexed, center after 6.6 years of followup. A possible limitation fluoroscopically confirmed knee radiograph, which is a of our study is potential health-based selection bias. The better radiographic method than the standing, weight- subjects in the present study had to be mobile enough to bearing, fully extended AP radiographs used in our study visit the research center at baseline and had to survive (7). In fully extended AP radiographs, it is not always the followup period or be healthy enough to visit the possible to acquire the fully extended position; pain and research center at followup. In other words, individuals stiffness of the joint make it harder to completely extend with the most severe symptoms were most likely not the knee joint, which provides a smaller joint space included. Therefore, it seems probable that in this width, which might overestimate disease progression younger and healthier population with less frequent (22). Therefore, it might be noteworthy that the re- lower-limb disability and pain, the prevalence of radio- ported relationships, although less strong, were detected graphic knee OA or progression of radiographic OA at despite the radiographic methods used. Finally, simple followup may have been underestimated. Based on the 1210 BROUWER ET AL

report by Sharma et al that malalignment is related to a Moreover, we thank the participating general practitioners, the faster functional decline in knee OA (7), we emphasize pharmacists, the many field workers at the research center in that the relationship reported in our study may be an Ommoord, and, of course, all of the participants. underestimation. Based on our analysis, we now know that mal- AUTHOR CONTRIBUTIONS alignment predates knee OA, and this might be attrib- Dr. Bierma-Zeinstra had full access to all of the data in the utable to genetic, posttraumatic, or developmental fac- study and takes responsibility for the integrity of the data and the accuracy of the data analysis. tors. Unfortunately, our study does not provide any data Study design. Brouwer, van Tol, Pols, Bierma-Zeinstra. on former knee injury and possible subsequent menis- Acquisition of data. Brouwer, van Tol, Bergink. cectomy; further studies on the background of malalign- Analysis and interpretation of data. Brouwer, van Tol, Bergink, Bernsen, Reijman, Bierma-Zeinstra. ment are necessary. Manuscript preparation. Brouwer, van Tol, Bergink, Belo, Bernsen, Our results show a stronger relationship between Reijman, Pols, Bierma-Zeinstra. malalignment and the risk of development of OA in the Statistical analysis. Brouwer, van Tol, Bernsen, Bierma-Zeinstra. overweight group than in the non-overweight group. The relationship might even be absent in the non-overweight REFERENCES group, suggesting that an unfavorable load is much less 1. Nationaal Kompas Volksgezondheid Web site. URL: http://www. harmful in non-overweight persons. However, our study rivm.nl/vtv/object_class/kom_artrose.html. showed a low number of knees with disease progression, 2. Guccione AA, Felson DT, Anderson JJ, Anthony JM, Zhang Y, Wilson PW, et al. The effects of specific medical conditions on the a common problem in population-based studies, and functional limitations of elders in the Framingham Study. Am J therefore the power was too low (too few cases in each Public Health 1994;84:351–8. 3. Ettinger WH, Davis MA, Neuhaus JM, Mallon KP. Long-term stratum, especially in the non-overweight group) to physical functioning in persons with knee osteoarthritis from compare the relationships between varus or valgus align- NHANES. I. Effects of comorbid medical conditions. J Clin ment and risk of progression of OA in the non- Epidemiol 1994;47:809–15. 4. Woolf AD, Pfleger B. Burden of major musculoskeletal conditions overweight, overweight, and obese groups. Therefore, [review]. Bull World Health Organ 2003;81:646–56. we do not yet know whether being overweight or obese 5. Kraus VB, Vail TP, Worrell T, McDaniel G. A comparative increases the relationship between malalignment and assessment of alignment angle of the knee by radiographic and physical examination methods. Arthritis Rheum 2005;52: progression of knee OA in a similar manner. Interest- 1730–5. ingly, Felson et al showed that high body weight in- 6. Cerejo R, Dunlop DD, Cahue S, Channin D, Song J, Sharma L. creases the risk of structural progression of knee OA, The influence of alignment on risk of knee osteoarthritis progression according to baseline stage of disease. Arthritis Rheum but its effect on progression appears to be limited to 2002;46:2632–6. knees with moderate malalignment (17). Together with 7. Sharma L, Song J, Felson DT, Cahue S, Shamiyeh E, Dunlop DD. the findings from our study, these results suggest that The role of knee alignment in disease progression and functional decline in knee osteoarthritis. JAMA 2001;286:188–95. these factors mutually influence each other. 8. Miyazaki T, Wada M, Kawahara H, Sato M, Baba H, Shimada S. The present results indicate a need to investigate Dynamic load at baseline can predict radiographic disease pro- the effectiveness of preventive interventions to reduce gression in medial compartment knee osteoarthritis. Ann Rheum Dis 2002;61:617–22. the risk of varus load, especially in overweight persons. 9. Belo JN, Berger MY, Reijman M, Koes BW, Bierma-Zeinstra SM. For secondary prevention, more active interventions Prognostic factors of progression of osteoarthritis of the knee: a (besides weight reduction) such as wedged insoles systematic review of observational studies. Arthritis Rheum 2007; 57:13–26. should be evaluated. 10. Dougados M, Gueguen A, Nguyen M, Thiesce A, Listrat V, Jacob In conclusion, we used FT angle measurements L, et al. Longitudinal radiologic evaluation of osteoarthritis of the to confirm relationships between malalignment and pro- knee. J Rheumatol 1992;19:378–84. 11. Hinman RS, May RL, Crossley KM. Is there an alternative to the gression of knee OA. This study is the first to show an full-leg radiograph for determining knee joint alignment in osteo- association between malalignment at baseline and the arthritis? Arthritis Rheum 2006;55:306–13. risk of development of knee OA; this association seems 12. Cicuttini F, Wluka A, Hankin J, Wang Y. Longitudinal study of the relationship between knee angle and tibiofemoral cartilage particularly applicable to overweight and obese persons. volume in subjects with knee osteoarthritis. Rheumatology (Ox- ford) 2004;43:321–4. 13. Reijman M, Hazes JM, Pols HA, Bernsen RM, Koes BW, ACKNOWLEDGMENTS Bierma-Zeinstra SM. Validity and reliability of three definitions of hip osteoarthritis: cross sectional and longitudinal approach. Ann We are very grateful to Dr. Odding for scoring the Rheum Dis 2004;63:1427–33. radiographs of the knee, and F. van Rooij, E. van der Heijden, 14. Kellgren JH, Lawrence JS. Radiological assessment of osteo- R. Vermeeren, and L. Verwey for collecting followup data. arthrosis. Ann Rheum Dis 1957;16:494–502. MALALIGNMENT AND DEVELOPMENT OF KNEE OA 1211

15. Moreland JR, Bassett LW, Hanker GJ. Radiographic analysis of 19. Tetsworth K, Paley D. Malalignment and degenerative arthro- the axial alignment of the lower extremity. J Bone Joint Surg Am pathy. Orthop Clin North Am 1994;25:367–77. 1987;69:745–9. 20. Tang WM, Zhu YH, Chiu KY. Axial alignment of the lower 16. Felson DT, Nevitt MC, Zhang Y, Aliabadi P, Baumer B, Gale D, extremity in Chinese adults. J Bone Joint Surg Am 2000;82-A: et al. High prevalence of lateral knee osteoarthritis in Beijing 1603–8. Chinese compared with Framingham Caucasian subjects. Arthritis 21. Ravaud P, Giraudeau B, Auleley GR, Chastang C, Poiraudeau S, Rheum 2002;46:1217–22. Ayral X, et al. Radiographic assessment of knee osteoarthritis: 17. Felson DT, Goggins J, Niu J, Zhang Y, Hunter DJ. The effect of reproducibility and sensitivity to change. J Rheumatol 1996;23: body weight on progression of knee osteoarthritis is dependent on 1756–64. alignment. Arthritis Rheum 2004;50:3904–9. 22. Mazzuca SA, Brandt KD, Buckwalter KA, Lequesne M. Pitfalls in 18. Sharma L, Lou C, Cahue S, Dunlop DD. The mechanism of the the accurate measurement of joint space narrowing in semiflexed, effect of obesity in knee osteoarthritis: the mediating role of anteroposterior radiographic imaging of the knee. Arthritis malalignment. Arthritis Rheum 2000;43:568–75. Rheum 2004;50:2508–15. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1212–1218 DOI 10.1002/art.22508 © 2007, American College of Rheumatology

Knee Alignment Does Not Predict Incident Osteoarthritis

The Framingham Osteoarthritis Study

David J. Hunter,1 Jingbo Niu,1 David T. Felson,1 William F. Harvey,1 K. Douglas Gross,1 Paula McCree,1 Piran Aliabadi,2 Burton Sack,1 and Yuqing Zhang1

Objective. To examine the relationship of knee mating equations, adjusting for age, sex, and body mass malalignment to the occurrence of knee osteoarthritis index (BMI). We used the same approach to assess the (OA) among subjects without radiographic OA at base- association between each alignment measurement and line to determine whether malalignment is a risk factor the risk of medial TF OA. for incident disease or simply a marker of increasing Results. Subjects in the case population were disease severity. older and had a higher BMI than the controls. The Methods. We selected 110 incident tibiofemoral alignment values were normally distributed and were (TF) OA case knees (76 subjects) and 356 random not different between the cases and the controls. After control knees (178 subjects) from among participants in adjustment for age, sex and BMI, there was no signifi- the Framingham Osteoarthritis Study. Case knees did cant increase in incident OA in the highest quartile not have OA at baseline (1992–1994 examination) but compared with the lowest quartile category for any of had developed OA (Kellgren/Lawrence grade >2) at the alignment measures (P for trend for anatomic axis followup (2002–2005 examination) (mean of 8.75 years and condylar tibial plateau angle was 0.83 and 0.80, between examinations). Control knees did not have OA respectively). Similar results were also observed for at baseline. Standardized digital radiographs of the medial compartment OA. fully extended knee with weight-bearing were read using Conclusion. a standard protocol and eFilm viewing software. We We found that baseline knee align- measured the anatomic axis, the condylar angle, the ment is not associated with either incident radiographic tibial plateau angle, and the condylar tibial plateau TF OA or medial TF OA. These results suggest that angle. The interobserver intraclass correlation coeffi- malalignment is not a risk factor for OA, but rather is a cient (ICC) ranged from 0.93 to 0.96 and the intraob- marker of disease severity and/or its progression. server ICC from 0.94 to 0.97. In a knee-specific analysis, we examined the relationship of each alignment mea- Osteoarthritis (OA) is widely believed to result surement to the risk of TF OA using generalized esti- from local mechanical factors acting within the context of systemic susceptibility. Previous studies have demonstrated that malalignment is a potent predictor of dis- Supported by NIH grants AG-18393 and AR-47785 and by ease progression in knee OA (1). However, these obser- the National Heart, Lung, and Blood Institute, Framingham Heart Study (contract N01-HC-25195). vations have only been made in populations with 1David J. Hunter, MBBS, PhD, Jingbo Niu, MD, David T. preexisting radiographic disease. While frontal plane Felson, MD, MPH, William F. Harvey, MD, K. Douglas Gross, PT, malalignment is clearly associated with increasing struc- PhD, Paula McCree, MSc, Burton Sack, MD, Yuqing Zhang, DSc: Boston University School of Medicine, Boston, Massachusetts; 2Piran tural damage in the knee joint, it is still unclear whether Aliabadi, MD: Brigham and Women’s Hospital, Boston, Massachu- the observed malalignment is a consequence or a cause setts. Address correspondence and reprint requests to David J. of worsening disease (2). In the effort to better under- Hunter, MBBS, PhD, Boston University School of Medicine, Clinical stand the etiology of knee OA and develop effective Epidemiology Research and Training Unit, 715 Albany Street A203, preventative interventions, it is essential that we clarify Boston, MA 02118. E-mail: [email protected]. Submitted for publication June 14, 2006; accepted in revised the role of knee malalignment in both the initial occur- form January 4, 2007. rence of the disease and its subsequent progression.

1212 KNEE ALIGNMENT AND INCIDENT OA 1213

There is good reason to believe that OA suscep- SUBJECTS AND METHODS tibility may be affected by differences in alignment. In The Framingham OA Study is a population-based theory, any shift from a neutral or collinear alignment of cohort study of risk factors for OA. The Framingham OA the hip, knee, and ankle affects load distribution at the Study includes a cohort of offspring of the original Framing- knee (3). The load-bearing mechanical axis is tradition- ham Heart Study, which was begun in 1948 (10). As part of a ally represented on radiographs by a line drawn from the study on the inheritance of OA, participants in the Framing- ham Offspring Cohort were originally examined between 1992 center of the femoral head to the center of the tibial and 1994 (examination 5 of the Framingham Heart Study). All spines and a second line from the center of the ankle surviving members of this group were contacted by telephone talus to the center of the tibial spines. In a varus and invited to participate in a more recent examination from malaligned knee, this load-bearing line passes medial to 2002 to 2005 (mean interval between visits 8.75 years). A the center of the knee, and an adduction moment arm is validated survey instrument (11), supplemented by questions about medication use, was used to exclude patients with created, which increases compressive force across the rheumatoid arthritis. medial compartment. In a valgus malaligned knee, the Radiographic assessments. During the Framingham load-bearing axis passes lateral to the knee center, and Offspring Cohort examination 5 visit (conducted during 1992– the resulting abduction moment arm increases force 1994), a radiograph of both knees in full extension with weight-bearing was obtained, using a standardized protocol across the lateral compartment (3). More recently, the that included outlines of the feet in order to keep the rotation intraarticular alignment of the femoral condyles and the of the knee the same at followup evaluations. Knee radio- tibial plateau has been proposed as a more valid indica- graphs were obtained at 0° and at 6° caudad, and the better of tor of how contact forces are distributed across the joint the 2 views (based on the optimal superimposition of the anterior and posterior margins of the medial tibial plateau) surfaces of the knee during weight bearing activities was selected for comparison with findings at the followup (4,5). Under conditions of normal alignment, the medial examination. compartment absorbs 60–70% of the compressive force After the subjects completed their followup examina- across the knee during weight bearing (6). This fact has tion, both baseline and followup radiographs were read independently by 2 study readers, one a bone and joint radiologist been offered as a possible explanation for why OA (PA), and the other a rheumatologist (BS). Knee radiographs affects the medial tibiofemoral compartment much were read in a paired manner, unblinded with regard to more often than the lateral compartment in both men chronological sequence. Readers graded each knee according and women (7). to the Kellgren/Lawrence (K/L) method (12) at both time points and evaluated the individual features and whether these In the presence of existing knee OA, abnormal features had changed over the followup period. Each knee was anatomic alignment is associated with accelerated struc- scored on a 0–3 scale for the presence of osteophytes and joint tural deterioration in the compartment under greatest space narrowing (JSN) using the Osteoarthritis Research compressive stress. Varus malalignment has been shown Society International atlas (13). For the K/L grade and its change, we used readings to lead to a 4-fold increase in focal medial knee OA from the bone and joint radiologist (PA). The adjudication progression, while valgus malalignment has been shown sessions were chaired by one of us (DTF). If there was a to predispose to a 2–5-fold increase in lateral OA difference in K/L grade based on osteophytes that led to a progression (1,8). Malalignment also appears to be a difference in the assignment of either prevalent or incident OA status, this was adjudicated. All adjudicated readings were major determinant of the size, location, and progression arrived at by consensus of the readers. Disagreements on of bone marrow lesions, with subsequent impact on the changes in the joint space were also adjudicated if the two rate of cartilage loss (9). However, these findings are readers disagreed and if adjudication of joint space change from studies of persons with preexisting disease. While altered the appropriateness of the K/L score. For the bone and joint radiologist, the intrareader kappa value for the K/L grade frontal-plane knee malalignment may be a potent risk was 0.82; the interreader kappa value was 0.74 (P Ͻ 0.001 for factor for disease progression and/or a useful clinical both comparisons). marker of increasing disease severity, it has not yet been Selection of case and control groups. Subjects eligible shown to be a risk factor for incident knee OA. There- for incident radiographic tibiofemoral (TF) OA were participants in the Framingham Offspring Cohort Study who had no fore, we performed a nested case–control study among radiographic TF OA (K/L grade Ͻ2) in both knees at baseline participants in the Framingham Osteoarthritis Study in visit (during 1992–1994) (12). During the study period, 110 order to evaluate whether baseline malalignment (both knees (from 76 subjects) developed incident radiographic TF Ն anatomic axis and condylar plateau angle) was associ- OA (K/L grade 2) at the followup visit (during 2002–2005). ϳ These knees served as the case group. The control group was ated with subsequent incident radiographic knee OA 9 randomly selected from among subjects who were eligible for years later. incident radiographic TF OA and were in the same age and 1214 HUNTER ET AL

these lines was 10 cm from the knee joint surfaces when included in the field of view on the radiograph. Given the possibility of discrepant findings between condylar tibial plateau alignment and the more traditional anatomic axis measure of knee alignment, we were careful to assess both. Varus angles were assigned negative values, and valgus angles were assigned positive values. All measurements were taken by a single reader (WFH) who was blinded to the study question and the outcome status of the subject. To evaluate for reader drift, we reassessed intrarater reliability by inserting 1 original reliability radiograph for every 10 new radiographs read. The intrarater intraclass correlation coefficient (ICC) ranged from 0.94 to 0.97, and the interrater ICC ranged from 0.93 to 0.96 for the different angular measures. Statistical analysis. We divided anatomic axis alignment, femoral condylar angle, and condylar tibial plateau angle into 4 categories according to the quartile distribution of each measurement among all subjects. Knees in the lowest category of specific alignment measurement were used as the referent group. We examined the relationship of each alignment measurement to the risk of incident TF OA, adjusting for age, BMI and sex. Previous studies have suggested that incident OA develops more frequently in knees that already have doubtful OA (K/L grade 1) (15,16). Similarly, increasing malalignment Figure 1. Measures of knee alignment in a varus knee. The anatomic has been associated with increasing disease severity (17). axis (AA) is the angle between lines drawn from the midpoint of the Rather than just assuming that the baseline K/L grade did not femur through the tibial spines and from the tibial spines to the effect either the subsequent risk of progression or the align- midpoint of the tibia. The condylar angle (CA) is the angle between ment, we stratified the knees according to their baseline K/L the mechanical or anatomic axis line of the femur and a line tangent to score (0 versus 1) and obtained a pooled effect estimate of the femoral condyles. The tibial plateau angle (PA) is the angle each alignment measurement on the risk of incident knee OA between the mechanical or anatomic axis line of the tibia and a line using a conditional logistic regression model with generalized tangent to the tibial plateau. The condylar tibial plateau (CP) angle is estimating equations to control for correlation between two the angle between the above-mentioned tangent lines. knees in these models. The variability of the tibial plateau angle measurement was small, and most values were close to the mean; therefore, we divided this measurement into tertile groups according to the distribution among all participants. body mass index (BMI) range as the case group. A total of 356 We used the same approach to assess the relationship between knees (from 178 subjects) served as controls. each alignment measurement and incident TF OA of the In a subgroup analysis aimed at determining the risk of medial compartment. We assessed for both linear and incident medial compartment OA, we further identified 80 U-shaped trends given the potential for risk in both valgus and case knees with isolated medial TF OA. Medial TF OA was varus directions in the incident TF OA analysis and tested for defined as medial joint space narrowing greater than lateral linear trends for the analysis of incident TF OA in the medial Ն joint space narrowing and a K/L grade 2 (including a definite compartment. osteophyte). Measurements of alignment. We measured knee alignment among all case and control knees. The radiographs were RESULTS digitized so that imaging software (eFilm Workstation version 2.0.0; Merge, Milwaukee, WI) could be used to compose Of 1,705 subjects seen at the initial examination, reference lines and calculate the following angular measures 1,279 (75.0%) obtained knee radiographs as part of the (see Figure 1): the anatomic axis alignment (in degrees from followup examination, with an average (ϮSD) interval 180°), the femoral condylar angle (in degrees from 90°), the Ϯ tibial plateau angle (in degrees from 90°), and the condylar of 8.75 1.0 years. Among the 2,259 knees undergoing tibial plateau angle (in degrees between the femoral condylar both baseline and followup examination, we identified and tibial plateau lines). 110 cases of incident TF OA (76 subjects). We randomly Our assessment of the anatomic axis is consistent with selected 356 control knees (178 subjects) from the that described by Kraus et al (14). The anatomic axis was defined as the angle formed by the intersection of 2 lines remaining participants in the Framingham Osteoarthri- originating from points bisecting the femur and tibia and tis Study. converging at the center of the tibial spine tips. The origin of The characteristics of the study population are KNEE ALIGNMENT AND INCIDENT OA 1215

Table 1. Characteristics of the study population Cases Controls (n ϭ 110 knees) (n ϭ 356 knees) Age, mean Ϯ SD (range) years 55.2 Ϯ 8.2 (32–74) 52.7 Ϯ 8.1 (32–73) Body mass index, mean Ϯ SD 30.5 Ϯ 5.6 27.1 Ϯ 4.7 % female 53.6 53.6 % of knees with K/L score of 1 at baseline* 35.5 7.3 % with history of knee injury 23.9 9.9 % who contributed 2 knees to the analysis 44.7 98.9 Measures of alignment, mean Ϯ SD (range) degrees† Anatomic axis 2.0 Ϯ 3.0 (Ϫ7 to 10) 2.2 Ϯ 3.2 (Ϫ7to10) Condylar angle 5.4 Ϯ 3.0 (Ϫ6 to 11) 5.2 Ϯ 3.0 (Ϫ3to13) Tibial plateau angle Ϫ1.1 Ϯ 1.2 (Ϫ5to3) Ϫ0.8 Ϯ 1.0 (Ϫ4to4) Condylar tibial plateau angle Ϫ2.4 Ϯ 1.8 (Ϫ6to3) Ϫ2.3 Ϯ 1.8 (Ϫ7to5) * K/L ϭ Kellgren/Lawrence. † Positive numbers indicate valgus angles; negative numbers indicate varus angles. displayed in Table 1. On average, subjects in the case ity of knees at followup were K/L grade Յ2 (91.8%), population were older, had a higher BMI, and were while a much smaller proportion were K/L grade 2 more likely to have had a prior knee injury than subjects (5.9%), grade 3 (1.7%), or grade 4 (0.6%). in the control population. All angular measures were The relationship of each knee alignment mea- similar between the two groups, with the exception of surement to the risk of incident knee OA is presented in the tibial plateau angle. The case knees had a smaller Table 2. The risk of incident knee OA did not increase tibial plateau angle than the control knees (P ϭ 0.009); with either increasing varus or increasing valgus ana- however, the alignment measurement derived from this, tomic axis malalignment. Using the lowest quartile as a the condylar tibial plateau angle, was not significantly reference group, the odds ratio was ϳ1.1 in each of the different between the two groups. second, third, and fourth quartiles of anatomic align- At followup, most of the incident knee OA cases ment (P ϭ 0.83 for trend) after adjusting for age, sex, were K/L grade 2 (73%). Of the remaining case knees and BMI. Similarly, the relative odds of incident knee 25% were K/L grade 3, and Ͻ3% were K/L grade 4. Of OA for the second, third, and fourth quartiles of the the control sample (without OA at baseline), the major- condylar tibial plateau angle measured 0.94, 0.71, and

Table 2. Relationship between alignment measurements and prevalence of incident osteoarthritis (adjusted for age, sex, and body mass index), by quartile of alignment* Quartile of alignment

1 (reference group) 2 3 4 P for trend Anatomic axis, degrees 5–10 valgus 3–4 valgus 0–2 valgus 1–7 varus No. of case knees 20 32 35 23 Prevalence 21.1 23.9 27.6 20.9 Adjusted OR (95% CI) 1 1.10 (0.56–2.19) 1.07 (0.53–2.16) 1.10 (0.52–2.30) Linear 0.83; U-shaped 0.84 Condylar angle, degrees Ϫ6to3 4to5 6to7 8to13 No. of case knees 26 22 36 26 Prevalence 20.0 21.0 29.0 24.3 Adjusted OR (95% CI) 1 0.90 (0.44–1.84) 1.31 (0.67–2.54) 0.97 (0.47–1.99) Linear 0.97; U-shaped 0.29 Tibial plateau angle, degrees Ϫ5toϪ2 Ϫ1toϪ1 0 to 4 No. of case knees 39 30 41 Prevalence 36.4 18.5 20.8 Adjusted OR (95% CI) 1 0.57 (0.31–1.05) 0.68 (0.38–1.24) Linear 0.045; U-shaped 0.12 Condylar tibial plateau angle, degrees Ϫ7toϪ4 Ϫ3toϪ3 Ϫ2toϪ2 Ϫ1to5 No. of case knees 29 26 16 39 Prevalence 24.8 26.8 17.0 24.7 Adjusted OR (95% CI) 1 0.94 (0.48–1.86) 0.71 (0.33–1.52) 1.12 (0.60–2.08) Linear 0.80; U-shaped 0.86 * Positive numbers indicate valgus angles; negative numbers indicate varus angles. Tibial plateau angle values were divided into tertiles because of the small variability of the values, with most lying close to the mean. OR ϭ odds ratio; 95% CI ϭ 95% confidence interval. 1216 HUNTER ET AL

Table 3. Baseline characteristics of the study population with medial tibiofemoral osteoarthritis at followup Cases Controls (n ϭ 80 knees) (n ϭ 356 knees) Age, mean Ϯ SD (range) years 56.2 Ϯ 7.7 (41–74) 52.7 Ϯ 8.1 (32–73) Body mass index, mean Ϯ SD 30.6 Ϯ 5.2 27.1 Ϯ 4.7 % female 46.3 53.6 % of knees with K/L score of 1 at baseline* 37.5 7.3 Measures of alignment, mean Ϯ SD (range) degrees† Anatomic axis 1.6 Ϯ 3.2 (Ϫ7 to 10) 2.2 Ϯ 3.2 (Ϫ7to10) Condylar angle 5.4 Ϯ 3.3 (Ϫ6 to 11) 5.2 Ϯ 3.0 (Ϫ3to13) Tibial plateau angle Ϫ1.2 Ϯ 1.3 (Ϫ5to3) Ϫ0.8 Ϯ 1.0 (Ϫ4to4) Condylar tibial plateau angle Ϫ2.6 Ϯ 1.7 (Ϫ6to1) Ϫ2.3 Ϯ 1.8 (Ϫ7to5) * K/L ϭ Kellgren/Lawrence. † Positive numbers indicate valgus angles; negative numbers indicate varus angles.

1.12 (P ϭ 0.80 for trend) after adjusting for age, sex, and DISCUSSION BMI. We found that baseline knee anatomic axis align- Of the 110 case knees with incident TF OA, 80 ment was not associated with either incident TF OA or (73%) had medial TF OA at followup. Similar to the incident medial TF OA. Similarly, the condylar tibial whole sample, the case subjects with medial TF OA were plateau angle did not predict incident knee OA. The older and had a higher BMI than the control population results suggest that malalignment may not be a primary (Table 3). The association between each angular mea- risk factor for incident knee OA, but rather, a marker of surement and the risk of incident medial OA is shown in disease severity and/or its progression. Table 4. The odds ratios for the risk of incident medial Varus and valgus malalignment have been shown knee OA for each quartile of the anatomic axis were 1.0, to increase the risk of medial and lateral knee OA 1.33, 1.35, and 2.04 (P ϭ 0.15 for trend) after adjusting progression (1). The effect of malalignment extends for age, sex, and BMI. The odds of incident medial knee beyond its direct effect on cartilage since it has been OA for each quartile of the condylar tibial plateau angle shown in association with the structural breakdown of were 1.0, 0.78, 0.51, and 0.95, respectively (P ϭ 0.78 for other knee tissues as well, such as the neighboring bone trend) after adjusting for age, sex, and BMI. marrow (18). Certain site-specific factors in the local

Table 4. Relationship of alignment measurements to prevalence of incident medial tibiofemoral osteoarthritis (adjusted for age, sex, and body mass index), by quartile of alignment* Quartile of alignment P for linear 1 (reference group) 2 3 4 trend Anatomic axis, degrees 5–10 valgus 3–4 valgus 0–2 valgus 1–7 varus No. of case knees 11 24 24 21 Prevalence 12.8 19.0 20.7 19.4 Adjusted OR (95% CI) 1 1.33 (0.62–2.87) 1.35 (0.59–3.10) 2.04 (0.85–4.92) 0.15 Condylar angle, degrees Ϫ6to3 4to5 6to7 8to13 No. of case knees 21 11 26 22 Prevalence 16.8 11.7 22.9 21.4 Adjusted OR (95% CI) 1 0.45 (0.19–1.11) 1.00 (0.47–2.11) 0.85 (0.39–1.87) 0.99 Tibial plateau angle, degrees Ϫ5toϪ2 Ϫ1toϪ1 0 to 4 No. of case knees 31 24 25 Prevalence 31.3 15.4 13.8 Adjusted OR (95% CI) 1 0.61 (0.31–1.19) 0.61 (0.31–1.21) 0.14 Condylar tibial plateau angle, degrees Ϫ7toϪ4 Ϫ3toϪ3 Ϫ2toϪ2 Ϫ1to5 No. of case knees 25 18 10 27 Prevalence 22.1 20.2 11.4 18.5 Adjusted OR (95% CI) 1 0.78 (0.36–1.65) 0.51 (0.21–1.23) 0.95 (0.48–1.87) 0.78 * Positive numbers indicate valgus angles; negative numbers indicate varus angles. Tibial plateau angle values were divided into tertiles because of the small variability of the values, with most lying close to the mean. OR ϭ odds ratio; 95% CI ϭ 95% confidence interval. KNEE ALIGNMENT AND INCIDENT OA 1217

joint environment, such as tibiofemoral congruence, protocol used in this study. The use of more sensitive anterior cruciate ligament integrity, and meniscal degen- imaging technology, such as magnetic resonance imag- eration and position also govern how load is distributed ing, may reveal different results. across the articular cartilage of a given joint. Previously, The mechanical axis measured on a full-limb Cooke et al (19) suggested that the loss of joint space radiograph is the gold standard method for assessing may account for much of the malalignment that has been knee alignment. All measures of alignment in this study observed in cases of knee OA. These observations were, were taken from a “short” radiograph of only the knee, however, never quantified. We recently conducted a as opposed to a full-limb radiograph (14). Measures on cross-sectional analysis which suggested that multiple the short radiograph do not capture proximal and distal factors, including cartilage loss, meniscal degeneration anatomy, but do avoid unwanted pelvic radiation, high and position, bone attrition, osteophytes, and ligament cost, and specialized equipment. The acquisition and damage, can contribute to knee malalignment (2). This measurement of the mechanical axis of the knee (de- analysis was performed in a population of persons with fined as the angle formed by the intersection of a line preexisting OA, in whom there is greater variability in from the center of the head of the femur to the center of measures of alignment than that seen in the analysis the tibial spines and a second line from the center of the presented herein. Understanding the role alignment ankle talus to the center of the tibial spines) is techni- plays in the etiology of OA is important, since the effect cally difficult and requires a full-limb radiograph. How- of standard risk factors for progression of knee OA, ever, a recent study demonstrated strong correlation including obesity (18), quadriceps strength (20), laxity between data obtained from full-limb measurements of (20), and stage of disease (1,8), are influenced by the the mechanical axis and short radiograph measurements presence or absence of malalignment. of the anatomic axis (14). In that study, the anatomic axis In contrast to all previous studies, we examined was offset a mean 4.21° of valgus from the mechanical the effect of malalignment on persons without knee OA axis (3.5° in women and 6.4° in men). The high correla- at baseline. We would posit, based on the findings of this tion suggests that the cumbersome full-length radio- study, that abnormalities in frontal plane knee alignment graphs could be exchanged for the more commonly are typically a consequence and not a primary cause of obtained, standard knee radiographs during the radio- OA. While increasing structural damage within the knee graphic assessment of knee alignment. However, while may help perpetuate a vicious circle of events by bring- they may be strongly correlated, there are differences, ing about malalignment, which in turn, can further strain already damaged tissue, the initial presence of malalign- and adequate exploration of the relationship between ment does not appear to significantly increase the risk alignment (mechanical axis) and incident OA should be posed to healthy knee tissue. This is consistent with pursued in a study where these measures are available. previous studies suggesting that knees with increasing Malalignment provides only a static impression of disease severity are more malaligned (17) and that the the expected uniplanar forces imparted on the joint association of alignment with progression differs accord- during activity. The dynamics of gait involve multiple ing to the severity of disease (8). The absence of a forces that stress the knee in several planes simulta- relationship between malalignment and incident knee neously. During the stance phase of gait, the ground OA persists whether alignment is determined between reaction force acting at the foot passes medial to the axis bony segments (anatomic axis alignment) or adjacent of the knee joint. The perpendicular distance from the joint surfaces (condylar tibial plateau angle). line of action of this force to the axis of the knee joint This study has a number of limitations which may constitutes a moment arm for the application of an impair the interpretation of the study findings. OA adduction moment to the knee (6). In a varus malaligned develops slowly, and the development of radiographic knee, the peak adduction moment is expected to in- signs can take many years. As a result, the number of crease, augmenting the load across the medial compart- incident cases of radiographic OA in our cohort may ment (3). Yet, static measures of alignment cannot tell have been limited by the relatively short period of us with certainty whether any dynamic moments around followup (mean of 8.75 years). In addition, radiographs the knee, including the adduction moment, are causative are unable to detect the earliest indications of OA; thus, of incident knee OA. The adduction moment might also many participants in this sample, both cases and con- be affected by habitual postures during locomotion or by trols, may have had more subtle signs of disease that more distal malalignments, such as tibial or calcaneal remained undetected by the radiographic examination varum. The possible role of distal malalignments in the 1218 HUNTER ET AL

etiology of knee OA is poorly understood and warrants comparison of Saudi Arabian and Canadian cases. Rheumatol Int 2002;22:160–4. a great deal more exploration. 5. Cooke TD. Definition of axial alignment of the lower extremity. Neither baseline anatomic axis alignment nor J Bone Joint Surg Am 2002;84-A:146–7. condylar tibial plateau angle is associated with incident 6. Andriacchi TP. Dynamics of knee malalignment [review]. Orthop Clin North Am 1994;25:395–403. TF OA. The absence of an association persists even 7. Ledingham J, Regan M, Jones A, Doherty M. Radiographic when only medial compartment disease is considered. patterns and associations of osteoarthritis of the knee in patients These results suggest that malalignment may not be a referred to hospital. Ann Rheum Dis 1993;52:520–6. 8. Cerejo R, Dunlop DD, Cahue S, Channin D, Song J, Sharma L. risk factor for incident OA, but rather, may serve as a The influence of alignment on risk of knee osteoarthritis progres- marker of disease severity and/or its progression. Fur- sion according to baseline stage of disease. Arthritis Rheum ther research is needed to confirm these findings. In 2002;46:2632–6. 9. Felson DT, McLaughlin S, Goggins J, LaValley MP, Gale ME, particular, we would recommend evaluation of the Totterman S, et al. Bone marrow edema and its relation to relationship between the mechanical axis and incident progression of knee osteoarthritis. Ann Intern Med 2003;139: knee OA. 330–6. 10. Felson DT, Naimark A, Anderson J, Kazis L, Castelli W, Meenan RF. The prevalence of knee osteoarthritis in the elderly: the Framingham Osteoarthritis Study. Arthritis Rheum 1987;30: ACKNOWLEDGMENTS 914–8. We would like to thank the study participants and the 11. Karlson EW, Sanchez-Guerrero J, Wright EA, Lew RA, Daltroy LH, Katz JN, et al. A connective tissue disease screening ques- staff of the Framingham Osteoarthritis Study. tionnaire for population studies. Ann Epidemiol 1995;5:297–302. 12. Kellgren JH, Lawrence JS, editors. The epidemiology of chronic rheumatism: atlas of standard radiographs of arthritis. Oxford: AUTHOR CONTRIBUTIONS Blackwell Scientific; 1963. Dr. Hunter had full access to all of the data in the study and 13. Altman RD, Hochberg M, Murphy WA Jr, Wolfe F, Lequesne M. takes responsibility for the integrity of the data and the accuracy of the Atlas of individual radiographic features in osteoarthritis. Osteo- data analysis. arthritis Cartilage 1995;3 Suppl A:3–70. Study design. Hunter, Harvey, Gross, Zhang, 14. Kraus VB, Vail TP, Worrell T, McDaniel G. A comparative Acquisition of data. Hunter, McCree, Aliabadi, Sack, assessment of alignment angle of the knee by radiographic and Analysis and interpretation of data. Hunter, Niu, Felson, Aliabadi, physical examination methods. Arthritis Rheum 2005;52:1730–5. Zhang, 15. Spector TD, Hart DJ, Doyle DV. Incidence and progression of Manuscript preparation. Hunter, Felson, Harvey, Gross, McCree, osteoarthritis in women with unilateral knee disease in the general Zhang, population: the effect of obesity. Ann Rheum Dis 1994;53:565–8. Statistical analysis. Hunter, Niu, Zhang. 16. Mazzuca SA, Brandt KD, German NC, Buckwalter KA, Lane KA, Katz BP. Development of radiographic changes of osteoarthritis in the “Chingford knee” reflects progression of disease or non- REFERENCES standardised positioning of the joint rather than incident disease. Ann Rheum Dis 2003;62:1061–5. 1. Sharma L, Song J, Felson DT, Cahue S, Shamiyeh E, Dunlop DD. 17. Wada M, Maezawa Y, Baba H, Shimada S, Sasaki S, Nose Y. The role of knee alignment in disease progression and functional Relationships among bone mineral densities, static alignment and decline in knee osteoarthritis [published erratum appears in dynamic load in patients with medial compartment knee osteoar- JAMA 2001;286:792]. JAMA 2001;286:188–95. thritis. Rheumatology (Oxford) 2001;40:499–505. 2. Hunter DJ, Zhang Y, Niu J, Tu X, Amin S, Goggins J, et al. 18. Sharma L, Lou C, Cahue S, Dunlop DD. The mechanism of the Structural factors associated with malalignment in knee osteoar- effect of obesity in knee osteoarthritis: the mediating role of thritis: the Boston osteoarthritis knee study. J Rheumatol 2005;32: malalignment. Arthritis Rheum 2000;43:568–75. 2192–9. 19. Cooke TD, Scudamore A, Greer W. Varus knee osteoarthritis: 3. Tetsworth K, Paley D. Malalignment and degenerative arthro- whence the varus? [review]. J Rheumatol 2003;30:2521–3. pathy [review]. Orthop Clin North Am 1994;25:367–77. 20. Sharma L, Dunlop DD, Cahue S, Song J, Hayes KW. Quadriceps 4. Cooke TD, Harrison L, Khan B, Scudamore A, Chaudhary MA. strength and osteoarthritis progression in malaligned and lax Analysis of limb alignment in the pathogenesis of osteoarthritis: a knees. Ann Intern Med 2003;138:613–9. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1219–1233 DOI 10.1002/art.22490 © 2007, American College of Rheumatology

Examining the Role of CD1d and Natural Killer T Cells in the Development of Nephritis in a Genetically Susceptible Lupus Model

Jun-Qi Yang,1 Xiangshu Wen,2 Hongzhu Liu,1 Gbolahan Folayan,2 Xin Dong,2 Min Zhou,2 Luc Van Kaer,3 and Ram Raj Singh4

Objective. CD1d-reactive invariant natural killer iNKT cells and iNKT cell responses were absent in T (iNKT) cells secrete multiple cytokines upon T cell CD1d0 BWF1 mice, the CD1d0 mice continued to have receptor (TCR) engagement and modulate many significant numbers of interferon-␥–producing NKT- immune-mediated conditions. The purpose of this study like (CD1d-independent TCR␤؉,NK1.1؉ and/or was to examine the role of these cells in the development DX5؉) cells. CD1d deficiency also influenced cytokine of autoimmune disease in genetically lupus-prone responses by conventional T cells: upon in vitro stimu- ؋ (NZB NZW)F1 (BWF1) mice. lation of splenocytes with Con A or anti-CD3, type 2 Methods. The CD1d1-null genotype was crossed cytokine levels were reduced, whereas type 1 cytokine onto the NZB and NZW backgrounds to establish 0 0 0 levels were increased or unchanged in CD1d mice as CD1d1-knockout (CD1d ) BWF1 mice. CD1d mice compared with their wild-type littermates. Additionally, and their wild-type littermates were monitored for numbers of thymic iNKT cells were lower in young the development of nephritis and assessed for cytokine wild-type BWF1 mice than in nonautoimmune strains. responses to CD1d-restricted glycolipid ␣-galactosyl- Conclusion. Germline deletion of CD1d exacer- ceramide (␣GalCer), anti-CD3 antibody, and con- bates lupus in BWF1 mice. This finding, together with canavalin A (Con A). Thymus and spleen cells were reduced thymic iNKT cells in young BWF1 mice as stained with CD1d tetramers that had been loaded with compared with nonautoimmune strains, implies a reg- ␣GalCer or its analog PBS-57 to detect iNKT cells, and ulatory role of CD1d and iNKT cells during the devel- the cells were compared between BWF1 mice and class opment of lupus. II major histocompatibility complex–matched nonauto- ؋ immune strains, including BALB/c, (BALB/c NZW)F1 (CWF1), and NZW. Systemic lupus erythematosus (SLE) is an auto- Results. CD1d0 BWF1 mice had more severe immune disease characterized by inflammation of mul- nephritis than did their wild-type littermates. Although tiple organs and uncontrolled production of autoantibodies (1). In humans and animals with SLE, diverse sets Dr. Van Kaer’s work was supported by NIH grant HL-68744. of T helper cells can promote autoantibody production Dr. Singh’s work was supported by NIH grants AR-47322 and AR- (2–4). The emergence of such autoreactive T helper 50797. cells in lupus is accompanied by either a loss or a 1Jun-Qi Yang, PhD, Hongzhu Liu, MD: University of Cincin- nati College of Medicine, Cincinnati, Ohio; 2Xiangshu Wen, PhD, defective induction of regulatory T cells (4–6). Elucidat- Gbolahan Folayan, BS, Xin Dong, PhD, Min Zhou, MD: David Geffen ing such impairments in regulatory networks would 3 School of Medicine, University of California, Los Angeles; Luc Van facilitate our understanding of the pathogenesis of lupus. Kaer, PhD: Vanderbilt University School of Medicine, Nashville, Tennessee; 4Ram Raj Singh, MD: Jonsson Comprehensive Cancer Studies in the late 1980s identified cells that Center, and David Geffen School of Medicine, University of Califor- express cell surface markers characteristic of natural nia, Los Angeles. Address correspondence and reprint requests to Ram Raj killer (NK) cells as well the CD3–T cell receptor (TCR) Singh, MD, University of California, Los Angeles, Division of Rheu- complex in the thymus, spleen, and bone marrow (7–10). matology, 1000 Veteran Avenue, Room 32-59 Rehab Center, Los Such natural killer T (NKT) cells rapidly produce Angeles, CA 90095-1670. E-mail: [email protected]. Submitted for publication October 18, 2006; accepted in interleukin-4 (IL-4) (11–13) and include 2 subsets, revised form December 21, 2006. CD4ϩ T cells and double-negative (DN) T cells. These

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cells express intermediate levels of TCR, with a highly are manipulated or on the nature of the underlying skewed TCR V␤ family (7,14) and an invariant TCR immune perturbation (35–38). It is important to unearth ␣-chain V␣14–J␣281 (now called V␣14–J␣18) (15,16), the full spectrum of these protective and pathogenic the expression of which was previously identified by use roles of iNKT cells in immune-mediated diseases, given of a panel of suppressor T cell hybridomas (17). Simul- the nonpolymorphic nature of CD1d that makes it an taneous studies identified the counterpart of murine attractive target of immune therapy on a mass scale. NKT cells in humans, V␣24–J␣Q TCR (now called To determine the role of iNKT cells in SLE, a few V␣24–J␣18), among DN peripheral blood T cells (18). studies have investigated alterations in the frequency Further studies showed that the development of NKT and functions of these cells in the peripheral blood of cells was independent of class II major histocompatibil- patients and healthy subjects. Two studies, one each ity complex (MHC) expression, but required the class I from Japan and Europe, found a significant reduction in MHC–like molecule CD1d (19). CD1d is an evolution- circulating TCR V␣24ϩ,V␤11ϩ DN T cells (composed ␤ ␤ arily conserved 2-microglobulin ( 2m)–associated proof iNKT cells as well as other T cells) in patients with tein (20), which is widely expressed on hematopoietic- SLE as compared with healthy subjects (39,40). Simul- derived cells (21). CD1d binds glycolipid antigens, such taneously, another study found that whereas invariant as ␣-galactosylceramide (␣GalCer), and activates NKT V␣24–J␣Q(V␣24–J␣18) TCR dominated DN V␣24ϩ T cells (22). cells at a high frequency in healthy subjects, this invari- Taken together, these studies suggest that NKT ant TCR was reduced to undetectable levels in DN cells are a separate T cell lineage characterized by V␣24ϩ T cells from patients with active SLE (41). The CD1d-restricted lipid antigen reactivity, an invariant invariant V␣24ϩ,J␣18ϩ T cells were restored to normal TCR (V␣14–J␣18 paired with V␤8.2/V␤7/V␤2 in mice levels when patients were successfully treated with and V␣24–J␣18 paired with V␤11 in humans), expression corticosteroids to achieve disease remission (41). More- of NK cell markers NK1.1/DX5, and rapid cytokine over, whereas V␣24ϩ,V␤11ϩ DN T cells from all response (20,23–25). Subsequent studies, however, led healthy subjects proliferated in response to ␣GalCer, 5 to the realization that NKT cells are a diverse group of of the 10 SLE patients tested exhibited no such response cells, which likely differ in their functions (26,27). For to ␣GalCer (39). These data clearly suggest that peri- example, some CD1d-restricted T cells, such as imma- pheral blood iNKT cells are impaired in patients with ture and recently activated cells, do not express NK SLE. markers. Notably, CD1d-restricted T cells from MRL- To understand the implications of these findings ϫ lpr/lpr (MRL-lpr), (NZB NZW)F1 (BWF1), and hy- in humans, we and other groups of investigators have drocarbon oil–induced animal models of SLE are mostly used animal models of SLE, such as BWF1, MRL-lpr, negative for NK1.1 or DX5 (28–31). In addition, some and hydrocarbon oil–injected mice (28–31,42). BWF1 CD1d-restricted T cells express diverse TCR ␣-chains mice spontaneously develop a T cell–dependent, instead of the invariant V␣14–J␣18 chain. Finally, some autoantibody-mediated lupus nephritis that mimics hu- T cells that express NK markers NK1.1 and DX5 are not man SLE (43). One group of investigators has treated CD1d-restricted. BWF1 mice with anti-CD1d antibody to suppress iNKT These observations led a panel of investigators to cells (42). This approach, however, may not achieve propose the following classification for NKT cells (26). complete neutralization of CD1d and its effects. Fur- Class I cells are invariant NKT (iNKT) cells, which are thermore, a recent study showed that CD1d ligation on also called classic NKT cells or type I NKT cells. They human monocytes leads to NF-␬B activation and IL-12 can best be detected using CD1d tetramers loaded with production (44). Thus, the anti-CD1d antibody that was glycolipids, such as ␣GalCer. Class II cells are variant used to neutralize CD1d might cause unintended conse- NKT cells, which are also called type II or nonclassic quences on various immune cells due to CD1d ligation, NKT cells. They are CD1d-dependent but express di- with ensuing effects on lupus. Hence, we introgressed a verse TCR ␣-chains. Class III cells are NKT-like cells. CD1d1-null (CD1d0) genotype into NZB and NZW They are CD1d-independent NK1.1/DX5ϩ T cells. strains to generate CD1d-deficient BWF1 mice that Ample evidence supports a protective role of have no iNKT cells. We monitored disease development iNKT cells against a variety of immune-mediated in- as well as responses of iNKT cells, NKT-like cells, and flammatory conditions (29,32–34). However, iNKT cells conventional T cells in these mice. In addition, we can also aggravate the same immune-mediated condi- assessed iNKT cell frequency at different stages of tions, depending on the stage of disease when iNKT cells disease development in wild-type BWF1 mice. We de- ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1221

scribe herein our findings and briefly review the current Flow cytometry. Single-cell suspensions were prepared understanding of the role of iNKT cells in lupus. in PBS buffer with 3% FCS, EDTA, and sodium azide after red blood cell lysis (spleen only) with PharmLyse (BD Biosciences, San Diego, CA). Spleen cells were incubated with anti- MATERIALS AND METHODS CD16/32 (2.4G2; PharMingen) to block Fc receptor ␥ II/III, followed by staining with conjugated anti-mouse mAb: anti- Mice. BWF1, NZB, NZW, and BALB/c mice were CD1d (1B1), anti-NK1.1, anti-CD4, and anti-B220; iNKT cells obtained from The Jackson Laboratory (Bar Harbor, ME). were detected using allophycocyanin-labeled anti-TCR␤ (all Some BALB/c ϫ NZW and NZB ϫ NZW mice were bred antibodies obtained from PharMingen) and phycoerythrin– ϫ ␣ locally to generate (BALB/c NZW)F1 (CWF1) and BWF1 labeled CD1d tetramer loaded with either GalCer (29) or the mice, respectively. The CD1d0 129 ϫ C57BL/6 mice (45) were ␣GalCer analog PBS-57 (47). Fluorescence-activated cell crossed onto the NZB and NZW backgrounds for 10 and 12 sorter (FACS) analysis was performed using FACSCalibur generations, respectively. At each backcross, the heterozygous (BD Biosciences), and data were analyzed using CellQuest ,CD1d؉/–) mice were identified by polymerase chain reaction (Becton Dickinson, San Jose, CA) or FlowJo (Tree Star) –/؉ analysis, as reported elsewhere (28). The N10 CD1d NZB Ashland, OR) software. Dead cells were excluded from the –/؉ mice were crossed with N12 CD1d NZW mice to establish analysis by electronic gating, based on forward and side .CD1d؉/؉ (CD1dϩ) and CD1d–/– (CD1d0) BWF1 mice. The light-scatter patterns (CD1d0 phenotypes were further confirmed by demonstrating Enrichment for T and NKT-like (TCR␤؉,DX5؉ the absence of CD1d on peripheral blood lymphocytes by flow cells. Splenic T cells from CD1d0 and CD1d؉ mice were cytometry using anti-CD1d monoclonal antibody (mAb) 1B1 enriched using mouse T cell enrichment columns (R&D (PharMingen, San Diego, CA). To confirm that mice from the Systems, Minneapolis, MN). In some experiments, purified T final backcross were indeed congenic, they were screened using cells were also incubated with anti-NK (DX5) microbeads a battery of simple sequence repeat markers (www.informatics. (Miltenyi Biotec, Auburn, CA) for 30 minutes on ice. After jax.org), all of which discriminated congenic strains from the washing once, cells were applied to the column for positive 129/B6 donors. selection of DX5ϩ T cells, and negative selection of Assessment of lupus disease. Survival and renal dis- TCR␤ϩ,DX5– cells. The purity of TCR␤ϩ,DX5ϩ cells and ease were assessed in the mice. Proteinuria was measured with TCR␤ϩ,DX5– cells ranged from 80–90% and 95–97%, respec- a colorimetric assay strip for albumin (Albustix; Bayer, tively. Elkhart, IN) and graded on a scale of 0–4ϩ, where 0 ϭ absent, In vivo TCR cross-linking for iNKT cell activation. As 1ϩϭՅ30–99 mg/dl (mild), 2ϩϭ100–299 mg/dl (moderate), described previously (13,45), mice were injected intravenously 3ϩϭ300–1,999 mg/dl (marked), and 4ϩϭՆ2,000 mg/dl with a single 1-␮g dose of anti-CD3 mAb (145-2C11; Phar- (severe). Severe proteinuria was defined as a value Ն300 mg/dl Mingen) and were euthanized after 90 minutes. Spleen cell on 2 consecutive examinations, as described previously (3). For suspensions (5 ϫ 106/ml) from these animals were cultured in renal histology, paraffin sections of kidneys were stained with complete RPMI 1640 medium (supplemented with 10% fetal hematoxylin and eosin, periodic acid–Schiff, and Masson’s calf serum, 2 ϫ 10–5M 2-mercaptoethanol, 20 mM HEPES, 1 trichrome, and scored for various histologic features in a mM sodium pyruvate, and 100 ␮g/ml of gentamicin). Superna- blinded manner, as described previously (46). Briefly, the tants were collected after 2 hours for use in cytokine assays. mean scores for individual pathologic features were summed In vitro cytokine detection. Spleen cells (1.5 ϫ 106/ml) to obtain the 4 main scores: the glomerular activity score, the were cultured in complete RPMI 1640 medium with 10–100 tubulointerstitial activity score, the chronic lesion score, and ng/ml of ␣GalCer or 2–10 ␮g/ml of concanavalin A (Con A; the vascular lesion score. These scores were converted into Sigma). Supernatants were collected at 24–48 hours and indices by dividing them by the number of individual features assayed for cytokines. In some experiments, purified T cells examined to obtain those scores. The indices thus obtained (TCR␤ϩ,DX5–) were stimulated with plate-bound anti-mouse were then averaged and summed to determine a composite CD3 antibody, and supernatants were collected after overnight kidney biopsy index. incubation. Measurement of anti-DNA antibody. IgG anti-DNA Interferon-␥ (IFN␥), IL-2, IL-4, IL-10, IL-12, IL-13, antibody was measured by enzyme-linked immunosorbent and transforming growth factor ␤1 (TGF␤1) were assayed by assay (ELISA), as described previously (6). Briefly, Costar sandwich ELISA using paired mAb of rat anti-mouse cyto- high-binding enzyme immunoassay/radioimmunoassay plates kines, as described previously (48). The following mAb pairs were coated with sonicated, nitrocellulose-filtered calf thymus were obtained from PharMingen: R4 6A2 and XMG1.2 for double-stranded DNA (Sigma, St. Louis, MO). After washing IFN␥, JES6-1A12 and JES6-5H4 for IL-2, 11B11 and BVD6- and blocking, plates were incubated overnight at 4°C with 24G2 for IL-4, JES5-2A5 and SXC-1 for IL-10, C15.6 and diluted test or control sera. After washing, alkaline- C17.8 for IL-12, and A75-2.1 and A75-3.1 for TGF␤1. Purified phosphatase–conjugated anti-mouse IgG (Southern Biotech- and biotinylated anti-mouse IL-13 mAb were obtained from nology Associates, Birmingham, AL) was added to the plate R&D Systems. After incubation with the biotinylated mAb, and incubated at room temperature for 1 hour. Plates were plates were incubated with alkaline phosphatase–conjugated developed with p-nitrophenyl phosphate substrate (Sigma) and streptavidin (Jackson ImmunoResearch, West Grove, PA), read at 405 nm in a Multiscan ELISA reader (Labsystems, developed with p-nitrophenyl phosphate substrate, and read at Helsinki, Finland). A positive reference standard of pooled 405 nm. serum from 1-year-old BWF1 mice was used to convert the Intracellular cytokine staining. Purified cells (1.5 ϫ anti-DNA antibody optical density values to units per milliliter. 106/ml) were stimulated with plate-bound anti-CD3 (mAb 1222 YANG ET AL

0 ϫ 0 ϩ ϭ Figure 1. Exacerbation of lupus nephritis in CD1d (NZB NZW)F1 (BWF1) mice. BWF1 mice (CD1d and CD1d littermates; n 17 per group) were monitored for proteinuria and then euthanized at 6 months of age to analyze renal histology (see Materials and Methods for details). a, Proteinuria grades (1ϩ to 4ϩ) in 20-week-old and 25-week-old female mice. Results are shown as the percentage of mice. b and c, Composite kidney biopsy index (KBI) (b) and the individual components of the KBI (glomerular activity score [GAS], tubulointerstitial activity score [TIAS], .ϭ P Ͻ 0.05 by Student’s t-test ء .chronic lesion score [CLS], and vascular lesion score [VLS]) (c) in female mice. Values are the mean and SEM d, Representative kidney sections from female mice stained with hematoxylin and eosin (left), periodic acid–Schiff (middle), and Masson’s trichrome (right). Kidney sections from CD1dϩ mice show glomerular proliferation (GP), focal mesangial proliferation (FM), and minimal interstitial fibrosis (IF; aniline blue staining), whereas sections from the CD1d0 mice show severe glomerular proliferation, infiltration and karyorrhexis (KR), severe glomerulosclerosis (GS), glomerular fibrosis with scarring (GFS), and dilated atrophic tubules (AT). e and f, Composite KBI (e) and the individual ;ϭ P Ͻ 0.05 ء .components of the KBI (f) in male mice at 8–9 months of age. Values are the mean and SEM of 10 CD1d0 and 8 CD1dϩ mice ϭ P ϭ 0.05–0.06, by Student’s t-test. g, Male BWF1 mice in another cohort were monitored for 13 months for survival. Results are shown as the ءء .ϭ P Ͻ 0.05 by log rank test ء .cumulative percentage survival in 23 CD1d0 (E) and 17 CD1dϩ (F) mice ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1223

Figure 2. Increased anti-DNA antibody production and enhanced lymphoid cellularity in CD1d0 (NZB ϫ 0 ϩ NZW)F1 (BWF1) mice. a, Serum IgG anti-DNA antibody (Ab) levels in 15 CD1d and 8 CD1d mice. Negative control values in 6 normal BALB/c mice were 3.5 Ϯ 0.8 units/ml (mean Ϯ SEM). b, IgG anti-DNA antibody in spleen cells from 12-week-old BWF1 mice (n ϭ 7 CD1d0 and n ϭ 4 CD1dϩ). Cells were cultured with lipopolysaccharide (LPS) for 5 days, supernatants were tested, and optical density (OD) values were determined. c, Enhanced lymphoid cellularity in CD1d0 BWF1 mice. Spleen cells were enumerated in 12-week-old female BWF1 mice (n ϭ 9 CD1d0 and n ϭ 5 CD1dϩ). Values are the mean Ϯ SEM. Results are from a representative experiment of 2 independent experiments performed using female .ϭ P Ͻ 0.05 by Mann-Whitney U test in a and Student’s t-test in b and c ء .mice

145-2C11) for 16 hours, followed by incubation with 3 ␮M its component chronic lesion score, in particular, were monensin for 4 hours. Cells were then stained with fluorescein also increased in CD1d0 mice (P Ͻ 0.05 by Student’s isothiocyanate–labeled anti-NK1.1 in the presence of 2.4G2 and washed twice, followed by fixation in 2% paraformalde- t-test) (Figures 1b and c). The individual components of hyde for 10 minutes at room temperature. The fixed cells were the chronic lesion score, including interstitial fibrosis then washed once and treated with FACS permeabilizing (P Ͻ 0.05), glomerular scarring (P Ͻ 0.02), tubular solution (Becton Dickinson) for 10 minutes at room tempera- atrophy (P Ͻ 0.05) and fibrous and cellular crescents ture. After washing, cells were stained with phycoerythrin- Ͻ 0 conjugated anti-mouse IL-2, IL-4, IL-10, or IFN␥ (PharMin- (P 0.05) were increased in the CD1d mice (data not gen) for 30 minutes on ice, and washed twice before flow shown). The glomerular activity score, tubulointerstitial cytometry analysis. activity score, and vascular lesion score were also in- Statistical analysis. Antibody and cytokine levels, lym- creased in CD1d0 mice, although the differences were phocyte percentages and numbers, and renal scores were ϭ compared using Student’s t-test or Mann-Whitney U test. not statistically significant (P 0.06–0.08) (Figure 1c). Frequencies of antibodies and proteinuria were compared Similar results were obtained in another cohort of using Fisher’s exact test (2-sided). Survival analysis was per- BWF1 mice (46 CD1d0 mice and 36 CD1dϩ mice) that –/formed using a log rank test. ؉ were established by intercrossing N10 CD1d NZW –/؉ mice with N8 CD1d NZB mice (data not shown). RESULTS Representative renal sections demonstrating 0 Acceleration of lupus nephritis in Cd1d-deficient more advanced kidney lesions in female CD1d mice are BWF1 mice. To investigate the effect of CD1d on shown in Figure 1d. A similar increase in renal disease 0 spontaneous lupus disease, we crossed mice with the was also observed in male CD1d BWF1 mice (Figures CD1d-null genotype onto mice with the NZB and NZW 1e and f). In male BWF1 mice that were monitored for 0 backgrounds and established CD1d0 BWF1 mice by 13 months, survival was significantly reduced in CD1d ϩ ؉/– ؉/– mice as compared with their CD1d littermates (Figure intercrossing N10 CD1d NZB mice with N12 CD1d NZW mice. The CD1d0 and CD1dϩ female BWF1 mice 1g). The cumulative frequency of severe proteinuria in (n ϭ 17 per group) were monitored for proteinuria and these mice showed a similar trend (data not shown). then euthanized at the age of 6 months to analyze renal These observations suggest that CD1d0 BWF1 mice histologic features (Figures 1a–d). have accelerated lupus nephritis, with a relatively rapid The frequency of severe proteinuria (grade Ն3ϩ) progression to chronic disease. was increased at 25 weeks of age in CD1d0 mice CD1d deficiency and increases in anti-DNA an- compared with CD1dϩ mice (P Ͻ 0.05 by Fisher’s exact tibody production and lymphoid cellularity. Consistent test) (Figure 1a). The composite kidney biopsy index and with increased renal disease, CD1d0 BWF1 mice had a 1224 YANG ET AL

0 ϫ 0 ϩ Figure 3. Reduced invariant natural killer T (iNKT) cell cytokine responses in CD1d (NZB NZW)F1 (BWF1) mice. CD1d and CD1d BWF1 littermates (3–4 months old) were analyzed for iNKT cells and their cytokine responses. a, Spleen cells were stained with phycoerythrin (PE)–labeled anti-mouse CD1d and fluorescein isothiocyanate (FITC)–labeled anti-mouse B220 monoclonal antibody (mAb) or with PE-labeled anti-mouse ␣-galactosylceramide (␣GalCer)–loaded CD1d tetramer (CD1d-␣GC tetramer) and FITC-labeled anti-mouse T cell receptor ␤ (TCR␤) mAb in the presence of anti-CD16/32 (2.4G2). Results are representative of 3 experiments, each with 3 mice per group. b, Effect of in vitro stimulation of spleen cells with the CD1d ligand ␣GalCer on cytokine responses. Spleen cells (n ϭ 5 mice per group) were cultured with the vehicle (Veh; 0.025% polysorbate-20 in phosphate buffered saline [PBS]) in which the glycolipids were dissolved or with 50 ng/ml of synthetic ␤GalCer (␤GC) or ␣GalCer (␣GC). Anti-CD1d mAb 1B1 (10 ␮g/ml) was added to some cultures containing ␣GalCer. Supernatants were collected at 48 hours and tested for interleukin-2 (IL-2) and IL-13. Addition of ␣GalCer, but not control glycolipid ␤GalCer or vehicle alone, induced strong cytokine responses in CD1dϩ BWF1 mice; such responses were absent in cultures containing anti-CD1d mAb or spleen cells from CD1d0 BWF1 mice. Values are the mean and SEM pg/ml. c, Responses of iNKT cells, as determined by rapid cytokine production by spleen cells upon brief in vivo stimulation with anti-CD3. Mice (n ϭ 3–5 per group) were injected intravenously with 1 ␮g of anti-CD3 mAb and were euthanized 90 minutes later. Suspensions of single spleen cells were cultured (5 ϫ 106/ml) for 2 hours, and supernatants were tested for IL-4 and interferon-␥ (IFN␥). Values are the mean and SEM pg/ml. No cytokines were detected in 2-hour culture supernatants from the control PBS-injected mice (data not shown). Results are from a representative ϭ P Ͻ 0.01 versus CD1dϩ ءء ;ϭ P Ͻ 0.05 ء .experiment of 2–4 independent experiments performed. Values are the mean and SEM pg/ml littermates, by Student’s t-test.

relatively rapid increase in serum IgG anti-DNA anti- previously reported in normal strains (45), we deter- body levels as compared with their CD1dϩ littermates mined iNKT cell numbers and functions in CD1d0 (Figure 2a), and their spleen cells spontaneously pro- BWF1 mice (Figure 3). As expected, CD1d expression duced higher levels of IgG anti-DNA antibody (Figure and ␣GalCer-loaded CD1d tetramer–positive T cells 2b). IgG anti-DNA antibody production was also in- (i.e., iNKT cells) were absent in CD1d0 BWF1 mice creased in lipopolysaccharide-stimulated spleen cells (Figure 3a). The CD1d-specific iNKT cell responses, as (Figure 2b). Lymphoid organ hypercellularity, another measured by cytokine production in response to feature of lupus, was also exacerbated in CD1d0 BWF1 ␣GalCer, were also absent in CD1d0 lupus mice (Figure mice (Figure 2c). 3b). In CD1d؉ BWF1 mouse spleen cells, ␣GalCer Effect of CD1d deficiency on iNKT cell responses stimulated the release of large amounts of type 1 (IFN␥, in BWF1 mice. To ensure that CD1d-reactive iNKT cell IL-2) and type 2 (IL-4, IL-13) cytokines, whereas neither responses are attenuated in CD1d0 BWF1 mice, as a control glycolipid (␤GalCer) nor the vehicle in which ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1225

Figure 4. Persistence of significant numbers of IFN␥-producing TCR␤ϩ,DX5ϩ cells (i.e., CD1d- independent NKT cells) in CD1d0 BWF1 mice. Spleen cells were stained for the indicated markers, and fluorescence-activated cell sorter (FACS) analysis was performed, gating on lymphocytes according to their forward and side light-scatter properties. Values shown in the plots are the percentages of cells in the respective gates. Representative FACS plots (n ϭ 4–7 mice per group) are shown. a, Percentages of conventional T cells (TCR␤ϩ,NK1.1– cells), NK cells (TCR␤–,NK1.1ϩ cells), and TCR␤ϩ,NK1.1ϩ cells in 4-month-old CD1d0 and CD1dϩ mice. b, Percentages of CD4ϩ,NK1.1ϩ cells in 3-month-old CD1d0 and CD1dϩ mice. c, The gated TCR␤ϩ,NK1.1ϩ cells from CD1dϩ BWF1 mice in a were further analyzed for cells positive for ␣GalCer-loaded CD1d tetramer and for DX5 expression. About 60% of TCR␤ϩ,NK1.1ϩ cells were found to be iNKT cells. d, Splenic lymphocytes from 4-month-old CD1dϩ BWF1 mice were analyzed for iNKT cells (TCR␤ϩ,␣GalCer-loaded CD1d tetramer positive), which were further analyzed for DX5 and NK1.1 expression. e and f, Spleen cells pooled from 3-month-old CD1d0 and CD1dϩ BWF1 mice (n ϭ 10 per group) were processed to enrich TCR␣/␤ϩ,DX5ϩ cells. Isolated cells (1.5 ϫ 106) were stimulated overnight with plate-bound anti-CD3 in 24-well plates, followed by incubation with monensin (3 ␮M) for an additional 4 hours. Cells were collected and stained for FITC-conjugated anti-mouse NK1.1, followed by intracellular cytokine staining. Percentages of IFN␥- and IL-4–producing cells were determined by flow cytometry (e). Cytokine responses in NK1.1ϩ and NK1.1– populations among the enriched DX5ϩ,TCR␣/␤ϩ cells are shown as the IFN␥:IL-4 ratio (f). Values are the mean. Results are from a representative experiment of 4 independent experiments performed. See Figure 3 for other definitions.

␣GalCer was dissolved induced significant cytokine re- mice. Thus, ␣GalCer-induced cytokine responses are sponses. Levels of active TGF␤1, IL-10, and IL-12 were mediated entirely by CD1d1 molecules in BWF1 mice low to undetectable in ␣GalCer-stimulated spleen cell and such responses are absent in the CD1d10 lupus- cultures in both wild-type and CD1d0 BWF1 mice. prone mice, as reported in normal mouse strains (45). Addition of an anti-CD1d mAb to the cultures inhibited Invariant NKT cells promptly produce cytokines the ␣GalCer-stimulated cytokine responses in wild-type in response to in vivo challenge with anti-CD3 (13). To 1226 YANG ET AL

examine the effect of CD1d deficiency on such iNKT cell responses in lupus-prone mice, CD1d0 and CD1dϩ BWF1 mice were injected with an anti-CD3 mAb and, after 90 minutes, were euthanized. Their spleen cells were cultured for 2 hours (Figure 3c). CD1d0 and CD1dϩ mice with the normal, non–lupus-prone background (B6/129) were used as controls. Consistent with previous reports of the findings in normal mouse strains (45), the rapid production of IL-4 was dramatically lower in CD1d0 BWF1 mice than in their wild-type littermates. Thus, the characteristic early IL-4 response by iNKT cells was decreased in CD1d0 BWF1 mice. The effect of CD1d deficiency on IFN␥ production, however, was less profound in BWF1 mice than in normal B6/129 mice (Figure 3c). Retention of significant numbers of IFN␥- secreting NKT-like cells (CD1d-independent TCR␤؉,NK1.1؉ and/or DX5؉ cells) in CD1d0 BWF1 mice. To begin to understand the mechanisms of disease exacerbation in CD1d0 mice, we first analyzed the phenotype of T cells in these mice (Figure 4). Although Figure 5. Conventional T cell cytokine responses in CD1d0 (NZB ϫ the levels were significantly reduced, CD1d0 mice had NZW)F1 (BWF1) mice. Whole spleen cells or purified splenic T cells considerable numbers of TCR␤ϩ,NK1.1ϩ cells (Figure from 3-month-old BWF1 mice were assayed for Th1 and Th2 cyto- 4a). The mean Ϯ SEM percentages of CD4ϩ,NK1.1ϩ kines. a, Spleen cells (1.5 ϫ 106 per ml) from CD1d0 or CD1dϩ BWF1 cells were 0.99 Ϯ 0.08% and 1.58 Ϯ 0.22% in CD1d0 and mice (n ϭ 5 per group) were cultured for 48 hours with or without ϩ ϭ Ͻ concanavalin A (Con A), and supernatants were tested for CD1d BWF1 mice, respectively (n 9 per group; P ␥ ␥ ϳ ␤ϩ ϩ interleukin-2 (IL-2), interferon- (IFN ), IL-4, IL-10, and IL-13. b, 0.05) (Figure 4b). Only 60% of TCR ,NK1.1 cells Purified splenic T cells pooled from CD1d0 or CD1dϩ BWF1 mice in wild-type BWF1 mice were iNKT cells (␣GalCer- (n ϭ 10 per group) were stimulated for 16 hours with plate-bound loaded CD1d tetramer positive) (Figure 4c). Intrigu- anti-mouse CD3 antibody, and supernatants were tested for IFN␥, ingly, only ϳ10% of TCR␤ϩ,NK1.1ϩ cells in wild-type IL-2, IL-4, IL-10, and IL-13. Values are the mean and SEM. Results ϭ ء mice expressed the pan-NK marker DX5 (Figure 4c), are from a representative experiment of 3 experiments performed. .ϭ P Ͻ 0.01, by Student’s t-test ءء ;P Ͻ 0.05 and Ͻ50% of TCR␤ϩ,DX5ϩ cells expressed NK1.1 (data not shown). Further analysis showed that among all iNKT cells (TCR␤ϩ,␣GalCer-loaded CD1d tetramer positive) in BWF1 mice, only 40% expressed any NK enumerated by intracellular cytokine staining (Figure marker (Figure 4d). In fact, only 15% of iNKT cells 4e). Among enriched TCR␤ϩ,DX5ϩ cells, 15.6% and expressed the pan-NK marker DX5. Thus, the majority 10.9% cells produced IFN␥ and 3.07% and 1.14% cells of TCR␤ϩ,DX5ϩ cells (85%) in wild-type BWF1 mice from CD1dϩ and CD1d0 BWF1 mice, respectively, will represent CD1d-restricted variant or type II NKT or produced IL-4. The mean ratios of IFN␥-producing to CD1d-independent NKT-like cells. In CD1d0 mice, all IL-4–producing TCR␤ϩ,NK1.1ϩ cells from 2 indepen- TCR␤ϩ,DX5ϩ cells will represent CD1d-independent dent experiments were higher in CD1d0 BWF1 mice NKT-like cells. (8:1) than in their CD1dϩ BWF1 littermates (4:1) To examine the phenotype of such NKT-like cells (Figure 4f). This finding suggests that whereas IL-4– in lupus-prone mice, we isolated TCR␤ϩ,DX5ϩ cells producing TCR␤ϩ,NK1.1ϩ cells are mostly CD1d- from spleen cells pooled from CD1d0 or CD1dϩ BWF1 restricted, TCR␤ϩ,NK1.1ϩ cells that develop in the mice (n ϭ 10 per group). Approximately 2.5 ϫ 105 or absence of CD1d may predominantly produce IFN␥. 1.5 ϫ 105 TCR␤ϩ,DX5ϩ cells that accounted for The latter (NKT-like) cells are relatively increased in ϳ0.6% or ϳ0.3% of spleen cells were isolated from each CD1d0 mice as compared with their wild-type BWF1 –CD1d؉ or CD1d0 BWF1 mouse, respectively. These littermates. Future studies will determine whether IL-4 cells were stimulated overnight with plate-bound anti- secreting TCR␤ϩ,NK1.1ϩ cells from wild-type BWF1 CD3, and the numbers of cytokine-producing cells were mice inhibit autoimmunity in CD1d0 BWF1 mice. ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1227

ϫ Figure 6. Invariant natural killer T (iNKT) cells in the thymus and spleen of (NZB NZW)F1 (BWF1) and control mice. a, Thymocytes and b, splenocytes were prepared from BWF1, BALB/c, and NZW mice of different ages (n ϭ 5–12 per group) and then stained for iNKT cells by ␣-galactosylceramide (␣GalCer)–loaded CD1d tetramer and T cell receptor ␤ (TCR␤). Values shown in the plots are the mean Ϯ SEM percentages. Results are representative of at least 5 independent experiments. ϭ P Ͻ 0.01 versus control mice, by Student’s t-test. See Table 1 for total numbers of thymic and splenic iNKT ءء ;ϭ P Ͻ 0.05 ء cells. c, Thymocytes and splenocytes from 8–14-week-old BWF1 mice or from the class II major histocompatibility ϫ ␤ complex–identical strain (BALB/c NZW)F1 (CWF1) were stained with allophycocyanin-labeled anti-TCR and phycoerythrin-labeled CD1d tetramer loaded with PBS-57, an analog of ␣GalCer. Percentages of TCR␤ϩ tetramer (iNKT) cells are shown at the upper right of the plots; mean Ϯ SEM percentages of iNKT cells from 2 experiments (n ϭ 2–4 mice per group per experiment) at the upper left.

Reduced type 2, but enhanced type 1, cytokine tate understanding of the role of CD1d in the pathogen- responses of in vitro Con A–stimulated spleen cells from esis of SLE. We therefore investigated whether CD1d CD1d0 BWF1 mice. In humans and mice with SLE, deficiency affects conventional T cell responses in BWF1 development of autoimmunity is accompanied by aber- mice. rant secretion of multiple cytokines, including IFN␥, Spleen cells from 3-month-old CD1d0 and IL-2, IL-4, IL-10, IL-13, and TGF␤ (1,43,46,49). Identi- CD1dϩ BWF1 littermates were cultured in the absence fying the cell types and delineating the mechanisms that or presence of Con A (2–10 ␮g/ml) for 48 hours. contribute to such cytokine abnormalities would facili- Supernatants were tested for cytokines by ELISA (Fig- 1228 YANG ET AL

ure 5a). All type 2 cytokines tested (IL-4, IL-10, and Table 1. Total numbers of CD1d/␣-galactosylceramide tetramer– IL-13) were significantly decreased in Con A–stimulated positive cells in the thymus and spleen of BALB/c and (NZB ϫ NZW)F mice of various age groups* cultures from CD1d0 mice as compared with wild-type 1 mice. Among type 1 cytokines, IL-2 was significantly Thymus Spleen 0 increased in CD1d mice, and IFN␥ levels were similar BALB/c in CD1d0 and CD1dϩ mice at low concentrations of Con 5–8 weeks old 124.6 Ϯ 7.9 52.8 Ϯ 6.4 Ϯ Ϯ A (2–5 ␮g/ml). At high concentrations of Con A (10 13–16 weeks old 58.5 6.9 52.1 7.6 32–39 weeks old 32.9 Ϯ 3.7 69.7 Ϯ 8.2 ␮ ␥ 0 ϫ g/ml) however, IFN was increased in CD1d mice as (NZB NZW)F1 compared with their CD1dϩ littermates. Levels of active 5–8 weeks old 54.2 Ϯ 7.3† 81.3 Ϯ 9.4 Ϯ Ϯ TGF␤1 and IL-12 were low to undetectable in all 13–16 weeks old 40.4 8.6 121.4 17.8 32–39 weeks old 24.3 Ϯ 18.2 118.4 Ϯ 21.6 cultures (data not shown). Thus, T cell stimulation with ϫ * Cells were obtained from (NZB NZW)F1 mice before (5–8 weeks Con A revealed that the potential for type 1 cytokine Ͼ 0 of age), during (13–18 weeks of age), and after ( 25 weeks of age) the production is elevated in CD1d BWF1 mice. onset of disease and from age-matched healthy BALB/c mice. Values Reduced type 2, but unchanged type 1, cytokine are the mean Ϯ SEM cell numbers ϫ104. responses of in vitro anti-CD3–stimulated T cells from † P Ͻ 0.01 versus BALB/c mice of the same age group, by Student’s t-test. CD1d0 BWF1 mice. To further investigate cytokine responses of conventional T cells in CD1d0 BWF1 mice, we purified TCRϩ,NK1.1– cells from the spleens of however, between diseased BWF1 mice (32–39 weeks CD1d0 and wild-type BWF1 mice and stimulated them old) and age-matched BALB/c mice. The percentages with plate-bound anti-CD3 for 16 hours or longer. As and total numbers of iNKT cells in the spleen were also shown in Figure 5b, levels of IL-4, IL-10 and IL-13 were not significantly different between BWF1 and BALB/c significantly reduced in CD1d0 mice as compared with or NZW mice at all ages tested (Figure 6b and Table 1). their wild-type littermates. IFN␥ levels were similar in Similar findings were obtained when iNKT cells were the 2 groups, and IL-2 was increased in CD1d0 as compared between BWF1 mice and class II MHC ϩ compared with CD1d mice. These observations suggest (H-2dz)–identical CWF1 mice (Figure 6c). Thus, iNKT that CD1d influences cytokine production by conven- cell numbers are reduced in the thymus of BWF1 mice at tional T cells, resulting in a pattern of reduced produc- early stages of disease development. tion of type 2 cytokines in CD1d0 BWF1 mice. Percentages and total numbers of iNKT cells in DISCUSSION the thymus and spleen of wild-type BWF1 mice. The above data suggest a protective role of CD1d in the In this study, we show that the proportions and development of lupus nephritis in the BWF1 mouse numbers of iNKT cells are reduced in the thymus of model. The V␣14–J␣18 TCR-expressing cells that dom- BWF1 mice at early stages of disease development. In inate the CD1d-reactive T cell repertoire (50) can be addition, CD1d deficiency since birth exacerbates lupus recognized by staining with ␣GalCer-loaded CD1d tet- nephritis in BWF1 mice, which is associated with an ramer (51). To monitor the frequency of these cells at increase in IFN␥-producing CD1d-independent NKT- different stages of lupus disease development, thymo- like (TCR␤ϩ,NK1.1ϩ and/or DX5ϩ) cells and a re- cytes from BWF1 mice before (5–8 weeks of age), during duced production of type 2 cytokines upon polyclonal (13–18 weeks of age), and after (Ͼ25 weeks of age) the stimulation of T cells. onset of disease and from age-matched healthy BALB/c Several studies have enumerated iNKT cells, as or NZW mice were stained with ␣GalCer-loaded CD1d defined by staining with ␣GalCer-loaded CD1d tet- tetramer. ramer, in lymphoid organs of animal models of lupus As shown in Figure 6a and Table 1, the percent- (Table 2). Consistent with data in this article, showing ages and total numbers of iNKT cells were significantly reduced iNKT cells in the thymus of young BWF1 mice lower in the thymus of BWF1 mice at the preclinical as compared with BALB/c and NZW mice, thymic iNKT stage (5–8 weeks old) than in age-matched BALB/c cells have been found to be reduced in all lupus-prone mice. Percentages, but not total numbers, of thymic strains examined thus far, including MRL-lpr, MRL-Fas, iNKT cells were also reduced in BWF1 mice at the early and pristane-injected BALB/c mice (28,29). The study by disease development stages (13–20 weeks old) of lupus Forestier et al (30), which reported expansion of iNKT than in age-matched BALB/c or NZW mice. Thymic cells in various organs with increasing age in BWF1 iNKT cell numbers were not significantly different, mice, also showed a lower proportion of thymic iNKT ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1229

Table 2. Prevalence of iNKT (␣GalCer-loaded CD1d tetramer–positive) cells in animal models of systemic lupus erythematosus* Animal strain Organ Author, year analyzed Experimental Control Prevalence of iNKT cells (ref.) Thymus Pristane-injected PBS-injected BALB/c Pristane-injected Ͻ PBS-injected (P Ͻ 0.01 for percentage; Yang et al, BALB/c P Ͻ 0.05 for total number) 2003 (28) Thymus MRL-lpr and C3H (MHCII-matched) and MRL-lpr ϭ MRL-Fas Ͻ BALB/c or C3H (P Ͻ 0.05 to P Yang et al, MRL-Fas BALB/c Ͻ 0.001) 2003 (29) Thymus BWF1 B6 BWF1 (0.2%) Ͻ B6 (0.5%) in 4–6-week-old mice; Forestier et al, percentage increased with increasing age in BWF1 mice 2005 (30) (0.6%, 1.5%, and 2.5% at ages 4, 14, and 34 weeks, respectively); for total number, BWF1 Ͼ B6 (P Ͻ 0.001) at age 30 weeks Thymus BWF1 BALB/c, NZW, and CWF1 BWF1 Ͻ BALB/c and NZW at preclinical and early Present study (MHCII-matched) clinical stages (ages 5–20 weeks); BWF1 ϭ BALB/c at advanced clinical stage (ages Ͼ32 weeks) Spleen MRL-lpr MRL-Fasϩ/ϩ, C3H MRL-lpr Ͻ MRL-Fas Ͻ C3H or BALB/c (P Ͻ 0.05 to P Yang et al, (MHCII-matched), and Ͻ 0.001) 2003 (29) BALB/c Spleen Pristane-injected PBS-injected BALB/c Pristane-injected ϭ PBS-injected (percentage and total Yang et al, BALB/c number) 2003 (28) Spleen BWF1 B6 No difference (8–12-week-old mice): absolute number Zeng et al, similar; percentage increased in BWF1 mice (statistical 2003 (31) significance not provided) Spleen BWF1 B6 BWF1 ϭ B6 at ages 4–6 weeks (percentage and total Forestier et al, number); BWF1 Ͼ B6 at ages 10–18 weeks (P Ͻ 0.05 2005 (30) for percentage and total number); at age 30 weeks, percentage reduced, but total number increased, in BWF1 versus B6 (P Ͻ 0.001 for total number) Spleen BWF1 BALB/c, NZW, and CWF1 No significant difference in percentage or total number at Present study (MHCII-matched) all ages tested (5–8 weeks, 13–16 weeks, and 32–39 weeks) Liver MRL-lpr and C3H (MHCII-matched) and MRL-lpr ϭ MRL-Fas Ͻ C3H Ͻ BALB/c (P Ͻ 0.05 to P Ͻ Yang et al, MRL-Fas BALB/c 0.001) 2003 (29) Liver BWF1 B6 BWF1 ϭ B6 in 4-week-old and 14-week-old mice; BWF1 Forestier et al, Ͼ B6 in 30-week-old mice 2005 (30) Kidney BWF1 B6 BWF1 ϭ B6 in 4-week-old mice; BWF1 Ͼ B6 at ages 14 Forestier et al, weeks and 30 weeks 2005 (30) Blood BWF1 B6 BWF1 Ͼ B6 in 4–6-week-old mice (P Ͻ 0.05) Forestier et al, 2005 (30) Lymph MRL-lpr and C3H (MHCII-matched) and MRL-lpr ϭ MRL-Fas ϭ C3H Ͻ BALB/c Yang et al, node MRL-Fas BALB/c 2003 (29)

* iNKT ϭ invariant natural killer T; ␣GalCer ϭ ␣-galactosylceramide; PBS ϭ phosphate buffered saline; MRL-lpr ϭ MRL-Faslpr/lpr; MRL-Fas ϭ ϩ/ϩ ϭ ϭ ϭ ϫ ϭ ϭ MRL-Fas ; C3H C3H.HeJ; MHCII class II major histocompatibility complex; BWF1 (NZB NZW)F1;B6 C57BL/6; CWF1 ϫ (BALB/c NZW)F1. cells in 4–6-week-old BWF1 mice than in control B6 absolute numbers of iNKT cells in the spleen of BWF1 mice (0.2% versus 0.5%, respectively). mice at 10–18 weeks of age. At later ages (25–35 weeks), In the spleen, however, we did not find any when BWF1 mice have splenomegaly and increased change in the numbers of iNKT cells in BWF1 mice. absolute numbers of iNKT cells, there was no propor- Similar data on splenic iNKT cells have been reported by tionate increase in iNKT cells as compared with B6 Zeng et al (31) in BWF1 mice and by our group (28) in mice. Thus, too few iNKT cells may be left to interact the hydrocarbon oil–induced model of lupus. Zeng et al with too many other cells in the lymphoid organs of (31) found no significant differences in the percentages these mice. Nevertheless, the Forestier study reported (mean 4.8 Ϯ 0.9% versus 3.5 Ϯ 0.5% of TCR␤ϩ cells) or an accumulation of iNKT cells in the liver and kidneys of absolute numbers (mean 989 Ϯ 88 ϫ 103 versus 923 Ϯ aged BWF1 mice (30). In contrast, MRL-lpr and MRL- 72 ϫ 103) of tetramer-positive cells in the spleen of Fasϩ/ϩ mice have a marked reduction in iNKT cells in BWF1 and B6 mice. In contrast to these 3 studies in the thymus, spleen, liver, and lymph nodes as compared BWF1 and hydrocarbon oil models, Forestier et al (30) with MHC-matched C3H mice (29). found a significant increase in the percentages and Overall, of the 5 studies that have investigated 1230 YANG ET AL

iNKT cells with the use of tetramers, 4 of them (refs. 28, also failed to detect a significant effect of CD1d defi- 29, 31 and the current study) have found either a ciency on the development of lupus nephritis in MRL- reduction or no significant difference in iNKT cells in lpr/lpr mice (54,55). Instead, lupus dermatitis is exacer- ␤ the lymphoid organs of lupus-prone mice. The fifth bated by 2m (54) and CD1d deficiency (55) in MRL- study, however, found a significant accumulation of lpr/lpr mice. these cells with age in various lymphoid organs (30). The Thus, different regulatory mechanisms may ac- reason for this discrepancy remains unclear. Environ- count for the different outcomes of iNKT cell manipu- mental factors, including diet, and the use of different lations in different models or in different stages of control strains (an MHC-unrelated control strain B6 in disease. Indeed, treatment of BWF1 mice with ␣GalCer, the Forestier study [30] and the Zeng study [31] versus which activates iNKT cells, delays the onset of renal MHC-matched strains, including BALB/c, NZW, and disease if given in the early, preclinical stages of auto- CWF1 mice in the present study) might partly account immunity, but it has no effect on the disease course for some of the differences. All studies to date on iNKT when given during the late stages of disease (Yang J-Q, cells in lupus-prone mice are summarized in Table 2. et al: unpublished observations). Treatment with In summary, a reduction in thymic iNKT cells appears to ␣GalCer during the late stages of disease can even be a common feature of lupus-prone mice, at least exacerbate nephritis and enhance IgM anti-DNA anti- during the first 6 weeks of life. A link between lupus body levels (31). Thus, iNKT cells may be unable to susceptibility and reduced thymic iNKT cells is sup- exert their regulatory effects in the presence of full- ported by a study showing that genetic control of thymic blown autoimmune disease, or a cytokine storm trig- iNKT cell numbers maps strongly to the robust lupus gered by activated iNKT cells might even further stim- susceptibility locus Bana3/Sle1/Nba2/Lbw7 region of ulate the already activated autoreactive T cells in certain chromosome 1 (52). conditions. Additionally, iNKT cells may exert different Such iNKT cell changes must be relevant to lupus effects during different manifestations of disease. For disease development, since germline deletion of CD1d, example, while iNKT cells may protect against lupus which causes a marked deficiency of iNKT cells (45), nephritis, they might exacerbate lupus-associated ath- also resulted in exacerbation of lupus nephritis in a erosclerosis (37). Further studies are required to clarify genetically autoimmune–susceptible model (Figure 1) as the mechanism of the disparate effects of iNKT cells well as in a hydrocarbon oil–induced model in otherwise during different stages or aspects of disease. Neverthe- normal BALB/c mice (28). Although we used N10–N12 0 less, several lines of evidence indicate a potent suppres- backcrossed CD1d BWF1 mice that are expected to carry Յ0.1% genes from the 129/B6 CD1d0 founders, sive role of CD1d-reactive iNKT cells in the early stages there remains the possibility that our results reflect the of autoimmune diseases. introduction or removal of a linked gene(s) during the The mechanisms by which CD1d-reactive T cells backcross of the mutated CD1d 129 locus onto the NZB contribute to protection against the development of ␥ and NZW backgrounds. Genotype analyses of our con- autoimmunity are unclear. We show that whereas IFN genic strains using simple sequence repeat markers, production was unaffected or was increased in the 0 however, did not suggest a replacement with donor CD1d BWF1 mice, production of type 2 cytokines was genes at any of the loci tested (data not shown). More- decreased upon in vitro stimulation of spleen cells with over, similar data have been obtained using different Con A or stimulation of purified T cells with anti-CD3 lupus models, namely CD1d0 BWF1 (Figure 1) and antibody (Figure 5). Conversely, iNKT cell activation by CD1d0 pristane-injected BALB/c mice (28), which is treatment of lupus-prone MRL-lpr mice with ␣GalCer evidence against the possibility that other lupus- has been shown to increase serum levels of IgE (29), susceptibility genes are responsible for our observations. which is a hallmark of the Th2 response. Although which Consistent with our data, a previous study has cytokines play vital roles in the development and pro- shown that in vitro coculture with NK1.1ϩ,CD3ϩ cells gression of SLE remains largely unresolved, several lines or their in vivo transfer also reduces anti-DNA antibody of evidence suggest that increased or stable T cell IFN␥ production in the B6-lpr/lpr model (53), which further production, along with reduced IL-4 production during supports the regulatory role of NKT cells in antibody- disease initiation, could contribute to disease exacerba- mediated autoimmunity. However, treatment of BWF1 tion. For example, IL-4 deficiency or its in vivo neutral- mice with an anti-CD1d mAb has been reported to delay ization increases antichromatin or anti-DNA antibody the onset of lupus (42). We and other investigators have production in animal models of lupus (46,56), and ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1231

transgenic overexpression of IL-4 can protect against the dicated a major role of CD1d-restricted CD4ϩ T cells in development of a lupus-like syndrome (57). IL-4 production and of CD1d-independent T cells in In addition to modulation of cytokine effects, IFN␥ production upon anti-CD3 stimulation (62). Con- CD1d deficiency and iNKT cells can modulate the sistent with our findings, a recent study showed that functions of other immune cell types (24,25). We have TGF␤ receptor II deficiency in T cells results in reduced also recently found that the addition of an iNKT cell iNKT cells, along with increased numbers of NKT-like ligand to spleen cells from the wild-type animals, but not cells that mediate fatal multisystem autoimmune disease to spleen cells from CD1d-deficient mice, can specifi- (63). cally suppress IgG autoantibody production, while acti- In summary, we have generated several lines of vating other B cell functions (58,59). However, the evidence in this and other studies (28,29,38,55,59) sug- effects of iNKT cells on anti-DNA antibody production gesting a protective role of iNKT cells in lupus, at least can only partly explain the disease-exacerbating effects during the early stages of development. However, iNKT of CD1d deficiency, since CD1d0 BWF1 mice had a cell activation can also aggravate lupus, such as during more profound increase in renal disease than in anti- the late stages of disease (31). One might argue that such DNA antibody levels. One possible explanation for this preventive therapy is not worth pursuing, since the finding may be the development of other autoantibodies patients do not present during the early preclinical in CD1d0 mice, which might perpetuate organ damage. stages. However, extrapolating our animal data to hu- For example, we have found that CD1d deficiency mans with SLE, ␣GalCer treatment given to patients results in the development of anti–ribosomal P and when they have achieved remission from other therapies anti-OJ antibodies, which are generally not induced in would prevent future relapses of disease. A clear under- pristane-injected wild-type BALB/c mice (28). Another standing of these roles may have implications for the possible explanation may be the activation of autoreac- development of iNKT cell–based therapies for the treat- tive T cells or other immune cells or increases in ment of complex lupus disease. Furthermore, ongoing cytokines and chemokines, which may directly perpetu- investigations might find other glycolipid agents that ate organ damage. Studies are under way to investigate confer protection at all stages of lupus disease. The idea the relative contribution of cytokine or other immune that iNKT cells may suppress lupus disease is particu- alterations induced by CD1d deficiency or iNKT cell larly appealing, because humans with SLE have reduced activation on the development and progression of lupus. numbers of circulating invariant TCR V␣24–J␣18– The mechanisms by which CD1d deficiency or positive T cells (41). In fact, treatments such as cortico- iNKT cell activation may influence type 1 versus type 2 steroids that suppress disease activity in SLE patients cytokine production remain unclear. In this regard, restore the numbers of circulating V␣24–J␣18 iNKT cells NK1.1ϩ,TCR␣/␤ϩ cells comprise a heterogeneous pop- (41). Clearly, a detailed analysis of the different roles of ulation of cells (29). Although most of these cells are iNKT cells in different stages and manifestations of CD1d-restricted, some NK1.1ϩ,TCR␣/␤ϩ cells are re- lupus in patients is warranted in order to utilize the full stricted by other MHC molecules (60,61). The restric- potential of these cells in therapeutics. tion elements of these CD1d-nonrestricted NKT-like cells are not clearly defined; however, recent reports ACKNOWLEDGMENTS ␣ have implicated roles for the TCR -chain connecting We thank Deijanira Albuquerque, Tam Bui, Seok- peptide domain or a fetal class I molecule in their mann Hong, and Jonathan Jacinto for technical assistance and positive selection or regulation, respectively (60,61). Laurence Morel for suggestions on genotyping the congenic Expansions or phenotype alterations of such NKT-like strains. Generation of the CD1d tetramer loaded with ␣ cells might contribute to enhanced autoimmunity in GalCer that was used in this study was supported by NIH 0 grant AI-042284 to Dr. Sebastian Joyce (Vanderbilt Univer- CD1d autoimmune-prone mice. In fact, the ratio of sity, Nashville, TN). The ␣GalCer was kindly provided by Kirin IFN␥-producing cells to IL-4–producing cells was higher Brewery, Ltd. (Gunma, Japan). The phycoerythrin-labeled among NKT-like cells in CD1d0 BWF1 mice (8:1) as CD1d tetramer loaded with PBS-57, an analog of ␣GalCer, compared with CD1dϩ BWF1 mice (4:1), which have was kindly provided by the NIH Tetramer Facility at Emory both “classic” iNKT cells and NKT-like cells (Figure 4). University (Atlanta, GA). Thus, CD1d-independent NKT-like cells may be respon- AUTHOR CONTRIBUTIONS sible in part for the increased IFN␥ production in CD1d0 Dr. Singh had full access to all of the data in the study and BWF1 mice. Similar inferences were made from studies takes responsibility for the integrity of the data and the accuracy of the ␤ in 2m/MHC class II–double-knockout mice, which in- data analysis. 1232 YANG ET AL

Study design. Yang, Van Kaer, Singh. specific for keyhole limpet hemocyanin. Int Immunol 1989;1: Acquisition of data. Yang, Wen, Liu, Folayan, Dong, Zhou, Singh. 557–64. Analysis and interpretation of data. Yang, Wen, Van Kaer, Singh. 18. Porcelli S, Yockey CE, Brenner MB, Balk SP. Analysis of T cell Manuscript preparation. Yang, Van Kaer, Singh. antigen receptor (TCR) expression by human peripheral blood Ϫ Ϫ Statistical analysis. Yang, Singh. CD4 8 ␣/␤ T cells demonstrates preferential use of several V␤ genes and an invariant TCR ␣ chain. J Exp Med 1993;178:1–16. 19. Bendelac A, Lantz O, Quimby ME, Yewdell JW, Bennink JR, REFERENCES Brutkiewicz RR. CD1 recognition by mouse NK1ϩ T lymphocytes. Science 1995;268:863–5. 1. Singh RR. SLE: translating lessons from model systems to human 20. Porcelli SA, Modlin RL. The CD1 system: antigen-presenting disease. Trends Immunol 2005;26:572–9. molecules for T cell recognition of lipids and glycolipids. Annu 2. Shivakumar S, Tsokos GC, Datta SK. T cell receptor ␣/␤ express- Rev Immunol 1999;17:297–329. ing double-negative (CD4Ϫ/Ϫ) and CD4ϩ T helper cells in 21. Brossay L, Jullien D, Cardell S, Sydora BC, Burdin N, Modlin RL, humans augment the production of pathogenic anti-DNA autoan- et al. Mouse CD1 is mainly expressed on hemopoietic-derived tibodies associated with lupus nephritis. J Immunol 1989;143: cells. J Immunol 1997;159:1216–24. 103–12. 22. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, et al. 3. Singh RR, Kumar V, Ebling FM, Southwood S, Sette A, Sercarz CD1d-restricted and TCR-mediated activation of V␣14 NKT cells EE, et al. T cell determinants from autoantibodies to DNA can by glycosylceramides. Science 1997;278:1626–9. upregulate autoimmunity in murine systemic lupus erythematosus. 23. Bendelac A, Rivera MN, Park SH, Roark JH. Mouse CD1-specific J Exp Med 1995;181:2017–27. NK1 T cells: development, specificity, and function. Annu Rev 4. Peng SL, Madaio MP, Hayday AC, Craft J. Propagation and Immunol 1997;15:535–62. regulation of systemic autoimmunity by ␥␦ T cells. J Immunol 24. Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. The 1996;157:5689–98. regulatory role of V␣14 NKT cells in innate and acquired immune 5. Singh RR, Ebling FM, Albuquerque DA, Saxena V, Kumar V, response. Annu Rev Immunol 2003;21:483–513. Giannini EH, et al. Induction of autoantibody production is 25. Kronenberg M. Toward an understanding of NKT cell biology: limited in nonautoimmune mice. J Immunol 2002;169:587–94. progress and paradoxes. Annu Rev Immunol 2005;23:877–900.

6. Fan GC, Singh RR. Vaccination with minigenes encoding VH- 26. Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van derived major histocompatibility complex class I-binding epitopes Kaer L. NKT cells: what’s in a name? Nat Rev Immunol 2004;4: activates cytotoxic T cells that ablate autoantibody-producing B 231–7. cells and inhibit lupus. J Exp Med 2002;196:731–41. 27. Behar SM, Cardell S. Diverse CD1d-restricted T cells: diverse 7. Fowlkes BJ, Kruisbeek AM, Ton-That H, Weston MA, Coligan phenotypes, and diverse functions. Semin Immunol 2000;12: JE, Schwartz RH, et al. A novel population of T-cell receptor 551–60. ␣␤-bearing thymocytes which predominantly expresses a single V␤ 28. Yang JQ, Singh AK, Wilson MT, Satoh M, Stanic AK, Park JJ, et gene family. Nature 1987;329:251–4. al. Immunoregulatory role of CD1d in the hydrocarbon oil- 8. Yankelevich B, Knobloch C, Nowicki M, Dennert G. A novel cell induced model of lupus nephritis. J Immunol 2003;171:2142–53. type responsible for marrow graft rejection in mice: T cells with 29. Yang JQ, Saxena V, Xu H, Van Kaer L, Wang CR, Singh RR. NK phenotype cause acute rejection of marrow grafts. J Immunol Repeated ␣-galactosylceramide administration results in expan- 1989;142:3423–30. sion of NK T cells and alleviates inflammatory dermatitis in 9. Sykes M. Unusual T cell populations in adult murine bone MRL-lpr/lpr mice. J Immunol 2003;171:4439–46. marrow: prevalence of CD3ϩCD4ϪCD8Ϫ and ␣␤ 30. Forestier C, Molano A, Im JS, Dutronc Y, Diamond B, Davidson TCRϩNK1.1ϩ cells. J Immunol 1990;145:3209–15. A, et al. Expansion and hyperactivity of CD1d-restricted NKT cells 10. Ballas ZK, Rasmussen W. NK1.1ϩ thymocytes: adult murine during the progression of systemic lupus erythematosus in (New ϫ CD4Ϫ, CD8Ϫ thymocytes contain an NK1.1ϩ, CD3ϩ, CD5hi, Zealand Black New Zealand White)F1 mice. J Immunol 2005; CD44hi, TCR–V␤8ϩ subset. J Immunol 1990;145:1039–45. 175:763–70. 11. Bendelac A, Schwartz RH. CD4ϩ and CD8ϩ T cells acquire 31. Zeng D, Liu Y, Sidobre S, Kronenberg M, Strober S. Activation of natural killer T cells in NZB/W mice induces Th1-type immune specific lymphokine secretion potentials during thymic maturation. responses exacerbating lupus. J Clin Invest 2003;112:1211–22. Nature 1991;353:68–71. 32. Hong S, Wilson MT, Serizawa I, Wu L, Singh N, Naidenko OV, et 12. Arase H, Arase N, Nakagawa K, Good RA, Onoe K. NK1.1ϩ al. The natural killer T-cell ligand ␣-galactosylceramide prevents CD4ϩ CD8Ϫ thymocytes with specific lymphokine secretion. Eur autoimmune diabetes in non-obese diabetic mice. Nat Med 2001; J Immunol 1993;23:307–10. pos pos 7:1052–6. 13. Yoshimoto T, Paul WE. CD4 , NK1.1 T cells promptly 33. Wang B, Geng YB, Wang CR. CD1-restricted NK T cells protect produce interleukin 4 in response to in vivo challenge with nonobese diabetic mice from developing diabetes. J Exp Med anti-CD3. J Exp Med 1994;179:1285–95. 2001;194:313–20. 14. Arase H, Arase N, Ogasawara K, Good RA, Onoe K. An NK1.1ϩ ϩ – 34. Dombrowicz D. Exploiting the innate immune system to control CD4 8 single-positive thymocyte subpopulation that expresses a allergic asthma. Eur J Immunol 2005;35:2786–8. highly skewed T-cell antigen receptor V␤ family. Proc Natl Acad 35. Morishima Y, Ishii Y, Kimura T, Shibuya A, Shibuya K, Hegab SciUSA1992;89:6506–10. AE, et al. Suppression of eosinophilic airway inflammation by 15. Lantz O, Bendelac A. An invariant T cell receptor ␣ chain is used treatment with ␣-galactosylceramide. Eur J Immunol 2005;35: by a unique subset of major histocompatibility complex class ϩ Ϫ Ϫ 2803–14. I–specific CD4 and CD4 8 T cells in mice and humans. J Exp 36. Jahng AW, Maricic I, Pedersen B, Burdin N, Naidenko O, Med 1994;180:1097–106. Kronenberg M, et al. Activation of natural killer T cells potentiates 16. Makino Y, Kanno R, Ito T, Higashino K, Taniguchi M. Predom- or prevents experimental autoimmune encephalomyelitis. J Exp inant expression of invariant V␣14ϩ TCR ␣ chain in NK1.1ϩ T Med 2001;194:1789–99. cell populations. Int Immunol 1995;7:1157–61. 37. Major AS, Singh RR, Joyce S, Van Kaer L. The role of invariant 17. Koseki H, Imai K, Ichikawa T, Hayata I, Taniguchi M. Predomi- natural killer T cells in lupus and atherogenesis. Immunol Res nant use of a particular ␣-chain in suppressor T cell hybridomas 2006;34:49–66. ROLE OF CD1d AND NKT CELLS IN NEPHRITIS IN A LUPUS MODEL 1233

38. Singh AK, Yang JQ, Parekh VV, Wei J, Wang CR, Joyce S, et al. 51. Matsuda JL, Naidenko OV, Gapin L, Nakayama T, Taniguchi M, The natural killer T cell ligand ␣-galactosylceramide prevents or Wang CR, et al. Tracking the response of natural killer T cells to promotes pristane-induced lupus in mice. Eur J Immunol 2005;35: a glycolipid antigen using CD1d tetramers. J Exp Med 2000;192: 1143–54. 741–54. 39. Kojo S, Adachi Y, Keino H, Taniguchi M, Sumida T. Dysfunction 52. Esteban LM, Tsoutsman T, Jordan MA, Roach D, Poulton LD, of T cell receptor AV24AJ18ϩ,BV11ϩ double-negative regula- Brooks A, et al. Genetic control of NKT cell numbers maps to tory natural killer T cells in autoimmune diseases. Arthritis major diabetes and lupus loci. J Immunol 2003;171:2873–8. Rheum 2001;44:1127–38. 53. Takeda K, Dennert G. The development of autoimmunity in 40. Van der Vliet HJ, von Blomberg BM, Nishi N, Reijm M, Voskuyl C57BL/6 lpr mice correlates with the disappearance of natural AE, van Bodegraven AA, et al. Circulating V␣24ϩ V␤11ϩ NKT killer type 1-positive cells: evidence for their suppressive action on cell numbers are decreased in a wide variety of diseases that are bone marrow stem cell proliferation, B cell immunoglobulin characterized by autoreactive tissue damage. Clin Immunol 2001; secretion, and autoimmune symptoms. J Exp Med 1993;177: 100:144–8. 155–64. 41. Oishi Y, Sumida T, Sakamoto A, Kita Y, Kurasawa K, Nawata Y, 54. Chan OT, Paliwal V, McNiff JM, Park SH, Bendelac A, Shlomchik ␣ ␣ ␤ et al. Selective reduction and recovery of invariant V 24J Q T cell MJ. Deficiency in 2-microglobulin, but not CD1, accelerates receptor T cells in correlation with disease activity in patients with spontaneous lupus skin disease while inhibiting nephritis in MRL- systemic lupus erythematosus. J Rheumatol 2001;28:275–83. Faslpr mice: an example of disease regulation at the organ level. 42. Zeng D, Lee MK, Tung J, Brendolan A, Strober S. Cutting edge: J Immunol 2001;167:2985–90. a role for CD1 in the pathogenesis of lupus in NZB/NZW mice. 55. Yang JQ, Chun T, Liu H, Hong S, Bui H, Van Kaer L, et al. CD1d J Immunol 2000;164:5000–4. deficiency exacerbates inflammatory dermatitis in MRL-lpr/lpr 43. Hahn BH, Singh RR. Animal models of systemic lupus erythem- mice. Eur J Immunol 2004;34:1723–32. atosus. In: Wallace DJ, Hahn BH, editors. Dubois’ lupus erythem- 56. Richards HB, Satoh M, Jennette JC, Croker BP, Yoshida H, atosus. 7th ed. Philadelphia: Lippincott Williams & Wilkins; 2007. Reeves WH. Interferon-␥ required for lupus nephritis in mice p. 299–355. treated with the hydrocarbon oil pristane. Kidney Int 2001;60: 44. Yue SC, Shaulov A, Wang R, Balk SP, Exley MA. CD1d ligation 2173–80. on human monocytes directly signals rapid NF-␬B activation and 57. Santiago ML, Fossati L, Jacquet C, Muller W, Izui S, Reininger L. production of bioactive IL-12. Proc Natl Acad SciUSA2005; Interleukin-4 protects against a genetically linked lupus-like auto- 102:11811–6. immune syndrome. J Exp Med 1997;185:65–70. 45. Mendiratta SK, Martin WD, Hong S, Boesteanu A, Joyce S, Van 58. Singh RR, Yang JQ. NKT cells suppress autoreactive B cells Kaer L. CD1d1 mutant mice are deficient in natural T cells that [abstract]. J Immunol 2006;176 Suppl:S151. promptly produce IL-4. Immunity 1997;6:469–77. 59. Singh RR, Wen X, Yang JQ. iNKT cells suppress autoreactive B 46. Singh RR, Saxena V, Zang S, Li L, Finkelman FD, Witte DP, et al. cells in a contact-dependent manner. Submitted for publication. Differential contribution of IL-4 and STAT6 vs STAT4 to the 60. Capone M, Troesch M, Eberl G, Hausmann B, Palmer E, Mac- development of lupus nephritis. J Immunol 2003;170:4818–25. Donald HR. A critical role for the T cell receptor ␣-chain 47. Liu Y, Goff RD, Zhou D, Mattner J, Sullivan BA, Khurana A, et connecting peptide domain in positive selection of CD1-indepen- al. A modified ␣-galactosyl ceramide for staining and stimulating dent NKT cells. Eur J Immunol 2001;31:1867–75. natural killer T cells. J Immunol Methods 2006;312:34–9. 61. Dang Y, Heyborne KD. Cutting edge: regulation of uterine NKT 48. Singh RR, Hahn BH, Sercarz EE. Neonatal peptide exposure can cells by a fetal class I molecule other than CD1. J Immunol prime T cells and, upon subsequent immunization, induce their 2001;166:3641–4. immune deviation: implications for antibody vs. T cell-mediated 62. Maeda M, Shadeo A, MacFadyen AM, Takei F. CD1d-indepen- ␤ autoimmunity. J Exp Med 1996;183:1613–21. dent NKT cells in 2-microglobulin-deficient mice have hybrid 49. Smolen JS, Steiner G, Aringer M. Anti-cytokine therapy in phenotype and function of NK and T cells. J Immunol 2004;172: systemic lupus erythematosus. Lupus 2005;14:189–91. 6115–22. 50. Park SH, Weiss A, Benlagha K, Kyin T, Teyton L, Bendelac A. 63. Marie JC, Liggitt D, Rudensky AY. Cellular mechanisms of fatal The mouse CD1d-restricted repertoire is dominated by a few early-onset autoimmunity in mice with the T cell-specific targeting autoreactive T cell receptor families. J Exp Med 2001;193: of transforming growth factor-␤ receptor. Immunity 2006;25: 893–904. 441–54. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1234–1241 DOI 10.1002/art.22497 © 2007, American College of Rheumatology

Structural Insertion/Deletion Variation in IRF5 Is Associated With a Risk Haplotype and Defines the Precise IRF5 Isoforms Expressed in Systemic Lupus Erythematosus

Sergey V. Kozyrev,1 Susanna Lewén,1 Prasad M. V. Linga Reddy,1 Bernardo Pons-Estel2 and the Argentine Collaborative Group, Torsten Witte3 and the German Collaborative Group, Peter Junker,4 Helle Laustrup,4 Carmen Gutiérrez,5 Ana Suárez,5 Maria Francisca González-Escribano,6 Javier Mart´ın7 and the Spanish Collaborative Group, and Marta E. Alarcon-Riquelme´ 1

Objective. To determine whether specific isoforms expression in relation to the genotypes of functional of IRF5 are transcribed in patients with systemic lupus SNPs was analyzed using quantitative polymerase chain erythematosus (SLE) who have risk genotypes in the reaction. Sequencing and genotyping of the IRF5 gene exon 1B donor splice site at single-nucleotide polymor- was performed. phism (SNP) no. rs2004640. Results. Sequencing of complementary DNA from Methods. Peripheral blood mononuclear cells individuals with different genotypes showed 4 basic were obtained from SLE patients and healthy controls isoforms transcribed from all 5؅-untranslated regions -from Argentina, Spain, and Germany and from trio (5؅-UTRs), suggesting no preferential isoform tran families from Spain and Denmark. A reporter assay was scription based on rs2004640 genotypes. Analysis of used to investigate the role of SNP no. rs2004640. IRF5 translation efficiency showed that exon 1A was the most efficient in initiating protein synthesis. We identified a Supported by the Swedish Research Council, the Swedish novel polymorphic insertion/deletion that defines the Association Against Rheumatism, the 80-Year Foundation of King pattern of expression of isoforms of IRF5. The insertion Gustaf V, the Marcus Borgstro¨ms Foundation, the Magnus Bergvalls Foundation, the Torsten and Ragnar So¨derbergs Foundation, and the consists of 4 repeats in exon 6 affecting the protein Danish Rheumatism Association. Dr. Kozyrev’s work was supported interaction domain. The insertion segregates in the risk by the Gurli and Edward Brunnbergs Foundation for Rheumatic Research. The German Collaborative Group’s work was supported by haplotype with the high expression allele of a poly(A) a grant from BMBF Kompetenznetz Rheuma C2.12. Dr. Alarcoń- site SNP no. rs10954213 and the exon 1B donor splice (Riquelme is supported by the Knut and Alice Wallenberg Foundation allele of the 5؅-UTR SNP no. rs2004640. The poly(A through a Royal Swedish Academy of Sciences award. 1Sergey V. Kozyrev, PhD, Susanna Leweń, MSc, Prasad M. V. polymorphism correlated with levels of IRF5 in cells Linga Reddy, MSc, Marta E. Alarcoń-Riquelme, MD, PhD: Rudbeck stimulated with interferon-␣. The SNP most strongly ؋ ؍ Laboratory, Uppsala University, Uppsala, Sweden; 2Bernardo Pons- P 3 associated with SLE was SNP no. rs2070197 ( 5.2 Estel, MD: Sanatorio Parque, Rosario, Argentina; Torsten Witte, ؊11 MD, PhD: Medical School Hannover, Hannover, Germany; 4Peter 10 ), which is a proxy of the risk haplotype, but does Junker, MD, Helle Laustrup, MD: Odense University Hospital, not appear to be functional. 5 Odense, Denmark; Carmen Gutie´rrez, MD, PhD, Ana Sua´rez, PhD: Conclusion. None of the functional variants inves- Hospital Universitario Central de Asturias, Universidad de Oviedo, Oviedo, Spain; 6Maria Francisca Gonza´lez-Escribano, PhD: Hospital tigated in this study is strongly associated with SLE, Virgen del Rocıó, Sevilla, Spain; 7Javier Martıń, MD, PhD: Instituto with the exception of the exon 1B donor splice site, and de Biomedicina Lo´pez Neyra, CSIC, Granada, Spain. Address correspondence and reprint requests to Marta E. its functional importance appears to be small. Our Alarcoń-Riquelme, MD, PhD, Department of Genetics and Pathology, results suggest that there may be other functional Rudbeck Laboratory, Uppsala University, Dag Hammarskjolds vag 20, polymorphisms, yet to be identified, in IRF5. We did not 751 85 Uppsala, Sweden. E-mail: [email protected]. Submitted for publication December 11, 2006; accepted in observe evidence of epistatic interaction between the revised form December 21, 2006. functional SNPs.

1234 SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5 1235

IRF5, a key component of the type I interferon 79 families was from Denmark (22 complete trios). All indi- pathway, has a strong genetic association with systemic viduals fulfilled the American College of Rheumatology 1982 lupus erythematosus (SLE). IRF5 is a transcription revised criteria for SLE (5). Control samples for expression analysis were obtained from healthy donors at the Uppsala factor involved in the transcriptional activation of vari- Academic Hospital Blood Bank, as previously described (4). ous proinflammatory cytokines and interferon-␣ (IFN␣) Stimulation of PBMCs. Freshly isolated PBMCs were (1) and is primarily involved in host defense against stimulated with 1,000 units of IFN␣ (RayBiotech, Norcross, viruses. The various isoforms of IRF5 have different GA) for 6 hours in RPMI 1640 medium supplemented with penicillin/streptomycin and 10% fetal calf serum. effects on the interferon system (2). Complementary DNA (cDNA) synthesis and quantita- The genetic association of IRF5 with SLE was tive polymerase chain reaction (PCR) of IRF5 UTR–specific recently identified (3), and we have previously described transcripts. Total RNA from individuals with SLE carrying the a mechanism through which IRF5 contributes to genetic various genotypes was purified from PBMCs with TRIzol ␮ susceptibility to SLE (4). The IRF5 gene has 4 alterna- (Invitrogen, San Diego, CA). We reverse-transcribed 2 gof total RNA with 2 units of MultiScribe reverse transcriptase in tive exons in the 5Ј-untranslated region (5Ј-UTR), PCR buffer II containing 5 mM MgCl2,1mM dNTPs, 0.4 units named exon 1A, exon 1B, exon 1C, and exon 1D. The T of RNase inhibitor, and 5 ␮M oligo(dT). All reagents were allele of the single-nucleotide polymorphism (SNP) no. from Applied Biosystems (Foster City, CA). Synthesis was rs2004640 introduces a donor splice site leading to the performed at 42°C for 45 minutes, followed by 95°C for 5 minutes. expression of exon 1B transcripts and the reduction of IRF5 isoforms with distinct 5Ј-UTRs were quantified exon 1C–derived transcripts (4). A second SNP located using TaqMan real-time PCR on an ABI Prism 7700 Sequence 5 kb downstream of IRF5, rs2280714, has a T allele that Detector (Applied Biosystems) and SDS version 1.9.1 soft- is strongly associated with high levels of IRF5 (4). ware. Primers used to distinguish PCR products with different Alleles at both SNP positions comprise a haplotype UTRs have been published previously (4). The exon 1D– specific primer was 5Ј-GCTCAGCCCGGATCTGCAG- associated with SLE (4). TTGCCAG-3Ј. We used a common reverse primer lying in Our primary hypothesis was that the 5Ј-UTRs exon 3 and a common TaqMan probe labeled with FAM and affected which of the alternative isoforms would be TAMRA. We performed 45 cycles of 2-step PCR (95°C for 15 seconds and 63°C for 1 minute) in buffer containing 1.5 mM expressed, and that the splice donor mutation repre- ␮ MgCl2, 200 M each dNTP, 0.5 units of Platinum Taq poly- sented by SNP no. rs2004640 changed their pattern of merase (Invitrogen), primer-probe mixture, and cDNA. Ex- expression. Identification of the major isoforms of IRF5 pression levels were normalized to levels of the human TBP in patients with SLE is of major importance in under- gene (Applied Biosystems). standing the mechanisms through which IRF5 leads to Cloning and sequence analysis of IRF5 isoforms. PCR the development of SLE. In the present study, we found amplification of diverse isoforms of IRF5 was performed with the same forward primers used for the TaqMan assay. The that a previously unknown structural insertion/deletion common reverse primer 5Ј-GCAGCCTTGTTATTGCAT- in exon 6 of the IRF5 gene leads to the precise expres- GCCAGCTG-3Ј was designed to allow amplification of the sion of specific isoforms in the risk haplotype associated full-length isoforms. Cycle conditions were 95°C for 3 minutes, with SLE. followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 1.5 minutes. PCRs were performed in a 25-␮l reaction volume with 0.5 units of High Fidelity Platinum PATIENTS AND METHODS Taq polymerase in the buffer supplied by the manufacturer (Invitrogen). Electrophoresis was performed on PCR products Patients and controls. Peripheral blood mononuclear on a 1% agarose gel. PCR products were subcloned in cells (PBMCs) were obtained from SLE patients and healthy pCR4-TOPO vector (Invitrogen), and 50 random positive controls from Argentina, Spain, and Germany. The groups of clones were analyzed by sequencing. PCR was also used for subjects from Argentina and Spain have been previously direct analysis of insertion/deletion patterns in cDNA pre- described (4). The group of subjects from Germany consisted pared from PBMCs. The following primers were used: forward of 288 SLE patients and 245 healthy controls from various 5Ј-TGCAGGAGAGGAGGAGGAAGAAGAG-3Ј, reverse centers in northern Germany. Samples from the German 5Ј-AACTTGATCTCCAGGTCGGTCA-3Ј. subjects were collected at the University of Hannover. The In vitro translation efficiency. To determine the effect study was conducted with the participation of the members of of alternative 5Ј-UTRs on protein synthesis, we amplified the Argentine, German, and Spanish Collaborative Groups full-length UTRs using a common reverse primer, as for the (Appendix A). quantitative PCR, and primers Ap1 and Ap2 and human Trio families were from 2 separate populations. Com- spleen BD Marathon-Ready cDNA (Clontech, Palo Alto, CA). plete trios consisted of both parents and an affected child, and PCR products were gel purified with a kit from Qiagen incomplete trios consisted of one parent and an affected child. (Chatsworth, CA) and subcloned in pCR4-TOPO vector for One set of 89 families was from Asturias in northern Spain and sequence analysis. The longest fragments were subcloned in the Madrid region (49 complete trios), and the second group of pGL3 promoter vector. Plasmid DNA was purified with the 1236 KOZYREV ET AL

EndoFree plasmid Maxi kit (Qiagen) and transfected with than other exon 1 transcripts in both stimulated and Lipofectamine 2000 into HEK 293 cells. To allow transfection unstimulated cells, while transcripts derived from exon normalization, pRL-TK vector (Promega, Madison, WI) was 1B or exon 1C were produced at levels 100 times lower cotransfected with the reporter constructs. After 48 hours of incubation, cells were harvested and lysed in a passive lysis (Figure 1a). Exon 1D transcripts initially cloned from buffer, according to the recommendations of the manufacturer human spleen cDNA were also found at very low levels. (Promega). Firefly and Renilla luciferase activity levels were We reasoned that despite the low levels of measured in duplicate with the Dual Luciferase Reporter mRNA for some isoforms, the corresponding protein Assay (Promega). Experiments were repeated 4 times with 2 levels might be different and dependent on 5Ј-UTR– independent DNA preparations. mediated translation efficiency. Therefore, we cloned Transterm (http://uther.otago.ac.nz/Transterm.html) Ј was used to analyze the 3Ј-UTR of IRF5 (6). cDNA for the 4 full-length 5 -UTRs in the luciferase Genotyping. For genotyping, we used the TaqMan reporter vector and analyzed their effect on in vitro SNP Genotyping Assay (Applied Biosystems), and detection protein expression. The most potent was the 5Ј-UTR was performed using an ABI 9700 real-time PCR system. The encoded by exon 1A. Exon 1B and exon 1D did not show primers and probes were designed by the Applied Biosystems any effect compared with the control (pGL3 promoter Assays-on-Demand service for allele discrimination with the vector), while the presence of exon 1C significantly 5Ј-nuclease assay and fluorogenic probes. The average genotype completeness for SNP no. rs10954213 and no. rs2070197 inhibited the efficiency of protein translation (Figure was 98.8% for the samples from Argentine subjects, 99.6% for 1b). Thus, mRNA for exons 1B, 1C, and 1D all have the samples from German subjects, and 86% for the samples poor translation efficiency as compared with that for from Spanish subjects. Genotyping for the insertion/deletion exon 1A. was performed by PCR analysis under the conditions speci- We then selected 18 individuals carrying the fied above, with the following primers: forward 5Ј- Ј Ј various genotypes of rs2004640. Using primers specific GCCGTCCACACGCACTCTCTGTAG-3 , reverse 5 -CTG- Ј AAGCCAGCAGGGTTGCCAG-3Ј. PCR products were re- for each 5 -UTR, we performed PCR amplification and solved on 2.5% agarose gels. cloned cDNA from those individuals and sequenced 50 Accession numbers. Sequences for the full-length 5Ј- clones for each. We observed that all 4 promoters and UTRs of IRF5 have been deposited in GenBank under 5Ј-UTRs led to expression of the same set of isoforms accession numbers DQ995491, DQ995492, DQ995493, and (V1, V4, V5, and V6 transcripts; results not shown). DQ995494. Accession numbers for the sequences of IRF5 with insertion and deletion are DQ995495 and DQ995496, respec- Hence, the effect of exons 1B, 1C, and 1D on the tively. production of IRF5 protein isoforms, as compared with Statistical analysis. Analyses of the genetic associa- exon 1A, is negligible, since they code for the same tions of the SNPs and haplotypes in patients and controls and isoform set as does exon 1A, which is expressed at far the family-based analysis were performed using Haploview, higher levels. version 3.2, which incorporates only complete trios. We used Family Based Association Testing software (7), which esti- Determination of isoform pattern by structural mates genotypes for the nongenotyped parents so that infor- insertion/deletion and alternative 3؅ acceptor splice mation from the incomplete trios can be used. The software sites. Interestingly and unexpectedly, based on the se- was used to analyze all Spanish and Danish trios jointly (n ϭ quences found in different samples, we could classify the 168) and corroborate the association results, taking into con- individuals into 3 separate groups: those who had V1 sideration all genetic information. Results were comparable with those obtained using Haploview. Five trios showed Men- and V4 isoforms, those who had V5 and V6 isoforms, delian inconsistencies and were excluded from the analysis. and those who expressed all 4 isoforms (Figure 1c), Odds ratios were calculated as previously described (4). Cor- suggesting Mendelian segregation and the effect of new relations of IRF5 messenger RNA (mRNA) levels were ana- structural genetic variation. We sequenced the complete lyzed using the t-test included in GraphPad software (Graph- IRF5 gene in 6 individuals from each of the 3 groups. Pad Software, San Diego, CA), as previously described (4). R language was used for logistic regression analysis using the case We identified a novel insertion/deletion, which control material. lacked 2 of 4 tandem repeats in exon 6 encoding for a proline-rich region within the putative PEST and interaction domains. The presence or absence of the repeats RESULTS determined the isoforms to be expressed, such that Low contribution of exon 1B transcripts to the individuals with 4 repeats (insertion) expressed isoforms overall levels of IRF5. We performed quantitative ana- V5 and V6, while individuals with 2 repeats (deletion) lysis of IRF5 gene expression in human PBMCs upon expressed isoforms V1 and V4 (Figure 1c). An alterna- stimulation with IFN␣. These studies clearly showed tive 3Ј acceptor splice site in exon 6 defined the expres- that exon 1A transcripts were expressed at higher levels sion of V1 or V4 and V5 or V6 isoforms, respectively SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5 1237

Figure 1. a, Expression levels of exon 1A, 1B, and 1C transcripts in unstimulated human peripheral blood mononuclear cells (PBMCs) and PBMCs stimulated with interferon-␣ (IFN␣). The data are typical of findings in individuals who have the T allele of rs2004640 and the A allele of rs10954213. Expression levels were normalized to levels of TBP. The expression level of the exon 1D transcript was detected with nested polymerase chain reaction (PCR) and is not shown on the plot. Values are the mean relative x-fold expression. b, Relative translation efficiency of the 4 alternative 5Ј-untranslated regions (5Ј-UTRs). HEK 293 cells were transfected with either pGL3 promoter vector (pGL3-prom) or constructs with alternative 5Ј-UTRs preceding luciferase gene. Firefly luciferase activity was measured with the Dual Luciferase Reporter Assay, and values were normalized to Renilla luciferase activity. Values are the mean Ϯ SD of the relative luciferase activity in 4 experiments. c, IRF5 gene structure. Solid boxes are protein coding exons. Open boxes are noncoding exons. Top, Location of the single-nucleotide polymorphisms and insertion/deletion (in-del) variation. Bottom, Two alternative 3Ј acceptor splice sites in exon 6, giving rise either to long or short isoforms with 2 repeats, V1 or V4, or to long or short isoforms with 4 repeats, V5 or V6. The PCR gel shows the cDNA expression pattern obtained with PCR amplification of the segment from exon 4 to exon 7, in 6 individuals with insertion/deletion genotypes having 2 repeats (2R), 4 repeats (4R), or both. Each lane represents 1 individual.

(Figure 1c). Further, all isoforms within each group (i.e., thus causing transcription to continue. Computational V1 and V4 in 1 group, V5 and V6 in 1 group, and V1, prediction with Transterm software revealed 2 strong V4, V5, and V6 in the heterozygous group) were equally AU-rich elements (AREs) located within the long 3Ј- expressed, according to our comprehensive sequencing UTR (6). analysis of transcripts in various individuals (results not We tested whether any of the SNPs of IRF5 shown). correlated with levels of IRF5 mRNA, and observed -Role of the length of the 3؅-UTR and SNP no. that, as expected, rs10954213, the 3Ј-UTR SNP, corre rs10954213 in determining the level of IRF5. Through lated with levels of IRF5 mRNA, in particular when cells bioinformatic analysis of the IRF5 gene, we found were stimulated with IFN␣ (Figure 2). The highest that SNP no. rs10954213 is located in the 3Ј-UTR correlations were observed with the A allele, but these polyadenylation site AAT(A/G)AA. Thus, SNP no. did not reach statistical significance due to the number rs10954213 potentially determines IRF5 expression lev- of samples used. No correlation was observed for els, with the A allele predicting mRNA with a short rs2004640, insertion/deletion, or the risk haplotype (data 3Ј-UTR, and the G allele disrupting the poly(A) site and not shown). 1238 KOZYREV ET AL

Figure 2. Effect of rs10954213 on expression of the IRF5 gene in unstimulated PBMCs (non-stim) and PBMCs stimulated with 1,000 units IFN␣. Diamonds represent individual samples, and bars represent the mean level of expression. The y-axis shows the expression levels normalized to TBP. See Figure 1 for other definitions.

Insertion in the risk haplotype. The insertion was divided the risk haplotype identified previously (4) into found in the risk haplotype defined by the nonfunctional smaller haplotypes, with one (TCA) clearly segregating SNP no. rs2070197, which is the SNP in IRF5 with the in patients and one (GTG) clearly protective (Table 1). highest level of association with SLE. SNP no. rs2070197 The 2 novel SNPs, SNP no. rs10954213 and no.

Table 1. Genetic association of the risk haplotype of IRF5* Case Control Haplotype Frequency Frequency ␹2 P OR (95% CI) Spanish samples TTA 0.432 0.402 1.55 0.28 – GTG† 0.217 0.288 16.344 5.2 ϫ 10Ϫ5 0.68 (0.57–0.82) GTA 0.142 0.152 0.522 0.4698 – TCA‡ 0.146 0.106 9.163 0.002 1.44 (1.13–1.84) Argentine samples TTA 0.309 0.303 0.042 0.836 – GTG† 0.363 0.431 5.616 0.0178 0.75 (0.59–0.95) GTA 0.094 0.129 3.636 0.0565 – TCA‡ 0.178 0.091 19.026 1.28 ϫ 10Ϫ5 2.18 (1.53–3.12) German samples TTA 0.374 0.343 1.073 0.300 – GTG† 0.274 0.331 3.979 0.046 0.76 (0.58–0.99) GTA 0.135 0.168 2.244 0.134 – TCA‡ 0.165 0.091 12.466 0.0004 1.99 (1.35–2.93) Combined samples TTA 0.381 0.363 1.599 0.206 – GTG† 0.271 0.334 22.279 2.35 ϫ 10Ϫ6 0.73 (0.65–0.83) GTA 0.125 0.147 5.058 0.02 – TCA‡ 0.159 0.102 34.189 5.0 ϫ 10Ϫ9 1.67 (1.40–1.99) TICA‡ 0.151 0.099 24.456 5.72 ϫ 10Ϫ8 – * The order of the single-nucleotide polymorphisms is rs2004640, rs2070197, and rs10954213. Odds ratios (ORs) and 95% confidence intervals (95% CIs) are shown for the protective and risk haplotypes only. † Protective haplotype. ‡ Risk haplotype. I ϭ insertion. SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5 1239

Table 2. Genetic association of the individual SNP risk alleles of IRF5 in combined samples* Associated Case Control SNP allele frequency frequency ␹2 P rs2004640 T 0.602 0.516 34.857 3.5 ϫ 10Ϫ9 rs2070197 C 0.166 0.096 43.083 5.2 ϫ 10Ϫ11 Insertion/deletion I 0.507 0.515 0.23 0.626 rs10954213 A 0.660 0.606 11.263 0.0008 * SNP ϭ single-nucleotide polymorphism. rs2070197, and the insertion/deletion were genotyped in individuals who were homozygous for rs2004640 and trios, patients, and controls. The risk haplotype (TCA) rs10954213 were heterozygous for the insertion (data was formed by the T allele of rs2004640, the C allele of not shown). rs2070197 (TϾC), the insertion, and the A allele of rs10954213 (GϾA) (i.e., TICA). DISCUSSION As shown in Table 1, the same risk haplotype was found in 3 separate sets of patients and controls, from We have identified a novel structural variation Spain, Germany, and Argentina, with a combined P determining the expression of the IRF5 isoforms. It was Ϫ value of 5 ϫ 10 9. SNP no. rs2070197 was by itself previously thought that isoforms in IRF5 resulted from Ϫ strongly associated with SLE (P ϭ 5.2 ϫ 10 11) (Table the 5Ј-UTR and splicing at exon 6 (2). However, in the 2), but bioinformatic analysis of the sequence did not present study we found that a polymorphic insertion/ provide any potential evidence that this SNP was func- deletion in exon 6 and the alternative 3Ј acceptor splice tional. SNP no. rs10954213 was weakly associated (P ϭ site in this exon determine the 2 major isoform groups in 0.0008), while the insertion/deletion was not by itself IRF5. Further, we showed that all 5Ј-UTRs of the IRF5 associated with SLE (Table 2). Of the functional SNPs, gene can lead to equal transcription of the 4 main rs2004640 was the most strongly associated with SLE isoform types, V1, V4, V5, and V6. Thus, the particular Ϫ9 (P ϭ 3.5 ϫ 10 ). Using a set of trio families, we exon 1 used makes no difference in terms of the protein confirmed the haplotype and the presence of the inser- identity, and only exon 1A transcripts are present at high tion in the risk haplotype (Table 3). Thus, individuals amounts in unstimulated cells and cells stimulated with with the risk haplotype (TICA) expressed V5 and V6 IFN␣. It is well known that 5Ј-UTRs play an important isoforms. The insertion was also present in a protective role in the regulation of gene expression (8,9). haplotype (GITG). Alternative UTRs with different indexes of trans- Using multivariate logistic regression, we tested lation efficiency can influence the amount of protein whether the 3 functional polymorphisms increased the expressed. Some structural elements in the 5Ј-UTR risk of developing SLE. We did not find evidence of this. determine the efficiency with which the protein synthesis The 3 SNPs together did not improve the association will be initiated and elongated. These include the length found for rs2004640 alone (data not shown). Most and composition of the UTR, secondary structure, presence of upstream initiation codons, and the conformity of the sequence surrounding the initiation codon, with Table 3. Genetic association of the risk haplotype of IRF5 in trio Kozak consensus defining the strength of the latter. In families* vitro analysis of the alternative 5Ј-UTRs of the IRF5 Transmitted/ gene showed that the exon 1A–encoded UTR had a ␹2 Haplotype† untransmitted Frequency P much higher translation potential than the other 3 TDTA 21.6/29.1 36.1 1.101 0.5491 UTRs, thus further supporting the hypothesis that the GITG 17.8/29.9 26.8 3.118 0.0189 IRF5 protein isoform pool is made up of mainly exon 1A GDTA 9.1/13.6 13.5 0.887 0.4028 TICA‡ 30.0/8.0 16.4 12.737 0.000137 transcripts and very little or no exon 1B that is used when the associated T allele is present. Taken together, * The order of the single-nucleotide polymorphisms is rs2004640, rs2070197, and rs10954213. The trios from Spain and Denmark were these data support the conclusion that SNP no. from a total of 168 families with 71 complete trios. Transmitted alleles rs2004640 is not functionally important in determining are expressed as absolute numbers. Haplotype frequencies and P the isoforms of IRF5. values were obtained using Family Based Association Testing software. †Dϭ deletion; I ϭ insertion. We also found, in the polyadenylation site of ‡ Risk haplotype. IRF5, an SNP (GϾA) determining the length of the 1240 KOZYREV ET AL

3Ј-UTR. The A allele leads to a short 3Ј-UTR, while the studies, in which haplotypes have not been unambigu- G allele encodes for a 1.5-kb long 3Ј-UTR and has 2 ously defined. Defining the risk haplotypes in disease strong AREs known to be responsible for short half-life will be necessary for understanding of the functional and rapid RNA degradation (10). Messenger RNA with genetics behind disease susceptibility. a shorter 3Ј-UTR bearing no such signals should be Addendum. Since the time this paper was submitted more stable and present at high levels. Indeed, we found for publication, an article describing the polyadenylation site that the A allele of rs10954213 correlated with high SNP of IRF5, no. rs10954213, has been published (Graham levels of IRF5, particularly when cells were stimulated DS, Manku H, Wagner S, Reid J, Timms K, Gutin A, et al. ␣ Association of IRF5 in UK SLE families identifies a variant with IFN . AREs are abundant in cytokines and growth involved in polyadenylation. Hum Mol Genet 2006. E-pub factor genes, for which rapid mRNA turnover is crucial ahead of print). for gene function (for review, see refs. 10 and 11). In mice, mutations disrupting AREs in the tumor necrosis ACKNOWLEDGMENTS factor ␣ (TNF␣) gene lead to elevated levels of circulating TNF␣ and hypersensitivity to stimulation with We would like to thank Hong Yin for technical assis- lipopolysaccharide (12,13). Thus, it is possible that SNP tance with the samples, and the Uppsala Genome Center for no. rs10954213 in the 3Ј-UTR poly(A) site of IRF5 could sequencing. We acknowledge Rob Graham and David Alt- shuler for sharing information on the typing of SNP no. be the functional mutation responsible for the level of rs10954213 and SNP no. rs2070197 prior to publication. SNP IRF5. The previously identified SNP no. rs2280714 is a no. rs10954213 was partly typed at the Broad Institute. We perfect proxy for rs10954213, but is located 5 kb 3Ј of thank Adriana I. Scollo, Armando M. Perichon, and Mariano IRF5 and therefore cannot clearly account for the high C. R. Tenaglia of CEDIM Diagno´stico Molecular y Forense levels of IRF5. SRL (Rosario, Argentina) for their help in DNA preparation The isoforms of IRF5 do not differ in their DNA of the Argentine samples, and Dr. Anne Voss for clinical help with the Danish samples. We would like to particularly thank binding sites, but differ in the PEST and interaction the Lupus Patient Association of Asturias for help in the domains (2). This may lead to the use of different collection of family samples. coactivator or inhibitor proteins acting in specific cells or tissue, leading in turn to the promotion of a particular AUTHOR CONTRIBUTIONS set of IRF5 targets. Exon 6 repeats forming the Dr. Alarcoń-Riquelme had full access to all of the data in the insertion/deletion polymorphism encode for the proline- study and takes responsibility for the integrity of the data and the rich region. Such structures, often present in transcrip- accuracy of the data analysis. tion factors, may define the variety and affinity of other Study design. Kozyrev, Pons-Estel, Junker, Laustrup, Alarcoń- cofactors during transcription activity (14–16). Whether Riquelme. Acquisition of data. Kozyrev, Leweń, Reddy, Witte, Junker, Laustrup, and how the insertion/deletion in the IRF5 gene influ- Gutie´rrez, Sua´rez, Gonza´lez-Escribano, Martıń. ences gene function remain to be shown. Analysis and interpretation of data. Kozyrev, Leweń, Reddy, Junker, IRF5 is clearly an SLE susceptibility gene, and in Laustrup, Martıń, Alarcoń-Riquelme. Manuscript preparation. Kozyrev, Pons-Estel, Witte, Junker, Laus- the present study we identified a risk haplotype, using trup, Alarcoń-Riquelme. samples from several populations. However, neither the Statistical analysis. Kozyrev, Reddy, Alarcoń-Riquelme. insertion nor the highly expressed allele of rs10954213 were associated with SLE or appear to explain the REFERENCES genetic effect of IRF5 on susceptibility. Also, rs2004640, the functional polymorphism with the strongest associa- 1. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N. IRF family of transcription factors as regulators of host defense. Annu Rev tion with SLE identified to date for IRF5, has a small Immunol 2001;19:623–55. functional impact. Therefore, it is highly conceivable 2. Mancl ME, Hu G, Sangster-Guity N, Olshalsky SL, Hoops K, that we have not identified all functional variation in Fitzgerald-Bocarsly P, et al. Two discrete promoters regulate the alternatively spliced human interferon regulatory factor-5 iso- IRF5 contributing to disease susceptibility and that one forms: multiple isoforms with distinct cell type-specific expression, or more additional risk haplotypes in linkage disequili- localization, regulation, and function. J Biol Chem 2005;280: brium with rs2004640 are yet to be found. Alternatively, 21078–90. 3. Sigurdsson S, Nordmark G, Goring HH, Lindroos K, Wiman AC, despite its lower effect on genetic risk, rs10954213 may Sturfelt G, et al. Polymorphisms in the tyrosine kinase 2 and have a stronger functional impact on disease expression. interferon regulatory factor 5 genes are associated with systemic This remains to be shown. lupus erythematosus. Am J Hum Genet 2005;76:528–37. 4. Graham RR, Kozyrev SV, Baechler EC, Reddy MV, Plenge RM, Our results may explain why apparently func- Bauer JW, et al. A common haplotype of interferon regulatory tional polymorphisms may not be replicated in some factor 5 (IRF5) regulates splicing and expression and is associated SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5 1241

with increased risk of systemic lupus erythematosus. Nat Genet MD (Hospital Interzonal General de Agudos Dr. Oscar Alende, Mar 2006;38:550–5. del Plata), Susana Gamron, MD, Cristina Drenkard, MD, Emilia 5. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield Menso, MD (UHMI 1, Hospital Nacional de Clıńicas, Universidad NF, et al. The 1982 revised criteria for the classification of systemic Nacional de Co´rdoba, Co´rdoba), Alberto Allievi, MD, Guillermo A. lupus erythematosus. Arthritis Rheum 1982;25:1271–7. Tate, MD (Organizacioń Me´dica de Investigacioń, Buenos Aires), Jose 6. Jacobs GH, Stockwell PA, Tate WP, Brown CM. Transterm— L. Presas, MD (Hospital General de Agudos Dr. Juań A. Fernandez, extended search facilities and improved integration with other Buenos Aires), Simon A. Palatnik, MD, Marcelo Abdala, MD, Mariela databases. Nucleic Acids Res 2006;34:D37–40. Bearzotti, PhD (Universidad Nacional de Rosario y Hospital Provin- 7. Horvath S, Xu X, Laird NM. The family based association test cial del Centenario, Rosario), Alejandro Alvarellos, MD, Francisco method: strategies for studying general genotype—phenotype Caeiro, MD, Ana Bertoli, MD (Hospital Privado, Centro Medico de associations. Eur J Hum Genet 2001;9:301–6. 8. Wang G, Guo X, Floros J. Differences in the translation efficiency Co´rdoba, Co´rdoba), Sergio Paira, MD, Susana Roverano, MD (Hos- and mRNA stability mediated by 5Ј-UTR splice variants of human pital Jose´ M. Cullen, Santa Fe), Cesar E. Graf, MD, Estela Bertero, SP-A1 and SP-A2 genes. Am J Physiol Lung Cell Mol Physiol PhD (Hospital San Martıń, Parana´), Cesar Caprarulo, MD, Griselda 2005;289:L497–508. Buchanan, PhD (Hospital Felipe Heras, Concordia, Entre Rıós), 9. Gray NK, Wickens M. Control of translation initiation in animals. Carolina Guilleroń, MD, Sebastian Grimaudo, PhD, Jorge Manni, MD Annu Rev Cell Dev Biol 1998;14:399–458. (Instituto de Investigaciones Me´dicas Alfredo Lanari, Buenos Aires), 10. Clark AR, Dean JL, Saklatvala J. Post-transcriptional regulation Luis J. Catoggio, MD, Enrique R. Soriano, MD, Carlos D. Santos, MD of gene expression by mitogen-activated protein kinase p38. FEBS (Hospital Italiano de Buenos Aires y Fundacioń Dr. Pedro M. Lett 2003;546:37–44. Catoggio para el Progreso de la Reumatologıá, Buenos Aires), Cristina 11. Khabar KS. The AU-rich transcriptome: more than interferons Prigione, MD, Fernando A. Ramos, MD, Sandra M. Navarro, MD and cytokines, and its role in disease. J Interferon Cytokine Res (Hospital Provincial de Rosario, Rosario), Guillermo A. Berbotto, 2005;25:1–10. MD, Marisa Jorfen, MD, Elisa J. Romero, PhD (Hospital Escuela Eva 12. Jacob CO, Lee SK, Strassmann G. Mutational analysis of TNF-␣ Peroń, Granadero Baigorria, Rosario), Mercedes A. Garcia, MD, Juan Ј gene reveals a regulatory role for the 3 -untranslated region in the C. Marcos, MD, Ana I. Marcos, MD (Hospital Interzonal General de genetic predisposition to lupus-like autoimmune disease. J Immu- Agudos General San Martıń, La Plata), Carlos E. Perandones, MD, nol 1996;156:3043–50. Alicia Eimon, MD (Centro de Educacioń Me´dica e Investigaciones 13. Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias Clıńicas, Buenos Aires), and Cristina G. Battagliotti, MD (Hospital de G. Impaired on/off regulation of TNF biosynthesis in mice lacking Ninõs Dr. Orlando Alassia, Santa Fe). TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 1999;10:387–98. Members of the German Collaborative Group are as follows: 14. Randall RA, Germain S, Inman GJ, Bates PA, Hill CS. Different K. Armadi-Simab, MD, Wolfgang L. Gross, MD (University Hospital Smad partners bind a common hydrophobic pocket in Smad2 via of Schleswig-Holstein, Campus Luebeck, Rheumaklinik Bad Bram- defined proline-rich motif. EMBO J 2002;21:145–56. stedt, Luebeck), Erika Gromnica-Ihle, MD (Rheumaklinik Berlin- 15. Swingler TE, Bess KL, Yao J, Stifani S, Jayaraman PS. The Buch, Berlin), Hans-Hartmut Peter, MD (Medizinische Universi- proline-rich homeodomain protein recruits members of the Grou- taetsklinik, Abteilung Rheumatologie und Klinische Immunologie, cho/Transducin-like enhancer of split protein family to co-repress Freiburg), Karin Manger, MD (Medizinische Klinik III derFAU transcription in hematopoietic cells. J Biol Chem 2004;279: Erlangen-Nuernberg, Erlangen), Sebastian Schnarr, MD, Henning 34938–47. Zeidler, MD (Abteilung Rheumatologie, Medizinische Hochschule 16. Prado F, Vincent G, Cardalda C, Beato M. Differential role of the Hannover, Hannover), and Reinhold E. Schmidt, MD (Abteilung proline-rich domain of nuclear factor 1-C splice variants in DNA Klinische Immunologie, Medizinische Hochschule Hannover, Han- binding and transactivation. J Biol Chem 2002;277:16383–90. nover). Members of the Spanish Collaborative Group are as follows: APPENDIX A: THE ARGENTINE, GERMAN, AND Norberto Ortego, MD (Hospital Clıńico San Cecilio, Granada), En- SPANISH COLLABORATIVE GROUPS rique de Ramoń, MD (Hospital Carlos Haya, Malaga), Juan Jimeńez- Alonso, MD (Hospital Virgen de las Nieves, Granada), Julio Sańchez- Members of the Argentine Collaborative Group are as fol- Romań, MD (Hospital Virgen del Rocio, Sevilla), and Miguel Angel lows: Hugo R. Scherbarth, MD, Pilar C. Marino, MD, Estela L. Motta, Lo´pez-Nevot, MD (Hospital Virgen de las Nieves, Granada). ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1242–1250 DOI 10.1002/art.22451 © 2007, American College of Rheumatology

Interleukin-6 and Chemokines in the Neuropsychiatric Manifestations of Systemic Lupus Erythematosus

H. Fragoso-Loyo, Y. Richaud-Patin, A. Orozco-Narva´ez, L. Da´vila-Maldonado, Y. Atisha-Fregoso, L. Llorente, and J. Sa´nchez-Guerrero

Objective. To define the cytokine and chemo- groups. All cytokines and chemokines, except TNF␣, kine profile in cerebrospinal fluid (CSF) from patients were significantly higher among the SLE patients with with neuropsychiatric systemic lupus erythematosus septic meningitis than among the NPSLE patients. Six (NPSLE). months later and in the absence of NP manifestations, Methods. Forty-two SLE patients who had been all elevated molecule levels, except RANTES, in patients hospitalized because of NP manifestations were studied. with NPSLE had decreased significantly, and no differ- Patients were evaluated at hospitalization and 6 months ences were noted between the NPSLE and non-NPSLE later; a CSF sample was obtained at each evaluation. As groups. controls, CSF from 6 SLE patients with septic meningi- Conclusion. A central nervous system response tis, 16 SLE patients with no history of NP manifesta- composed of IL-6 and chemokines, but not Th1/Th2 tions (non-NPSLE), and 25 patients with nonauto- cytokines, is associated with NP manifestations in SLE immune diseases were also studied. Soluble molecules, patients. including cytokines (interleukin-2 [IL-2], IL-4, IL-6, ␣ ␣ IL-10, tumor necrosis factor [TNF ], and Systemic lupus erythematosus (SLE) is a chronic ␥ ␥ interferon- [IFN ]) and chemokines (monocyte che- inflammatory autoimmune disease of unknown etiology motactic protein 1 [MCP-1], RANTES, IL-8, monokine with heterogeneous clinical manifestations, including ␥ ␥ induced by IFN [MIG], and interferon- –inducible diverse neuropsychiatric (NP) manifestations (1). The 10-kd protein [IP-10]), were measured with the use of prevalence of NP manifestations in patients with SLE cytometric bead array kits. (NPSLE) ranges between 14% and 75%, depending on Results. CSF levels of the following molecules the ascertainment method used (2). A wide array of NP were significantly increased in NPSLE patients as com- manifestations has been described, which encompass pared with non-NPSLE and nonautoimmune diseases common features, such as headache and mood disor- control patients, respectively: IL-6 (32.7 versus 3.0 and ders, as well as rarer events, such as seizures and 2.96 pg/ml), IL-8 (102.8 versus 29.97 and 19.7 pg/ml), psychosis. However, the attribution of NP manifesta- IP-10 (888.2 versus 329.7 [P not significant] and 133.6 tions in these patients is complex because SLE and pg/ml), RANTES (3.8 versus 2.5 and 2.2 pg/ml), MCP-1 non-SLE factors often coexist (3). Several studies have (401.7 versus 257.9 [P not significant] and 136.9 pg/ml), reported that NP manifestations are the most common and MIG (35.4 versus 11.4 and 3.5 pg/ml). Low levels of cause of irreversible damage in SLE (4–7). IL-2, IL-4, IL-10, TNF␣, and IFN␥ were found in all Despite these facts, an accurate indicator of lupus involvement of the central nervous system (CNS) has H. Fragoso-Loyo, MD, Y. Richaud-Patin, BS, A. Orozco- Narvaéz, MD, L. Da´vila-Maldonado, MD, Y. Atisha-Fregoso, MD, L. not yet been identified. A variety of clinical, laboratory, Llorente, MD, J. Sańchez-Guerrero, MD, MS: Instituto Nacional de and radiographic findings have been reported to be Ciencias Me´dicas y Nutricioń Salvador Zubirań, Me´xico, DF, Me´xico. potentially useful. However, because of the multiple Address correspondence and reprint requests to J. Sańchez- Guerrero, MD, MS, Department of Immunology and Rheumatology, pathogenic mechanisms underlying the NP manifesta- Instituto Nacional de Ciencias Me´dicas and Nutricioń Salvador Zubi- tions in SLE (8), the search for an accurate diagnostic rań, Vasco de Quiroga 15, Tlalpan, Mexico, Federal District 14000, test is of utmost importance. Mexico. E-mail: [email protected]. Submitted for publication July 14, 2006; accepted in revised Some studies have shown that cytokines, such as form December 12, 2006. interleukin-6 (IL-6), and chemokines, such as IL-8, are

1242 IL-6 AND CHEMOKINES IN NP MANIFESTATIONS OF SLE 1243

useful for diagnosing NPSLE (9–13). A few other re- controls, we also studied CSF samples obtained from 6 SLE ports have described increased levels of IL-1, IL-10, patients with septic meningitis, 16 SLE patients without cur- tumor necrosis factor ␣ (TNF␣), and interferon-␥ rent or past NP manifestations (non-NPSLE group), and 25 ␥ patients with nonautoimmune diseases and without NP mani- (INF ) in the cerebrospinal fluid (CSF) of patients with festations (nonautoimmune diseases group) who, during the NPSLE (9,11,12). More recently, a role for monocyte study period, underwent elective surgery that required spinal chemotactic protein 1 (MCP-1)/CCL2, interferon-␥– block and gave their written permission for collection of a CSF inducible 10-kd protein (IP-10)/CXCL10, and fractal- sample. kine (CX CL1) has been implicated in NPSLE, suggest- CSF samples were centrifuged at 12,000g, and the 3 supernatant was immediately (Ͻ30 minutes in all instances) ing a chemokine-mediated inflammatory reaction in the collected and frozen at –86°C until assayed for cytokine and CNS of NPSLE patients (14–16). Overall, the direct and chemokine contents. indirect effects of several mediators of inflammation The study was approved by the Institutional Commit- have been emphasized as possible contributors in the tee of Biomedical Research. All patients gave their written pathogenesis of NPSLE. informed consent. Flow cytometric detection of cytokines and chemo- The aim of the present study was to measure the kines. Soluble molecules were measured with the use of levels of representative inflammatory molecules, includ- cytometric bead array kits (BD Biosciences, San Diego, CA) ing cytokines (IL-2, IL-4, IL-6, IL-10, TNF␣, and IFN␥) according to the manufacturer’s recommendations. These soluble molecules included Th1 and Th2 cytokines (IL-2, IL-4, and chemokines (MCP-1/CCL2, IL-8/CXCL8, IP-10/ ␣ ␥ ␥ IL-6, IL-10, TNF , and IFN ) and chemokines (CCL2/MCP-1, CXCL10, monokine induced by IFN (MIG)/CXCL9, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL10/ and RANTES/CCL5), in CSF samples from NPSLE IP-10). patients in order to identify the most reliable mole- Bead flow cytometry allows the simultaneous quantifi- cule(s) associated with CNS involvement. cation of various proteins in the same test. These assays use beads of the same size that can be distinguished by different fluorescence intensities. Each cluster of the same fluorescence PATIENTS AND METHODS intensity is coated with an antibody against the target molecule. The reaction is revealed by the corresponding secondary Study population. We studied 42 patients with SLE antibody conjugated with a different fluorochrome. Fifty mi- diagnosed according to the American College of Rheumatol- croliters of CSF per test was used. Samples were analyzed in a ogy (ACR) criteria (17) who had been hospitalized at our FACScan flow cytometer (Becton Dickinson, San Jose, CA) institution between February 1, 2003 and June 30, 2005 using the BD Cytometric Bead Array software (BD Bio- because of NP manifestations and from whom a CSF sample sciences). Results are expressed as picograms per milliliter. had been obtained. All NPSLE patients were evaluated ac- Statistical analysis. Categorical variables were com- cording to a standardized protocol by the participating rheu- pared using chi-square or Fisher’s exact test. Continuous matologists and neurologists at the time of hospitalization and variables were analyzed using Student’s t-test, Mann-Whitney 6 months later. At the time of hospitalization, information U test, Wilcoxon’s signed rank test, paired t-test, or one-way about sociodemographic features, SLE characteristics (i.e., age analysis of variance. Cytokine and chemokine values are at diagnosis [defined as the date the fourth lupus criterion was presented as the median and interquartile range. P values less met], disease duration at the index hospitalization, number of than 0.05 (2-tailed) were considered significant. Analysis was SLE criteria accumulated, autoantibody profile, etc.), and performed using the SPSS 12.0 computer program (SPSS, treatment was gathered using a standardized format. Disease Chicago, IL). activity was assessed at baseline and 6 months later using the Systemic Lupus Erythematosus Disease Activity Index 2000 update (SLEDAI-2K) (18). The medical records of all patients RESULTS were reviewed to collect additional information, including the SLE course and chronic damage accrual according to the Characteristics of the study population. The Systemic Lupus International Collaborating Clinics/ACR mean Ϯ SD age of the study patients was 30.5 Ϯ 11.5 Damage Index (19). years in the NPSLE group, 37.8 Ϯ 9.8 years in the Neuropsychiatric manifestations were classified ac- non-NPSLE group, 29.0 Ϯ 10.9 years in the SLE with cording to the ACR nomenclature and case definitions for Ϯ neuropsychiatric lupus syndromes (20). Manifestations were septic meningitis group, and 38.6 17.0 years in the attributed to SLE based on the absence of exclusion factors for nonautoimmune diseases group. Disease characteristics the attribution of the NP manifestations (20), and none of the of the SLE patient groups are shown in Table 1. patients had minor NP events that have previously been The NP manifestations observed were seizure reported to occur at a comparable frequency in the normal disorders in 14 patients, severe refractory headache in 8, population (21). A CSF sample was obtained from all patients upon acute confusional state in 8, cerebrovascular disease in 4, their admission to the hospital. A second CSF sample was mononeuritis multiplex in 3, psychosis in 2, transverse obtained from 30 of the 42 NPSLE patients 6 months later. As myelitis in 1, polyneuropathy in 1, and pseudotumor 1244 FRAGOSO-LOYO ET AL

Table 1. Demographic and clinical characteristics of the SLE study patients at the time of hospitalization* SLE patients with NPSLE patients Non-NPSLE patients septic meningitis (n ϭ 42) (n ϭ 16) (n ϭ 6) P Age, years 30.5 Ϯ 11.5 37.8 Ϯ 9.8 29 Ϯ 10.9 0.07 No. male/female 6/36 2/14 0/6 – SLE duration, years 3.8 Ϯ 4.4 8.8 Ϯ 6.5 3.8 Ϯ 3.7 0.009 No. of SLE criteria met 6.0 Ϯ 1.9 6.3 Ϯ 1.9 5.5 Ϯ 1.4 0.69 SLEDAI-2K score 14.1 Ϯ 8.7 7.0 Ϯ 5.9 11.5 Ϯ 7.0 0.013 SLICC/ACR Damage Index 0.7 Ϯ 1.1 1.0 Ϯ 1.2 0.3 Ϯ 0.5 0.40 % taking prednisone 82 81 60 0.51 Prednisone dosage, mg/day 16.7 Ϯ 14.2 16.6 Ϯ 18.8 16.5 Ϯ 25.0 0.99 % taking immunosuppressants 59 38 40 0.33 * Except where indicated otherwise, values are the mean Ϯ SD. SLE ϭ systemic lupus erythematosus; NPSLE ϭ neuropsychiatric SLE; SLEDAI-2K ϭ Systemic Lupus Erythematosus Disease Activity Index 2000 update; SLICC/ACR ϭ Systemic Lupus International Collaborating Clinics/American College of Rheumatology. cerebri in 1 patient. SLE was considered to be the likely CXCL9 showing the greatest differences (P ϭ 0.0001), cause of these NP events; in 22 patients (52%), no followed by RANTES/CCL5 (P ϭ 0.0004) and MCP-1/ associated factors for the NP manifestations were CCL2 (P ϭ 0.0014) (Table 3). identified, and in 20 patients (48%), concurrent, nonexclusion factors (i.e., severe infections, metabolic Table 2. Cytokine levels in cerebrospinal fluid from NPSLE patients abnormalities, thrombotic thrombocytopenic purpura, and controls* antiphospholipid syndrome, high doses of steroids, Cytokine, patient group Median (IQR) pg/ml valvular heart disease, and arterial hypertension) were Interleukin-2 identified (20). NPSLE 0 (0–2.2) Nonautoimmune diseases patients underwent Non-NPSLE 0 (0–1.7) SLE with septic meningitis 2.7 (1.8–71)† elective surgery because of the following conditions: Nonautoimmune diseases 1.3 (0–2.3) bone marrow donation in 7 patients, hysterectomy in 6, Interleukin-4 NPSLE 0 (0–8) Tenckhoff catheter placement in 3, hydrocele in 2, lower Non-NPSLE 0 (0–2.3) limb amputation due to type 2 diabetes mellitus in 2, SLE with septic meningitis 34 (7–251)† saphenectomy in 2, circumcision in 1, umbilical hernia in Nonautoimmune diseases 1.4 (0–3.7) Interleukin-6 1, and inguinal hernioplasty in 1 patient. NPSLE 32 (3.8–129) The microorganisms responsible for septic men- Non-NPSLE 3 (1.3–5.7)† ingitis in the 6 SLE patients were as follows: Streptococ- SLE with septic meningitis 453 (61–8,468) Nonautoimmune diseases 2.9 (2–3.9)‡ cus pneumoniae in 2 patients, Listeria monocytogenes in Interleukin-10 1, Cryptococcus neoformans in 1, Mycobacterium tuber- NPSLE 1.5 (0–6.3) culosis in 1, and Staphylococcus species in 1 patient. Non-NPSLE 1.4 (0–2.1) SLE with septic meningitis 9.2 (4.6–30.8)§ Chemokine and cytokine levels. NPSLE patients Nonautoimmune diseases 1.5 (0–2.2) versus patients with nonautoimmune diseases. Since there Interferon-␥ are no CSF reference values for the molecules studied, NPSLE 0 (0–7.7) Non-NPSLE 0 (0–2.1) we first compared the NPSLE patient group with the SLE with septic meningitis 12 (3–1,044)§ group of patients with nonautoimmune diseases. Of all Nonautoimmune diseases 1.6 (0–2.5) ␣ cytokines measured, only IL-6 showed a significant Tumor necrosis factor NPSLE 0 (0–1.3) difference, being higher in the NPSLE patients (P ϭ Non-NPSLE 0 (0–0) 0.0001). The levels of IL-2, IL-4, IL-10, TNF␣, and IFN␥ SLE with septic meningitis 1.1 (0–56) were either low or undetectable in most patients (Table 2). Nonautoimmune diseases 0 (0–1.7) In stark contrast with the above, all chemokines * NPSLE ϭ neuropsychiatric systemic lupus erythematosus; IQR ϭ we analyzed were significantly higher in the NPSLE interquartile range. † P Ͻ 0.01 versus NPSLE patients. group than in the nonautoimmune diseases control ‡ P Ͻ 0.0001 versus NPSLE patients. group, with IP-10/CXCL10, IL-8/CXCL8, and MIG/ § P Ͻ 0.05 versus NPSLE patients. IL-6 AND CHEMOKINES IN NP MANIFESTATIONS OF SLE 1245

Table 3. Chemokine levels in cerebrospinal fluid from NPSLE pa- (P ϭ 0.058), MIG/CXCL9 (P ϭ 0.03), RANTES/CCL5 tients and controls* (P ϭ 0.015), IL-8 (P ϭ 0.0158) (Tables 2 and 3). Chemokine, Median (IQR) Figure 1 shows the data for the most representa- patient group pg/ml tive chemokines and IL-6 in individual patients in all IL-8/CXCL8 subgroups. NPSLE 102 (35–272) Cytokine and chemokine levels at the time of Non-NPSLE 29 (21–48)† SLE with septic meningitis 516 (155–955)‡ hospitalization and 6 months later in NPSLE patients. Nonautoimmune diseases 19 (13–24)§ Six months after hospitalization, a second CSF sample MIG/CXCL9 was obtained from 30 of the 42 NPSLE patients. The NP NPSLE 35 (9–513) Non-NPSLE 11 (5–36)‡ manifestations observed in these 30 patients were sei- SLE with septic meningitis 1,073 (61–2,223)‡ zure disorders in 11, severe refractory headache in 5, Nonautoimmune diseases 3 (2–6)§ acute confusional state in 5, cerebrovascular disease in 2, IP-10/CXCL10 NPSLE 888 (276–4,407) mononeuritis multiplex in 3, psychosis in 1, transverse Non-NPSLE 329 (190–583) myelitis in 1, polyneuropathy in 1, and pseudotumor SLE with septic meningitis 5,107 (4,520–6,236)† cerebri in 1. All but 2 patients received prednisone (or Nonautoimmune diseases 133 (84–164)§ MCP-1/CCL2 its equivalent) at a dosage of 0.5 mg/kg or higher, NPSLE 401 (123–1,263) including 7 patients who received intravenous pulses of Non-NPSLE 257 (165–391) methylprednisolone; 5 patients also received intrave- SLE with septic meningitis 2,803 (401–4,713) Nonautoimmune diseases 136 (88–177)† nous pulses of cyclophosphamide. At the time of the RANTES/CCL5 6-month followup visit, NP manifestations had resolved NPSLE 3.8 (2.9–8.2) in all patients (mean Ϯ SD SLEDAI-2K score 5.1 Ϯ 6.4). Non-NPSLE 2.4 (2–3.3)¶ SLE with septic meningitis 9.4 (5.5–15.8)‡ As at the time of hospitalization, CSF levels of Nonautoimmune diseases 2.1 (1.8–4.1)¶ IL-2, IL-4, IL-10, TNF␣, and IFN␥ at the 6-month assessment were either low or undetectable, and there * NPSLE ϭ neuropsychiatric systemic lupus erythematosus; IQR ϭ interquartile range; IL-8 ϭ interleukin-8; MIG ϭ monokine induced was no significant difference between the values ob- by interferon-␥; IP-10 ϭ interferon-␥–inducible 10-kd protein; MCP- tained at the 2 analyses (data not shown). A clear-cut ϭ 1 monocyte chemotactic protein 1. decrease in nearly all of the previously elevated levels of † P Ͻ 0.01 versus NPSLE patients. ‡ P Ͻ 0.05 versus NPSLE patients. the study molecules was observed (Table 4). An impor- § P Ͻ 0.0001 versus NPSLE patients. tant result that should be emphasized is that in the Ͻ ¶ P 0.001 versus NPSLE patients. second CSF sample, the levels of IL-6 and chemokines in the NPSLE patients were similar to those in the non- NPSLE patients. However, the levels in the NPSLE NPSLE patients versus non-NPSLE patients. To patients at 6 months were still not as low as those in the establish whether these values were attributable to the nonautoimmune diseases patients at baseline, at least in SLE or specifically to the neurologic activity of SLE, we terms of the values for IL-8 (P Ͻ 0.001), MIG (P Ͻ compared the NPSLE patients with the non-NPSLE 0.0001), IP-10 (P Ͻ 0.0001), and MCP-1 (P Ͻ 0.0001). patients. This comparison showed differences in IL-6 Figure 2 shows the individual data for the mole- (P ϭ 0.0016), IL-8/CXCL8 (P ϭ 0.0016), MIG/CXCL9 cules whose levels decreased significantly between base- (P ϭ 0.04), and RANTES/CCL5 (P ϭ 0.0006) levels. line and 6 months. There was also a tendency toward statistical significance for the differences in IP-10/CXCL10 (P ϭ 0.064) and DISCUSSION IFN␥ (P ϭ 0.057) levels (Tables 2 and 3). NPSLE patients versus SLE patients with septic The mechanisms responsible for the brain dam- meningitis. Finally, we compared the NPSLE group with age that occurs during NPSLE have not been clarified. the SLE with septic meningitis group as a possible Our results show that despite the diversity and hetero- positive control for a clear inflammatory process. Indeed geneous nature of the NP manifestations of SLE, the all cytokine and chemokine levels, except TNF␣, were levels of several molecules are increased in the CSF of higher among the SLE patients with septic meningitis most patients during the outbreak of NP activity. Thus, than among the NPSLE patients: IL-2 (P ϭ 0.01), IL-4 high levels of IL-6 and the chemokines IL-8/CXCL8, (P ϭ 0.002), IL-6 (P ϭ 0.099), IL-10 (P ϭ 0.04), IFN␥ MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, and (P ϭ 0.018), IP-10/CXCL10 (P ϭ 0.005), MCP-1/CCL2 RANTES/CCL5 were consistently found in NPSLE pa- 1246 FRAGOSO-LOYO ET AL

Figure 1. Levels of interleukin-6 (IL-6) and chemokines in cerebrospinal fluid from patients with neuropsychiatric systemic lupus erythematosus (NPSLE), non-NPSLE, SLE with septic meningitis (SM), and nonautoimmune diseases (non-AI). Each data point represents an individual patient. Horizontal lines show the mean. MIG ϭ monokine induced by interferon-␥; IP-10 ϭ interferon-␥–inducible 10-kd protein; MCP-1 ϭ monocyte chemotactic protein 1. IL-6 AND CHEMOKINES IN NP MANIFESTATIONS OF SLE 1247

Table 4. Cytokine and chemokine levels in cerebrospinal fluid from function in the damaged CNS that is distinct from its 30 NPSLE patients, as determined at baseline and 6 months* role in proinflammatory events (27). Cytokine or Baseline, 6 months, These observations, together with our results, chemokine median (IQR) pg/ml median (IQR) pg/ml which did not show any increase in cytokines (with the IL-6 17 (3.8–121) 3.1 (2.2–4.4)† exception of IL-6), makes it feasible to speculate that IL-8/CXCL8 106.8 (33.7–272) 27 (20.5–38.8)‡ increased chemokine levels, acting either in an autocrine MIG/CXCL9 48 (9.4–568) 11.5 (7.1–27)† or a paracrine manner, can be involved in the CNS IP-10/CXCL10 888 (313–4,673) 407 (313–903)§ MCP-1/CCL2 401 (184–1,655) 298 (214–397)¶ damage in NPSLE, whether by activation and signaling RANTES 4 (3.2–8.2) 3.8 (3–7.2) systems or even by other functions of which we are not * The baseline evaluation was at the time of hospitalization. NPSLE ϭ yet aware. In this regard, recent studies have indicated neuropsychiatric systemic lupus erythematosus; IQR ϭ interquartile that in addition to chemotactic activity for leukocytes, range; IL-6 ϭ interleukin-6; MIG ϭ monokine induced by interfer- MCP-1/CCL2 also plays a role in tumor metastasis and on-␥; IP-10 ϭ interferon-␥–inducible 10-kd protein; MCP-1 ϭ monocyte chemotactic protein 1. angiogenesis, in the development of CNS, immune, and † P ϭ 0.0001 versus baseline. vascular systems, as well as in the modulation of cell ‡ P ϭ 0.001 versus baseline. proliferation, apoptosis, and protein synthesis (28–32). § P ϭ 0.002 versus baseline. ¶ P ϭ 0.044 versus baseline. Even though MCP-1 has been the best-characterized chemokine so far, it is not unreasonable to suppose that the other chemokines evaluated will also show functions tients. The presence of IL-6 and IL-8/CXCL8 in the CSF other than inflammatory or cell recruitment, and this of patients with NPSLE is one of their commonly might suggest that inflammation is not an obligatory described traits (9–13). More recently, MCP-1/CCL2 component of the events that initiate or follow NP and IP-10/CXCL10 have been reported in SLE with manifestations in SLE patients. CNS involvement (14,15). To our knowledge, MIG/ In a high percentage of NPSLE patients, it was CXCL9 and RANTES/CCL5 have not previously been possible to obtain a second CSF sample after the NP described in the CSF of patients with NPSLE. With manifestations had resolved. Thus, we were able to show regard to the functions of these molecules, it is worth that the levels of practically all of the molecules that had noting that even though IP-10/CXCL10 and MIG/ originally shown high concentrations had decreased sig- CXCL9 are predominantly chemoattractive for Th1 nificantly, although not as low as the levels found in the cells, MCP-1/CCL2 for Th2 cells, and RANTES/CCL5 patients with nonautoimmune diseases. Rather, they for both cell subpopulations (22), we found almost no reached a range of values that could be defined as the production of the characteristic cytokines by any of these basal level in SLE, since they were similar to the levels cells in the NPSLE patients. Th1 and Th2 cytokines were found in the non-NPSLE patients. This information detected only in the patients with SLE with septic could prove to be of importance, because SLE patients meningitis, where there is a clear inflammatory setting show neurologic damage over the long-term, which and disruption of the blood–brain barrier. manifests itself as a decrease in brain volume on mag- That only IL-6 and chemokines are expressed netic resonance imaging (MRI) studies and as a wors- during the clinical activity of NPSLE clearly reflects a ening of cognitive function clinically (7,33). Even though quite different scenario from that found in the periph- this worsening is greater in patients with a history of eral blood, where, besides chemokines, cytokines are NPSLE, it is also found in patients without previous NP conspicuous (23,24). This difference suggests that the manifestations (34). There is no recognized etiology of production of these molecules occurs in situ and that the these alterations; however, it is possible that this slight, damage seen in NPSLE does not require the involve- but persistent, increase in chemokine levels in the CSF ment of factors derived from blood. In fact, evidence has of SLE patients elicits this damage by maintaining a been obtained suggesting that neurons, microglia, and chronic low-level response. astroglia can all serve as both targets and sources of In this regard, previous reports have indicated chemokines (25,26), which seems to represent an innate that in patients with subtle cognitive impairment, there immune response. Moreover, previous reports suggest is an increase in IP-10/CXCL10, MCP-1/CCL2, and that enhanced expression of proinflammatory cytokines IL-8/CXCL8 levels (35). Even though current knowl- such as TNF␣, IL-1␤, and IL-6 is not required for edge does not enable us to establish a cause-and-effect neuronal and glial responses to injury and that chemo- relationship between the increase in chemokine levels kines, particularly MCP-1/CCL2, may have another and the occurrence of neurologic damage, the link 1248 FRAGOSO-LOYO ET AL

Figure 2. Changes in levels of interleukin-6 (IL-6) and chemokines in cerebrospinal fluid (CSF) from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) between hospitalization (baseline) and 6 months. Paired samples of CSF were obtained from 30 NPSLE patients during active disease and after 6 months, when there was no evidence of NP manifestations. Each line represents an individual patient. MIG ϭ monokine induced by interferon-␥; MCP-1 ϭ monocyte chemotactic protein 1; IP-10 ϭ interferon-␥–inducible 10-kd protein. IL-6 AND CHEMOKINES IN NP MANIFESTATIONS OF SLE 1249

between them is amply suggestive that this indeed could non-SLE factors, and their contribution to the develop- be the case. Consistent with this possibility is a recent ment of the hospitalization event is uncertain. Since this study showing that the presence of intrathecal IL-6 and is a complex issue, some misclassification in terms of the IL-8 led to the synthesis of matrix metalloproteinase 9, attribution could be present. Our results apply to pa- which potentially culminated in an insult to the brain tients with NPSLE manifestations who needed to be parenchyma, resulting in the release of neuronal and hospitalized for diagnosis and/or treatment. However, astrocytic degradation products that terminated in MRI- they would not apply to nonhospitalized patients or to verifiable lesions and clinical states of brain deficiency (13). chronic serious manifestations (e.g., seizures). Because Our results confirm previous hypotheses about this study was conducted in a single center with limited the intrathecal presence of cytokines and chemokines in ethnic variation among the patients, one must be cir- the CSF of patients with NPSLE. However, several cumspect about extrapolating the results to all patients issues remain unexplained that deserve consideration. with SLE. First, what triggers the response observed in NPSLE? Much remains to be learned about the pathoge- Second, why is this response not self-perpetuated like netic mechanisms that lead to NP manifestations in SLE the ones found elsewhere (e.g., the kidney)? Third, why patients. The absence of experimental models and the do non-NPSLE patients with relatively high CSF levels difficult access to the CNS are barriers that will have to of chemokines not show overt clinical manifestations of be cleared through other means. The quantification of CNS? Fourth, does the presence of chemokines in the IL-6 and chemokines in CSF however, seems to be an CSF of non-NPSLE patients represent an inflammatory accurate indicator of neurologic involvement in SLE response or an attempt to restore homeostasis against a that could be useful in the followup and evaluation of persistent stimulus? Certainly, many more questions treatment response in these patients. It is important to could be posed. However, what seems to be conclusive is do our utmost to understand and possibly prevent these that overproduction of IL-6 and chemokines in the CSF clinical manifestations of SLE, which undoubtedly, are plays a key role in the pathogenesis and emergence of still one of the major causes of morbidity and irrevers- NPSLE, although strictly speaking, this is not sufficient ible damage. to explain it. Our study has several limitations. Although a AUTHOR CONTRIBUTIONS relatively large number of patients with NP manifesta- Dr. Sańchez-Guerrero had full access to all of the data in the tions were included, we studied the cytokine and che- study and takes responsibility for the integrity of the data and the mokine profile for NP manifestations in general but accuracy of the data analysis. were unable to define the profile for specific NP mani- Study design. Fragoso-Loyo, Orozco-Narvaéz, Da´vila-Maldonado, Llorente, Sańchez-Guerrero. festations, since the study was not adequately powered Acquisition of data. Fragoso-Loyo, Richaud-Patin, Orozco-Narvaéz, for such an analysis. Given that we did not have a control Da´vila-Maldonado, Atisha-Fregoso, Llorente, Sańchez-Guerrero. group of patients with nonautoimmune diseases and Analysis and interpretation of data. Fragoso-Loyo, Richaud-Patin, Orozco-Narvaéz, Da´vila-Maldonado, Atisha-Fregoso, Llorente, similar NP manifestations, we could not determine Sańchez-Guerrero. whether the abnormal levels of IL-6 and chemokines Manuscript preparation. Fragoso-Loyo, Richaud-Patin, Da´vila- found were specific for NPSLE patients or were reflec- Maldonado, Atisha-Fregoso, Llorente, Sańchez-Guerrero. Statistical analysis. Fragoso-Loyo, Atisha-Fregoso, Sańchez- tive of the NP event itself, regardless of the etiology. We Guerrero. tried to include only patients with NP manifestations that were attributable to SLE, rejecting patients with exclusion factors for the attribution of the NP manifes- REFERENCES tations (20) as well as patients with minor NP events that 1. Ruiz-Irastorza G, Khamashta MA, Castellino G, Hughes GR. have a comparable frequency in the normal population Systemic lupus erythematosus. Lancet 2001;357:1027–32. 2. Brey RL, Holliday SL, Saklad AR, Navarrete MG, Hermosillo- (21). In the latter study, all patients with headaches, both Romo D, Stallworth CL, et al. Neuropsychiatric syndromes in tension-type and migraine, were excluded, but no pa- lupus: prevalence using standardized definitions. Neurology 2002; tient with severe refractory headache was seen. Our 58:1214–20. 3. Hanly JG, McCurdy G, Fougere L, Douglas JA, Thompson K. study included patients with severe refractory headache, Neuropsychiatric events in systemic lupus erythematosus: attribu- as defined in the SLEDAI-2K, because it is considered a tion and clinical significance. J Rheumatol 2004;31:2156–62. clinical manifestation of lupus activity and because this 4. Rivest C, Lew RA, Welsing PM, Sangha O, Wright EA, Partridge AL, et al. Association between clinical factors, socioeconomic was the reason for their hospitalization. status, and organ damage in recent onset systemic lupus erythem- In 48% of our patients, there were concurrent atosus. J Rheumatol 2000;27:680–4. 1250 FRAGOSO-LOYO ET AL

5. Alarcon GS, McGwin G Jr, Bartolucci AA, Roseman J, Lisse J, Rheumatology Damage Index for systemic lupus erythematosus. Fessler BJ, et al, for the LUMINA Study Group. Systemic lupus Arthritis Rheum 1996;39:363–9. erythematosus in three ethnic groups. IX. Differences in damage 20. ACR Ad Hoc Committee on Neuropsychiatric Lupus Nomencla- accrual. Arthritis Rheum 2001;44:2797–806. ture. The American College of Rheumatology nomenclature and 6. Jonsen A, Bengtsson AA, Nived O, Ryberg B, Sturfelt G. Out- case definitions for neuropsychiatric lupus syndromes. Arthritis come of neuropsychiatric systemic lupus erythematosus within a Rheum 1999;42:599–608. defined Swedish population: increased morbidity but low mortal- 21. Ainiala H, Hietaharju A, Loukkola J, Peltola J, Korpela M, ity. Rheumatology (Oxford) 2002;41:1308–12. Metsanoja R, et al. Validity of the new American College of 7. Mikdashi J, Handwerger B. Predictors of neuropsychiatric damage Rheumatology criteria for neuropsychiatric lupus syndromes: a in systemic lupus erythematosus: data from the Maryland lupus population-based evaluation. Arthritis Rheum 2001;45:419–23. cohort. Rheumatology (Oxford) 2004;43:1555–60. 22. Zlotnik A, Yoshie O. Chemokines: a new classification system and 8. Jennekens FG, Kater L. The central nervous system in systemic their role in immunity. Immunity 2000;12:121–7. lupus erythematosus. Part 2. Pathogenetic mechanisms of clinical 23. Mok CC, Lau CS. Pathogenesis of systemic lupus erythematosus. syndromes: a literature investigation [review]. Rheumatology (Ox- J Clin Pathol 2003;56:481–90. ford) 2002:41:619–30. 24. Lit LC, Wong CK, Tam LS, Li EK, Lam CW. Raised plasma 9. Alcocer-Varela J, Aleman-Hoey D, Alarcon-Segovia D. Interleu- concentration and ex vivo production of inflammatory chemokines kin-1 and interleukin-6 activities are increased in the cerebrospinal in patients with systemic lupus erythematosus. Ann Rheum Dis 2006;65:209–15. fluid of patients with CNS lupus erythematosus and correlate with 25. Giulian D, Vaca K, Corpuz M. Brain glia release factors with local late T-cell activation markers. Lupus 1992;1:111–7. opposing actions upon neuronal survival. J Neurosci 1993;13: 10. Hirohata S, Tanimoto K, Ito K. Elevation of cerebrospinal fluid 29–37. interleukin-6 activity in patients with vasculitides and central 26. Giulian D, Li J, Leara B, Keenen C. Phagocytic microglia release nervous system involvement. Clin Immunol Immunopathol 1993; cytokines and cytotoxins that regulate the survival of astrocytes 66:225–9. and neurons in culture. Neurochem Int 1994;25:227–33. 11. Trysberg E, Carlsten H, Tarkowski A. Intrathecal cytokines in 27. Little AR, Benkovic SA, Miller DB, O’Callaghan JP. Chemically systemic lupus erythematosus with central nervous system involve- induced neuronal damage and gliosis: enhanced expression of the ment. Lupus 2000;9:498–503. proinflammatory chemokine, monocyte chemoattractant protein 12. Svenungsson E, Andersson M, Brundin L, van Vollenhoven R, (MCP)-1, without a corresponding increase in proinflammatory Khademi M, Tarkowski A, et al. Increased levels of proinflamma- cytokines. Neuroscience 2002;115:307–20. tory cytokines and nitric oxide metabolites in neuropsychiatric 28. Liss C, Fekete MJ, Hasina R, Lam CD, Lingen MW. Paracrine lupus erythematosus. Ann Rheum Dis 2001;60:372–9. angiogenic loop between head-and-neck squamous-cell carcino- 13. Trysberg E, Blennow K, Zachrisson O, Tarkowski A. Intrathecal mas and macrophages. Int J Cancer 2001;93:781–5. levels of matrix metalloproteinases in systemic lupus erythemato- 29. Rezaie P, Trillo-Pazos G, Everall IP, Male DK. Expression of sus with central nervous system engagement. Arthritis Res Ther ␤-chemokines and chemokine receptors in human fetal astrocyte 2004;6:R551–6. and microglial co-cultures: potential role of chemokines in the 14. Ikuni N, Okamoto H, Yoshio T, Sato E, Kamitsuji S, Iwamoto T, developing CNS. Glia 2002;37:64–75. et al. Raised monocyte chemotactic protein-1 (MCP-1)/CCL2 in 30. Sasayama S, Okada M, Matsumori A. Chemokines and cardiovas- cerebrospinal fluid of patients with neuropsychiatric lupus. Ann cular disease. Cardiovasc Res 2000;45:267–9. Rheum Dis 2006;65:253–6. 31. Luther SA, Cyster JG. Chemokines as regulators of T cell differ- 15. Okamoto H, Katsumata Y, Nishimura K, Kamatani N. Interferon- entiation. Nat Immunol 2001;2:102–7. inducible protein 10/CXCL10 is increased in the cerebrospinal 32. Salcedo R, Ponce MI, Young HA, Wasserman K, Ward JM, fluid of patients with central nervous system lupus. Arthritis Kleinman HK, et al. Human endothelial cells express CCR2 and Rheum 2004;50:3731–2. respond to MCP-1: direct role of MCP-1 in angiogenesis and 16. Yajima N, Kasama T, Isozaki T, Odai T, Matsunawa M, Negishi tumor progression. Blood 2000;96:34–40. M, et al. Elevated levels of soluble fractalkine in active systemic 33. Appenzeller S, Rondina JM, Li LM, Costallat LT, Cendes F. lupus erythematosus: potential involvement in neuropsychiatric Cerebral and corpus callosum atrophy in systemic lupus erythem- manifestations. Arthritis Rheum 2005;52:1670–5. atosus. Arthritis Rheum 2005;52:2783–9. 17. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield 34. Kozora E, Arciniegas DB, Filley CM, Ellison MC, West SG, NF, et al. The 1982 revised criteria for the classification of systemic Brown MS, et al. Cognition, MRS neurometabolites, and MRI lupus erythematosus. Arthritis Rheum 1982;25:1271–7. volumetrics in non-neuropsychiatric systemic lupus erythemato- 18. Gladman DD, Ibanez D, Urowitz MB. Systemic Lupus Erythem- sus: preliminary data. Cogn Behav Neurol 2005;18:159–62. atosus Disease Activity Index 2000. J Rheumatol 2002;29:288–91. 35. Galimberti D, Schoonenboom N, Scheltens P, Fenoglio C, Bouw- 19. Gladman D, Ginzler E, Goldsmith C, Fortin P, Liang M, Urowitz man F, Venturelli E, et al. Intrathecal chemokine synthesis in mild M, et al. The development and initial validation of the Systemic cognitive impairment and Alzheimer disease. Arch Neurol 2006; Lupus International Collaborating Clinics/American College of 63:538–43. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1251–1262 DOI 10.1002/art.22510 © 2007, American College of Rheumatology

Reproductive and Menopausal Factors and Risk of Systemic Lupus Erythematosus in Women

Karen H. Costenbader,1 Diane Feskanich,2 Meir J. Stampfer,3 and Elizabeth W. Karlson1

Objective. Systemic lupus erythematosus (SLE) ciated with an increased risk of SLE among women in occurs predominantly in women, and hormones may the younger (NHSII) cohort. Age at first birth, parity, play a role in its etiology. This study was carried out to and total duration of breastfeeding were not associated examine associations between female reproductive and with SLE. menopausal factors and the development of SLE. Conclusion. Early age at menarche, oral contra- Methods. A cohort of 238,308 women was prospec- ceptive use, early age at menopause, surgical meno- tively examined. Subjects were older women (ages 30–55 pause, and postmenopausal use of hormones were each years at start) and younger women (ages 25–42 years at associated with an increased risk of SLE. These associ- start) from the Nurses’ Health Study (NHS) and NHSII ations may point to the mechanisms underlying the cohorts. Incident SLE diagnosed between 1976 and 2003 pathogenesis of SLE. was confirmed by medical record review. The relative risk (RR) of SLE was estimated separately in each Systemic lupus erythematosus (SLE) is an inflam- cohort using Cox proportional hazards models, and matory rheumatic disease of immunologic origin, which then pooled using meta-analysis random effects models. is characterized by autoantibody production and protean Results. Two hundred sixty-two incident cases of clinical manifestations. The etiology of SLE is unknown; SLE were confirmed among the women. In multivariable similar to many autoimmune diseases, it is thought that models adjusted for reproductive and other risk factors, environmental factors trigger the disease in genetically < age 10 years at menarche (pooled RR 2.1, 95% confi- predisposed individuals. Given that 80–90% of SLE dence interval [95% CI] 1.4–3.2), oral contraceptive use occurs in women, reproductive factors are potentially (pooled RR 1.5, 95% CI 1.1–2.1), and use of postmeno- important in its pathogenesis, but a clear picture of their pausal hormones (RR 1.9, 95% CI 1.2–3.1) significantly role has not emerged. increased the risk of SLE. An elevation of SLE risk was Epidemiologic relationships between reproduc- observed among postmenopausal women primarily after tive factors and SLE have been mainly examined in surgical menopause (RR 2.3, 95% CI 1.2–4.5), and also case–control studies, and the results have been ambigu- among women with earlier age at natural menopause ous and conflicting. Both early and late menarche occa- (P for trend < 0.05). Menstrual irregularity was asso- sionally, but not consistently, have been associated with Supported by the NIH (grants CA-87969, R01-AR-49880, increased risk of SLE (1–4). Oral contraceptive use was K12-HD-051959, and K24-AR-0524-01). Dr. Costenbader is recipient found to be unrelated to SLE in 2 case–control studies of an Arthritis Foundation/American College of Rheumatology Ar- (1,4), whereas another study showed protective effects thritis Investigator Award and a Katherine Swan Ginsburg Memorial Award. from the progesterone-only pill (5). Menstrual irregular- 1Karen H. Costenbader, MD, MPH, Elizabeth W. Karlson, ity was associated with increased risk of SLE in a MD: Robert B. Brigham Arthritis and Musculoskeletal Diseases Japanese case–control study (2). Both short and long Clinical Research Center, and Brigham and Women’s Hospital, Bos- ton, Massachusetts; 2Diane Feskanich, ScD: Brigham and Women’s menstrual cycles were associated with increased risk of Hospital, Boston, Massachusetts; 3Meir J. Stampfer, MD, DrPH: SLE in the Carolina Lupus Study, a population-based Brigham and Women’s Hospital, and Harvard School of Public Health, Boston, Massachusetts. case–control study (4). Although some findings suggest Address correspondence and reprint requests to Karen H. that women with SLE experience increased disease Costenbader, MD, MPH, Brigham and Women’s Hospital, 75 Francis activity during pregnancy (6–8), parity has not been Street, Boston, MA 02115. E-mail: [email protected]. Submitted for publication June 12, 2006; accepted in revised linked to increased risk of SLE (1,4,9), and age at first form January 4, 2007. birth has not been investigated. Earlier age at meno-

1251 1252 COSTENBADER ET AL

pause was found among women with SLE in the Caro- received a positive response, and as “possible” if 3 of the lina Lupus Study, but postmenopausal hormone use was questions were given a positive response; in the latter case we not associated with risk of SLE in that study (4). mailed screening questionnaires to the participant in subsequent years. The only prospective cohort study to investigate In total, after 5 mailings, we received a response from these relationships has been the Nurses’ Health Study 80% of the women with a self-reported diagnosis of SLE or any (NHS) (10,11), in which a tendency toward increased other connective tissue diseases (10,331 in the NHS and 4,276 susceptibility to SLE was observed among users of oral in the NHSII; total of 14,607 women). After excluding subjects who denied having SLE (n ϭ 2,777), who reported having SLE contraceptives (11) and those taking postmenopausal diagnosed before the start of their participation in the cohort hormones (10). Our aim in the present study was to (n ϭ 1,669), who denied permission for a review of their reexamine these associations among a larger number of medical records (n ϭ 1,356), or who provided negative re- incident SLE cases during 10 additional years of fol- sponses on the connective tissue diseases screening question- ϭ lowup, and to extend the investigations to include a naire regarding SLE symptoms (n 3,958), we requested medical records from 4,847 women, resulting in a total of 3,563 range of reproductive and menopausal factors among records (74%) with adequate information. the women in both the NHS and NHSII cohorts, com- Two board-certified rheumatologists (KHC and EWK) prising the largest cohorts of women who have been who are trained in chart abstraction independently conducted followed up prospectively for rheumatic disease. a medical record review in which they examined the charts for the ACR diagnostic criteria for SLE (13,14). We used 2 definitions of confirmed SLE, requiring the presence of 1) at SUBJECTS AND METHODS least 4 ACR classification criteria documented in the medical record or 2) at least 3 ACR criteria and reviewers’ consensus Study population. The NHS database includes a pro- on the diagnosis of SLE. Because the results of the analyses spectively followed cohort of 121,700 female nurses (ages with the 2 definitions were very similar, we chose to report the 30–55 years in 1976 when the study began) (12), while the results applying the second definition. Using the first defini- NHSII was established in 1989, when 116,608 female nurses tion, we confirmed 241 cases of incident SLE, and using the (ages 25–42 years) completed a baseline questionnaire about second definition, we confirmed 262 cases of incident SLE, their medical histories and lifestyles. Ninety-four percent of diagnosed between 1976 and 2003, yielding a case- the NHS participants from 1976–2002 and 95% of the NHSII confirmation rate of 69% of the medical records reviewed and participants from 1989–2003 have remained in active followup 7% of the original self-reports of SLE. (5–6% of the women no longer respond to questionnaires and Population for analysis. For all analyses, we excluded have not been confirmed as dead). Both studies were designed prevalent cases of SLE diagnosed before the start of each to prospectively examine the effects of hormonal, reproduc- cohort. Women who reported having any connective tissue tive, and other lifestyle factors on chronic diseases, in partic- disease that was not subsequently confirmed to be SLE by ular cancer and cardiovascular disease. Information regarding medical record review were censored from the analysis at the diseases, lifestyle, and health practices is collected from the time of the self-report. In addition, women were censored if subjects in both cohorts via biennial questionnaires. All aspects they no longer responded to the biennial questionnaires, since of this study were approved by the Brigham and Women’s incident cases could not be identified. Thus, the final group Hospital Institutional Review Board. included 103,818 women who were followed up from 1976 to Identification of SLE. From 1976 to 1982, participants 2002 in the NHS, and 114,021 women who were followed up self-reported a diagnosis of SLE or other connective tissue from 1989 to 2003 in the NHSII. disease, including rheumatoid arthritis, mixed connective tis- Hormonal and reproductive factors. All exposure in- sue disease, scleroderma, polymyositis, dermatomyositis, or formation was self-reported on the mailed questionnaires that Sjo¨gren’s syndrome, which was reported in a write-in section of were administered every 2 years since 1976 in the NHS and the NHS questionnaire. Beginning in 1982 in the NHS and every 2 years since 1989 in the NHSII. Data on age at 1989 in the NHSII, participants have been asked specifically menarche were collected at baseline in both cohorts. Parity whether they have a physician’s diagnosis of SLE. For this and age at first birth were assessed on every questionnaire in study, we contacted 3,958 women (1,891 in the NHS, 2,067 in the NHSII cohort, but only from baseline through 1984 in the the NHSII) who gave a self-reported diagnosis of SLE and NHS cohort due to the older ages of these women. Total 14,282 women (11,748 in the NHS, 2,534 in NHSII) who lifetime breastfeeding history was assessed in 1986 in the NHS reported having other connective tissue diseases on any of the cohort and in 1997 in the NHSII cohort. With regard to the biennial questionnaires from 1976–2002 (NHS) and 1989–2003 regularity of menses, the women were asked (in 1988 in the (NHSII). We requested permission to review their medical NHS and in 1989 in the NHSII) to indicate “the regularity of records and asked that each participant complete the connec- natural menstrual periods between the ages 18 and 22 when tive tissue diseases screening questionnaire, which included 13 you were neither pregnant nor taking oral contraceptives”; questions concerning symptoms of SLE (all 11 of the American responses were categorized as either regular or irregular College of Rheumatology [ACR] criteria for the classification menses. In addition, in 1982, participants in the NHS were of SLE [13,14] as well as alopecia and Raynaud’s phenomenon asked about the regularity of their menses at ages 20–35 years, [15]). This questionnaire was scored as “positive,” i.e., indicat- classified as very regular, somewhat regular, somewhat irreg- ing a diagnosis of SLE, if 4 of the questions on symptoms ular, and very irregular. REPRODUCTIVE AND MENOPAUSAL FACTORS IN SLE 1253

Information on oral contraceptive use was collected potential confounder of the relationship with age at menarche from 1976 to 1982 in the NHS cohort (after which the ages of (18), was reported by the participants in 1992 in the NHS and the women were 36–61 years and use of oral contraceptives in 1991 in the NHSII. In a validation study by Michels et al (19), this age range was rare) and from 1989 to 2001 in the younger, the Spearman’s correlation between birth weight as reported NHSII cohort, and was categorized as never, past, or current by participants in the NHS and the NHSII and that reported by use. Current use of oral contraceptives was defined as use their own mothers was 0.77. In addition, in 1988 in the NHS within 1 month of the date of questionnaire return, whereas and in 1989 in the NHSII, participants were asked to identify past use was defined as use that ceased at least 1 month before the body shape that most closely matched their own at ages 5 the date of questionnaire return. Periods of use were summed and 10 years, with reference to 10 body-shape outlines ranging to calculate the total duration of oral contraceptive use, and from very thin/underweight to obese. Remote recall of body for past users, the time since last use was also determined. In shape using these outlines has been shown to be a valid a validation study, detailed telephone interviews regarding oral method, with Pearson’s correlation coefficients from 0.5 to 0.8 contraceptive use were conducted in a subsample of 215 for recall of body shape at young ages (20). NHSII participants using a structured life events calendar. Alcohol intake was reported at least every 4 years Spearman’s correlation coefficients for the duration of oral starting in 1980 in the NHS and 1991 in the NHSII. Physical contraceptive use calculated between the 2 methods was 0.94 activity at ages 18–22 years was assessed with the following (12). Detailed data concerning type and preparation of oral retrospective question in 1988 in the NHS and 1989 in the contraceptives were collected from the nurse participants in NHSII: “During ages 18–22, how often did you participate in the NHSII cohort. At baseline in 1989, these women were strenuous (aerobic) physical activity at least twice per week?”. asked to record the number of months they had taken specific Since all of the women in both cohorts are nurses, the range of types of oral contraceptive, identified from a booklet with socioeconomic status is limited. Husband’s education level was color photographs of all oral contraceptive brands. These data assessed in 1992 in the NHS and 1999 in the NHSII and was were updated in each 2-year cycle, in the same manner. chosen as a proxy for socioeconomic status. On each of the NHS and NHSII biennial question- Statistical analysis. All analyses were initially con- naires, participants were asked whether their menstrual peri- ducted separately in the 2 cohorts. Person-years of followup ods had ceased permanently and, if so, at what age and for accrued from the date of return of the baseline questionnaire what reason (occurring naturally or after chemotherapy, radi- until the date of diagnosis of SLE (as defined in the medical ation, or surgery). In addition, those who had undergone record) or date of any reported connective tissue disease that surgery were asked if they had one ovary or both ovaries was not confirmed as SLE, death, or loss to followup (defined removed. The first report of age at menopause was analyzed. as no further return of questionnaires). Age-adjusted relative Menopausal status was categorized as premenopausal, post- risks (RRs) were calculated after the participants were strati- menopausal, or unsure/missing. Self-reported menopausal sta- fied into 5-year age categories. Cox proportional hazards tus and age at menopause are highly reproducible in our regression models were utilized to study associations with SLE cohorts; in a validation study of a subsample of NHS partici- (developing from age 25 years to age 78 years), with simulta- pants, 82% of naturally postmenopausal women reported the neous adjustments for the reproductive variables of interest as same age at menopause (within 1 year) on each of 2 question- well as for age (in months), smoking status, birth weight, and naires that were mailed 2 years apart (16). Total ovulatory body size at ages 5 and 10 years. The current BMI, the BMI at years were calculated as the age at menopause minus the age age 18 years, race, physical activity at age 18 years, alcohol at menarche minus the number of childbirths (12 months each) intake, and husband’s education level were not associated with minus the years of oral contraceptive use. the risk of SLE, and additional adjustments for these variables Data concerning postmenopausal use of hormone re- did not affect the risk estimates in any of the multivariable placement were collected at baseline and updated every 2 models in either cohort; they were therefore not included in years in both cohorts. Past and current use of hormone the final models. In this type of updated analysis, time-varying replacement, as well as total duration of use and time since last information from each 2-year questionnaire is used to analyze use, were defined in the same manner as for oral contraceptive the risk of SLE in the next 2-year cycle. Tests for linear trend use. Menopausal status was included as a covariate in these in the Cox models were performed by entering continuous analyses in both cohorts, whereas postmenopausal hormone variables in the multivariable models, using the median value use was not included in the NHSII analyses because of the within each category when appropriate. small number of women (8% during the years of these Analyses investigating the associations of the risk of analyses) who were postmenopausal in the cohort. SLE with the age at first birth and the total duration of Covariate information. Age was updated in each cycle. breastfeeding were restricted to the parous women in each Race was assessed in 1992 in the NHS and 1989 in the NHSII, cohort. In a subanalysis, we investigated the associations and categorized as Caucasian, African, or Hispanic origin. between reproductive factors and the risk of SLE among Information on smoking was also collected every 2 years, and women in the NHS cohort who were ages 30–42 years at the cigarette smoking status was classified as never, past, or start of this cohort, analogous to the age of participants in the current and included as a covariate in the models, since current NHSII cohort. smoking has been associated with the risk of SLE (17). Body For analyses involving menopause as a variable of mass index (BMI) was computed for each 2-year time interval interest, we did not include participants with an indetermin- using the most recent weight (in kilograms divided by height in able or missing age at onset of menopause (e.g., women who square meters) as reported at baseline. Personal birth weight, reported undergoing surgical hysterectomy with unilateral which was included as a possible confounder, in particular a oophorectomy). We restricted analyses of postmenopausal 1254 COSTENBADER ET AL

Table 1. Reproductive and menopausal factors, adjusted for age, and risk of systemic lupus erythematosus in women from the NHS and the NHSII* Oral contraceptive use

NHS NHSII

Never Ever Never Ever (n ϭ 54,746) (n ϭ 48,300) (n ϭ 16,502) (n ϭ 91,450) Age, mean years 58.3 51.1 36.4 36.7 Caucasian, % 98 98 97 97 Smoking status, % Past smoker 37 39 15 8 Current smoker 17 18 24 13 Age at menarche Յ10 years, % 6688 Irregular menses, ages 18–22 years, % 16 20 22 24 Age Ͻ22 years at first birth, % 8 10 7 11 Total lifetime breastfeeding Ն12 months, %† 18 17 42 32 Postmenopausal, % 70 67 2 3 Current use of postmenopausal hormones, %‡ 25 32 48 60 * Characteristics of the women in the initial Nurses’ Health Study (NHS) (ages 44–69 years) in 1990, and in the second NHS (NHSII) (ages 27–44 years) in 1991. † Among parous women. ‡ Among postmenopausal women.

hormone use to postmenopausal women. In analyses of age at cles. Thirty-two percent of oral contraceptive users, menopause and total ovulatory years, we censored women who compared with 42% of nonusers, reported breastfeeding reported any type of non-natural menopause (e.g., surgical, Ն chemical, or radiation-induced). We performed a sensitivity for 12 months within the younger (NHSII) cohort analysis of menopause-related exposures that was limited to (Table 1). In both cohorts, the likelihood of postmeno- premenopausal women and naturally postmenopausal women pausal hormone use was higher among postmenopausal who had never taken any postmenopausal hormones. We also women who had taken oral contraceptives in the past. performed an analysis of the association of postmenopausal There were no important differences in the characteris- hormone use and risk of SLE stratified by ages above or below the mean age at menopause in the cohort. Chi-square tests tics (listed in Table 1) of the women who responded to were used to compare the age at menopause, and 2-sided our case-validation mailings compared with those who t-tests were used to compare rates of postmenopausal hormone did not. use among different groups of women. One hundred sixty-four cases of incident SLE To increase the precision of risk estimates and to obtain a single summary from the NHS and NHSII cohorts, the were confirmed in the NHS cohort and 98 cases in the RR results from the 2 cohorts were pooled by meta-analysis NHSII cohort. The overall incidence rate of SLE in the using a random effects model (21). Data were combined only NHS cohort was 164 cases per 2,829,458 person-years of when there was no significant evidence of heterogeneity be- followup, or 5.8/100,000. In the NHSII cohort, the tween the results from the 2 cohorts. SAS (version 9) was used incidence rate was 98 cases per 1,636,737 person-years of for all analyses (22). followup, or 6.0/100,000. This incidence of SLE, ϳ6in 100,000, among women ages 25–81 years is consistent RESULTS with recent estimates of the incidence of SLE in US The characteristics of the women participating in women (23). In both NHS cohorts, the rates were the NHS in 1990 (approximate midpoint of followup) highest in the youngest women and declined with age. and in the NHSII in 1991 by categories of oral contra- The presenting manifestations of SLE among the ceptive use are shown in Table 1. The majority of the incident cases and the proportions of women with women in both cohorts were of Caucasian ancestry. serologic and hematologic abnormalities, nephritis, and Among women in the NHS cohort, those who had ever arthritis at initial diagnosis are shown in Table 2. Few taken oral contraceptives were younger than never users, women with SLE had renal involvement at initial diag- but this was not true in the NHSII cohort. In both nosis. We did not review the updated medical records cohorts, slightly more women who had ever taken oral for each case, although this would have allowed us to contraceptives reported having irregular menstrual cy- determine the cumulative ACR criteria over time. Most REPRODUCTIVE AND MENOPAUSAL FACTORS IN SLE 1255

Table 2. Characteristics, at diagnosis, of women in the NHS and the risks were very similar to those reported for the entire NHSII who had incident systemic lupus erythematosus (SLE)* cohort, albeit with wider confidence intervals (e.g., RR NHS NHSII of SLE among ever users of oral contraceptives 1.8, 95% (n ϭ 164) (n ϭ 98) CI 1.1–3.0). Age at diagnosis, mean Ϯ SD years 52.4 Ϯ 8.3 41.1 Ϯ 6.2 Irregular menses at ages 18–22 years conferred Race an increased risk of SLE in the NHSII cohort, but not in Caucasian 157 (96) 96 (98) African 5 (3) 1 (1) the older (NHS) women. A more detailed assessment of Hispanic 1 (0.6) 1 (1) menstrual cycle regularity at ages 20–35 years, which was Antinuclear antibody positive† 154 (94) 98 (100) available only for the NHS cohort, also showed no Anti-dsDNA antibody positive† 23 (14) 50 (51) No. of ACR SLE criteria met, mean 4.7 Ϯ 1.1 4.7 Ϯ 1.2 association with the risk of SLE (data not shown). Parity, Ϯ SD age at first birth, and total length of breastfeeding were Arthritis 30 (18) 65 (66) all unrelated to the risk of SLE. Hematologic involvement 26 (16) 57 (58) Renal involvement 4 (2) 10 (10) Age-adjusted and multivariable analyses of Diagnosed by ACR member physician 119 (73) 88 (90) menopausal factors in the NHS cohort are shown in * Except where indicated otherwise, values are the number (%) of Table 4. (NHSII participants were not included in these subjects. NHS ϭ Nurses’ Health Study; NHSII ϭ second NHS; analyses, because only 8% were postmenopausal by the ϭ ϭ anti-dsDNA anti–double-stranded DNA; ACR American College end of followup.) The risk of SLE was elevated among of Rheumatology. † Positive by medical record review. postmenopausal women, primarily after surgical menopause (RR 2.3, 95% CI 1.2–4.5). The age-adjusted risk of SLE was elevated among all postmenopausal women in the NHS (RR 2.4, 95% CI 1.5–4.0). However, in a cases in both cohorts were diagnosed by a physician who univariate, crude model, the risk of SLE among post- was a member of the ACR. More than 95% of the menopausal women was not elevated (RR 0.9, 95% CI women with SLE in both cohorts were of Caucasian 0.7–1.3); indeed, the incidence of SLE was higher among ancestry, reflecting the racial composition of the co- premenopausal women. Among the postmenopausal horts. women in the NHS cohort, the use of postmenopausal Results of age-adjusted and multivariable analy- hormones was associated with a significant, 90% in- ses of the reproductive factors in both cohorts are shown creased risk of developing SLE (RR 1.9, 95% CI 1.2– in Table 3. Early age at menarche (age 10 years or younger, compared with age 12 years) was strongly 3.1), compared with those who had never taken post- associated with the risk of SLE in the NHS cohort and menopausal hormones. However, the risk of SLE did not was also associated, to a smaller extent, with the risk of appear to be related to duration of postmenopausal SLE in the NHSII cohort; the pooled results for both hormone use or time since last use. cohorts revealed a significant, doubled risk of SLE The age at onset of menopause in women who Ϯ Ϯ (pooled RR 2.1, 95% confidence interval [95% CI] later developed SLE was 51.6 3.9 years (mean SD), Ϯ 1.4–3.2). Ever use of oral contraceptives was also asso- whereas it was 52.7 4.3 years in the rest of the cohort Ͻ ciated with an increased risk of developing SLE, with a (P 0.01 by t-test). We observed a trend of increasing pooled RR of 1.5 (95% CI 1.1–2.1). The risk of SLE was risk of SLE in relation to younger age at onset of natural Ͻ the highest among women with a short duration (Ͻ2 menopause (P 0.05). Compared with women who years) of oral contraceptive use (RR 1.9, 95% CI experienced natural menopause at age Ն53 years, 1.3–2.8), and the effects of oral contraceptive use were women who underwent natural menopause before age long lasting (in those reporting Ն10 years since last use 47 years were at greater than twice the risk of incident of oral contraceptives, RR 1.7, 95% CI 1.2–2.4). SLE (RR 2.2, 95% CI 0.9–5.4), even after adjustment Analysis of the type of hormones and the hor- for postmenopausal hormone use and other potential mone potency of oral contraceptives taken by women in confounders in multivariable models. The age at onset of the NHSII cohort did not reveal any particular relation- menopause was significantly younger in those women ship between either the type or the potency of estrogen whose menopause was induced by surgery (bilateral or progestin and the risk of SLE (data not shown). In a oophorectomy) compared with those who had experi- subanalysis limited to younger women in the NHS enced natural menopause (mean Ϯ SD 47.8 Ϯ 5.3 years cohort (those ages 30–42 years at the start of the compared with 52.1 Ϯ 3.2 years; P Ͻ 0.001), and women cohort), there were 89 cases of incident SLE and the with surgical menopause were more likely to have taken 1256 COSTENBADER ET AL

Table 3. Reproductive factors and risk of systemic lupus erythematosus among women in the NHS 1976–2002 and the NHSII 1989–2003* No. of cases Person-years Age-adjusted RR (95% CI) Multivariable RR (95% CI) NHS Age at menarche, years Յ10 22 168,833 2.6 (1.5–4.4) 2.5 (1.5–4.2) 11 29 466,967 1.3 (0.8–2.1) 1.2 (0.8–2.0) 12 37 753,576 1.0 (referent) 1.0 (referent) 13 47 872,757 1.1 (0.7–1.7) 1.1 (0.7–1.7) Ն14 29 567,326 1.1 (0.7–1.8) 1.1 (0.7–1.8) P for trend† 0.02 0.02 Menstrual regularity, ages 18–22 years Regular 107 166,3145 1.0 (referent) 1.0 (referent) Irregular 27 481,004 0.9 (0.6–1.3) 0.9 (0.6–1.3) Parity Nulliparous 16 190,067 1.0 (referent) 1.0 (referent) Parous 147 2,602,964 0.7 (0.4–1.1) 0.8 (0.5–1.3) Number of children‡ 0 16 190,067 1.0 (referent) 1.0 (referent) 1 8 208,643 0.4 (0.2–1.0) 0.5 (0.2–1.1) 2 39 780,254 0.6 (0.3–1.0) 0.6 (0.3–1.1) 3 50 762,144 0.8 (0.4–1.3) 0.8 (0.5–1.4) Ն4 50 851,924 0.7 (0.4–1.3) 0.8 (0.4–1.4) P for trend† 0.1 0.2 Age at first birth, years§ Ͻ22 17 277,505 1.0 (0.6–1.7) 0.9 (0.5–1.6) 22 to Ͻ25 73 1,182,350 1.0 (referent) 1.0 (referent) 25 to Ͻ29 39 825,158 0.8 (0.6–1.2) 0.8 (0.6–1.2) Ͼ29 18 326,333 1.0 (0.6–1.6) 1.1 (0.6–1.8) P for trend† 0.6 0.3 Breastfeeding§ None 46 8,522,009 1.0 (referent) 1.0 (referent) Յ3 months 38 528,335 1.4 (0.9–2.1) 1.3 (0.8–2.0) 4–11 months 28 499,625 1.0 (0.6–1.6) 1.0 (0.6–1.6) 12–23 months 15 280,570 1.0 (0.5–1.7) 1.0 (0.6–1.9) Ն24 months 9 154,808 1.0 (0.5–2.1) 1.2 (0.6–2.5) P for trend† 0.2 0.7 Oral contraceptive use Never 65 1,447,252 1.0 (referent) 1.0 (referent) Ever 95 1,277,560 1.4 (1.0–2.0) 1.6 (1.1–2.2) Past 95 1,255,240 1.4 (1.1–2.0) 1.6 (1.1–2.2) Current 0 22,320 – – Duration of oral contraceptive use Never 85 1,447,252 1.0 (referent) 1.0 (referent) Ͻ2 years 42 467,818 1.7 (1.2–2.6) 1.8 (1.2–2.7) 2toϽ5 years 23 336,920 1.2 (0.7–2.1) 1.5 (0.9–2.4) Ն5 years 21 410,796 1.0 (0.6–1.7) 1.1 (0.7–1.8) P for trend† 0.08 0.2 Time since last oral contraceptive use Never 65 1,447,252 1.0 (referent) 1.0 (referent) Ͼ10 years 52 648,553 1.6 (1.1–2.3) 1.6 (1.1–2.4) 5toϽ10 years 23 337,425 1.3 (0.8–2.1) 1.5 (0.9–2.4) Ͻ5 years 10 172,178 1.1 (0.6–2.2) 1.3 (0.7–2.6) Current user 0 22,320 – – P for trend† 0.3 0.3 NHSII Age at menarche, years Յ10 11 128,588 1.6 (0.8–3.2) 1.6 (0.8–3.3) 11 15 271,802 1.0 (0.5–1.9) 1.0 (0.6–2.0) 12 27 493,938 1.0 (referent) 1.0 (referent) 13 29 449,344 1.2 (0.7–2.0) 1.1 (0.7–2.0) Ն14 16 239,064 1.0 (0.6–1.8) 0.9 (0.5–1.6) P for trend† 0.6 0.3 Menstrual regularity, ages 18–22 years Regular 64 1,208,298 1.0 (referent) 1.0 (referent) Irregular 31 380,578 1.5 (1.0–2.4) 1.5 (1.0–2.4) REPRODUCTIVE AND MENOPAUSAL FACTORS IN SLE 1257

Table 3. (Cont’d) No. of cases Person-years Age-adjusted RR (95% CI) Multivariable RR (95% CI) Parity Nulliparous 21 329,951 1.0 (referent) 1.0 (referent) Parous 73 1,200,473 1.0 (0.6–1.6) 1.0 (0.6–1.6) Number of children‡ 0 21 329,951 1.0 (referent) 1.0 (referent) 1 15 239,969 1.0 (0.5–2.0) 1.0 (0.5–1.9) 2 36 577,477 1.0 (0.6–1.8) 1.1 (0.6–1.9) 3 11 284,871 0.8 (0.4–1.7) 0.9 (0.4–1.7) Ն4 5 98,156 1.0 (0.4–2.6) 1.1 (0.4–2.7) P for trend† 0.6 0.6 Age at first birth, years§ Ͻ22 7 167,697 0.6 (0.2–1.3) 0.6 (0.2–1.3) 22 to Ͻ25 21 278,654 1.0 (referent) 1.0 (referent) 25 to Ͻ29 28 428,687 0.9 (0.5–1.5) 0.9 (0.5–1.7) Ͼ29 17 332,594 0.7 (0.4–1.3) 0.8 (0.4–1.4) P for trend† 0.8 0.8 Breastfeeding§ None 13 172,571 1.0 (referent) 1.0 (referent) Յ3 months 13 164,776 1.0 (0.5–2.1) 1.0 (0.5–2.3) 4–11 months 18 271,802 0.8 (0.4–1.6) 0.9 (0.5–1.9) 12–23 months 13 230,617 0.7 (0.3–1.5) 0.8 (0.4–1.8) Ն24 months 9 179,097 0.5 (0.3–1.5) 0.8 (0.4–2.0) P for trend† 0.3 0.6 Oral contraceptive use Never 6 214,058 1.0 (referent) 1.0 (referent) Ever 90 1,342,919 2.4 (1.1–5.5) 2.3 (1.0–5.0) Past 80 1,200,031 2.4 (1.1–5.6) 2.3 (1.1–5.2) Current 10 79,760 2.5 (0.9–7.3) 1.9 (0.7–5.2) Duration of oral contraceptive use Never 6 214,058 1.0 (referent) 1.0 (referent) Ͻ2 years 25 339,622 2.7 (1.1–6.5) 2.5 (1.0–6.1) 2toϽ5 years 26 390,841 2.4 (1.0–5.8) 2.2 (0.9–5.5) Ն5 years 36 535,051 2.4 (1.0–5.7) 2.1 (1.0–5.1) P for trend† 0.8 0.9 Time since last oral contraceptive use Never 6 214,058 1.0 (referent) 1.0 (referent) Ͼ10 years 43 708,843 2.3 (1.0–5.3) 2.2 (0.9–5.3) 5toϽ10 years 13 207,934 2.2 (0.8–5.9) 1.9 (0.7–5.1) Ͻ5 years 21 250,775 3.0 (1.2–7.7) 2.5 (1.0–6.4) Current user 10 142,888 2.5 (0.9–7.3) 1.9 (0.7–5.3) P for trend† 0.9 0.9 Pooled results (NHS ϩ NHSII) Age at menarche, years Յ10 – – – 2.1 (1.4–3.2) 11 – – – 1.2 (0.8–1.7) 12 – – – 1.0 (referent) 13 – – – 1.1 (0.8–1.6) Ն14 – – – 1.0 (0.7–1.5) P for trend† 0.02 Menstrual regularity, ages 18–22 years Regular – – – 1.0 (referent) Irregular – – – 1.1 (0.8–1.5) Parity Nulliparous – – – 1.0 (referent) Parous – – – 0.9 (0.6–1.3) Number of children‡ 0 – – – 1.0 (referent) 1 – – – 0.8 (0.4–1.3) 2 – – – 0.8 (0.6–1.3) 3 – – – 0.8 (0.5–1.3) Ն4 – – – 0.9 (0.5–1.4) P for trend† – – – 0.7 1258 COSTENBADER ET AL

Table 3. (Cont’d) No. of cases Person-years Age-adjusted RR (95% CI) Multivariable RR (95% CI) Age at first birth, years§ Ͻ22 – – – 0.8 (0.5–1.3) 22 to Ͻ25 – – – 1.0 (referent) 25 to Ͻ29 – – – 0.9 (0.6–1.2) Ͼ29 – – – 0.9 (0.6–1.4) P for trend† – – – 0.7 Breastfeeding§ None – – – 1.0 (referent) Յ3 months – – – 1.2 (0.8–1.8) 4–11 months – – – 1.0 (0.7–1.5) 12–23 months – – – 0.9 (0.6–1.5) Ն24 months – – – 1.0 (0.6–1.8) P for trend† – – – 0.5 Oral contraceptive use Never – – – 1.0 (referent) Ever – – – 1.5 (1.1–2.1) Past – – – 1.7 (1.2–2.3) Current – – – – Duration of oral contraceptive use Never – – – 1.0 (referent) Ͻ2 years – – – 1.9 (1.3–2.8) 2toϽ5 years – – – 1.6 (1.1–2.5) Ն5 years – – – 1.3 (0.8–2.0) P for trend† – – – 0.3 Time since last oral contraceptive use Never – – – 1.0 (referent) Ն10 years – – – 1.7 (1.2–2.4) 5toϽ10 years – – – 1.6 (1.0–2.4) Ͻ5 years – – – 1.6 (1.0–2.9) Current user – – – – P for trend† – – – 0.5 * Risk values are the relative risk (RR) (95% confidence interval [95% CI]) in the Nurses’ Health Study (NHS) and second NHS (NHSII) as well as pooled results from Cox proportional hazards models, adjusted for age, body shape at ages 5 and 10 years, age at menarche, menstrual regularity, parity, personal birth weight, oral contraceptive use (or duration of oral contraceptive use or time since last oral contraceptive use), cigarette smoking (never, past, current), and menopausal status. NHS analyses were additionally adjusted for postmenopausal hormone use (never, past, or current). Risk estimates were pooled using the Dersimonian and Laird random effects model. † All P values for trend exclude the referent group. ‡ Model adjusts for above list of covariates, substituting parity categorized as number of children in 5 categories for parity as nulliparous/parous. § Cox proportional hazards model limited to parous women only, adjusted for same covariates as well as age at first birth and total duration of breastfeeding.

postmenopausal hormones (80% versus 46%; P Ͻ DISCUSSION 0.001). In these 2 large, prospective cohorts of women, In a sensitivity analysis limited to premenopausal women and naturally postmenopausal women who had we have found that estrogen-related exposures, includ- not taken hormones, there were 50 cases of incident ing early menarche and exogenous estrogen use (both SLE. Moreover, the risk estimate for SLE was 3.3 (95% oral contraceptives and postmenopausal hormones), CI 1.3–8.8 in the multivariable model) in women who were associated with susceptibility to SLE. Our results were younger than age 47 years at onset of menopause, provide evidence that the timing of menarche and although the trend for age at menopause was no longer menopause and the use of exogenous estrogens are statistically significant (P for trend ϭ 0.07 in the multi- associated with the risk of SLE in women. Although no variable model). In an analysis stratified by age at dose responses were observed for exogenous estrogens, menopause (Յ51 years compared with Ն52 years), we the risk of SLE remained elevated for more than 10 did not observe any difference in the estimates of the years after the cessation of oral contraceptives and at risk of SLE, nor was there any association with ever use least 5 years after cessation of postmenopausal hor- of postmenopausal hormones (data not shown). mones. REPRODUCTIVE AND MENOPAUSAL FACTORS IN SLE 1259

Table 4. Menopause-related factors and risk of systemic lupus erythematosus among women in the NHS 1976–2002 cohort* No. of cases Person-years Age-adjusted RR (95% CI) Multivariable RR (95% CI) Menopausal status Premenopausal 52 872,617 1.0 (referent) 1.0 (referent) Postmenopausal 94 1,676,712 2.4 (1.5–4.0) 1.8 (1.0–3.4) Natural menopause 47 995,418 1.6 (0.8–3.3) 1.5 (0.8–2.9) Surgical menopause† 35 326,210 3.5 (2.0–6.1) 2.3 (1.2–4.5) Other menopause‡ 11 347,647 2.1 (0.2–18.4) 1.3 (0.5–3.3) PMH use§ Never 23 550,867 1.0 (referent) 1.0 (referent) Ever 65 916,454 1.9 (1.2–3.1) 1.9 (1.2–3.1) Past 27 359,311 2.4 (1.3–4.2) 2.2 (1.3–3.9) Current 38 557,143 1.7 (1.0–2.9) 1.7 (0.9–2.9) Duration of PMH use§ Never 23 550,867 1.0 (referent) 1.0 (referent) Ͻ5 years’ use 34 441,661 1.7 (1.0–3.0) 1.8 (1.0–3.0) Ն5 years’ use 31 474,793 2.1 (1.2–3.8) 2.0 (1.1–3.6) P for trend¶ – – 0.8 0.7 Time since last PMH use§ Never 23 550,867 1.0 (referent) 1.0 (referent) Ն5 years 10 140,856 2.7 (1.3–6.0) 2.3 (1.1–5.0) Ͻ5 years 16 144,954 2.8 (1.5–5.4) 2.8 (1.5–5.4) Current 38 557,143 1.7 (1.0–2.9) 1.7 (1.0–2.9) P for trend¶ – – 0.1 0.3 Age at menopause# Ͻ47 years 9 86,076 2.4 (1.1–5.1) 2.2 (0.9–5.4) 47–49 years 16 213,649 1.9 (1.0–3.5) 1.8 (0.9–3.6) 50–52 years 26 510,142 1.4 (0.8–2.6) 1.4 (0.8–2.5) Ն53 years 30 798,592 1.0 (referent) 1.0 (referent) P for trend – – Ͻ0.01 Ͻ0.05 Total ovulatory years#** Ͻ24 years 18 232,401 2.7 (0.9–8.2) 1.2 (0.4–3.4) 24–29 years 15 179,910 4.5 (1.8–11.4) 1.6 (0.6–4.2) 30–34 years 12 319,314 1.6 (0.6–4.0) 1.1 (0.4–2.8) Ͼ34 years 10 406,406 1.0 (referent) 1.0 (referent) P for trend – – Ͻ 0.01 0.9 * Risk values are the relative risk (RR) (95% confidence interval [95% CI]) from multivariable Cox proportional hazards models adjusted for age, body shape at ages 5 and 10 years, age at menarche, menstrual regularity, parity, personal birth weight, oral contraceptive use, cigarette smoking (never, past, current), menopausal status, and postmenopausal hormone (PMH) use (never, past, current). NHS ϭ Nurses’ Health Study. † Includes only hysterectomy with bilateral removal of ovaries. ‡ Includes radiation- and medication-induced menopause. § Analyses including postmenopausal women only. ¶ P for trend excludes never users. # Multivariable models censoring women who had types of menopause other than natural menopause. ** Age at menarche was not included in multivariable models because it is part of the definition of total ovulatory years.

Often viewed as a surrogate for the duration of and size, growth trajectory in early life, age at menarche, exposure to estrogen, age at menarche has been exam- and the risks of these diseases are complex (32–34). We ined as a risk factor for SLE in past case–control studies controlled for birth weight and body shape at ages 5 and (1–4). Menarche reflects maturation of the 10 years in all of our multivariable models, but this did hypothalamic–pituitary axis and the onset of pulsatile not affect our results, which further supports the notion secretion of gonadotropin-releasing hormone. Age at of an association between early menarche and increased menarche is determined, in part, by genetics (24) and risk of SLE. However, we did not find any association race (25) and, in part, by environmental factors such as between number of ovulatory years and the risk of SLE, nutritional status, BMI, and physical activity (26–28). suggesting that it may not be duration of estrogen Menarchal age is related to birth weight and size and exposure, but rather the timing of estrogen exposure, early childhood growth (18), and has been associated that is related to SLE risk. with risk of endometriosis (29) and breast cancer Sex steroid hormones, including 17␤-estradiol, (30,31), although the relationships between birth weight testosterone, prolactin, progesterone, and dehydroepi- 1260 COSTENBADER ET AL

androsterone (DHEA), influence the regulation of the Polymorphisms in the estrogen receptor gene immune system (35–38) and the severity of disease in ER␣ have been investigated in relation to age at disease patients with SLE (39–44). Grimaldi and colleagues onset and manifestations of SLE (50–52), and in relation have demonstrated that estrogens play an important role to age at menopause. In a case–control study in Greece, in B cell maturation, selection, and activation, and the ER␣ codon 594 genotype was associated with potentially have an effect on the breakdown of immune younger age at SLE onset and with malar rash, mouth tolerance seen in SLE (35,45). A meta-analysis of case– ulcers, and serositis (52). In a Swedish case–control control studies of hormonal levels showed significantly study, the rare PvuII C and XbaI G alleles were associ- lower levels of androgens (testosterone and DHEA) and ated with later SLE onset, skin manifestations, and higher levels of estradiol and prolactin among women milder disease (50). These polymorphisms have been with existing SLE than in controls (46). However, asso- associated with susceptibility to the effects of estrogen ciations between hormone levels in unaffected women (53,54), age at menarche (55), and, in one report, age at and the risk of SLE have not been assessed. Recent menopause (56), supporting a role for interactions be- randomized controlled trials in the US (47) and Mexico tween genetics, estrogen, and the estrogen receptor in (48) demonstrated that use of oral contraceptives was the pathogenesis of SLE. The current model for the not associated with an increase in lupus flares in women development of autoimmune diseases such as SLE in- with mild, stable SLE, whereas a year of treatment with volves multiple different susceptibility genes interacting postmenopausal hormones did lead to a small increase with a variety of potential environmental exposures. in the rate of flares (49). The role of female hormones in Potential interactions between estrogen receptor poly- the development of SLE is undoubtedly complex and morphisms, other genes, and exposure to endogenous challenging to study, given the relative rarity of the and exogenous estrogens at various points in the life- disease in the population. cycle may be responsible for producing a range of SLE In the Carolina Lupus Study, women with SLE phenotypes. experienced earlier menopause than did control subjects The strengths of our study include the large (4). In the NHS cohort, we also found that women who number of incident SLE cases in these combined co- later developed SLE experienced menopause slightly horts, the repeated assessment of exposures allowing for earlier than their counterparts, and that the risk of SLE time-varying covariates, prospective assessment of most was higher with earlier age at menopause. The increased exposures, and the long duration of followup. We used a risk of SLE in postmenopausal women was mainly very thorough 2-stage process for validation of the SLE among those women who had experienced surgical diagnosis, including careful medical record review; menopause (bilateral oophorectomy), and these women moreover, all women who self-reported any connective were both younger at the onset of menopause and more tissue disease that we were unable to confirm as definite likely to have taken postmenopausal hormones. The risk SLE were excluded from all analyses. We also defined of SLE was further attenuated in multivariable models, SLE as the presence of Ն3 ACR criteria and the suggesting that increased and earlier use of postmeno- reviewers’ consensus on SLE, since we identified 21 pausal hormones may be responsible for much of the women who the experts agreed had definite SLE but did risk. We found that total number of ovulatory years was not meet the ACR criteria for SLE (for example, having not independently associated with the risk of SLE. The SLE nephritis and positivity for antinuclear antibodies total duration of ovulation is based on age at menarche and anti–double-stranded DNA). The results were es- and age at menopause, both of which are related to the sentially unchanged when a definition of at least 4 ACR development of SLE but likely act through separate criteria was used. A high proportion of the SLE cases biologic mechanisms. were diagnosed by ACR member physicians, supporting Results of past studies have suggested a potential our method of case validation. relationship between menstrual irregularity and in- Limitations of this study include the fact that creased risk of SLE (2), and we also observed a border- some of the data, including birth weight, age at men- line increase in SLE risk associated with menstrual arche, menstrual regularity, physical activity at ages irregularity in the cohort of younger women, but not in 18–22 years, and BMI at age 18 years, relied upon the older women or in the pooled results. Increased total participant recall. We used self-reported exposure data duration of breastfeeding was inversely associated with and our study design was observational. Unfortunately, the risk of SLE in the Carolina Lupus Study (4), but we we have no additional information concerning the pre- did not observe a similar association. cise indications for surgical hysterectomies in women in REPRODUCTIVE AND MENOPAUSAL FACTORS IN SLE 1261

our cohorts (e.g., endometriosis, uterine or ovarian 2. Minami Y, Sasaki T, Komatsu S, Nishikori M, Fukao A, Yoshi- cancers, menorrhagia), and this subject deserves further naga K, et al. Female systemic lupus erythematosus in Miyagi Prefecture, Japan: a case-control study of dietary and reproductive investigation. In addition, because of the prospective factors. Tohoku J Exp Med 1993;169:245–52. design, we excluded women who developed SLE at a 3. Nagata C, Fujita S, Iwata H, Kurosawa Y, Kobayashi K, Kobayashi younger age, prior to the start of the cohorts, which was M, et al. Systemic lupus erythematosus: a case-control epidemiologic study in Japan. Int J Dermatol 1995;34:333–7. potentially related to earlier estrogen exposure. Partici- 4. Cooper GS, Dooley MA, Treadwell EL, St.Clair EW, Gilkeson pants in the NHS cohorts are a select group of mainly GS. Hormonal and reproductive risk factors for development of Caucasian, educated women, and are not among the systemic lupus erythematosus: results of a population-based, case–control study. Arthritis Rheum 2002;46:1830–9. groups at highest risk of severe SLE; hence, the gener- 5. Petri M, Thompson E, Abusuwwa R, Huang J, Garrett E. BALES: alizability of our findings to Hispanic Americans and The Baltimore Lupus Environmental Study [abstract]. Arthritis African Americans and those of lower socioeconomic Rheum 2001;44 Suppl 9:S331. status may be limited (57,58). For example, in the US, 6. Wong KL, Chan FY, Lee CP. Outcome of pregnancy in patients with systemic lupus erythematosus: a prospective study. Arch menarche occurs earlier among African American girls Intern Med 1991;151:269–73. as compared with white girls (25), but these cohorts did 7. Urowitz MB, Gladman DD, Farewell VT, Stewart J, McDonald J. not contain enough nonwhite subjects to investigate the Lupus and pregnancy studies. Arthritis Rheum 1993;36:1392–7. 8. Petri M. Prospective study of systemic lupus erythematosus preg- relationships between race, age at menarche, and risk of nancies. Lupus 2004;13:688–9. SLE. An alternative explanation not captured on the 9. Hochberg MC, Kaslow RA. Risk factors for the development of annual questionnaires, i.e., that some unmeasured con- systemic lupus erythematosus [abstract]. Clin Res 1983;31:732A. founder such as another characteristic (hormonal, be- 10. Sanchez-Guerrero J, Liang MH, Karlson EW, Hunter DJ, Colditz GA. Postmenopausal estrogen therapy and the risk for developing havioral, genetic, or other) of those women who had systemic lupus erythematosus. Ann Intern Med 1995;122:430–3. early menarche or had taken exogenous estrogens, could 11. Sanchez-Guerrero J, Karlson EW, Liang MH, Hunter DJ, Speizer be responsible for the observed associations and must be FE, Colditz GA. Past use of oral contraceptives and the risk of developing systemic lupus erythematosus. Arthritis Rheum 1997; considered. 40:804–8. In this prospective epidemiologic study, we have 12. Hunter DJ, Manson JE, Colditz GA, Chasan-Taber L, Troy L, shown potentially important associations between Stampfer MJ, et al. Reproducibility of oral contraceptive histories and validity of hormone composition reported in a cohort of US estrogen-associated reproductive and menopausal fac- women. Contraception 1997;56:373–8. tors and the risk of incident SLE among women. The 13. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield complex relationships between reproductive hormones NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. and the development of SLE clearly warrant further 14. Hochberg MC, for the Diagnostic and Therapeutic Criteria Com- study. mittee of the American College of Rheumatology. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis ACKNOWLEDGMENTS Rheum 1997;40:1725. 15. Karlson EW, Sanchez-Guerrero J, Wright EA, Lew RA, Daltroy We gratefully acknowledge the participants in the NHS LH, Katz JN, et al. A connective tissue disease screening ques- and the NHSII for their continuing cooperation. We also thank tionnaire for population studies. Ann Epidemiol 1995;5:297–302. Frank Speizer, Walter Willett, and Francine Grodstein for 16. Colditz GA, Stampfer MJ, Willett WC, Stason WB, Rosner B, their expert advice, and Susan Malspeis, Karen Corsano, and Hennekens CH, et al. Reproducibility and validity of self-reported Jae Hee Kang for their technical assistance. menopausal status in a prospective cohort study. Am J Epidemiol 1987;126:319–25. 17. Costenbader KH, Kim DJ, Peerzada J, Lockman S, Nobles-Knight AUTHOR CONTRIBUTIONS D, Petri M, et al. Cigarette smoking and the risk of systemic lupus erythematosus: a meta-analysis. Arthritis Rheum 2004;50:849–57. Dr. Costenbader had full access to all of the data in the study 18. Adair LS. Size at birth predicts age at menarche. Pediatrics and takes responsibility for the integrity of the data and the accuracy 2001;107:e59–66. of the data analysis. 19. Michels KB, Trichopoulos D, Robins JM, Rosner BA, Manson JE, Study design. Costenbader, Stampfer, Karlson. Hunter DJ, et al. Birthweight as a risk factor for breast cancer. Acquisition of data. Costenbader, Karlson. Lancet 1996;348:1542–6. Analysis and interpretation of data. Costenbader, Feskanich, 20. Must A, Willett WC, Dietz WH. Remote recall of childhood Stampfer, Karlson. height, weight, and body build by elderly subjects. Am J Epidemiol Manuscript preparation. Costenbader, Feskanich, Stampfer, Karlson. 1993;138:56–64. Statistical analysis. Costenbader, Feskanich, Stampfer, Karlson. 21. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177–88. REFERENCES 22. SAS. 9th ed. Cary (NC): SAS Institute; 1990. 23. Uramoto KM, Michet CJ Jr, Thumboo J, Sunku J, O’Fallon WM, 1. Grimes DA, LeBolt SA, Grimes KR, Wingo PA. Systemic lupus Gabriel SE. Trends in the incidence and mortality of systemic erythematosus and reproductive function: a case-control study. lupus erythematosus, 1950–1992. Arthritis Rheum 1999;42:46–50. Am J Obstet Gynecol 1985;153:179–86. 24. Loesch DZ, Huggins R, Rogucka E, Hoang NH, Hopper JL. 1262 COSTENBADER ET AL

Genetic correlates of menarcheal age: a multivariate twin study. 44. Chang DM, Lan JL, Lin HY, Luo SF. Dehydroepiandrosterone Ann Hum Biol 1995;22:470–90. treatment of women with mild-to-moderate systemic lupus ery- 25. Anderson SE, Must A. Interpreting the continued decline in the thematosus: a multicenter randomized, double-blind, placebo- average age at menarche: results from two nationally representa- controlled trial. Arthritis Rheum 2002;46:2924–7. tive surveys of U.S. girls studied 10 years apart. J Pediatr 2005; 45. Grimaldi CM, Hill L, Xu X, Peeva E, Diamond B. Hormonal 147:753–60. modulation of B cell development and repertoire selection. Mol 26. Leenstra T, Petersen LT, Kariuki SK, Oloo AJ, Kager PA, ter Immunol 2005;42:811–20. Kuile FO. Prevalence and severity of malnutrition and age at 46. McMurray RW, May W. Sex hormones and systemic lupus ery- menarche: cross-sectional studies in adolescent schoolgirls in thematosus: review and meta-analysis [review]. Arthritis Rheum western Kenya. Eur J Clin Nutr 2005;59:41–8. 2003;48:2100–10. 27. Ersoy B, Balkan C, Gunay T, Egemen A. The factors affecting the relation between the menarcheal age of mother and daughter. 47. Petri M, Kim MY, Kalunian KC, Grossman J, Hahn BH, Sam- Child Care Health Dev 2005;31:303–8. maritano LR, et al, for the OC-SELENA Trial. Combined oral 28. Torstveit MK, Sundgot-Borgen J. Participation in leanness sports contraceptives in women with systemic lupus erythematosus. but not training volume is associated with menstrual dysfunction: N Engl J Med 2005;353:2550–8. a national survey of 1276 elite athletes and controls. Br J Sports 48. Sanchez-Guerrero J, Uribe AG, Jimenez-Santana L, Mestanza- Med 2005;39:141–7. Peralta M, Lara-Reyes P, Seuc AH, et al. A trial of contraceptive 29. Missmer SA, Hankinson SE, Spiegelman D, Barbieri RL, Malspeis methods in women with systemic lupus erythematosus. N Engl S, Willett WC, et al. Reproductive history and endometriosis J Med 2005;353:2539–49. among premenopausal women. Obstet Gynecol 2004;104(5 Pt 49. Buyon JP, Petri MA, Kim MY, Kalunian KC, Grossman J, Hahn 1):965–74. BH, et al. The effect of combined estrogen and progesterone 30. Garland M, Hunter DJ, Colditz GA, Manson JE, Stampfer MJ, hormone replacement therapy on disease activity in systemic lupus Spiegelman D, et al. Menstrual cycle characteristics and history of erythematosus: a randomized trial. Ann Intern Med 2005;142(12 ovulatory infertility in relation to breast cancer risk in a large Pt 1):953–62. cohort of US women. Am J Epidemiol 1998;147:636–43. 50. Johansson M, Arlestig L, Moller B, Smedby T, Rantapaa-Dahl- 31. Colditz GA. Epidemiology of breast cancer: findings from the qvist S. Oestrogen receptor ␣ gene polymorphisms in systemic Nurses’ Health Study. Cancer 1993;71(4 Suppl):1480–9. lupus erythematosus. Ann Rheum Dis 2005;64:1611–7. 32. Ahlgren M, Melbye M, Wohlfahrt J, Sorensen TI. Growth patterns 51. Lee YJ, Shin KS, Kang SW, Lee CK, Yoo B, Cha HS, et al. and the risk of breast cancer in women. N Engl J Med 2004;351: Association of the oestrogen receptor ␣ gene polymorphisms with 1619–26. disease onset in systemic lupus erythematosus. Ann Rheum Dis 33. Dos Santos Silva I, de Stavola BL, Hardy RJ, Kuh DJ, McCormack 2004;63:1244–9. VA, Wadsworth ME. Is the association of birth weight with 52. Kassi E, Vlachoyiannopoulos PG, Kominakis A, Kiaris H, Mout- premenopausal breast cancer risk mediated through childhood sopoulos HM, Moutsatsou P. Estrogen receptor ␣ gene polymor- growth? Br J Cancer 2004;91:519–24. phism and systemic lupus erythematosus: a possible risk? Lupus 34. De Stavola BL, dos Santos Silva I, McCormack V, Hardy RJ, Kuh 2005;14:391–8. DJ, Wadsworth ME. Childhood growth and breast cancer. Am J 53. Kobayashi N, Fujino T, Shirogane T, Furuta I, Kobamatsu Y, Epidemiol 2004;159:671–82. Yaegashi M, et al. Estrogen receptor ␣ polymorphism as a genetic 35. Grimaldi CM, Cleary J, Dagtas AS, Moussai D, Diamond B. marker for bone loss, vertebral fractures and susceptibility to Estrogen alters thresholds for B cell apoptosis and activation. estrogen. Maturitas 2002;41:193–201. J Clin Invest 2002;109:1625–33. 54. Suuriniemi M, Mahonen A, Kovanen V, Alen M, Lyytikainen A, 36. Peeva E, Michael D, Cleary J, Rice J, Chen X, Diamond B. Wang Q, et al. Association between exercise and pubertal BMD is Prolactin modulates the naive B cell repertoire. J Clin Invest modulated by estrogen receptor ␣ genotype. J Bone Miner Res 2003;111:275–83. 2004;19:1758–65. 37. Lahita RG. Sex steroids and the rheumatic diseases. Arthritis 55. Stavrou I, Zois C, Ioannidis JP, Tsatsoulis A. Association of Rheum 1985;28:121–6. ␣ 38. Lahita RG, Bradlow HL, Kunkel HG, Fishman J. Increased 16 polymorphisms of the oestrogen receptor gene with the age of ␣-hydroxylation of estradiol in systemic lupus erythematosus. menarche. Hum Reprod 2002;17:1101–5. J Clin Endocrinol Metab 1981;53:174–8. 56. Weel AE, Uitterlinden AG, Westendorp IC, Burger H, Schuit SC, 39. Vera-Lastra O, Jara LJ, Espinoza LR. Prolactin and autoimmu- Hofman A, et al. Estrogen receptor polymorphism predicts the nity. Autoimmun Rev 2002;1:360–4. onset of natural and surgical menopause. J Clin Endocrinol Metab 40. Chapel TA, Burns RE. Oral contraceptives and exacerbation of 1999;84:3146–50. lupus erythematosus. Am J Obstet Gynecol 1971;110:366–9. 57. Alarcon GS, McGwin G Jr, Sanchez ML, Bastian HM, Fessler BJ, 41. Lahita RG. The importance of estrogens in systemic lupus ery- Friedman AW, et al, for the LUMINA Study Group. Systemic thematosus. Clin Immunol Immunopathol 1992;63:17–8. lupus erythematosus in three ethnic groups. XIV. Poverty, wealth, 42. Petri M, Robinson C. Oral contraceptives and systemic lupus and their influence on disease activity. Arthritis Rheum 2004;51: erythematosus [review]. Arthritis Rheum 1997;40:797–803. 73–7. 43. Petri MA, Lahita RG, van Vollenhoven RF, Merrill JT, Schiff M, 58. Alarcon GS, Calvo-Alen J, McGwin G, Uribe AG, Toloza SM, Ginzler EM, et al, for the GL701 Lupus Study Group. Effects of Roseman J, et al, for the LUMINA Study Group. Systemic lupus prasterone on corticosteroid requirements of women with systemic erythematosus in a multiethnic cohort: LUMINA XXXV. Predic- lupus erythematosus: a double-blind, randomized, placebo-con- tive factors of high disease activity over time. Ann Rheum Dis trolled trial. Arthritis Rheum 2002;46:1820–9. 2006;65:1168–74. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1263–1272 DOI 10.1002/art.22505 © 2007, American College of Rheumatology

Histopathologic and Clinical Outcome of Rituximab Treatment in Patients With Cyclophosphamide-Resistant Proliferative Lupus Nephritis

Iva Gunnarsson,1 Birgitta Sundelin,1 Thorunn Jonsd´ ottir,´ 1 Stefan H. Jacobson,2 Elisabet Welin Henriksson,1 and Ronald F. van Vollenhoven1

Objective. Rituximab is a monoclonal antibody clinical improvement was noted, with a reduction in directed against the CD20 marker of B cells. Because of SLEDAI scores (from a mean of 15 to 3), anti–double- its ability to deplete B lymphocytes, it has been sug- stranded DNA antibody levels (from a mean of 174 gested that the drug could be of benefit in B cell– IU/ml to 56 IU/ml), and anti-C1q antibody levels (from dependent diseases, including systemic lupus erythem- a mean of 35 units/ml to 22 units/ml). On repeat renal atosus (SLE). The purpose of this study was to biopsy, improvement in the histopathologic class of investigate the histopathologic and clinical effects of nephritis occurred in a majority of patients, and a combination treatment with rituximab and cyclo- decrease in the renal activity index was noted (from 6 to phosphamide (CYC) in patients with CYC-resistant 3). A reduction in the number of CD3, CD4, and CD20 proliferative lupus nephritis. cells in the renal interstitium was noted in 50% of the Methods. Seven female patients with proliferative patients on repeat biopsy. lupus nephritis were treated with rituximab in combi- Conclusion. At 6 months of followup, all patients nation with CYC. Renal biopsies were performed before had responded both clinically and histopathologically to treatment and during followup. SLE activity was evalu- combination therapy. For patients with proliferative ated by the Systemic Lupus Erythematosus Disease lupus nephritis who fail to respond to conventional Activity Index (SLEDAI) and the British Isles Lupus immunosuppressive therapy including CYC, combined Assessment Group index. In 6 of the 7 patients, immu- treatment with rituximab and CYC may constitute a new nostaining of lymphocyte subpopulations in the renal treatment option. tissue was performed before treatment and during followup. Results. At 6 months of followup, significant Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease in which B cell hyper- reactivity and autoantibody formation are prominent Supported by a grant from King Gustaf V’s 80th Birthday features. In patients with severe organ involvement, Fund and by funds from the Karolinska Institute. The fee to register the study with the Swedish Medical Product Agency was paid by Roche treatment with cyclophosphamide (CYC) has generally AB, Stockholm, Sweden. been regarded as standard treatment, especially in lupus 1 Iva Gunnarsson, MD, PhD, Birgitta Sundelin, MD, PhD, nephritis (1). Classic studies have shown that CYC- Thorunn Jo´nsdo´ttir, MD, Elisabet Welin Henriksson, RN, PhD, Ronald F. van Vollenhoven, MD, PhD: Karolinska University Hospi- based treatment regimens for severe SLE nephritis are tal at Solna, Stockholm, Sweden; 2Stefan H. Jacobson, MD, PhD: significantly more successful in maintaining kidney func- Danderyd University Hospital, Stockholm, Sweden. tion than glucocorticoids alone, and such treatment Drs. Gunnarsson, Jo´nsdo´ttir, and van Vollenhoven have received honoraria (less than $10,000 each author) from Roche. Dr. regimens have become the “gold standard” (2–4). Infec- Henriksson has received speaker’s fees (less than $10,000 each) from tious complications during CYC therapy cannot be Wyeth and Abbott. Address correspondence and reprint requests to Iva Gunnars- neglected, and the use of newer, less toxic treatments, son, MD, PhD, Department of Rheumatology, D2:01, Karolinska such as mycophenolate mofetil, are needed (5). How- University Hospital at Solna, S-171 76 Stockholm, Sweden. E-mail: ever, despite cytotoxic therapy, some patients are poor [email protected]. Submitted for publication April 27, 2006; accepted in revised responders and are thus at risk of organ damage and form January 4, 2007. renal function impairment. Therefore, it remains an

1263 1264 GUNNARSSON ET AL

Table 1. Main clinical characteristics, treatment, and nephritis data in patients with lupus nephritis before initiation of rituximab treatment* Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Age, years 33 25 26 19 32 43 29 Disease duration, years 11 12 10 3 8 22 1 Main manifestations during N, Cut., A, Hem. N, Cut., A N, Cut., A, Ser. N, Cut., A, Hem. N, Cut., N, Cut., N, Ser., A disease course A, CNS A, Ser. Previous therapies other AM, AZA, MTX AM, AZA, CSA AZA, CSA, MTX, AM, AZA, MTX, AM, AZA AM, AZA MMF than CYC MMF MMF WHO class at first onset of IIIb IVc IVb IVa IVc IVa IVb nephritis Activity index at onset 6 3 6 4 13 3 6 Chronicity index at onset 6 2 0 0 3 0 1 Total cumulative dose of 6,150 9,400 7,850 8,100 12,000 14,700 5,800 CYC, mg Time from nephritis onset 10 35 22 20 31 39 11 to rituximab therapy, months *Nϭ nephritis; Cut. ϭ cutaneous manifestations; A ϭ arthritis; Hem. ϭ hematologic manifestations; Ser. ϭ serositis; CNS ϭ central nervous system manifestations; CYC ϭ cyclophosphamide; AM ϭ antimalarial agents; AZA ϭ azathioprine; MTX ϭ methotrexate; CSA ϭ cyclosporin A; MMF ϭ mycophenolate mofetil; WHO ϭ World Health Organization. issue of major concern to identify and develop alterna- potential of this new therapy in patients with severe and tive treatment modalities in patients whose disease is refractory renal SLE; a brief report on 2 patients who refractory to conventional immunosuppressive treat- are included in the present study has recently been ments. published (19). The aim of the present study was to Treatment with the monoclonal antibody ritux- evaluate the efficacy and safety of a treatment protocol imab (anti-CD20) is commonly used in patients with B consisting of the combination of rituximab and CYC in cell lymphomas and has in recent years also been tested a group of treatment-resistant patients with lupus ne- in several rheumatologic diseases, such as rheumatoid phritis. We describe herein the results seen in our study arthritis (6,7), Wegener’s granulomatosis (8–11), and patients with respect to the clinical manifestations of the Sjo¨gren’s syndrome (12). Reports of the beneficial ef- renal disease, the renal response criteria, and the his- fects of rituximab in patients with proliferative lupus topathologic outcome. nephritis have also been published during the last few years (13–24). We have previously shown that renal responses, PATIENTS AND METHODS when assessed clinically, usually by improvements in Patients. Seven female patients with severe SLE and serum creatinine levels and proteinuria grades, do not renal involvement (mean age 30 years [range 19–43 years]) always reflect diminished histopathologic activity in the who had failed to respond to conventional immunosuppressive renal tissue (25). In that study, we found that a substan- therapy including CYC were included in this study of rituximab plus CYC. Treatment failure was defined as persistence of tial proportion of patients had ongoing inflammatory histopathologic findings of active proliferative lupus nephritis activity in the renal tissue despite the appearance of in a recent renal biopsy despite immunosuppressive therapy. being clinically quiescent. Accordingly, repeated renal At initiation of rituximab treatment, the patients had a mean biopsies may be fundamental in the evaluation of ne- SLE duration of 10 years (range 1–22 years). All patients phritis patients to determine the ongoing inflammatory fulfilled the American College of Rheumatology (ACR) crite- process in the target organ. Although some of the ria for SLE (26). Patients received rituximab in combination with CYC, accompanied by a temporary increase in the studies cited above have documented a beneficial clini- glucocorticoid dosage. cal response to rituximab in patients with lupus nephri- The study was approved by the Local Ethics Commit- tis, detailed information regarding renal outcomes, in- tee and by the Swedish Medical Product Agency. The clinical cluding repeated renal biopsy data, was not presented. characteristics, previous treatments, and nephritis data in the Furthermore, data regarding depletion of lymphocyte patient cohort are presented in Table 1. Treatment protocol. On day 1, methylprednisolone was cell subsets in the tissue of lupus patients during ritux- given intravenously at a dosage of 250 mg, followed by imab therapy are not available. intravenous CYC at a dosage of 0.5 gm/m2 of body surface At our center, we have chosen to investigate the area. On day 2, hydrocortisone was given intravenously at a RITUXIMAB TREATMENT OF CYC-RESISTANT PROLIFERATIVE LUPUS NEPHRITIS 1265

Figure 1. Changes from baseline evaluation to the 6-month followup in A, the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, B, the serum creatinine (s-creatinine) level (normal Ͻ120 ␮moles/liter), C, the serum albumin (s-albumin) level (normal Ͼ36 gm/liter), and D, the anti–double-stranded DNA (anti-dsDNA) antibody level (normal Ͻ15 IU/ml, by fluorescence enzyme .ϭ P Ͻ 0.01 ءء ;ϭ P Ͻ 0.05 ء .(immunoassay dosage of 100 mg, followed by intravenous rituximab at a etry. The limit for detection of CD19ϩ B cells was Ͻ0.01 ϫ dosage of 375 mg/m2 of body surface area. On days 9 and 16, 109/liter (normal range 0.12–0.38). hydrocortisone and rituximab were administered at the same Serology and complement levels. Serum IgG anti– dosages as on day 2. On day 23, methylprednisolone, CYC, and double-stranded DNA (anti-dsDNA) antibodies were mea- rituximab were administered at the same dosages as before. sured by a fluorescence enzyme immunoassay (FEIA) method After the first rituximab infusion, the oral prednisolone (Pharmacia, Uppsala, Sweden). The cutoff value for normal dosage was increased to 1 mg/kg of body weight during the first levels in the FEIA analysis was Ͻ15 IU/ml, but values below week, tapered to 0.75 mg/kg during the second week, and then this level were also recorded for use in statistical calculations. to 0.5 mg/kg during the third week. After termination of the Anti-C1q antibodies were analyzed by an enzyme-linked im- fourth rituximab infusion, the patients continued taking their munosorbent assay method (29), with a normal level being Ͻ16 pretreatment prednisolone dosage, with subsequent tapering units/ml. during followup as clinically allowed. After the course of Analysis of complement component C1q was per- rituximab treatment, no other immunosuppressive treatment formed by rocket electrophoresis (30) using rabbit anti-C1q as was given before the renal biopsy was repeated. the antibody. Levels of C1q were expressed as the percentage Followup evaluation. After the treatment course was of the levels in normal blood donors (normal range 76–136%). completed, the patients were followed up every 1–2 months C3 and C4 levels were determined by nephelometry (normal according to a standardized protocol. Each visit included a range 0.5–1.2 gm/liter for C3 and 0.1–0.4 gm/liter for C4). clinical examination and blood sampling. The overall disease Renal evaluation and histopathologic assessment. Re- activity was evaluated using the Systemic Lupus Erythemato- nal evaluation at the start of the study and at the 6-month sus Disease Activity Index (SLEDAI) (27). Disease activity on followup included a urinalysis (dip slide procedure), a urinary an organ-based level for renal involvement and outcome was sediment assessment, and an investigation of albumin excre- assessed using the British Isles Lupus Assessment Group tion in a 24-hour urine collection. The glomerular filtration (BILAG) index (28). rate (GFR) was determined by the clearance of iohexol, Blood sampling included investigation of serum creat- according to routine clinical procedures of the laboratory. inine and serum albumin levels, serologic findings, and com- Repeat renal biopsies were performed during fol- plement levels. Circulating B cells in the peripheral blood were lowup. All biopsy samples were graded according to the World evaluated by the detection of CD19ϩ B cells on flow cytom- Health Organization (WHO) classification system described in 1266 GUNNARSSON ET AL

Table 2. Histopathologic features, disease activity, ongoing therapy, and laboratory variables in patients with lupus nephritis before rituximab treatment, at initiation of rituximab treatment, after 6 months of followup, and at repeat renal biopsy* Assessment Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 At biopsy before rituximab WHO class IIIb IVc IVb IVc IIIb IVb IVb Activity index 5 5576710 Chronicity index 8 713441 Time from biopsy to initiation of rituximab, months 2.5 11 1 3 1 0.5 1.5 At initiation of rituximab SLEDAI score 6 8 19 8 32 12 19 Renal BILAG score A AAAAAA Prednisolone, mg/day 5 20 15 15 12.5 50† 15 Ongoing therapy CYC CSA CYC CYC CYC AZA CYC Serum creatinine, ␮moles/liter 84 100 63 77 85 111 99 Albuminuria, gm/24 hours 0.2 1.6 4.6 2.9 1.4 5.9 2.4 GFR, ml/minute 60 70 104 95 71 52 76 At 6 months of followup Renal BILAG score C B C B B D C At repeat biopsy WHO class Ib Vb IVa/Vb IIIb IIb IIb IIb Activity index 1 324242 Chronicity index 8 723243 Time between biopsies, months 3 and 12‡ 5 12 10 7 7 7 * WHO ϭ World Health Organization; SLEDAI ϭ Systemic Lupus Erythematosus Disease Activity Index; BILAG ϭ British Isles Lupus Assessment Group (scores are defined as follows: A ϭ requires urgent disease-modifying therapy, B ϭ demands close attention, often symptomatic therapy, C ϭ stable, controlled with current therapy, and D ϭ no involvement); CYC ϭ cyclophosphamide; CSA ϭ cyclosporin A; AZA ϭ azathioprine; GFR ϭ glomerular filtration rate. † Patient had a recent increase in prednisolone dosage. ‡ A repeat biopsy performed 12 months after the first biopsy yielded the same results as the second biopsy performed at 3 months.

1995 (31). In addition, activity and chronicity indices were B lymphocytes were depleted and after B cells had returned to determined (32). The histopathologic findings at first onset of the peripheral circulation. In the other patients, repeat biop- nephritis, the cumulative dose of CYC given before rituximab sies were performed 5–12 months after rituximab treatment, treatment, and the time from first onset of nephritis to and in all cases repopulation of peripheral blood B lympho- initiation of rituximab therapy are presented in Table 1. cytes had already occurred. Evaluation of renal response and remission. Remis- Immunohistochemical stainings were performed using sion of nephritis at the 6-month followup was defined as a Benchmark XT immunostaining instrument from Ventana normal serum creatinine and serum albumin levels, inactive Medical Systems (Tucson, AZ) according to routine clinical urinary sediment, and a 24-hour urinary albumin level Ͻ0.5 procedures at the Department of Pathology and Cytology, gm; partial renal remission was defined as a Ն50% improve- Karolinska University Hospital. The cells were stained with the ment in all renal parameters that were abnormal at baseline, following antibodies: CD3 (RM 9107-S; NeoMarkers, obtained without a deterioration in any of them (23). For further from AH Diagnostics, Stockholm, Sweden), CD4 (NCL-CD4- evaluation of the renal response, the recently published ACR 1F6; Novocastra, obtained from Immunokemi, Stockholm, response criteria for proliferative and membranous renal dis- Sweden), CD8 (M7103; Dako, Copenhagen, Denmark), CD20 ease in SLE clinical trials (33) were used. Consistent with these (M0755; Dako), CD79a (M0750; Dako), and CD38 (NCL- criteria, evaluation of renal function (the GFR), measurement CD38-290; Novocastra). of the urinary protein level (assessed by the urinary excretion Statistical analysis. For comparison of variables at of albumin in a 24-hour urine collection), and assessment of baseline and followup, paired Student’s t-test was used. P abnormalities in the urinary sediment were performed, and values less than 0.05 were considered significant. StatView adverse events were recorded. software (SAS Institute, Cary, NC) was used for statistical Lymphocyte subpopulations. Immunohistochemical calculations. stainings with investigation of the number and localization of lymphocyte subpopulations were performed on renal biopsy samples from 6 of the 7 patients. Analyses of T cells (CD3, RESULTS CD4, CD8), B cells (CD20, CD79a), and plasma cells (CD38) were also performed. In patient 1, two repeat biopsies were Clinical response and treatment. At inclusion in performed during the followup period, the first at 3 months the study, the mean SLEDAI score was 15 (range 6–32), from initiation of rituximab therapy and the second at 12 months. In this patient, B lymphocytes were depleted for 9 and there was a significant decrease at the 6-month months from the peripheral circulation. Accordingly, we were followup (mean score 3 [range 0–10]; P ϭ 0.0022) here able to analyze the cell subsets during both the time when (Figure 1A). A reduction in the prednisolone dosage was RITUXIMAB TREATMENT OF CYC-RESISTANT PROLIFERATIVE LUPUS NEPHRITIS 1267

Table 3. Lymphocyte subpopulations in the renal interstitial infiltrate at baseline (0 months) and at repeat biopsy* Lymphocyte subpopulation Patient, time Time to B cell of biopsy CD3 CD4 CD8 CD20 CD79a CD38 repopulation Patient 1 Baseline ϩϩ ϩϩϩ ϩ/ϩϩϩϩϩ9 months 3 months (ϩ)(ϩ)(ϩ)––– 12 months ϩϩ ϩϩ ϩ (ϩ)/ϩ (ϩ)(ϩ)/ϩ Patient 2 Baseline ϩ – ϩ – – – 2 months 5 months (ϩ)– ϩ ––– Patient 3 Baseline (ϩ)(ϩ)(ϩ)(ϩ)–(ϩ) 2 months 12 months – – – – – (ϩ) Patient 4 Baseline (ϩ)(ϩ) ϩ (ϩ)– ϩ 8 months 10 months (ϩ)– (ϩ)–(ϩ) ϩ Patient 5 Baseline ND ND ND ND ND ND 4 months 7 months ND ND ND ND ND ND Patient 6 Baseline ϩϩ ϩϩ – ϩϩϩϩ6 months 7 months ϩϩ ϩϩ ϩϩ ϩ ϩ ϩ/ϩϩ Patient 7 Baseline ϩ (ϩ)/ϩ (ϩ) – – – 4 months 7 months (ϩ)(ϩ)(ϩ)– –(ϩ) * Lymphocyte subsets were scored as follows: – ϭ none, (ϩ) ϭ occasional, ϩϭfew, ϩϩ ϭ moderate, and ϩϩϩ ϭ many. ND ϭ not determined.

seen in 6 of the 7 patients at 6 months, from a mean of At study inclusion, the mean Ϯ SD C3 level was 18.9 mg/day (range 5–50) to a mean of 8.3 mg/day (range 0.6 Ϯ 0.25 gm/liter (range 0.37–1.05), the mean Ϯ SD C4 2.5–17.5) (P ϭ 0.13). At baseline, all patients had a level was 0.13 Ϯ 0.11 gm/liter (range 0.02–0.36), and the BILAG score of A for renal involvement (requires mean Ϯ SD C1q level was 61 Ϯ 46% (range 8–152). An urgent disease-modifying therapy), and at followup, all increase in both C3 and C4 levels was noted at 6 months patients showed improvement (Table 2). of followup (P ϭ 0.0003 for C3 and P ϭ 0.0024 for C4) Before treatment, the mean serum creatinine (Figures 2B and C). For the C1q levels, an improvement ␮ level was 88 moles/liter (range 63–111), and at 6 was noted in 5 of the 7 patients, and these 5 patients had ␮ ϭ months, it was 73 moles/liter (range 62–86) (P 0.035) normal levels at 6 months of followup (P not significant). (Figure 1B). The serum albumin level increased from a Results of renal evaluation and histopathologic mean of 27 gm/liter (range 18–39) at baseline to 34 assessment. Hematuria, as determined by urinary sedi- gm/liter at 6 months (range 26–44) (P ϭ 0.024) (Fig- ment, was graded on a scale of 0–4 for the statistical ure 1C). calculations, where 0 ϭ 0–3 erythrocytes/high-power Reappearance of CD19ϩ B cells was detected field (hpf) (normal), 1 ϭ 4–10 erythrocytes/hpf, 2 ϭ after a mean of 5 months from initiation of combination ϭ therapy with rituximab and CYC (median 4 months 11–20 erythrocytes/hpf, and 3 21–50 erythrocytes/hpf. [range 2–9 months]) (Table 3). Before rituximab treatment, hematuria was present in all Changes in serologic and complement levels. At patients: 4 had grade 1, 1 had grade 2, and 2 had grade study entry, all patients except patient 1 had detectable 3. At 6 months, 3 patients had no signs of hematuria ϭ anti-dsDNA antibodies, as determined by FEIA. A (grade 0), 2 had grade 1, and 2 had grade 2 (P 0.078). significant decrease in the anti-dsDNA antibody level None of the patients had detectable white blood cells in was seen at the 6-month evaluation (P ϭ 0.035) (Figure the urine before study treatment. Occurrence of granu- 1D). In 5 of the 7 patients, anti-C1q antibody levels were lar casts was noted in 3 patients at study entry, but at the above the reference range at study entry, and at 6 6-month followup, granular casts were no longer present months, these levels had significantly decreased in these in these patients (P ϭ 0.078). 5 patients (P ϭ 0.016) (Figure 2A). The mean level of albuminuria in a 24-hour urine 1268 GUNNARSSON ET AL

Figure 2. Changes from baseline evaluation to the 6-month followup for A, the anti-C1q antibody level (normal Ͻ16 units/ml), B, the C3 ءءء ;ϭ P Ͻ 0.01 ءء ;ϭ P Ͻ 0.05 ء .level (normal 0.5–1.2 gm/liter), C, the C4 level (normal 0.1–0.4 gm/liter), and D, the renal activity index ϭ P Ͻ 0.001. collection at study entry was 2.7 gm (range 0.2–5.9), and (see Tables 1 and 2). Table 2 shows the histopathology at 6 months, this value had decreased to 0.8 gm (range results at pretreatment and repeat renal biopsies. The 0.1–1.8; P ϭ 0.055). At study entry, the mean GFR was time from treatment to B cell repopulation is shown in 75 ml/minute (range 52–104), and at 6 months, it was 88 Table 3. ml/minute (range 54–115), with improvement in 5 of the Renal response and renal remission. At 6 months 7 patients (P ϭ 0.16). of followup, 3 patients had reached complete renal A repeat renal biopsy was performed in all pa- remission and 1 patient had a partial remission, accord- tients 3–12 months (median 7 months) after rituximab ing to the renal remission criteria described by Sfikakis treatment. One patient had 2 repeat biopsies, one at 3 et al (23). Three patients (patients 2, 4, and 5) improved months and the other at 12 months. Improvement in the but did not achieve the criteria for partial remission histopathologic findings, with transformation of WHO because of persistent albuminuria and hypoalbuminemia classes, was observed in all patients. The renal activity (Ͻ50% improvement). Applying the recently published index decreased significantly between the first and the response criteria for proliferative and membranous re- repeat biopsy (P ϭ 0.002) (Figure 2D). Interestingly, nal disease in SLE (33), an improvement in the GFR was only 2 patients had an increase in the chronicity index on seen in 2 patients, the GFR was unchanged in 4, and 1 repeat renal biopsy after rituximab treatment (P ϭ 0.77). patient had a worsened GFR. A complete response of This contrasts with the changes in chronicity indices the urinary protein was noted in 2 patients, a partial between the biopsy obtained at the time nephritis was response in 1, and no change in 4. Urinary sediment diagnosed and the pre-rituximab biopsy performed after improved in the 3 patients with detectable granular casts treatment failure with CYC. Between these 2 biopsies, at study entry, the other 4 patients (without granular which were obtained after a longer interval than for the casts) did not fulfill the criteria for active sediment at pre- and post-rituximab biopsies, an increase in the renal study entry, since white blood cells were absent in all chronicity index was seen in all but 1 patient (P ϭ 0.015) (Table 4). RITUXIMAB TREATMENT OF CYC-RESISTANT PROLIFERATIVE LUPUS NEPHRITIS 1269

Table 4. Renal response and adverse events at 6 months* Patient GFR Proteinuria Urinary sediment Adverse events 1 NC NC –† Photosensitive eruption 2 Improved NC –† Herpes zoster (limited) 3 NC Partial response Improved None 4 NC NC –† None 5 Worsened NC Improved None 6 Improved Complete response –† Neutropenic fever‡ 7 NC Complete response Improved Urinary tract infection * Proteinuria values were calculated from the 24-hour urinary albumin determination. GFR ϭ glomerular filtration rate; NC ϭ no change. † Urinary sediment was inactive at study entry. ‡ Onset after second infusion of cyclophosphamide.

Lymphocyte subpopulations in renal tissue. pheral blood. In animal studies, treatment with mono- Lymphocyte subpopulations were mainly present in the clonal antibodies against CD20 resulted in B cell deple- renal interstitium, and only occasional cells were found tion from the peripheral lymph nodes and bone marrow in the glomeruli, vessels, or tubuli (data not shown). In as well as the peripheral blood (34). However, despite patient 1, who had 2 repeat renal biopsies performed, a depletion of B cells from the circulation, lymph nodes, decrease in the numbers of all lymphocyte subpopula- and peritoneal cavity, a relative organ-specific resistance tions was noted in the biopsy performed at 3 months, to anti-CD20 therapy affecting marginal-zone compart- with the numbers increasing again in the biopsy per- ments, germinal centers, and Peyer’s patches was shown formed at 12 months (after repopulation of peripheral in mice (37). blood B lymphocytes). In the other 5 patients evaluated, Our study is the first to demonstrate that in most all of whom had regained peripheral blood B lympho- of the patients who had detectable CD20ϩ cells in renal cytes at the time of the second renal biopsy, a slight tissue, a reduction of these cells was seen after treat- reduction in CD3 and CD4 cells was noted in 3 of the 5 ment. However, in all but 1 patient, repopulation of biopsy samples. CD20 cells decreased in 2 of these 5 peripheral blood B cells had occurred at the time of patients, but no differences were observed for CD8, repeated biopsy. The findings in patient 1, who had CD79a, or CD38 cells (Table 3). biopsies performed at the time of B cell depletion as well Adverse events related to treatment. The adverse as repopulation, showing an initial disappearance and events observed during the study are presented in Table 4. then a recurrence of B cells, strongly indicate that B cells are temporarily depleted from the renal tissue after DISCUSSION rituximab treatment. In addition, a simultaneous reduc- ϩ ϩ Treatment with rituximab in combination with tion or disappearance of CD3 and CD4 lymphocyte CYC was found to be effective and safe in the 7 patients subpopulations was noted in a majority of the patients. with therapy-resistant proliferative lupus nephritis eval- Interestingly, Sfikakis et al (23) recently reported a uated in the present study. Improvement was achieved in reduction in T cell subsets as well as T helper cell all patients at 6 months of followup, both with regard to activation after rituximab treatment, thus demonstrating clinical variables and with regard to renal histopathology that B cell depletion not only affects B cell populations on repeat biopsies. Despite the fact that no treatment but may have other regulatory effects on mediators of other than glucocorticoids was given during the followup inflammation and functions of the immune system. period, a reduction in the use of prednisolone was Serum levels of immunoglobulins are generally achieved in most patients. maintained after rituximab therapy, which further sug- The chimeric monoclonal antibody rituximab gests that B lymphocytes in the central lymphoid organs binds to the CD20 epitope that is present on all mature are not eliminated completely and that long-lived B lymphocytes, and this drug has been extensively plasma cells may maintain antibody production in the studied in the treatment of B cell lymphomas (34–36). bone marrow. In studies of patients with rheumatoid Rituximab is capable of inducing cell death as well as a arthritis, rituximab treatment was associated with a complete depletion of B lymphocytes from the peri- decrease in levels of rheumatoid factor; however, levels 1270 GUNNARSSON ET AL

of total serum immunoglobulin did not change substan- deficient knockout mice, demonstrating that comple- tially (6). This finding was further confirmed in a study ment activation is fundamental for rituximab efficacy by Cambridge et al (38), in which decreases in both (42). In the present study, no relation to the initial rheumatoid factor and anti–cyclic citrullinated peptide complement levels and renal response could be demon- titers were seen, while the general levels of immuno- strated (data not shown). The exact mechanism of action globulins were less affected. The same phenomenon was in the treatment of lupus patients has not been resolved, seen in a patient with Wegener’s granulomatosis, show- but it seems likely that different mechanisms of B cell ing a decreased titer of cytoplasmic antineutrophil cyto- depletion may be operative. Further studies addressing plasmic antibodies after rituximab treatment (8,10). this issue are important in understanding the modes of Consistent with these findings, a significant decrease in action of rituximab. anti-dsDNA antibody levels was noted in the present Although a number of reports have described the study, as also shown by other investigators (24), provid- clinical effects of rituximab in patients with lupus ne- ing additional evidence that autoreactive B lymphocytes phritis, almost no data regarding repeat renal biopsies may be targeted by this combination therapy with some have been published to date. We previously demon- degree of selectivity. Our study is also the first to strated that patients with lupus nephritis may still have demonstrate a decrease in levels of anti-C1q antibodies, histopathologically active disease after immunosuppres- which are of special interest since they are associated sive treatment, despite apparent clinical improvement with proliferative lupus nephritis and renal disease ac- (25). Therefore, to identify patients with ongoing renal tivity (39,40). inflammation, the performance of repeat renal biopsies Rituximab may induce B cell depletion by several may be fundamental. In this patient cohort, the his- mechanisms, including induction of apoptosis (41), acti- topathologic results, while favorable overall, allowed us vation of complement, which has been implicated in to more accurately distinguish between the excellent certain settings (34,42), and antibody-dependent cell- response seen in 5 of the 7 patients and the more modest mediated cytotoxicity (34). Several factors may influence histologic improvements in the remaining 2 patients. the individual response to treatment, and serum concen- The patients with a less favorable histopathologic out- trations of rituximab have been shown to influence the come could not be identified through use of the recently variability in B cell depletion (43), although it was published ACR response criteria for proliferative and administered in a low-dose regimen and without CYC. membranous renal disease in SLE clinical trials (33), The data on B cell depletion obtained in the present and this further strengthens the importance of perform- study show that patients who had the shortest duration ing repeat biopsies in the evaluation of renal response. of B cell depletion (2 months) showed a trend toward a Our finding that renal chronicity remained relatively less favorable histopathologic outcome (WHO classes V unchanged from that in pretreatment biopsies suggests and IV/V) compared with patients who had more long- that rituximab therapy may prevent the development of standing B cell depletion times. Since only 7 patients irreversible organ damage, at least over the short term. were studied, it is not possible to draw any firm conclu- We conclude that rituximab therapy in patients sions from this finding, but similar observations of short with treatment-resistant proliferative lupus nephritis was B cell depletion times and unresponsiveness to treat- effective and well tolerated. For lupus nephritis patients ment have been described by others (23). Interestingly, a who fail to respond to conventional immunosuppressive correlation between short B cell depletion times and therapy including CYC, combined treatment with ritux- high numbers of circulating B cells before treatment has imab and CYC can be very effective and constitutes a also been shown in a larger patient population (44). promising new treatment possibility. Thus, the total levels of circulating B cells may influence the B cell depletion time and thereby possibly affect the ACKNOWLEDGMENTS therapeutic outcome. The importance of Fc␥ receptor IIIa genotype in We thank Eva Jemseby for management of blood samples and Eleanor Gullstro¨m for assistance with the pa- B cell depletion has recently been addressed in lupus tients. patients treated with rituximab, showing that patients carrying a high-affinity V allele had a greater depletion AUTHOR CONTRIBUTIONS of B cells than did patients with a low-affinity genotype Dr. Gunnarsson had full access to all of the data in the study (43). In animal models, the activity of rituximab was and takes responsibility for the integrity of the data and the accuracy completely abolished when administered to C1q- of the data analysis. RITUXIMAB TREATMENT OF CYC-RESISTANT PROLIFERATIVE LUPUS NEPHRITIS 1271

Study design. Gunnarsson, van Vollenhoven. berg DA. An open study of B lymphocyte depletion in systemic Acquisition of data. Gunnarsson, Jońsdo´ttir, Jacobson, Henriksson. lupus erythematosus. Arthritis Rheum 2002;46:2673–7. Analysis and interpretation of data. Gunnarsson, Sundelin, Jońsdo´ttir, 18. Ten Cate R, Smiers FJ, Bredius RG, Lankester AC, van Suijle- Jacobson, van Vollenhoven. kom-Smit LW, Huizinga TW, et al. Anti-CD20 monoclonal anti- Manuscript preparation. Gunnarsson, Sundelin, Jońsdo´ttir, Jacobson, body (rituximab) for refractory autoimmune thrombocytopenia in van Vollenhoven. a girl with systemic lupus erythematosus [letter]. Rheumatology Statistical analysis. Gunnarsson. (Oxford) 2004;43:244. 19. Van Vollenhoven RF, Gunnarsson I, Welin-Henriksson E, Sun- delin B, Osterborg A, Jacobson SH, et al. Biopsy-verified response REFERENCES of severe lupus nephritis to treatment with rituximab (anti-CD20 monoclonal antibody) plus cyclophosphamide after biopsy-docu- 1. Boumpas D, Austin HA III, Vaughn E, Klippel J, Steinberg A, mented failure to respond to cyclophosphamide alone. Scand Yarboro C, et al. Controlled trial of pulse methylprednisolone J Rheumatol 2004;33:1–6. versus two regimens of pulse cyclophosphamide in severe lupus 20. Gottenberg JE, Guillevin L, Lambotte O, Combe B, Allanore Y, nephritis. Lancet 1992;340:741–5. Cantagrel A, et al. Tolerance and short term efficacy of rituximab 2. Steinberg A, Kaltreider B, Staples P, Goetzl E, Talal N, Decker J. in 43 patients with systemic autoimmune diseases. Ann Rheum Dis Cyclophosphamide in lupus nephritis: a controlled trial. Ann 2005;64:913–20. Intern Med 1971;75:165–71. 21. Anolik JH, Barnard J, Cappione A, Pugh-Bernard AE, Felgar RE, 3. Balow JE, Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Looney RJ, et al. Rituximab improves peripheral B cell abnormal- Klippel JH, et al. Effect of treatment on the evolution of renal ities in human systemic lupus erythematosus. Arthritis Rheum abnormalities in lupus nephritis. N Engl J Med 1984;311:491–5. 2004;50:3580–90. 4. Austin HA III, Klippel JH, Balow JE, le Riche NG, Steinberg AD, 22. Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend Plotz PH, et al. Therapy of lupus nephritis: controlled trial of LJ, et al. B cell depletion as a novel treatment for systemic lupus prednisone and cytotoxic drugs. N Engl J Med 1986;314:614–9. erythematosus: a phase I/II dose-escalation trial of rituximab. 5. Ginzler EM, Dooley MA, Aranow C, Kim MY, Buyon J, Merrill Arthritis Rheum 2004;50:2580–9. JT, et al. Mycophenolate mofetil or intravenous cyclophosphamide 23. Sfikakis PP, Boletis JN, Lionaki S, Vigklis V, Fragiadakai KG, for lupus nephritis. N Engl J Med 2005;353:2219–28. Iniotaki A, et al. Remission of proliferative lupus nephritis follow- 6. Edwards JC, Cambridge G. Sustained improvement in rheumatoid ing B cell depletion therapy is preceded by down-regulation of the arthritis following a protocol designed to deplete B lymphocytes. T cell costimulatory molecule CD40 ligand: an open-label trial. Rheumatology (Oxford) 2001;40:205–11. Arthritis Rheum 2005;52:501–13. 7. Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, 24. Leandro MJ, Cambridge G, Edwards JC, Ehrenstein MR, Isen- Emery P, Close DR, et al. Efficacy of B-cell–targeted therapy with berg DA. B-cell depletion in the treatment of patients with rituximab in patients with rheumatoid arthritis. N Engl J Med systemic lupus erythematosus: a longitudinal analysis of 24 pa- 2004;350:2572–81. tients. Rheumatology (Oxford) 2005;44:1542–5. 8. Specks U, Fervenza FC, McDonald TJ, Hogan MC. Response of 25. Gunnarsson I, Sundelin B, Heimburger M, Forslid J, van Vollen- Wegener’s granulomatosis to anti-CD-20 chimeric monoclonal hoven R, Lundberg IE, et al. Repeated renal biopsy in prolifera- antibody therapy. Arthritis Rheum 2001;44:2836–40. tive lupus nephritis: predictive role of serum C1q and albuminuria. 9. Eriksson P. Nine patients with anti-neutrophil cytoplasmic anti- J Rheumatol 2002;29:693–9. body-positive vasculitis successfully treated with rituximab. J In- 26. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield tern Med 2005;257:540–8. NF, et al. The 1982 revised criteria for the classification of systemic 10. Keogh KA, Ytterberg SR, Fervenza FC, Kimberly A, Carlson A, lupus erythematosus. Arthritis Rheum 1982;25:1271–7. Schroeder DR, et al. Rituximab for refractory Wegener’s granu- 27. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang DH, lomatosis. Am J Respir Crit Care Med 2006;173:180–7. and the Committee on Prognosis Studies in SLE. Derivation of the 11. Omdal R, Wildhagen K, Hansen T, Gunnarsson R, Kristoffersen SLEDAI: a disease activity index for lupus patients. Arthritis G. Anti-CD20 therapy of treatment-resistant Wegener’s granulo- Rheum 1992;35:630–40. matosis: favourable but temporary response. Scand J Rheumatol 28. Hay EM, Bacon PA, Gordon C, Isenberg DA, Maddison P, Snaith 2005;34:229–32. ML, et al. The BILAG index: a reliable and valid instrument for 12. Pijpe J, van Imhoff GW, Spijkervet FK, Roodenburg JL, Wolbink measuring clinical disease activity in systemic lupus erythemato- GJ, Mansour K, et al. Rituximab treatment in patients with sus. QJM 1993;86:447–58. primary Sjo¨gren’s syndrome: an open-label phase II study. Arthri- 29. Martensson U, Thiel S, Jensenius J, Sjoholm A. Human autoan- tis Rheum 2005;52:2740–50. tibodies against C1q: lack of cross-reactivity with the collectins 13. Anolik JH, Campbell D, Felgar R, Rosenblatt J, Young F, Looney mannan-binding protein, lung surfactant protein A and bovine RJ. B lymphocyte depletion in the treatment of systemic lupus conglutin. Scand J Immunol 1996;43:314–20. (SLE): phase I/II trial of rituximab (Rituxan) in SLE [abstract]. 30. Laurell AB, Martensson U, Sjoholm AG. The development of Arthritis Rheum 2002;46:S289. simple tests for C1q, C1r, C1s and C2, and the determination of 14. Fra GP, Avanzi GC, Bartoli E. Remission of refractory lupus properdin. Stuttgart: Georg Thieme Publishers; 1978. nephritis with a protocol including rituximab. Lupus 2003;12: 31. Churg J, Bernstein J, Glassock RJ. Renal disease: classification 783–7. and atlas of glomerular diseases. 2nd ed. New York: Igaku-Shoin; 15. Weide R, Heymanns J, Pandorf A, Koppler H. Successful long- 1995. term treatment of systemic lupus erythematosus with rituximab 32. Austin HA III, Muenz LR, Joyce KM, Antonovych TA, Kullick maintenance therapy. Lupus 2003;12:779–82. ME, Klippel JH, et al. Prognostic factors in lupus nephritis: 16. Perotta S, Locatelli F, La Manna A, Cennamo L, De Stefano P, contribution of renal histologic data. Am J Med 1983;75:382–91. Nobili B. Anti-CD20 monoclonal antibody (rituximab) for life- 33. Renal Disease Subcommittee of the American College of Rheu- threatening autoimmune haemolytic anaemia in a patient with matology Ad Hoc Committee on Systemic Lupus Erythematosus systemic lupus erythematosus. Br J Haematol 2002;116:465–7. Response Criteria. The American College of Rheumatology cri- 17. Leandro MJ, Edwards JC, Cambridge G, Ehrenstein MR, Isen- teria for proliferative and membranous renal disease in systemic 1272 GUNNARSSON ET AL

lupus erythematosus clinical trials. Arthritis Rheum 2006;54: patients with active systemic lupus erythematosus. Br J Rheumatol 421–32. 1997;36:32–7. 34. Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab 40. Marto N, Bertolaccini M, Calabuig E, Hughes G, Khamashta M. R, et al. Depletion of B cells in vivo by a chimeric mouse human Anti-C1q antibodies in nephritis: correlation between serum titres monoclonal antibody to CD20. Blood 1994;83:435–45. and renal disease activity and positive predictive value in systemic 35. Maloney DG. Mechanism of action of rituximab. Anticancer lupus nephritis. Ann Rheum Dis 2005;64:444–8. Drugs 2001;12 Suppl 2:S1–4. 41. Shan D, Ledbetter JA, Press OW. Signaling events involved in 36. Davis TA, White CA, Grillo-Lopez AJ, Velasquez WS, Link B, anti-CD20-induced apoptosis of malignant human B cells. Cancer Maloney DG, et al. Single-agent monoclonal antibody efficacy in Immunol Immunother 2000;48:673–83. bulky non-Hodgkin’s lymphoma: results of a phase II trial of 42. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani rituximab. J Clin Oncol 1999:1851–7. 37. Gong Q, Ou Q, Ye S, Lee WP, Cornelius J, Diehl L, et al. E, et al. Complement activation determines the therapeutic activ- Importance of cellular microenvironment and circulatory dynam- ity of rituximab in vivo. J Immunol 2003;171:1581–7. ics in B cell immunotherapy. J Immunol 2005;174:817–26. 43. Anolik JH, Campbell D, Felgar RE, Young F, Sanz I, Rosenblatt ␥ 38. Cambridge G, Leandro MJ, Edwards JC, Ehrenstein MR, Salden J, et al. The relationship of Fc RIIIa genotype to degree of B cell M, Bodman-Smith M, et al. Serologic changes following B lym- depletion by rituximab in the treatment of systemic lupus erythem- phocyte depletion therapy for rheumatoid arthritis. Arthritis atosus. Arthritis Rheum 2003;48:455–9. Rheum 2003;48:2146–54. 44. Vallerskog T, Gunnarsson I, Widhe M, Risselada A, Klareskog L, 39. Gunnarsson I, Ronnelid J, Huang Y, Rogberg S, Nilsson B, van Vollenhoven R, et al. Treatment with rituximab affects both Lundberg I, et al. Association between ongoing anti-C1q antibody the cellular and the humoral arm of the immune system in patients production in peripheral blood and proliferative nephritis in with SLE. Clin Immmunol 2007;122:62–74. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1273–1285 DOI 10.1002/art.22491 © 2007, American College of Rheumatology

The Clinical Continuum of Cryopyrinopathies

Novel CIAS1 Mutations in North American Patients and a New Cryopyrin Model

Ivona Aksentijevich,1 Christopher D. Putnam,2 Elaine F. Remmers,1 James L. Mueller,2 Julie Le,1 Richard D. Kolodner,2 Zachary Moak,1 Michael Chuang,1 Frances Austin,1 Raphaela Goldbach-Mansky,1 Hal M. Hoffman,2 and Daniel L. Kastner1

Objective. The cryopyrinopathies are a group of cryopyrin or involved in the caspase 1/interleukin-1␤ rare autoinflammatory disorders that are caused by signaling pathway. CIAS1 and other candidate genes mutations in CIAS1, encoding the cryopyrin protein. were sequenced, models of cryopyrin domains were However, cryopyrin mutations are found only in 50% of constructed using structurally homologous proteins as patients with clinically diagnosed cryopyrinopathies. templates, and disease-causing mutations were mapped. This study was undertaken to investigate the structural Results. Forty patients were mutation positive, effect of disease-causing mutations on cryopyrin, in and 7 novel mutations, V262A, C259W, L264F, V351L, order to gain better understanding of the impact of F443L, F523C, and Y563N, were found in 9 patients. No disease-associated mutations on protein function. mutations in any candidate genes were identified. Most Methods. CIAS1 We tested for mutations in 22 mutations mapped to an inner surface of the hexameric patients with neonatal-onset multisystem inflammatory ring in the cryopyrin model, consistent with the hypothe- disease/chronic infantile neurologic, cutaneous, articu- sis that the mutations disrupt a closed form of lar syndrome, 12 with Muckle-Wells syndrome (MWS), cryopyrin, thus potentiating inflammasome assembly. 18 with familial cold-induced autoinflammatory syn- Disease-causing mutations correlated with disease se- drome (FCAS), and 3 probands with MWS/FCAS. In a verity only for a subset of known mutations. subset of mutation-negative patients, we screened for Conclusion. Our modeling provides insight into mutations in proteins that are either homologous to potential molecular mechanisms by which cryopyrin mutations can inappropriately activate an inflamma- Supported by the National Institute of Arthritis and Muscu- tory response. A significant number of patients who are loskeletal and Skin Diseases Intramural Research Program, the Na- tional Institute of Allergy and Infectious Diseases (grant R01-AI- clinically diagnosed as having cryopyrinopathies do not 52430), and the Ludwig Institute of Cancer Research. Dr. Putnam is have identifiable disease-associated mutations. recipient of a Postdoctoral Fellowship from the Damon Runyon Cancer Research Foundation and the Robert Black Charitable Trust. 1Ivona Aksentijevich, MD, Elaine F. Remmers, PhD, Julie The cryopyrinopathies (also known as cryopyrin- Le, BS, Zachary Moak, BS, Michael Chuang, BS, Frances Austin, BS, associated periodic syndromes [CAPS]) are rare autoin- Raphaela Goldbach-Mansky, MD, Daniel L. Kastner, MD, PhD: flammatory diseases that have been recently character- National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland; 2Christopher D. Putnam, PhD, James L. Mueller, ized at the molecular level with the identification of BS, Richard D. Kolodner, PhD, Hal M. Hoffman, MD: University of mutations in the gene CIAS1 (cold-induced autoinflam- California San Diego School of Medicine, La Jolla. matory syndrome 1). They include familial cold autoin- Drs. Aksentijevich and Putnam contributed equally to this work; Drs. Hoffman and Kastner contributed equally to this work. flammatory syndrome (FCAS), Muckle-Wells syndrome Dr. Hoffman has received consulting fees (more than (MWS), and neonatal-onset multisystem inflammatory $10,000) from Regeneron Pharmaceuticals. Address correspondence and reprint requests to Ivona Ak- disease (NOMID; also known as chronic infantile neu- sentijevich, MD, Genetics and Genomics Branch, National Institutes rologic, cutaneous, articular syndrome [CINCA syn- of Health, Building 9, Room 1N132, 9000 Rockville Pike, Bethesda, drome]). Although these disorders have been classified MD 20892-1820. E-mail: [email protected]. Submitted for publication September 26, 2006; accepted in as distinct entities, patients often present with overlap- revised form December 21, 2006. ping symptoms that include fevers, urticarial skin rash,

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varying degrees of arthralgia/arthritis, neutrophil- NACHT-associated domain). Evolutionary analysis has mediated inflammation, and an intense acute-phase shown that cryopyrin is highly conserved among pri- response. In general, patients with NOMID/CINCA mates and other mammals, including the mouse. With syndrome (MIM no. 607115) have the most severe the exceptions of V198, V262, and M662, all amino acids clinical phenotype, patients with FCAS (MIM no. mutated in CAPS patients are conserved in the primate 120100) have the least severe phenotype, and patients and mouse proteins, suggesting that the functions of with MWS (MIM no. 191100) present with an interme- these genes have been under strong evolutionary selec- diate phenotype (1). Several patients have overlapping tion (9). phenotypes, e.g., MWS/FCAS and MWS/NOMID. To date, Ͼ40 known disease-associated muta- NOMID/CINCA syndrome is characterized by tions have been reported, and most of them are found in nearly continuous symptoms of inflammation presenting exon 3 of CIAS1, encoding the NACHT subdomains and first during the neonatal period or early infancy, with a NAD domain (10). Recently, the first non–exon 3 migratory and nonpruritic urticaria-like rash and fever. mutations in patients with NOMID/CINCA syndrome Later, patients develop symptoms of central nervous (G755R, G755A, and Y859C) were identified (11–13). system (CNS) inflammation, including chronic aseptic All but 1 of the known CIAS1 mutations are missense meningitis, increased intracranial pressure, cerebral at- changes, suggesting that loss-of-function mutations have rophy, seizures, and sometimes mental retardation. Ap- a different phenotype. Saito and coworkers described a proximately 80% of patients develop progressive senso- NOMID/CINCA syndrome patient with somatic mosa- rineural hearing loss and ocular changes, including icism for a mutation in exon 3 of CIAS1, with varying conjunctivitis, anterior and posterior uveitis, optic disc frequencies of the mutant allele in whole blood and edema, and optic nerve atrophy with progressive vision leukocytes. Those authors proposed that somatic mosa- loss. Another feature of the disease is a highly charac- icism may account for at least some of the undetected teristic and sometimes disabling arthropathy caused by mutations in CIAS1 mutation–negative patients (14). overgrowth of the patella and epiphyses of the long Cryopyrin is part of a multiprotein complex bones. Approximately 20% of patients with NOMID/ termed the inflammasome (15,16) that plays a critical CINCA syndrome die before reaching adulthood (2). role in the regulation of intracellular host defense in MWS usually presents with more episodic attacks response to bacterial toxins and RNA, small antiviral of inflammation associated with a generalized urticaria- compounds (imiquimod [R837] and resiquimod [R848]), like rash, fever, malaise, arthralgia, and progressive and uric acid crystals. Cryopyrin is essential for activa- hearing loss. Twenty-five percent of patients also de- tion of caspase 1 and processing of interleukin-1␤ (IL- velop serum amyloid A amyloidosis (3,4). FCAS (also 1␤) and IL-18 cytokines (17–20). The mechanism by known as familial cold urticaria) is characterized by which cryopyrin mutations cause inflammatory disease is cold-induced, daylong episodes of fever associated with still elusive, although in vitro studies suggest that the rash, arthralgia, headaches, and less frequently conjunc- mutant protein is constitutively active (21,22). Func- tivitis, but without other signs of CNS inflammation. tional studies of macrophages from NOMID and MWS Symptoms begin within the first 6 months of life, and patients show a constitutive increase in IL-1␤ and IL-18 generalized cold exposure is the predominant trigger of secretion (7,16,23). As a result of this finding a new the inflammatory attacks (1). therapy targeting the IL-1␤ pathway, the IL-1 receptor The cryopyrinopathies are caused by dominantly antagonist anakinra, has been initiated in patients with inherited or de novo mutations in CIAS1, a gene that these disorders (24), and has caused a rapid and dra- encodes a protein called cryopyrin (also known as matic improvement of symptoms and a decrease in levels PYPAF1 or NALP3) (3–7). Cryopyrin belongs to the of acute-phase reactants (12,25–31). family of NLR (nucleotide-binding domain, leucine-rich In this study we analyzed 55 NOMID/MWS/ repeat) proteins (also known as CATERPILLER, FCAS probands who have not been previously reported, NALP, or PYPAF), with 14 identified members (8). and identified 7 novel mutations. Fifteen patients were These proteins have 3 common structural domains: PYD mutation negative and the rest were carriers for previ- (an N-terminal pyrin domain), a central NACHT do- ously reported mutations. We also summarized the main (also known as NBD or NOD [nucleotide-binding results of genetic testing for CIAS1 mutations in the or oligomerization domain]), and a C-terminal leucine- North American cohort of 195 patients clinically diag- rich repeat (LRR) domain. The NACHT domain con- nosed as having NOMID/MWS/FCAS. In the absence of sists of 2 or 3 NACHT subdomains and NAD (a an experimentally determined cryopyrin structure, we CLINICAL CONTINUUM OF CRYOPYRINOPATHIES 1275

Table 1. Newly identified CIAS1 mutations and clinical symptoms of patients with NOMID/CINCA syndrome, MWS, and FCAS* Cold- Deforming Diagnosis/age triggering Skin CNS Joint arthro- CIAS1 Control (years)/sex factor rash involvement† involvement‡ pathy mutation chromosomes FCAS/57/M Yes Yes No Yes No C259W 0/196 (777 TϾG) NOMID/11/F No Yes Yes Yes No V262A 0/950 (785 TϾC) NOMID/1.5/F No Yes Yes Yes No L264F 0/924 (790 CϾT) NOMID/9/F No Yes Yes Yes Yes L264F 0/924 (790 CϾT) NOMID/13/F No Yes Yes Yes No V351L 0/924 (1051 GϾC) NOMID/16/M No Yes Yes Yes No F443L 0/930 (1329 CϾG) MWS/7/M No Yes No Yes No F523C 0/296 (1568 TϾG) FCAS/62/F Yes Yes No Yes No Y563N 0/188 (1687 TϾA) FCAS/40/F Yes Yes No Yes No Y563N 0/188 (1687 TϾA) * NOMID/CINCA syndrome ϭ neonatal-onset multisystem inflammatory disease/chronic infantile neurologic, cutaneous articular syndrome; MWS ϭ Muckle-Wells syndrome; FCAS ϭ familial cold-induced autoinflammatory syndrome; CNS ϭ central nervous system. † Eye involvement and/or sensorineural hearing loss and/or chronic meningitis. ‡ Arthralgia and/or arthritis. used modeling to explore the nature of CAPS-associated African American patients in the New York Cancer Project mutations on cryopyrin. panel). Sequence analysis and modeling of 3-dimensional structure. Sequences of exon 3 of cryopyrin were aligned with PATIENTS AND METHODS NACHT and NAD homologies of NALP1, NALP6, NALP10, and NALP12, using Sequoia (33). The consensus secondary Patients. Patients included in this study fulfilled clini- structure was determined by comparing the predictions for all cal criteria for NOMID/CINCA syndrome, MWS, or FCAS 5 proteins using multiple programs (34–37). Models were (31,32). For participation in the study all patients (or parents generated by threading the cryopyrin sequence onto a known or legal guardians if the patient was a minor child) provided structure with Swiss-Model (38). Models for both the pyrin written informed consent as approved by the respective institutional review boards. Among the NOMID/CINCA syndrome domain and the LRR domain were generated from known patients, 10 were Caucasian, 10 were Hispanic, 1 was African sequence homologs; however, no structures were available for American, and 1 was Asian. All FCAS and MWS patients were obvious homologs of the NACHT and NAD domains of of Caucasian ancestry. cryopyrin. Using threading algorithms (39,40), multiple mem- Molecular analysis. All coding exons of CIAS1 were bers of the AAAϩ ATPase family were top hits for the first screened for mutations, using primers that have been described NACHT subdomain for cryopyrin, NALP2, NALP3, CARD4, previously (5,7). Mutation detection was performed by fluo- and the mouse MHCII transactivator. These models were the rescent sequencing with BigDye Terminator version 3.1 chem- basis of cycles of automated and manual threading and struc- istry (Applied Biosystems, Foster City, CA) on an ABI 3100 or ture minimization with Swiss-Model and the Swiss 3700 automated sequencer. The sequencing data were ana- PDB Viewer. All molecular images were generated using lyzed with Sequencher 4.5 (Gene Codes, Ann Arbor, MI). The PyMol (41). allele frequencies of novel mutations were evaluated in a panel of 376 or 750 Caucasian control DNA samples from the North American Rheumatoid Arthritis Collection, using mass spec- RESULTS trometry (homogeneous MassExtend assay; Sequenom, San Diego, CA) or in a panel of 100 normal control DNA samples Novel CIAS1 mutations. We analyzed 22 patients from a North American blood bank (5), by automated se- with NOMID/CINCA syndrome, 12 with MWS, 18 with quencing. The allele frequencies for novel single-nucleotide FCAS, and 3 probands with MWS/FCAS overlap, for polymorphisms (SNPs) identified in the candidate genes were evaluated in different panels of control samples (from Cauca- mutations in CIAS1. The initial screening included se- sian patients in the North American Rheumatoid Arthritis quencing the 1,753-bp exon 3 of CIAS1, where most of Collection, from the Coriell human diversity panel, and from the previously reported mutations were found. The exon 1276 AKSENTIJEVICH ET AL

3 mutation–negative patients were screened for muta- Ten patients, including 1 with NOMID/CINCA tions in the other 8 exons of CIAS1. syndrome, were found to carry the Q703K (c.2107CϾA) We identified 7 novel CIAS1 mutations, occur- missense change (allele frequency 0.04). We evaluated ring in 5 patients with NOMID/CINCA syndrome, 1 whether Q703K may be a novel disease-associated mu- with MWS, and 3 with FCAS (Table 1). Seven NOMID/ tation with variable expressivity. We found 1 homozy- CINCA syndrome patients were carriers for previously gous subject and 34 carriers for the 2107CϾA nucleotide described CIAS1 mutations (4 patients with the D303N transversion in a panel of 374 control Caucasian DNA mutation and 1 patient each with the T436N, G569R, samples (allele frequency 0.05). and L632F mutations). Ten patients were mutation We investigated the allele frequency for one of negative. Their clinical presentations were similar to the earliest reported CIAS1 mutations, V198M, in a those of mutation-positive patients, and they all had an large collection of DNA samples from Caucasian sub- excellent response to treatment with anakinra. Eight jects. This mutation was originally identified in an FCAS MWS probands had previously described mutations, family that was later found to also have an E525K including 2 with R260W and 6 with T348M. Three MWS mutation (5), and was subsequently reported in Span- patients were mutation negative. Three FCAS patients ish (42) and French (4) FCAS families, in a British were carriers for 2 novel mutations (Table 1), while 2 family with MWS and 2 British patients with unchar- lacked mutations. The remaining 13 FCAS probands acterized periodic fevers (3), and in 3 German pa- were carriers for L353P (8 patients), M659K (1 patient), tients with atypical periodic fevers (43). V198M has A439V (2 patients), and L305P (2 patients). Three been considered to be a reduced-penetrance CIAS1 patients had MWS/FCAS overlap phenotypes; 2 carried mutation because it was found in asymptomatic family R260W and 1 had D303N. members and in control DNA samples. We did not Potential reduced-penetrance CIAS1 mutations. identify V198M in any of 125 patients with clinically In a family presenting with an atypical autoinflammatory uncharacterized autoinflammatory disease. We syndrome we identified the R488K mutation, which was screened a panel of 742 Caucasian DNA samples and previously reported in a Spanish family with FCAS-like found 11 carriers among 1,484 control chromosomes symptoms (42). Further investigation of the family in the (allele frequency 0.0074). present study revealed 2 asymptomatic family members Candidate gene screening in mutation-negative who were carriers for R488K, supporting the notion that NOMID/CINCA syndrome patients. In our entire cohort this is a reduced-penetrance CIAS1 mutation as proof NOMID/CINCA syndrome patients, 49% were neg- posed earlier (42). We screened 740 Caucasian control ative for CIAS1 mutations. Based on the report by Saito chromosomes and found R488K in 1 of 740; we thus et al (14), we considered the possibility that some estimated an R488K allele frequency of 0.0014 in the mutations might be missed due to undetected somatic healthy Caucasian population. mosaicism. However, we found no evidence of somatic The National Institutes of Health (NIH) Clinical mosaicism in our CIAS1 mutation–negative patients; Research Center and the Department of Pediatrics at therefore, we believe it is an uncommon mechanism for the University of California San Diego are 2 major US the cryopyrinopathies (13). referral centers for patients with various autoinflamma- In a subset of CIAS1 mutation–negative tory diseases, including cryopyrinopathies, familial Med- NOMID/CINCA syndrome patients, we further iterranean fever (MIM no. 249100), tumor necrosis screened for mutations in the NACHT domains of 5 factor receptor–associated periodic syndrome (MIM no. NALP proteins that are either highly homologous to 1420680), hyperimmunoglobulinemia D with periodic cryopyrin or have similar tissue expression profiles. We fever syndrome (MIM no. 260920), and the syndrome of also performed mutational analysis of other genes that pyogenic arthritis with pyoderma gangrenosum and acne encode proteins that are either described as part of the (MIM no. 604416). We screened 125 unrelated patients inflammasome protein complex or are involved in regu- with clinically uncharacterized autoinflammatory dis- lation of the caspase 1/IL-1␤ signaling pathway (Table ease for mutations in exon 3 of CIAS1. This was a cohort 2). We identified many missense substitutions in these of patients who were referred to the NIH periodic fever genes, including 10 novel nonsynonymous nucleotide clinic with a history of recurrent fevers with or without changes, but all of them proved to be polymorphisms localized inflammation persisting for Ͼ6 months, and (Table 2). SNPs identified in African American patients who tested negative for mutations in the clinically indi- with NOMID/CINCA syndrome were evaluated in sam- cated periodic fever genes. ples from African American controls. LNCLCNIUMO CRYOPYRINOPATHIES OF CONTINUUM CLINICAL Table 2. Candidate genes screened for mutations in a subset of CIAS1 mutation–negative patients*

Nonsynonymous SNPs and deletions identified in RefSeq No. of NOMID/CINCA syndrome Candidate gene DNA† Exons screened patients patients‡ Allele frequencies of novel SNPs in control chromosomes§

NALP1/DEFCAP NM_033004.2 Exon 4 (1,705 bp) 7 T246S (rs11651595; c.737CϾG) I601F (c. 1801AϾT)¶ I601F: T allele frequency in 348 P1 samples ϭ 0.02 T allele frequency in 94 P2 samples ϭ 0.02 T allele frequency in 135 P3 samples ϭ 0.15 T782S (c.2345CϾG)¶ T782S: G allele frequency in 375 P1 samples ϭ 0.06 G allele frequency in 95 P2 samples ϭ 0.03 G allele frequency in 146 P3 samples ϭ 0.10 NALP2/PYPAF2 NM_017852.1 Exon 6 (1,567 bp) 7 T221M (rs17699678; c.662CϾT) R364K (rs4306647; c.1091 GϾA) T529A (c. 1585AϾG)¶ T529A: G allele frequency in 371 samples ϭ 0.23 G allele frequency in 89 P2 samples ϭ 0.10 G allele frequency in 134 P3 samples ϭ 0.16 NALP4/PYPAF4 NM_134444.3 Exon 3 (1,576 bp) 4 A144T (rs441827; c.430GϾA) E383D (rs17857373; c.1149GϾC) NALP6/PYPAF5 NM_138329.1 Exon 4 (1,756 bp) 7 M163L (rs6421985; c.487AϾC) P244A (c.730CϾG)¶ P244A: G allele frequency in 376 P1 samples ϭ 0.18 G allele frequency in 85 P2 samples ϭ 0.12 G allele frequency in 140 P3 samples ϭ 0.05 NALP10/PYNOD NM_176821.3 Exons 1–2 4 Nonsynonymous SNPs not identified NALP12/PYPAF7 NM_144687.1 Exon 3 (1,702 bp) 5 D142N (c.414GϾA)¶ D142N: A allele frequency in 371 P1 samples ϭ 0.002 A allele frequency in 95 P2 samples ϭ 0.01 A allele frequency in 143 P3 samples ϭ 0.04 F402L (c.1196CϾG)¶ F402L: G allele frequency in 363 P1 samples ϭ 0.07 CARD8/CARDINAL/TUCAN NM_014959 Exons 1–8 12 Ex1: C10X (rs2043211; c.30TϾA) Ex3: I68V (rs11881179; c.202AϾG) Ex2: c.126_127insAA¶ c.126_127insAA all frequency in 371 P1 samples ϭ 0.05 Ex4: A200T (c.598GϾA)¶ A200T: A allele frequency in 375 P1 samples ϭ 0.001 Ex4: P219R (c.656CϾG)¶ P219R: G allele frequency in 352 P1 samples ϭ 0.007 G allele frequency in 143 P3 samples ϭ 0.11 Ex5: P280L (c.839CϾT)¶ P280L: no carriers in 304 P1 samples no carriers in 94 P2 samples T allele frequency in 141 P3 samples ϭ 0.007 ICEBERG NM_021571.2 Exon 1 7 Nonsynonymous SNPs not identified IPAF/CARD12/NOD3 NM_021209.3 Exon 5 6 Nonsynonymous SNPs not identified IL1-RN NM_000577.3 Exons 1–5 5 Nonsynonymous SNPs not identified PSEUDO-ICE/COP NM_052889.2 Exons 1–3 10 Nonsynonymous SNPs not identified PYCARD/ASC NM_013258.3 Exons 1–3 9 Nonsynonymous SNPs not identified

* The following single-nucleotide polymorphisms (SNPs) were identified in African American patients with neonatal-onset multisystem inflammatory disease/chronic infantile neurologic, cutaneous articular syndrome (NOMID/CINCA syndrome): I601F and T782S (NALP1), D142N (NALP12), P219R and P280L (CARD8). SNPs P244A (NALP6), F402L (NALP12), and A200T, and c.126_127insAA (CARD8) were identified in Caucasian patients with NOMID/CINCA syndrome. † Uses a GenBank file as reference sequence (coding DNA).

‡ Using a dbSNP identifier as a reference; “c.” indicates a coding DNA sequence. 1277 §P1ϭ Caucasian control samples (376 samples); P2 ϭ Human Diversity Panel control samples (96 samples); P3 ϭ African American samples (150 samples). ¶ Novel nonsynonymous SNPs that were ruled out (proved to be polymorphisms) by screening control chromosomes. 1278 AKSENTIJEVICH ET AL

Figure 1. Sequence alignment of cryopyrin with NALP1, NALP6, NALP10, and NALP12 for most of exon 3 of cryopyrin encoding the NACHT and NACHT-associated domains. Asterisks below the alignments show positions with perfect identity. AAAϩ domain functional motifs that are identifiable in cryopyrin are shown in red. Disease-causing mutations are shown above the sequence; boxed mutations are those newly identified in this study. The consensus secondary structure prediction for all proteins from multiple prediction programs is depicted using bars for ␣-helices (␣1–␣22) and arrows for ␤-strands (␤1–␤6). Secondary structural elements shown in dark green correspond to the first subdomain of NACHT, those shown in light green correspond to the second (and possibly third) subdomain(s) of NACHT, and those shown in yellow-green correspond to the NACHT-associated domain.

Multiple sequence alignment of the NACHT do- CAPS-associated CIAS1 mutations on cryopyrin func- main of cryopyrin with homologous human proteins. tion, using a molecular modeling approach. The vast We investigated the potential molecular impact of majority of mutations underlying FCAS, MWS, and CLINICAL CONTINUUM OF CRYOPYRINOPATHIES 1279

NOMID are missense mutations in the NACHT and 4-helix bundles, and the secondary structural predictions NACHT-associated (NAD) domains of cryopyrin. Al- would be consistent with this (Figure 1), although the though there are several mutation clusters in exon 3 of possibility of inserted secondary structural elements in CIAS1, suggesting the presence of functionally impor- the NACHT family cannot be ruled out. Notably, the tant sites, there is no apparent correlation between pattern of disease-causing mutations affecting amino mutation cluster position and disease severity. For in- acids at conserved surfaces might continue outside of the stance, the mutation cluster adjacent to the conserved modeled regions. For example, the mutations causing ϩ Walker B motif (Mg2 -binding site) at residues 300–303 ␣-helix 12 amino acid substitutions (T436P, T436N, includes NOMID-, MWS-, and FCAS-associated muta- T436I, A439V, A439T, A439P, F443L [Figure 1]) are all tions (Figures 1 and 2A). spaced by 3 or 4 residues and would define one face of Phylogenetic studies have identified human this ␣-helix. NALP1, NALP6, NALP10, and NALP12 proteins as Most AAAϩ proteins assemble into functional being closely related to cryopyrin (9). Recent studies hexamers (47). To gain additional insight into the have shown that NALP1, NALP6, and NALP12 are disease-causing amino acid substitutions, we superim- expressed in hematopoietic tissue; similar to findings in posed the NACHT subdomain 1 model onto the struc- cryopyrin, overexpression of these proteins shows that ture of the HsIU chaperone, which is one of the AAAϩ they also regulate caspase 1 activity (8). Thus, we aligned proteins whose structure was experimentally determined the protein sequence of the cryopyrin NACHT domain in a hexamer assembly (48). Surprisingly, the surface with its most homologous human proteins, NALP1, containing most of the disease-causing amino acid sub- NALP6, NALP10, and NALP12, to evaluate whether stitutions in the monomer model mapped to a single the CAPS-associated mutations affect amino acids that inner surface of the assembled hexameric surface, which are conserved between these proteins (Figure 1). The would be consistent with the notion of functional mul- sequence alignment showed that only 5 CAPS- timerization of cryopyrin through the NACHT domain associated mutations affected highly conserved amino (Figure 2C). acid residues (C259W, G301D, D303N, A352V, and Close analysis of the NOMID mutations affecting L632F); 3 mutations were associated with the most the LRR domains, G755R and G755A, indicated that severe NOMID phenotype, while 2 were associated with these mutations may function similarly to NACHT do- the mild FCAS phenotype. main mutations, by inappropriately exposing LRRs to Molecular impact of CIAS1 mutations on protein assemble inflammasomes (Figure 2B). The internal function, determined based on predicted modeling for packing between leucine-rich repeats of the LRR do- cryopyrin. CIAS1 mutations (Figure 2A) were placed main forces every second helix to accommodate a side onto 3-dimensional structural models of human chain from an adjacent helix in a pocket lined by a cryopyrin threaded onto templates of homologous struc- glycine. Any other residue at the glycine position would tures (Figure 2B). The modeling of the N-terminal part be larger and would disrupt the LRR packing, poten- of the NACHT homology (NACHT subdomain 1) was tially kinking the arc of the LRR domain or generating possible because of the robust identification of this a flexible joint in the LRR at the repeat containing the portion of the NACHT homology as a member of the substitution. G755 is the first glycine in this position in AAAϩ family of proteins, and is consistent with previ- the LRR domain, and amino acid substitutions may ous models generated for this portion of the NACHT decouple the orientation of the LRR domain from the domain (44,45). These models demonstrate that the closed/open status of the NACHT domains (Figure 2D). majority of mutations that can be placed in the model The fact that mutations of equivalent glycines in later are located on one face of subdomain 1; for most AAAϩ repeats have not been observed may suggest that they proteins, this face is where additional domains from the may be too disruptive to the LRR binding surface or same protein can interact with subdomain 1 (46). This may not displace enough of the surface to direct inflam- intraprotein domain–domain interaction could result in masome assembly. a closed or nonfunctional state of a modular protein such as cryopyrin. DISCUSSION More C-terminal portions of the NACHT and NACHT-associated domains do not robustly identify The cryopyrinopathies represent a spectrum of any domain by sequence homology or threading; how- autoinflammatory diseases that are caused by mutations ever, the second NACHT of AAAϩ proteins is typically in a single gene, and in which severity of the phenotype 1280 AKSENTIJEVICH ET AL

Figure 2. A, The domain and exon arrangements of cryopyrin displayed with the positions of point mutations implicated in familial cold-induced autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and other phenotypes. Domains are displayed as colored boxes; exons are numbered below and delineated by horizontal lines. Point mutations responsible for various clinical syndromes are displayed from the weakest phenotype (FCAS) on the light yellow portion of the gradient to the strongest phenotype (NOMID) on the red portion of the gradient. B, Three-dimensional models of the pyrin domain, subdomain 1 of NACHT, and the leucine-rich repeats (LRRs). Disease-causing mutations are displayed as spheres at the C␣ positions, color-coded as yellow for FCAS, orange for MWS, red for NOMID, and gray for other. For amino acid positions identified in multiple syndromes, the most severe phenotype is indicated. The pyrin domain of cryopyrin was threaded onto the pyrin domain of human ASC (Protein Data Bank [PDB] code no. 1ucp [52]). The first domain of NACHT was generated using the structure of apoptotic protease–activating factor 1 (APAF1; PDB code no. 1z6t [53]). APAF1 and cryopyrin deviate between ␣5 and ␤4 and were modeled using consensus secondary structure predictions. This region includes the positions of the G326E and S331R mutations (red spheres that do not map to the common mutation surface). The LRR domains were modeled using the porcine ribonuclease inhibitor structure (PDB code no. 2bnh [54]). C, Superimposition of the model for NACHT subdomain 1 on the hexameric assembly of the AAAϩ ATPase HslU (PDB code no. 1doo [48]). Most of the mutations (colored as in B) map to the inner, concave surface of the hexameric ring. In other AAAϩ ATPases, this surface is where AAAϩ subdomains 2 and 3 tend to contact subdomain 1 (46). As in other AAAϩ-ATPases, the hexameric assembly places nucleotide-binding sites at the interfaces between adjacent protomers. D, Location of the disease-causing mutations in cryopyrin, suggesting a model for inappropriate inflammasome assembly. In wild-type cryopyrin, opening of the protein to expose the pyrin and LRR domains is normally triggered due to an external stimulus. The open state of cryopyrin is presumably the active one that assembles a functional inflammasome. With mutations in the NACHT domain, the closed state is destabilized due to mutations at the interface of the hinge, tending to favor the activated and open state. The G755 mutations might similarly drive inflammasome assembly by producing a “kink” in the regular LRR structure that inappropriately exposes the LRR domains either to more readily open the structure or to become competent to directly assemble the inflammasome. CLINICAL CONTINUUM OF CRYOPYRINOPATHIES 1281

is most likely influenced by a number of other genetic 130 UK Caucasians, 0 of 150 Spanish Caucasians, and 2 and environmental modifiers. We report 7 novel of 48 Asian Indians (3,5,42), and thus have raised the NOMID/MWS/FCAS-causing mutations in exon 3 of question of whether V198M is a true disease-associated CIAS1. All of these mutations cause missense nucleotide mutation. R488K was not identified in any of 150 substitutions and have not been found in any of several Spanish control DNA samples; however, it was found in hundred control chromosomes. Thus, with this report 1 unaffected member of a Spanish FCAS family (42). In the total number of CIAS1 disease-associated mutations the current study, which is the largest survey of healthy is 56 (10). Caucasian controls to date, neither variant was found at Our entire NOMID/CINCA syndrome cohort the 1% allele frequency required to be considered includes 39 patients who fulfill clinical criteria for diag- formally as a polymorphism. Nevertheless, given that the nosis. In the past, we reported 8 mutation-positive and 9 V198M allele frequency approaches this level and that mutation-negative patients (7,49,50); herein we report we have recently observed a patient with V198M and an additional 12 mutation-positive and 10 mutation- periodic fever that did not respond to anakinra (Kastner negative cases. The mutation-negative patients were DL, et al: unpublished observations), we would ap- screened for mutations in all exons of CIAS1, thus ruling proach the clinical significance of this variant with out mutations in the coding part of cryopyrin as a cause caution. Considering that these substitutions have been of the disease. However, our mutation screening associated with nonspecific autoinflammatory pheno- method, although extensive, did not rule out the pres- types, in vitro functional assays may be useful in under- ence of mutations in the noncoding regulatory se- standing their pathogenic consequences. Based on the quences of CIAS1. Thus, CIAS1 mutations were identi- similar allele frequency of Q703K in patients and control fied in only 51% of cases (20 of 39), suggesting cohorts (0.04 versus 0.05; P ϭ 0.84), we conclude that significant genetic heterogeneity among patients with Q703K is unlikely to be pathogenic. NOMID/CINCA syndrome. The vast majority of mutations underlying FCAS, Similarly, 25% (3 of 12) of the probands in our MWS, and NOMID are dominant mutations. This is MWS cohort lacked mutations in the coding sequence of consistent with the notion that cryopyrinopathies are CIAS1 exons. In the remaining patients, we found 3 caused by gain-of-function mutations (21) and is quite known CIAS1 mutations (R260W, A352V, and T348M). different from the spectrum of frameshift and nonsense Interestingly, the prevalence of MWS appears to be mutations observed in diseases driven by loss of func- lower in North America than that reported in Europe. tion. We thus sought to understand the structural impact This may result from phenotype assignment preference of these activating mutations. Our modeling of the first in the disease continuum or the presence of larger NACHT subdomain yielded essentially similar results families with MWS in Europe and larger families with using 2 different templates (Cdc6p from Pyrobaculum FCAS in North America, resulting in a selection bias. aerophilum [Protein Data Bank code no. 1fnn] and Over the past few years we have identified 137 apoptotic protease–activating factor 1 [Protein Data FCAS patients who belong to 24 families. The vast Bank code no. 1z6t]). The findings were also consistent majority of them (114 patients from 12 families) are with results of previous studies (44,45) in that most of carriers for the L353P mutation. At least 3 of these the mutations in this subdomain, including C259W, families share a common ancestor, and 1 family has been V262A, L264F, and V351L identified in this study, fall confirmed by genotyping to be independent (51). Of the on one face of the monomer (Figure 2B). Mapping these remaining patients, 21 are carriers for the following mutants onto a model of a cryopyrin hexamer revealed CIAS1 mutations: C259W, L305P, A439V, E525K, the surprising result that a single, solvent-exposed sur- Y563N, E627G, and M659K. Two FCAS patients are face is affected (Figure 2C). This suggests that the mutation negative. primary impact of these mutations is to disrupt a surface In this study, we evaluated allele frequencies for and differs from previous proposals that mutations 3 CIAS1 mutations that may be considered polymor- function via defects in nucleotide hydrolysis, conforma- phisms, V198M, R488K, and Q703K, in a large panel of tional changes, or multimerization. DNA samples from Caucasian controls. For V198M and Although the possibility that the mutated surface R488K, we found allele frequencies of 0.0074 and in the hexamer interacts with the LRRs cannot be ruled 0.0014, respectively, among 742 and 370 Caucasians. out (8), comparisons with other AAAϩ proteins suggest Previous studies identified V198M in control DNA that other NACHT subdomains may interact at this samples from 0 of 109 North American Caucasians, 1 of surface. Interaction between NACHT subdomains is 1282

Table 3. Genotype/phenotype of reported CIAS1 mutations* Other FCAS MWS/FCAS MWS MWS/NOMID NOMID

aa Mutation Families Patient Families Patient Families Patient Families Patient Families Patient Families Patient Refs. R488 K 1† 2 1 1 42, P V198 M 4 5‡ 2†§ 3 1¶ 3 3, 5, 42, 43 L305 P 4 19 3, 42, P L353 P 12# 114 51, P M659 K 2 4 10, P A439 V 3** 7 - 3 - 2 5, 10, P T 14 4 P 11 10 R260 W 1 12 4 13 6 34 4, 10, 16, 27, P L 22 45 P 1145 D303 N 1 2 1 1 1 1 9 10 4, 6, 7, 10, 23, 42, 45, P G 1145 T348 M 10 18 5 5 4, 10, 28, 42, 45, P G569 R 1 2 1 1 4, 25 F523 L 111 17 C 11 P G326 E 111 110 L632 F 111 110 L264 F 111 1P H 117 T436 I 11 10 N 126 P 1110 F309 S 2 2 6, 10 G755 R 1112 A 1113 Y570 C 6 6 7, 10, 14 F 1110 * Amino acid (aa) residues for which mutations are only reported once are excluded. Family data are the total number of reported families with Ն1 affected patient; patient data are the total number of reported patients. P ϭ present study (see Table 1 for other definitions). † Unaffected family members with mutation.

‡ Uncharacterized periodic fevers. AL ET AKSENTIJEVICH § One family also carried E525K. ¶ Patients in family with features of FCAS, MWS, and NOMID. # Three families confirmed (others presumed) to have common ancestor; 1 family confirmed independent. **One family with intrafamilial variable phenotype. CLINICAL CONTINUUM OF CRYOPYRINOPATHIES 1283

consistent with patterns of mutations in other NACHT structural modeling of cryopyrin suggests a common domains that suggest that another surface is disrupted disease mechanism. But most importantly, several lines (Figure 1). Similarly, the effect of the G755A/R muta- of evidence presented here, including the finding of tions in the LRRs may be quite similar to those in patients without cryopyrin mutations, the lack of a clear NACHT subdomains, even if the mutations affect genotype/phenotype correlation for many mutations, cryopyrin in a structurally different way (Figure 2D). and the potential reduced penetrance of V198M and These models are consistent with the notion that R488K, strongly suggest the presence of additional known mutations share a common mechanism whereby genetic factors that initiate or modulate the cryopyri- a closed and inactive conformation of cryopyrin is nopathies. Identification of these genes will be impor- disrupted, leading to activation of the inflammasome tant for understanding innate immunity as well as pro- complex and subsequent activation of caspase 1 and viding new targets for controlling these diseases. increased IL-1␤ and IL-18 secretion (Figure 2D). The fact that most of the disease-causing mutations occur in the NACHT and NACHT-associated domains suggests ACKNOWLEDGMENTS that the gain of function requires intact pyrin and LRR We would like to thank all of the physicians who domains, or that most mutations in the pyrin and LRR referred their patients to us for molecular diagnostic testing. domains cannot activate cryopyrin. These disease- We would also like to acknowledge Dr. Peter Gregersen for causing mutations do not need to completely disrupt the sharing the North American Rheumatoid Arthritis Collection and New York Cancer Project control samples with us, and inactive state of cryopyrin, but rather could simply Amir Misaghi and Scott Anderson for technical support. influence the normal equilibrium between active and inactive forms of the molecule and the ease by which inflammasome assembly is activated by external stimulus. AUTHOR CONTRIBUTIONS Our models, however, cannot directly address the Dr. Aksentijevich had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy clinically important problem of the severity of any of the data analysis. particular mutation. And the question remains as to Study design. Aksentijevich, Putnam, Remmers, Goldbach-Mansky, whether the CAPS mutations show a clear correlation Hoffman, Kastner. Acquisition of data. Aksentijevich, Remmers, Mueller, Le, Moak, between genotype and phenotype. If so, then their Chuang, Austin Goldbach-Mansky, Hoffman. defects can be understood primarily in the context of Analysis and interpretation of data. Aksentijevich, Putnam, Remmers, cryopyrin structure–function relationships. If not, then Mueller, Le, Kolodner Goldbach-Mansky, Hoffman, Kastner. Manuscript preparation. Aksentijevich, Putnam, Remmers, Mueller, other genetic factors likely play a role in the disease Kolodner Goldbach-Mansky, Hoffman, Kastner. process. Addressing the genotype/phenotype correlation Statistical analysis. Aksentijevich, Remmers. is complicated by the small number of patients, report- Modeling of cryopyrin structure. Putnam. ing bias, and variable phenotyping by clinicians. Despite these limitations, the phenotypes of many patients with REFERENCES known mutations have been adequately described in the 1. Stojanov S, Kastner DL. Familial autoinflammatory diseases: literature and can be used as a basis for this analysis. genetics, pathogenesis and treatment. Curr Opin Rheumatol 2005; Analysis of all known mutations indicates that 17:586–99. mutational severity is not correlated with mutation 2. Prieur AM, Griscelli C, Lampert F, Truckenbrodt H, Guggenheim MA, Lovell DJ, et al. A chronic, infantile, neurological, cutaneous cluster position (Figure 1), the specific residue mutated and articular (CINCA) syndrome: a specific entity analyzed in 30 (for example, R260 in Table 3), or conservation among patients. Scand J Rheumatol Suppl 1987;66:57–68. CIAS1 orthologs (Figure 1). Thus, we focused solely on 3. Aganna E, Martinon F, Hawkins PN, Ross JB, Swan DC, Booth DR, et al. Association of mutations in the NALP3/CIAS1/ multiply observed mutations (Table 3). At both ends of PYPAF1 gene with a broad phenotype including recurrent fever, the disease severity continuum, there are mutations with cold sensitivity, sensorineural deafness, and AA amyloidosis. Ar- relatively consistent phenotypes. For example, L353P thritis Rheum 2002;46:2445–52. 4. Dode C, Le Du N, Cuisset L, Letourneur F, Berthelot JM, and L305P tend to be associated with milder pheno- Vaudour G, et al. New mutations of CIAS1 that are responsible types, whereas F309S and Y570C tend to be associated for Muckle-Wells syndrome and familial cold urticaria: a novel with more severe phenotypes. Other mutations, includ- mutation underlies both syndromes. Am J Hum Genet 2002;70: 1498–506. ing those affecting A439, R260, and D303, have signifi- 5. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner cantly variable severities, strongly suggesting that other RD. Mutation of a new gene encoding a putative pyrin-like protein genetic factors can play a role in disease severity. causes familial cold autoinflammatory syndrome and Muckle- Wells syndrome. Nat Genet 2001;29:301–5. In conclusion, we have identified a number of 6. Feldman J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis S, novel disease-causing mutations in CIAS1, and our et al. Chronic, infantile, neurological, cutaneous and articular 1284 AKSENTIJEVICH ET AL

syndrome is caused by mutations in CIAS1, a gene highly ex- anakinra in a de novo case of neonatal-onset multisystem inflam- pressed in polymorphonuclear cells and chondrocytes. Am J Hum matory disease. Arthritis Rheum 2004;50:2708–9. Genet 2002;71:198–203. 26. Hoffman HM, Rosengren S, Boyle DL, Cho JY, Nayer J, Mueller 7. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, JL, et al. Prevention of cold-associated acute inflammation in Hofmann SR, et al. De novo CIAS1 mutations, cytokine activa- familial cold autoinflammatory syndrome by interleukin-1 recep- tion, and evidence for genetic heterogeneity in patients with tor antagonist. Lancet 2004;364:1779–85. neonatal-onset multisystem inflammatory disease (NOMID): a 27. Alexander T, Klotz O, Feist E, Ruther K, Burmester GR, Pleyer new member of the expanding family of pyrin-associated autoin- U. Successful treatment of acute visual loss in Muckle-Wells flammatory diseases. Arthritis Rheum 2002;46:3340–8. syndrome with interleukin 1 receptor antagonist. Ann Rheum Dis 8. Tschopp J, Martinon F, Burns K. NALPs: a novel protein family 2005;64:1245–6. involved in inflammation. Nat Rev Mol Cell Biol 2003;4:95–104. 28. Seitz M, Kamgang RK, Simon HU, Villiger PM. Therapeutic 9. Anderson JP, Mueller JL, Rosengren S, Boyle DL, Schaner P, interleukin (IL) 1 blockade normalises increased IL1␤ and de- Cannon SB, et al. Structural, expression, and evolutionary analysis creased tumour necrosis factor ␣ and IL-10 production in blood of mouse CIAS1. Gene 2004;338:25–34. mononuclear cells of a patient with CINCA syndrome. Ann 10. Infevers database. URL: http://fmf.igh.cnrs.fr/infevers. Rheum Dis 2005;64:1802–3. 11. Frenkel J, van Kempen MJ, Kuis W, van Amstel HK. Variant 29. Boschan C, Witt O, Lohse P, Foeldvari I, Zappel H, Schweigerer chronic infantile neurologic, cutaneous, articular syndrome due to L, et al. Neonatal-onset multisystem inflammatory disease (NO- a mutation within the leucine-reach repeat domain of CIAS1 MID) due to a novel S331R mutation of the CIAS1 gene and [letter]. Arthritis Rheum 2004;50:2719–20. response to interleukin-1 receptor antagonist treatment. Am J 12. Matsubayashi T, Sugiura H, Arai T, Oh-Ishi T, Inamo Y. Anakinra Med Genet 2006;140:883–6. therapy for CINCA syndrome with a novel mutation in exon 4 of 30. Rynne M, Maclean C, Bybee A, McDermott MF, Emery P. the CIAS1 gene. Acta Paediatr 2006;95:246–9. Hearing improvement in a patient with variant Muckle-Wells 13. Aksentijevich I, Remmers EF, Goldbach-Mansky R, Reiff A, syndrome in response to interleukin 1 receptor antagonist. Ann Kastner DL. Mutational analysis in neonatal-onset multisystem Rheum Dis 2006;65:533–4. inflammatory disease: comment on the articles by Frenkel et al 31. Goldbach-Mansky R, Dailey NJ, Canna SW, Gelabert A, Jones J, and Saito et al [letter]. Arthritis Rheum 2006;54:2703–4. Rubin BI, et al. Neonatal-onset multisystem inflammatory disease 14. Saito M, Fujisawa A, Nishikomori R, Kambe N, Nakata-Hizume responsive to interleukin-1␤ inhibition. N Engl J Med 2006;355: M, Yoshimoto M, et al. Somatic mosaicism of CIAS1 in a patient 581–92. with chronic infantile neurologic, cutaneous, articular syndrome. 32. Hoffman HM, Wanderer AA, Broide DH. Familial cold autoin- Arthritis Rheum 2005;52:3579–85. flammatory syndrome: phenotype and genotype of an autosomal 15. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular dominant periodic fever. J Allergy Clin Immunol 2001;108:615–20. platform triggering activation of inflammatory caspases and pro- 33. Bruns CM, Hubatsch I, Ridderstrom M, Mannervik B, Tainer JA. cessing of proIL-1␤. Mol Cell 2002;10:417–26. Human glutathione transferase A4-4 crystal structures and mu- 16. Agostini L, Martinon F, Burns K, McDermott M, Hawkins PN, tagenesis reveal the basis of high catalytic efficiency with toxic lipid Tschopp J. NALP3 forms an IL-1␤-processing inflammasome with peroxidation products. J Mol Biol 1999;288:427–39. increased activity in Muckle-Wells autoinflammatory disorder. 34. Rost B, Sander C. Combining evolutionary information and neural Immunity 2004;20:319–25. networks to predict protein secondary structure. Proteins 1994;19: 17. Kanneganti T, Ozoren N, Body-Malapel M, Amer A, Park JH, 55–72. Franchi L, et al. Bacterial RNA and small antiviral compounds 35. McGuffin LJ, Bryson K, Jones DT. The PSIPRED protein struc- activate caspase-1 through cryopyrin/Nalp3. Nature 2006;440: ture prediction server. Bioinformatics 2000;16:404–5. 233–6. 36. Cuff JA, Barton GJ. Application of enhanced multiple sequence 18. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, alignment profiles to improve protein secondary structure predic- Roose-Girma M, et al. Cryopyrin activates the inflammasome in tion. Proteins 2000;40:502–11. response to toxins and ATP. Nature 2006;440:228–32. 37. Ouali M, King RD. Cascaded multiple classifiers for secondary 19. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout- structure prediction. Protein Sci 2000;9:1162–76. associated uric acid crystals activate the NALP3 inflammasome. 38. Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an Nature 2006;440:237–41. automated protein homology-modeling server. Nucl Acid Res 20. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichten- 2003;31:3381–5. berger GS, Grant EP, et al. Critical role for NALP3/CIAS1/ 39. Kelley LA, MacCallum RM, Sternberg MJ. Enhanced genome cryopyrin in innate and adaptive immunity through its regulation annotation using structural profiles in the program 3D-PSSM. J of caspase-1. Immunity 2006;24:317–27. Mol Biol 2003;299:499–520. 21. Dowds TA, Masumoto J, Zhu L, Inohara N, Nunez G. Cryopyrin- 40. Shi J, Blundell TL, Mizuguchi K. FUGUE: sequence-structure induced interleukin 1␤ secretion in monocytic cells: enhanced homology recognition using environment-specific substitution ta- activity of disease-associated mutants and requirement for ASC. bles and structure-dependent gap penalties. J Mol Biol 2001;310: J Biol Chem 2004;279:21924–8. 243–57. 22. Yu JW, Wu J, Zhang Z, Datta P, Ibrahimi I, Taniguchi S, et al. 41. DeLano WL. The PyMOL molecular graphics system. URL: Cryopyrin and pyrin activate caspase-1, but not NF-␬B, via ASC http://www.pymol.org. oligomerization. Cell Death Differ 2006;13:236–49. 42. Arostegui JI, Aldea A, Modesto C, Jesus Rua M, Arguellas F, 23. Janssen R, Verhard E, Lankester A, ten Cate R, van Dissel JT. Gonzalez-Ensenat MA, et al. Clinical and genetic heterogeneity Enhanced interleukin-1␤ and interleukin-18 release in a patient among Spanish patients with recurrent autoinflammatory syn- with chronic infantile neurologic, cutaneous, articular syndrome. dromes associated with the CIAS1/PYPAF1/NALP3 gene. Arthri- Arthritis Rheum 2004;50:3329–33. tis Rheum 2004;50:4045–50. 24. Hawkins PN, Lachmann HJ, McDermott MF. Interleukin-1-recep- 43. Porksen G, Lohse P, Rosen-Wolff A, Heyden S, Forster T, tor antagonist in the Muckle-Wells syndrome. N Engl J Med Wendisch J, et al. Periodic fever, mild arthralgias, and reversible 2003;348:2583–4. moderate and severe organ inflammation associated with the 25. Hawkins PN, Bybee A, Aganna E, McDermott MF. Response to V198M mutation in the CIAS1 gene in three German patients— CLINICAL CONTINUUM OF CRYOPYRINOPATHIES 1285

expanding phenotype of CIAS1 related autoinflammatory syn- of a child with CINCA/NOMID syndrome improved during and drome. Eur J Haematol 2004;73:123–7. after treatment with thalidomide. Scand J Rheumatol 2005;34: 44. Albrecht M, Domingues FS, Schreiber S, Lengauer T. Structural 246–9. localization of disease-associated sequence variations in the 50. Matsubara T, Hasegawa M, Shiraishi M, Hoffman HM, Ichiyama NACHT and LRR domains of PAYPAF1 and NOD2. FEBS Lett T, Tanaka T, et al. A severe case of chronic infantile neurologic, 2003;554:520–8. cutaneous, articular syndrome treated with biologic agents. Arthri- 45. Neven B, Callebaut I, Prieur AM, Feldmann J, Bodemer C, tis Rheum 2006;54:2314–20. Lepore L, et al. Molecular basis of the spectral expression of 51. Hoffman HM, Gregory S, Mueller J, Tresierras M, Broide D, CIAS1 mutations associated with phagocytic cell-mediated auto- Wanderer A, et al. Fine structure mapping of CIAS1: identifica- inflammatory disorders CINCA/NOMID, MWS, and FCU. Blood tion of an ancestral haplotype and a common FCAS mutation 2004;103:2809–15. L353P. Hum Genet 2003;112:209–16. 46. Putnam CD, Clancy CB, Tsuruta H, Gonzalez S, Wetmur JG, 52. Liepinsh E, Barbals R, Dahl E, Sharipo A, Staub E, Otting G. The Tainer JA. Structure and mechanism of the RuvB Holliday death-domain fold of the ASC pyrin domain, presenting a basis of junction branch migration motor. J Mol Biol 2001;311:297–310. pyrin/pyrin recognition. J Mol Biol 2003;332:1155–63. 47. Hanson PI, Whitehart SW. AAAϩ proteins: have engine, will 53. Riedel SJ, Li W, Chao Y, Schwarzenbacher R, Shi Y. Structure of work. Nat Rev Mol Cell Biol 2005;6:519–29. the apoptotic protease-activating factor 1 bound to ADP. Nature 48. Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD, 2005;434:926–33. Huber R. The structures of HslU and the ATP-dependent pro- 54. Kobe B, Deisenhofer J. Crystal structure of porcine ribonuclease tease HslU-HslV. Nature 2000;403:800–5. inhibitor, a protein with leucine-rich repeats. Nature 1993;366: 49. Kallinich T, Hoffman HM, Roth J, Keitzer R. The clinical course 751–6. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1286–1291 DOI 10.1002/art.22444 © 2007, American College of Rheumatology

Positive Association of SLC26A2 Gene Polymorphisms With Susceptibility to Systemic-Onset Juvenile Idiopathic Arthritis

Rebecca Lamb,1 Wendy Thomson,1 the British Society of Paediatric and Adolescent Rheumatology, Emma M. Ogilvie,2 and Rachelle Donn1

Objective. To investigate SLC26A2, the gene that systemic-onset JIA. Our findings also highlight causes diastrophic dysplasia, in juvenile idiopathic ar- systemic-onset JIA as being a distinctly different disease thritis (JIA). from that in the other JIA subgroups. Methods. Nine polymorphisms across the SLC26A2 gene locus were investigated using MassArray Juvenile idiopathic arthritis (JIA) is the most genotyping in 826 UK Caucasian JIA cases and 617 common chronic rheumatic disease of childhood and is ethnically matched healthy controls. characterized by persistent idiopathic inflammation of 1 Results. Significant associations between multiple or more joints, with an onset before the age of 16 years single-nucleotide polymorphisms (SNPs) across and a duration of 6 weeks or more (1). JIA is clinically SLC26A2 and systemic-onset JIA were found. In each heterogeneous. Systemic-onset JIA accounts for 10– case, homozygosity for the minor allele conferred the 20% of JIA. Patients are often extremely unwell, with increased risk of disease susceptibility: rs1541915 (odds evidence of systemic inflammation, as manifested by ratio [OR] 2.3, 95% confidence interval [95% CI] 1.4– fever, rash, serositis, and hyperferritinemia, in addition ؍ P ؍ P 3.7, 0.0003), rs245056 (OR 2.8, 95% CI 1.7–4.6, to arthritis, which may occur at presentation or later in ؍ P 0.00002), rs245055 (OR 2.5, 95% CI 1.2–5.0, 0.004), the disease course (1). ؍ P rs245051 (OR 2.3, 95% CI 1.4–3.7, 0.0005), As part of a strategy of taking candidate genes ؍ P rs245076 (OR 2.7, 95% CI 1.3–5.4, 0.0015), and from loci of monogenic syndromes with features in ؍ P rs8073 (OR 2.3, 95% CI 0.9–5.6, 0.04). common with JIA, we have previously shown WISP3, the Conclusion. These findings show the value of gene that causes progressive pseudorheumatoid dyspla- using monogenic disease loci as candidates for investi- sia, to confer susceptibility to polyarticular disease- gation in JIA. We identified a subgroup-specific associ- SLC26A2 course JIA (2). Continuing this approach in the present ation between SNPs within the gene and study, we investigated the gene for diastrophic dysplasia (DTD), SLC26A2 (solute carrier family 26 [sulfate trans- Supported by the Epidemiology Unit Core Support program porter] member 2; also called DTD sulfate transporter of the Arthritis Research Campaign, UK. Ms Lamb’s work was supported by an MRC PhD studentship (G78/7554). [DTDST]), in a well-defined cohort of UK JIA cases. 1Rebecca Lamb, PhD, Wendy Thomson, PhD, Rachelle Mutations within the SLC26A2 gene (locus 5q32– Donn, MB ChB, PhD: University of Manchester, Manchester, UK; q31) are known to cause a clinical spectrum of osteo- 2Emma M. Ogilvie, MMed: University College London, London, UK. Contributors to the British Society of Paediatric and Adoles- chondrodysplasias, with disease phenotypes ranging cent Rheumatology are as follows: Dr. M. Abinun, Dr. M. Becker, Dr. from severe/fatal disease to milder phenotypes (3). This A. Bell, Professor A. Craft, Dr. E. Crawley, Dr. J. David, Dr. H. Foster, range of conditions includes achondrogenesis type IB, a Dr. J. Gardener-Medwin, Dr. J. Griffin, Dr. A. Hall, Dr. M. Hall, Dr. A. Herrick, Dr. P. Hollingworth, Dr. L. Holt, Dr. S. Jones, Dr. G. fatal skeletal dysplasia, atelosteogenesis type II (4), Pountain, Dr. C. Ryder, Professor T. Southwood, Dr. I. Stewart, Dr. H. recessive multiple epiphyseal dysplasia (5,6), and the Venning, Dr. L. Wedderburn, Professor P. Woo, and Dr. S. Wyatt. mildest phenotype, DTD, an autosomal-recessive chon- Address correspondence and reprint requests to Rachelle Donn, MB ChB, PhD, Arthritis Research Campaign Epidemiology drodysplasia (7). While many of the clinical aspects of Unit and Centre for Molecular Medicine, University of Manchester, the osteochondrodysplasias are evidently nonoverlap- Stopford Building, Oxford Road, Manchester M13 9PT, UK. E-mail: ping with JIA, the associated early degenerative changes [email protected]. Submitted for publication September 12, 2006; accepted in of joints occur in both (8). The osteochondrodysplasias revised form December 6, 2006. arise through loss-of-function mutations within the

1286 SLC26A2 SNPs AND SUSCEPTIBILITY TO SYSTEMIC-ONSET JIA 1287

Figure 1. Schematic representation of the SLC26A2 gene locus on chromosome 5q32–q33.1, showing exon locations and single-nucleotide polymorphisms (SNPs; denoted by their rs numbers). The length of the gene is 27 kb. SNP annotation was based on the National Center for Biotechnology Information dbSNP database refSNP and using genomic build 34.

SLC26A2 gene that result in absent or severely defective desorption ionization–time-of-flight mass spectrometry (Se- protein synthesis. It was our hypothesis that milder quenom, San Diego, CA). Amplifications were conducted genetic changes (single-nucleotide polymorphisms according to the manufacturer’s protocol. Polymerase chain reaction primers and probe sequences are available from the [SNPs]) give rise to an altered, but less dramatic, quan- authors on request. tity and/or quality of the SLC26A2 protein, and that this Statistical analysis. Prior to analysis, the raw data were contributes to the oligogenetic basis of JIA. “cleaned” by the removal of data for DNA samples that failed to genotype for Ն20% of the SNPs assayed, as well as by removal of data for SNPs that failed to genotype for Ն20% of PATIENTS AND METHODS the samples. Hardy-Weinberg equilibrium was determined sepa- Patients and controls. Blood samples were obtained rately in the cases and controls with the use of Stata version 8 following informed consent. Ethics approval was obtained software (Stata, College Station, TX). SNPs were removed from the Multi-centre Research Ethics Committee (MREC from analysis if deviation from Hardy-Weinberg equilibrium 99/8/84) and the University of Manchester Committee on the (P Յ 0.05) was observed in the controls. Ethics of Research on Human Beings (8/92/[i] [b]). DNA Single-point analysis. Association of the SLC26A2 SNPs samples from JIA patients were collected as part of the British was investigated using the chi-square test, and odds ratios Society of Paediatric and Adolescent Rheumatology national (ORs) with 95% confidence intervals (95% CIs) were calcu- repository for JIA. All cases were classified according to the lated using Stata version 8 software. P values less than or equal International League of Associations for Rheumatology clas- to 0.05 were considered significant. Initially, genotype frequen- sification criteria (1). cies for each SNP were compared between the JIA cases (as a DNA samples from 826 UK Caucasian JIA cases were whole) and the controls. Then, subgroup analysis was per- available for genotyping. The total number of JIA cases formed in which each JIA subgroup was individually compared available for genotyping per subgroup was as follows: 133 with with the controls. systemic-onset JIA, 215 with persistent oligoarticular JIA, 127 Multipoint analysis. Linkage disequilibrium was calcu- with extended oligoarticular JIA, 176 with rheumatoid factor lated, and the association of varying-length haplotypes was (RF)–negative polyarticular JIA, 53 with RF-positive polyar- tested using the expectation-maximization algorithm. This was ticular JIA, 58 with enthesitis-related JIA, 57 with psoriatic implemented using HelixTree software (http://www.goldenhelix. JIA, and 7 with unclassifiable JIA. In addition, a control com/index.jsp). population consisting of 617 unrelated and ethnically matched Correction for multiple testing. To limit Type II error, 2 healthy individuals was used. The controls were blood donors methods were used. First, permutation testing was used, and or had been recruited via general practitioners’ registers. empirical P values were generated. This was instigated in Genotyping. Nine SNPs were genotyped across the Clump (http://linkage.rockefeller.edu/soft/clump.html), a SLC26A2 locus, giving an average spacing between SNPs of Monte Carlo–based method for assessing significance in case– ϳ300 bp (Figure 1). Genotyping was performed using high- control association studies. One hundred thousand simulations throughput MassArray DNA analysis with matrix-assisted laser were performed. Empirical P (Pe) values less than or equal to 1288 LAMB ET AL

Table 1. Genotype frequencies in JIA cases versus controls for SLC26A2 SNPs* JIA cases Controls P for SNP, Sample P for Genotype Sample P for Genotype genotype genotype size HWE frequency, no. (%) size HWE frequency, no. (%) association rs1541915 AA 664 0.63 215 (32.4) 563 0.73 195 (34.6) AC 332 (50.0) 277 (49.2) CC 117 (17.6) 91 (16.2) 0.64 rs245056 AA 682 0.48 228 (33.4) 559 0.42 213 (38.1) AT 341 (50.0) 272 (48.7) TT 113 (16.6) 74 (13.2) 0.12 rs245055 TT 724 0.57 396 (54.7) 585 0.91 342 (58.5) TC 275 (38) 212 (36.2) CC 53 (7.3) 31 (5.3) 0.21 rs245051 TT 746 0.76 276 (37.0) 595 0.6 234 (39.3) TC 352 (47.2) 284 (47.8) CC 118 (15.8) 77 (12.9) 0.31 rs245076 AA 648 0.19 355 (54.8) 557 0.81 332 (59.6) AG 240 (37.0) 195 (35.0) GG 53 (8.2) 30 (5.4) 0.08 rs3756307 GG 697 1 509 (73.0) 574 0.48 428 (74.6) GA 174 (25.0) 138 (24.0) AA 14 (2.0) 8 (1.4) 0.64 rs30832 GG 659 1 644 (97.7) 563 0.06 552 (98.0) GA 15 (2.3) 10 (1.8) AA 0 (0.0) 1 (0.2) 0.46 rs3776070 AA 591 0.05 516 (87.3) 490 0.03 425 (86.8) AT 69 (11.7) 59 (12.0) TT 6 (1.0) 6 (1.2) – rs8073 AA 716 0.06 465 (64.9) 586 0.89 396 (67.6) AC 214 (29.9) 171 (29.2) CC 37 (5.2) 19 (3.2) 0.21 * The single-nucleotide polymorphisms (SNPs) are listed as they occur in the SLC26A2 gene, 5Ј to 3Ј. The sample size represents the number of individuals with a genotyping result for each SNP. JIA ϭ juvenile idiopathic arthritis; HWE ϭ Hardy-Weinberg equilibrium.

0.05 were considered significant. Second, the Bonferroni cor- Genotype frequencies of the remaining SNPs rection was applied. Correction for the 8 SNPs analyzed and were compared between the JIA cases and the controls. the 7 subgroups of JIA investigated (n ϭ 56) was used. No significant associations (P Յ 0.05) were observed for Corrected P (Pcorr) values less than or equal to 0.05 were considered significant. any of the SNPs (Table 1). To investigate the possibility that SLC26A2 SNPs confer susceptibility to specific RESULTS subgroups of JIA, genotype frequencies of SNPs in Nine SNPs (rs1541915, rs245056, rs245055, controls were compared with those in each JIA subgroup rs245051, rs245076, rs3756307, rs30832, rs3776070, and individually (Table 2). rs8073) were genotyped across the SLC26A2 locus. Highly significant associations between 5 SNPs SLC26A2 SNP rs3776070 deviated from Hardy- across the SLC26A2 gene and systemic-onset JIA were Weinberg equilibrium in both the JIA cases (P ϭ 0.05) observed: rs1541915 (P ϭ 0.0015), rs245056 (P ϭ and the controls (P ϭ 0.03). Additionally, the success 0.0001), rs245055 (P ϭ 0.0052), rs245051 (P ϭ 0.0015), rate for this SNP was Ͻ80% in both the JIA cases (76%) and rs245076 (P ϭ 0.0018). When a recessive mode of and the controls (79%), and this SNP was therefore inheritance was applied to the data, where 2 copies of excluded from further analysis. the minor allele confer susceptibility, the level of signif- SLC26A2 SNPs AND SUSCEPTIBILITY TO SYSTEMIC-ONSET JIA 1289

Table 2. Genotype frequencies in JIA subgroups versus controls for SLC26A2 SNPs* Persistent Extended RF-negative RF-positive Enthesitis- Systemic-onset JIA oligoarticular oligoarticular polyarticular polyarticular related JIA Psoriatic JIA patients JIA patients JIA patients JIA patients JIA patients patients patients No. (%) SNP, of genotype No. (%) P No. (%) P No. (%) P No. (%) P No. (%) P No. (%) P No. (%) P controls rs1541915 AA 30 (27.0) 65 (34.2) 30 (31.9) 45 (34.1) 16 (39.5) 15 (33.3) 13 (26.5) 195 (34.6) AC 47 (42.4) 95 (50.0) 43 (45.8) 69 (52.3) 16 (42.1) 27 (60.0) 33 (67.4) 277 (49.2) CC 34 (30.6) 0.0015† 30 (15.8) 0.98 21 (22.3) 0.34 18 (13.6) 0.72 7 (18.4) 0.61 3 (6.7) 0.18 3 (6.1) 0.03† 91 (16.2) rs245056 AA 28 (26.2) 69 (37.3) 31 (30.4) 53 (36.3) 12 (32.4) 18 (37.5) 16 (30.2) 213 (38.1) AT 47 (43.9) 93 (50.3) 50 (49.0) 73 (50.0) 18 (48.7) 24 (50.0) 34 (64.1) 272 (48.7) TT 32 (29.9) 0.0001† 23 (12.4) 0.92 21 (20.6) 0.1 20 (13.7) 0.92 7 (18.9) 0.57 6 (12.5) 0.98 3 (5.7) 0.08 74 (13.2) rs245055 TT 57 (47.1) 122 (61.3) 61 (55.4) 89 (60.1) 16 (43.2) 24 (47.1) 25 (46.3) 342 (58.5) TC 49 (40.5) 64 (32.2) 40 (36.4) 51 (34.5) 19 (51.4) 25 (49.0) 26 (48.1) 212 (36.2) CC 15 (12.4) 0.0052† 13 (6.5) 0.52 9 (8.2) 0.48 8 (5.4) 0.92 2 (5.4) 0.14 2 (3.9) 0.21 3 (5.6) 0.22 31 (5.3) rs245051 TT 38 (29.9) 78 (38.6) 38 (35.2) 69 (43.1) 15 (37.5) 20 (38.5) 17 (32.7) 234 (39.3) TC 57 (44.9) 94 (46.5) 49 (45.4) 74 (46.3) 21 (50.0) 25 (48.0) 30 (57.7) 284 (47.8) CC 32 (25.2) 0.0015† 30 (14.9) 0.79 21 (19.4) 0.19 17 (10.6) 0.59 5 (12.5) 0.91 7 (13.5) 0.99 5 (9.6) 0.41 77 (12.9) rs245076 AA 56 (47.1) 112 (60.5) 45 (51.7) 79 (58.9) 13 (44.8) 23 (53.5) 25 (53.2) 332 (59.6) AG 47 (39.5) 59 (31.9) 34 (39.1) 47 (35.1) 14 (48.3) 19 (44.2) 19 (40.4) 195 (35.0) GG 16 (13.4) 0.0018† 14 (7.6) 0.47 8 (9.2) 0.22 8 (6.0) 0.96 2 (6.9) 0.22 1 (2.3) 0.48 3 (6.4) 0.62 30 (5.4) rs3756307 GG 84 (71.8) 132 (69.1) 75 (72.8) 106 (73.6) 29 (82.9) 41 (80.4) 39 (75.0) 428 (74.6) GA 28 (23.9) 58 (30.4) 23 (22.3) 36 (25.0) 6 (17.1) 9 (17.6) 13 (25.0) 138 (24.0) AA 5 (4.3) 0.1281 1 (0.5) 0.17 5 (4.9) 0.06 2 (1.4) 0.95 0 (0.0) 0.64 1 (2.0) 0.41 0 (0.0) 0.93 8 (1.4) rs30832 GG 108 (98.2) 182 (98.9) 85 (98.8) 137 (97.9) 35 (97.2) 43 (95.6) 50 (92.6) 552 (98.0) GA 2 (1.8) 2 (1.1) 1 (1.2) 3 (2.1) 1 (2.8) 2 (4.4) 4 (7.4) 10 (1.8) AA 0 (0.0) 1 0 (0.0) 0.8 0 (0.0) 1 0 (0.0) 0.78 0 (0.0) 0.53 0 (0.0) 0.28 0 (0.0) 0.05† 1 (0.2) rs8073 AA 77 (62.1) 139 (70.9) 60 (56.1) 98 (67.1) 25 (65.8) 32 (62.7) 32 (64.0) 396 (67.6) AC 38 (30.6) 45 (23.0) 41 (38.3) 0.06 42 (28.8) 11 (28.9) 18 (35.3) 17 (34.0) 171 (29.2) CC 9 (7.3) 0.0944 12 (6.1) 0.07 6 (5.6) 6 (4.1) 0.87 2 (5.3) 0.69 1 (2.0) 0.68 1 (2.0) 0.77 19 (3.2) * Persistent oligoarticular juvenile idiopathic arthritis (JIA) affects Յ4 joints; extended oligoarticular JIA affects Ն5 joints after 6 months. Rheumatoid factor (RF)–negative and RF-positive polyarticular JIA affects Ն5 joints within the first 6 months. All JIA patients were classified according to the criteria of the International League of Associations for Rheumatology (1). SNPs ϭ single-nucleotide polymorphisms. † Statistically significant P value versus controls.

ϭ ϭ icance increased, conferring more than a 2-fold in- rs245055 (Pe 0.00588), rs245051 (Pe 0.00069), ϭ ϭ creased risk of disease: rs1541915 (odds ratio [OR] 2.3, rs245076 (Pe 0.00296), and rs8073 (Pe 0.044). For 95% confidence interval [95% CI] 1.4–3.7, P ϭ 0.0003), psoriatic JIA significant association was maintained with ϭ ϭ rs245056 (OR 2.8, 95% CI 1.7–4.6, P 0.00002), rs1541915: Pe 0.037. rs245055 (OR 2.5, 95% CI 1.2–5.0, P ϭ 0.004), rs245051 Furthermore, when the Bonferroni correction, a (OR 2.3, 95% CI 1.4–3.7, P ϭ 0.0005), rs245076 (OR highly conservative test which assumes independence of 2.7, 95% CI 1.3–5.4, P ϭ 0.0015), and rs8073 (OR 2.3, all the parameters tested, was applied, the associations 95% CI 0.9–5.6, P ϭ 0.04). with psoriatic JIA were lost. The associations with A weak association-by-genotype of 2 of the SNPs systemic-onset JIA, however, remained significant for 3 ϭ ϭ and psoriatic JIA was also seen: rs1541915 (P 0.03) of the SNPs tested: rs1541915 (Pcorr 0.0168), rs245056 ϭ ϭ ϭ and rs30832 (P 0.05). (Pcorr 0.0011), and rs245051 (Pcorr 0.028). The positive associations with systemic-onset JIA Significant linkage disequilibrium was observed were maintained following permutation testing: across the SLC26A2 SNPs examined (i.e., between the ϭ ϭ Ј Ј rs1541915 (Pe 0.00052), rs245056 (Pe 0.00005), studied SNPs located most 5 [rs1541915] and most 3 1290 LAMB ET AL

[rs8073], with DЈϭ0.87 and r2 ϭ 0.49). However, no ever, no systemic-onset JIA–specific linkage peaks, haplotype association of an order of magnitude greater which would be indicative of potential candidate loci, than the single-point association was observed. were identified due to the very limited patient sample size (n ϭ 11) studied (15). DISCUSSION Alternatively, our findings could suggest heterogeneity in the pathogenesis of the cartilage involvement SLC26A2 is the gene that causes a family of in JIA, with SLC26A2 contributing to the cartilage osteochondrodysplasias. All of these are characterized damage in systemic-onset JIA only. Currently, there are by abnormal growth and remodeling of cartilage and no histopathologic or imaging studies to address this bone (9). Because of the clinical overlap between these point. osteochondrodysplasias and JIA, we hypothesized that While JIA is the most common chronic rheu- SNPs within the same gene, SLC26A2, and not the matic disease of childhood, it is a rare condition, affect- previously described mutations, may confer susceptibil- ing 1 in 10,000 children under the age of 16 years. The Ј ity to JIA. Significant associations within the 5 region of clinical subgrouping means that the numbers of patients the SLC26A2 gene and systemic-onset JIA were identi- with any 1 clinical presentation are relatively small for fied, which remained after correction for multiple test- conducting association-based genetic studies. With this ing. The most significant association observed was with in mind, we tried to overcome the potential problem that SNP rs245056 within the first intron of the SLC26A2 the observed positive associations between SLC26A2 gene, which confers an almost 3-fold increased risk of and systemic-onset JIA might be false-positive ones by disease susceptibility, with an OR of 2.8 (95% CI using both permutation testing and Bonferroni correc- 1.7–4.6) at P ϭ 0.00005 and P ϭ 0.0011. e corr tion. Additional support for the importance of these SLC26A2 encodes an anion exchanger that trans- observations is required, and functional studies to ad- ports extracellular sulfate into the cytoplasm for the dress the consequence of the SNP polymorphisms within sulfation of proteoglycans in the extracellular matrix of relevant cell types should now be conducted. cartilage (10). Within cartilage, the proteoglycan mole- In summary, we studied a very large group of UK cules form a highly hydrated, gel-like substance in which Caucasian JIA cases and ethnically matched healthy the fibrous proteins are embedded; this resists compres- controls and found significant associations between sive forces on the matrix while allowing the rapid SLC26A2 and systemic-onset disease. These findings diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells (11). Despite the reiterate the value of investigating loci that cause mo- expression of SLC26A2 in numerous different tissues nogenic syndromes as JIA susceptibility genes. Further (12), mutations in this gene are only known to function- studies in different populations of systemic-onset JIA ally affect cartilage. should now be conducted in an attempt to replicate the Our finding of significant associations with just 1 findings of SLC26A2 as a susceptibility gene for subgroup of JIA, the systemic-onset JIA subgroup, was systemic-onset JIA. somewhat unexpected. In the osteochondrodysplasias, some residual activity of the mutated SLC26A2 protein, AUTHOR CONTRIBUTIONS combined with the use of alternative sources of sulfate, Drs. Lamb and Donn had full access to all of the data in the appears to be sufficient to supply the needs of cell types study and take responsibility for the integrity of the data and the other than chondrocytes. This may be how cartilage- accuracy of the data analysis. Study design. Thomson, Donn. specific defects arise (13,14). In systemic-onset JIA, a Acquisition of data. Lamb, Thomson, Ogilvie, Donn. complex genetic disease, alternative sources of sulfate or Analysis and interpretation of data. Lamb, Donn. sulfate transporters may also be defective. The combi- Manuscript preparation. Lamb, Thomson, Ogilvie, Donn. nation of subtle changes in more than one gene involved Statistical analysis. Lamb, Donn. in sulfate transportation may contribute to the systemic manifestations that are characteristic of systemic-onset REFERENCES JIA. One way to determine whether multiple such loci 1. Petty RE, Southwood TR, Baum J, Bhettay E, Glass DN, Manners contribute to systemic-onset JIA would be to examine P, et al. Revision of the proposed classification criteria for juvenile data generated from whole-genome studies. Recently, idiopathic arthritis: Durban, 1997. J Rheumatol 1998;25:1991–4. 2. Lamb R, Thomson W, Ogilvie E, Donn R. Wnt-1–inducible Thompson et al (15) performed a genome-wide scan of signaling pathway protein 3 and susceptibility to juvenile idio- sibling pairs with juvenile rheumatoid arthritis. How- pathic arthritis. Arthritis Rheum 2005;52:3548–53. SLC26A2 SNPs AND SUSCEPTIBILITY TO SYSTEMIC-ONSET JIA 1291

3. Rossi A, Superti-Furga A. Mutations in the diastrophic dysplasia 10. Habuchi O. Diversity and functions of glycosaminoglycan sulfo- sulfate transporter (DTDST) gene (SLC26A2): 22 novel muta- transferases. Biochim Biophys Acta 2000;1474:115–27. tions, mutation review, associated skeletal phenotypes, and diag- 11. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Cell nostic relevance. Hum Mutat 2001;17:159–71. junctions, cell adhesion, and the extracellular matrix. In: Molecu- 4. Hastbacka J, Superti-Furga A, Wilcox WR, Rimoin DL, Cohn lar biology of the cell. New York: Garland Science; 2002. p. DH, Lander ES. Atelosteogenesis type II is caused by mutations in 949–1010. the diastrophic dysplasia sulfate-transporter gene (DTDST): evi- 12. Haila S, Hastbacka J, Bohling T, Karjalainen-Lindsberg ML, Kere dence for a phenotypic series involving three chondrodysplasias. J, Saarialho-Kere U. SLC26A2 (diastrophic dysplasia sulfate trans- Am J Hum Genet 1996;58:255–62. porter) is expressed in developing and mature cartilage but also in other tissues and cell types. J Histochem Cytochem 2001;49: 5. Sheffield EG. Double-layered patella in multiple epiphyseal dys- 973–82. plasia: a valuable clue in the diagnosis. J Pediatr Orthop 1998;18: 13. Superti-Furga A, Rossi A, Steinmann B, Gitzelmann R. A chon- 123–8. drodysplasia family produced by mutations in the diastrophic 6. Ballhausen D, Bonafe L, Terhal P, Unger SL, Bellus G, Classen M, dysplasia sulfate transporter gene: genotype/phenotype correla- et al. Recessive multiple epiphyseal dysplasia (rMED): phenotype tions. Am J Med Genet 1996;63:144–7. delineation in eighteen homozygotes for DTDST mutation 14. Esko JD, Elgavish A, Prasthofer T, Taylor WH, Weinke JL. R279W. J Med Genet 2003;40:65–71. Sulfate transport-deficient mutants of Chinese hamster ovary cells: 7. Walker BA, Scott CI, Hall JG, Murdoch JL, McKusick VA. sulfation of glycosaminoglycans dependent on cysteine. J Biol Diastrophic dwarfism. Medicine (Baltimore) 1972;51:41–59. Chem 1986;261:15725–33. 8. Petty RE. Classification of childhood arthritis: a work in progress. 15. Thompson SD, Moroldo MB, Guyer L, Ryan M, Tombragel EM, Baillieres Clin Rheumatol 1998;12:181–90. Shear ES, et al. A genome-wide scan for juvenile rheumatoid 9. Rimoin DL. The chondrodystrophies. Adv Hum Genet 1975;5: arthritis in affected sibpair families provides evidence of linkage. 1–118. Arthritis Rheum 2004;50:2920–30. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1292–1294 DOI 10.1002/art.22509 © 2007, American College of Rheumatology

Interstitial Pneumonitis in Blau Syndrome With Documented Mutation in CARD15

Mara L. Becker,1 Tammy M. Martin,2 Trudy M. Doyle,2 and Carlos D. Rose´3

This is the first report of a CARD15 mutation– Although disease expression of both Blau syn- positive patient with Blau syndrome who exhibited drome and early-onset sarcoidosis involves granuloma- interstitial lung disease, a feature historically consid- tous infiltration of several organs, distinguishing both ered absent from Blau syndrome, while typical of the from adult sarcoidosis has historically relied on clinical adult form of sarcoidosis. This case illustrates the features. Both Blau syndrome and early-onset sarcoid- continued evolution of the phenotype of a disease ini- osis usually lack constitutional symptoms, hilar adeno- tially conceived as a familial inflammatory granuloma- pathy, and lung involvement. Blau syndrome and early- tous disease limited to the triad of synovitis, dermatitis, onset sarcoidosis present in the first 5 years of life; and uveitis. sarcoidosis presents later in the teen years and adulthood. Long-term morbidity in Blau syndrome and early- Blau syndrome is an autosomal-dominant disease onset sarcoidosis involves unremitting eye and joint historically described as a “classic triad” of granuloma- disease, while in sarcoidosis, pulmonary involvement tous polyarthritis, uveitis, and papuloerythematous rash dominates the phenotype. CARD15 mutations are not (1). Further reported cases of familial granulomatous associated with sarcoidosis, further distinguishing it from diseases with phenotypic expression beyond this “classic Blau syndrome and early-onset sarcoidosis (10). This is triad” were restricted from the diagnosis due to this the first report of a CARD15 mutation–positive patient strict definition (2,3). Two reports surfaced of visceral with Blau syndrome who exhibited interstitial pneumo- granulomas outside of the triad in patients with Blau nitis and granulomatous lymphadenitis, features usually syndrome (4,5), and the previously strict definition of identified with the adult disease, sarcoidosis. Blau syndrome was later challenged. The genetic expression of Blau syndrome was mapped to chromosome 16 CASE REPORT (6) and then found to be associated with mutations in the NACHT domain of CARD15 (7). The patient is 1 of 4 affected family members Another granulomatous disease of childhood, with Blau syndrome. He initially presented at age 1 year early-onset sarcoidosis, is clinically similar to Blau syn- with unilateral hip arthritis, which rapidly progressed to drome but without autosomal-dominant transmission. an exuberant symmetric polyarthritis. He was diagnosed Recent work demonstrated the same de novo mutation as having nongranulomatous bilateral anterior uveitis at in CARD15 in select patients with early-onset sarcoidosis age 3 years. He was treated with steroids and methotrex- (8,9). ate for the diagnosis of juvenile rheumatoid arthritis. Noncaseating granulomas were discovered on synovial Dr. Martin and Ms Doyle’s work was supported by the NIH biopsy at age 6 years, which led to the diagnosis of Blau (grant EY-013139). Dr. Martin’s work was also supported by a Career syndrome. His mother and 2 younger siblings are also Development Award from Research to Prevent Blindness. 1Mara L. Becker, MD: Children’s Mercy Hospitals and Clin- affected, most notably with polyarthritis and anterior ics, Kansas City, Missouri; 2Tammy M. Martin, PhD, Trudy M. Doyle, uveitis. All affected family members are genetically BS: Oregon Health and Science University, Portland; 3Carlos D. Rose´, confirmed to have Blau syndrome with a substitution of MD, CIP: duPont Hospital for Children, Wilmington, Delaware. Address correspondence and reprint requests to Mara L. glutamine for arginine at position 334 of CARD15 Becker, MD, Section of Rheumatology, Children’s Mercy Hospitals (R334Q) (11). The patient and his 2 younger affected and Clinics, 2401 Gillham Road, Kansas City, MO 64108. E-mail: brothers have been treated successfully with the anti– [email protected]. ␣ Submitted for publication August 17, 2006; accepted in tumor necrosis factor agent infliximab, with steroid- revised form January 4, 2007. sparing effects over the past several years.

1292 INTERSTITIAL PNEUMONITIS IN BLAU SYNDROME 1293

Figure 1. Biopsy tissue from the left posterior auricular lymph node, revealing discrete non- necrotizing granulomas with a multinucleated giant cell (original magnification ϫ 200).

At age 16 years, the patient experienced a grad- Epstein-Barr virus were suggestive of past infection. A ual onset of painless enlarged cervical lymphadeno- purified protein derivative test yielded negative results. pathy. The lymphadenopathy was intermittent. It usually Computed tomography (CT) of the chest re- involved the left side, but was at times bilateral. Initially, vealed several enlarged axillary lymph nodes, the largest he had no fever, weight loss, or further constitutional measuring 1.8 cm on the left and 2.5 cm on the right. symptoms. His primary care physician prescribed pred- Minimal mediastinal adenopathy was noted, including a nisone 10 mg/day for 5 days, after which the lymph- 1-cm right hilar node and several 4-mm pretracheal adenopathy resolved. However, once the therapy was nodes. Several small areas of ground-glass opacity were completed, his symptoms immediately returned. After seen in the medial segment of the right middle lobe and his regularly scheduled infliximab infusions, the lymph bilateral lower lobes. nodes would regress in size, with subsequent enlarge- We performed a biopsy of the left posterior ment between treatments. Over the course of several auricular node and included surrounding islands of months, he experienced an ϳ15-pound weight loss and salivary gland tissue. Histologically, the lymph node night sweats. retained its architecture, and there were mature lympho- On examination, an enlarged firm posterior au- cytes and scattered histiocytes within the lymph node. ricular node 1 cm in diameter was noted on the left. This node was matted and fixed to the skin, making it difficult Discrete, well-organized, and non-necrotizing granulo- to distinguish from the tail of the parotid gland. Multiple mas were noted (Figure 1). The salivary gland also small, mobile, nontender posterior cervical nodes were contained non-necrotizing granulomas. The tissue was also observed bilaterally. No axillary or inguinal lymph- stained for acid-fast bacilli and fungus, both of which adenopathy was noted. Boggy symmetric polyarthritis were absent. of the large joints, including the shoulders, arms, and Because of the above-noted new disease manifes- wrists, was unchanged. tations, prednisone treatment (10 mg/day) was reinsti- An initial laboratory evaluation for infections tuted. The infliximab dosage was increased to 10 mg/kg revealed negative titers for Bartonella henselae and Tox- every 8 weeks for disease control. This treatment course oplasma gondii. Antibody titers for cytomegalovirus and has resulted in resolution of his lymphadenopathy and 1294 BECKER ET AL

improvement in his arthritis. A repeat CT scan is AUTHOR CONTRIBUTIONS pending. Dr. Becker had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. DISCUSSION Study design. Becker, Rose´. Acquisition of data. Martin, Doyle. In 1985, Douglas Jabs and Edward Blau sepa- Analysis and interpretation of data. Becker, Martin, Rose´. rately reported 2 multigenerational families with granu- Manuscript preparation. Becker, Martin, Rose´. lomatous disease. Subsequent reports of families exhib- iting granulomatous synovitis, uveitis, and rash REFERENCES confirmed Blau syndrome as a distinct clinical entity. The clinical manifestations of the disease are the “classic 1. Blau EB. Familial granulomatous arthritis, iritis, and rash. J Pe- diatr 1985;107:689–93. triad” of proliferative polyarthritis, described often as 2. Jabs DA, Houk JL, Bias WB, Arnett FC. Familial granulomatous boggy or cyst-like; panuveitis with multifocal choroiditis synovitis, uveitis, and cranial neuropathies. Am J Med 1985;78: (12); and papuloerythematous rash transmitted in an 801–4. autosomal-dominant pattern. 3. Rotenstein D, Gibbas DL, Majmudar B, Chastain EA. Familial granulomatous arteritis with polyarthritis of juvenile onset. N Engl Clinically indistinguishable from Blau syndrome, J Med 1982;306:86–90. but lacking the autosomal-dominant transmission, is 4. Saini SK, Rose CD. Liver involvement in familial granulomatous early-onset sarcoidosis. Early-onset sarcoidosis has been arthritis (Blau syndrome). J Rheumatol 1996;23:396–9. genetically linked to Blau syndrome by evidence of de 5. Ting SS, Ziegler J, Fischer E. Familial granulomatous arthritis (Blau syndrome) with granulomatous renal lesions. J Pediatr novo CARD15 mutations. Although granulomatous in- 1998;133:450–2. volvement is generally limited to the joints, skin, and 6. Tromp G, Kuivaniemi H, Raphael S, Ala-Kokko L, Christiano A, eyes, there are reports of widespread granuloma inva- Considine E, et al. Genetic linkage of familial granulomatous inflammatory arthritis, skin rash, and uveitis to chromosome 16. sion in early-onset sarcoidosis, leading to morbidity and Am J Hum Genet 1996;59:1097–107. mortality secondary to multiorgan failure in end-stage 7. Miceli-Richard C, Lesage S, Rybojad M, Prieur AM, Manouvrier- disease (13,14). These findings occurred in terminally Hanu S, Hafner R, et al. CARD15 mutations in Blau syndrome. advanced cases and were described before the mutation Nat Genet 2001;29:19–20. 8. Kanazawa N, Matsushima S, Kambe N, Tachibana T, Nagai S, of CARD15 was known. Blau syndrome and early-onset Miyachi Y. Presence of a sporadic case of systemic granulomatosis sarcoidosis contrast with the adult-like form of sarcoid- syndrome with a CARD15 mutation. J Invest Dermatol 2004;122: osis, which presents in older children and adolescents, 851–2. clinically manifesting with systemic features of fever, 9. Rose CD, Doyle TM, McIlvain-Simpson G, Coffman JE, Rosen- baum JT, Davey MP, et al. Blau syndrome mutation of CARD15/ weight loss, hilar adenopathy, and pulmonary infiltra- NOD2 in sporadic early onset granulomatous arthritis. J Rheuma- tion. tol 2005;32:373–5. There have been sporadic reports of visceral 10. Schurmann M, Valentonyte R, Hampe J, Muller-Quernheim J, involvement in Blau syndrome, including granulomatous Schwinger E, Schreiber S. CARD15 gene mutations in sarcoidosis. Eur Respir J 2003;22:748–54. liver, kidney, and vascular infiltration (4,5,15). However, 11. Rose CD, Wouters CH, Meiorin S, Doyle TM, Davey MP, the clinical features of granulomatous lymphadenitis and Rosenbaum JT, et al. Pediatric granulomatous arthritis: an inter- interstitial pneumonitis exhibited by our patient have national registry. Arthritis Rheum 2006;54:3337–44. not, to our knowledge, been previously reported in a 12. Latkany PA, Jabs DA, Smith JR, Rosenbaum JT, Tessler H, Schwab IR, et al. Multifocal choroiditis in patients with familial familial case with documented mutation. juvenile systemic granulomatosis. Am J Ophthalmol 2002;134: As clinical information is collected in the Inter- 897–904. national Registry for Pediatric Granulomatous Arthritis 13. Fink CW, Cimaz R. Early onset sarcoidosis: not a benign disease. J Rheumatol 1997;24:174–7. (11) and confirmed by genetic analysis, we propose that 14. Hafner R, Vogel P. Sarcoidosis of early onset: a challenge for the interstitial pneumonitis, lymphadenopathy, hepatic in- pediatric rheumatologist. Clin Exp Rheumatol 1993;11:685–91. volvement, and large-vessel vasculitis be considered part 15. Wang X, Kuivaniemi H, Bonavita G, Mutkus L, Mau U, Blau E, et of the disease phenotype. All of these clinical expres- al. CARD15 mutations in familial granulomatosis syndromes: a study of the original Blau syndrome kindred and other families sions have been associated with substitutions in or near with large-vessel arteritis and cranial neuropathy. Arthritis Rheum the NACHT domain of CARD15. 2002;46:3041–5. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1295–1303 DOI 10.1002/art.22506 © 2007, American College of Rheumatology

Clinical and Immunogenetic Features of Patients With Autoantibodies to Asparaginyl–Transfer RNA Synthetase

Michito Hirakata,1 Akira Suwa,2 Tetsuya Takada,1 Shinji Sato,1 Sonoko Nagai,3 Ekkehard Genth,4 Yeong W. Song,5 Tsuneyo Mimori,3 and Ira N. Targoff6

Objective. We have previously described anti-KS identified from restriction fragment length polymor- autoantibodies and provided evidence that they are phism of polymerase chain reaction–amplified genomic directed against asparaginyl–transfer RNA (tRNA) syn- DNA. thetase (AsnRS). The aim of the present study was to Results. Anti-AsnRS antibodies were identified in identify patients with anti-AsnRS autoantibodies and the sera of 8 patients (5 Japanese, 1 American, 1 elucidate the clinical significance of this sixth antisyn- German, and 1 Korean) by IP of the same distinctive set thetase antibody. In particular, we studied whether it of tRNA and protein that differed from those precipi- was associated with the syndrome of myositis (polymy- tated by the other 5 antisynthetases, and these antibod- ositis or dermatomyositis [DM]), interstitial lung dis- ies showed specific inhibition of AsnRS activity. Two of ease (ILD), arthritis, and other features that had been these patients had DM, but 7 of 8 (88%) had ILD. Four previously associated with the 5 other anti–aminoacyl– patients (50%) had arthritis, and 1 had Raynaud’s tRNA synthetase autoantibodies. phenomenon. This antisynthetase was very rare among Methods. More than 2,500 sera from patients with myositis patients (present in 0% of Japanese myositis connective tissue disease (including myositis and ILD) patients), but it was found in 3% of Japanese ILD and controls were examined for anti-AsnRS autoanti- patients. Thus, most patients with anti-AsnRS had bodies by immunoprecipitation (IP). Positive and con- chronic ILD with or without features of connective trol sera were tested for the ability to inhibit AsnRS by tissue disease. Interestingly, all 4 Japanese patients preincubation of the enzyme source with the serum. The tested had DR2 (DRB1*1501/1502), compared with 33% HLA class II (DRB1, DQA1, DQB1, DPB1) alleles were of healthy controls. Conclusion. These results indicate that anti- Supported in part by grants from the Japanese Ministry of AsnRS autoantibodies, like anti–alanyl–tRNA syn- Education, Science, Culture, Sports, and Technology, the Japanese thetase autoantibodies, have a stronger association with Ministry of Health, Labor, and Welfare, and the Keio University School of Medicine. Dr. Targoff’s work was supported in part by the ILD than with myositis and may be associated with the US Department of Veterans Affairs. DR2 phenotype. 1Michito Hirakata, MD, Tetsuya Takada, MD, Shinji Sato, MD: Keio University School of Medicine, Tokyo, Japan; 2Akira Suwa, MD: Tokai University School of Medicine, Isehara, Japan; 3Sonoko The aminoacyl–transfer RNA (aminoacyl-tRNA) Nagai, MD, Tsuneyo Mimori, MD: Kyoto University Graduate School synthetases are a family of cytoplasmic enzymes that of Medicine, Kyoto, Japan; 4Ekkehard Genth, MD: Clinic and Re- search Institute for Rheumatic Diseases Aachen, Aachen, Germany; catalyze the formation of aminoacyl-tRNA from a spe- 5Yeong W. Song, MD: Seoul National University Hospital, Seoul, cific amino acid and its cognate tRNA and play a crucial Korea; 6Ira N. Targoff, MD: Veterans Affairs Medical Center, Uni- role in protein synthesis. Autoantibodies to certain of versity of Oklahoma Health Sciences Center, and Oklahoma Medical Research Foundation, Oklahoma City. these synthetases (histidyl–, threonyl–, alanyl–, Dr. Targoff serves as a technical consultant to the Oklahoma isoleucyl–, and glycyl–tRNA synthetases) have been Medical Research Foundation Clinical Immunology Laboratory. identified in patients with inflammatory myopathies Address correspondence and reprint requests to Michito Hirakata, MD, Section of Rheumatology, Department of Internal (1–6). Among these “antisynthetase autoantibodies,” Medicine, Keio University School of Medicine, 35 Shinanomachi, the most common is anti–Jo-1 (anti–histidyl–tRNA syn- Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: mhirakat@ thetase [anti-HisRS]), found in 20% of patients with sc.itc.keio.ac.jp. Submitted for publication September 25, 2005; accepted in polymyositis/dermatomyositis (PM/DM) (7–11). Anti– revised form January 4, 2007. PL-7 (anti–threonyl–tRNA synthetase [anti-ThrRS])

1295 1296 HIRAKATA ET AL

and anti–PL-12 (anti–alanyl–tRNA synthetase [anti- of ILD with arthritis and/or myositis. Immunoprecipita- AlaRS]) autoantibodies are less common, found in tion (IP) and aminoacylation inhibition studies with sera 3–4% of all patients with PM/DM (4,5,11–13), while from these patients provide additional evidence that anti-OJ (anti–isoleucyl–tRNA synthetase [anti-IleRS]) anti-KS (anti-AsnRS) reacts with asparaginyl–tRNA and anti-EJ (anti–glycyl–tRNA synthetase [anti-GlyRS]) synthetase. autoantibodies are the least common, occurring in Ͻ2% (6,14,15), although the frequency may vary in different PATIENTS AND METHODS populations (16). Characteristic clinical features have been found Sera. Serum samples from a collection of sera from in patients with anti-HisRS and other antisynthetase ϳ800 patients seen at the current or previous collaborating autoantibodies (1,9,10). These features include myositis, centers of the authors (Keio University, Tokyo, Japan; Kyoto University, Kyoto, Japan; Seoul National University, Seoul, interstitial lung disease (ILD), arthritis, Raynaud’s phe- Korea; Clinic and Research Institute for Rheumatic Diseases nomenon, fever with exacerbations, and the skin lesion Aachen, Aachen, Germany; University of Oklahoma Health of the fingers referred to as mechanic’s hands, and they Sciences Center, Oklahoma City; National Institutes of appear to form a distinct syndrome referred to as the Health, Bethesda, MD) or sera referred there for testing were “antisynthetase syndrome” (8–11). Although the similar- stored at Ϫ20°C and were tested for the presence of anti- ity of the clinical features associated with different AsnRS autoantibodies. Sera from the following patients were included: 1) patients with PM or DM according to the criteria antisynthetases is impressive (17,18), certain differences described by Bohan and Peter (24,25); 2) patients with a have been noted, which must be considered preliminary condition suggesting the clinical diagnosis of myositis; 3) due to the small reported number of patients with patients with ILD who had no evidence of myositis and did not non-HisRS antisynthetases (1,9,19). Patients with anti- meet criteria for other connective tissue diseases; and 4) AlaRS appear to be more likely than those with anti- patients with serum anticytoplasmic antibodies, regardless of HisRS to have ILD and/or arthritis either without diagnosis. Approximately 1,700 other sera have also been tested, including sera from patients with other conditions myositis or with little evidence of muscle disease. Ab- including systemic lupus erythematosus, systemic sclerosis, and sence of significant myositis over the full disease course rheumatoid arthritis, as well as sera from normal subjects. in patients with anti-HisRS is rare (Ͻ5%), although it Many of the sera were tested in studies of other autoantibod- may occur. Clinically significant myositis was seen in ies. All samples were obtained after the patients gave their 60% of US patients with anti-AlaRS (13), whereas none informed consent, as approved by the corresponding institutional review boards. Stored sera known to contain autoanti- of 6 Japanese patients with anti-AlaRS autoantibodies bodies against synthetases for histidine, threonine, alanine, fulfilled criteria for myositis (20). Among patients with glycine, and isoleucine were used as controls. anti-IleRS, 2 of 10 had ILD without evidence of myosi- ILD was considered to be present if an interstitial tis, and 1 had ILD with subclinical myositis (14). In infiltrate was observed on chest radiography. DM was consid- addition, antisynthetases may occur in either PM or DM, ered to be present if a heliotrope rash and/or Gottron’s papules were observed. but PM is usually more common with anti-HisRS IP. IP from HeLa cell extracts was performed as (10,16,21), and DM is usually more common with other previously described (6,10). Ten microliters of patient sera was antisynthetases, especially anti-GlyRS (15,22). mixed with 2 mg of protein A–Sepharose CL-4B (Pharmacia We recently described anti-KS autoantibodies Biotech, Uppsala, Sweden) in 500 ␮l of IP buffer (10 mM Tris and provided evidence that the KS antigen is HCl at pH 7.5, 500 mM NaCl, 0.1% Nonidet P40 [NP40]) and incubated with end-over-end rotation (Labquake shaker; Lab asparaginyl–tRNA synthetase (AsnRS) (23). This sixth Industries, Berkeley, CA) for 2 hours at 4°C. The IgG-coated antisynthetase was found in sera from 3 patients with Sepharose was washed 4 times in 500 ␮l of IP buffer using ILD and/or inflammatory arthritis without evidence of 10-second spins in a microfuge tube, and resuspended in 400 ␮l myositis. It immunoprecipitated a 65-kd protein and a of NET-2 buffer (50 mM Tris HCl at pH 7.5, 150 mM NaCl, unique tRNA that was distinct from that precipitated by 0.05% NP40). For analysis of RNAs, this suspension was incubated any previously described antisynthetase or other re- with 100 ␮l of extracts, derived from 6 ϫ 106 cells, on the ported tRNA-related antibody. Each of the 3 sera and rotator for 2 hours at 4°C. The antigen-bound Sepharose was their IgG fractions showed significant inhibition of then collected with a 10-second centrifugation in the mi- AsnRS activity, but did not inhibit any of the other 19 crofuge, washed 4 times with NET-2 buffer, and resuspended ␮ ␮ aminoacyl–tRNA synthetase activities. in 300 l of NET-2 buffer. To extract bound RNAs, 30 lof 3.0M sodium acetate, 30 ␮l of 10% sodium dodecyl sulfate In this report, we describe the clinical and immu- (SDS), and 300 ␮l of phenol/chloroform/isoamyl alcohol (50: nogenetic features of 5 additional patients with anti- 50:1; containing 0.1% 8-hydroxyquinoline) were added to the AsnRS autoantibodies, most of whom had the syndrome Sepharose beads. After agitation in a Vortex mixer and CLINICAL SIGNIFICANCE OF ANTI-AsnRS AUTOANTIBODIES 1297

spinning for 1 minute, RNAs were recovered in the aqueous on mild exertion. Chest radiography and computed tomo- phase after ethanol precipitation and dissolved in 20 ␮lof graphy (CT) scanning showed bilateral basilar infiltrates. The electrophoresis sample buffer, composed of 10M urea, 0.025% patient had hypoxemia, with a restrictive pattern on pulmonary bromphenol blue, and 0.025% xylene cyanol FF (Bio-Rad, function tests. No muscle weakness was observed, and the Hercules, CA) in Tris–borate–EDTA buffer (90 mM Tris HCl creatine kinase (CK) level was normal (67 IU/liter). A lung at pH 8.6, 90 mM boric acid, and 1 mM EDTA). The RNA biopsy specimen obtained by video-assisted thoracic surgery samples were denatured at 65°C for 5 minutes and then showed mild interstitial chronic inflammation and interstitial resolved by 7M urea–10% polyacrylamide gel electrophoresis fibrosis lacking a temporal heterogeneity pattern, and a diag- (PAGE), with silver staining (Bio-Rad). nosis of fibrotic nonspecific interstitial pneumonia was made. For protein studies, antibody-coated Sepharose was Patient 2. The patient, a 51-year-old German woman, mixed with 400 ␮lof35S-methionine–labeled HeLa extract developed a nonproductive cough and dyspnea on exertion. derived from 2 ϫ 105 cells and rotated at 4°C for 2 hours. After Chest radiography showed bibasilar interstitial fibrosis, and 4 washes with IP buffer, the Sepharose was resuspended in pulmonary function tests showed a restrictive pattern with SDS sample buffer (2% SDS, 10% glycerol, 62.5 mM Tris HCl decreased diffusing capacity for carbon monoxide (DLco). A at pH 6.8, 0.005% bromphenol blue). After heating at 90°C for diagnosis of ILD was made, and the patient’s pulmonary 5 minutes, the proteins were fractionated by 10% SDS-PAGE, function remained stable throughout her disease course. She enhanced with 0.5M sodium salicylate, and dried. Labeled had polyarthralgia and developed erythema and keratosis of proteins were analyzed by autoradiography. the palms and fingers consistent with mechanic’s hands, but no Aminoacylation. Aminoacylation inhibition reactions cutaneous scleroderma, Raynaud’s phenomenon, or DM rash were performed as described previously, with minor modifica- (Gottron’s papules or heliotrope rash) was observed. No tion (6,26). Six microliters of HeLa cell extract diluted 1:10 in muscle weakness was found, and the CK level was normal (56 Tris buffered saline was incubated with 3 ␮l of a 1:10 dilution IU/liter at the first visit) each time it was measured. When the of serum for 2 hours at 4°C. This was combined with 17 ␮lof patient was age 58 years, ovarian carcinoma was found, and reaction solution (50 mM Tris HCl at pH 7.5, 0.02M NaCl, surgery with subsequent irradiation was performed. She died

0.01M MgSO4,1mM dithiothreitol) containing 8 units of of metastatic ovarian carcinoma at age 63 years. yeast tRNA, 3 ␮lof14C-asparagine or other 3H-labeled amino Patient 3. The patient, a 72-year-old American woman, acid, and 1 ␮l of 200 mM cold amino acid. Ten-microliter developed an itchy red eczematous rash that was thought to be aliquots were tested at 10 minutes and 20 minutes, spotted due to a medication for hypertension. The rash was soon onto filter paper treated with 5% trichloroacetic acid (TCA), accompanied by progressive weakness, myalgias, mild dyspnea, washed 5 times with 5% TCA, then with ethanol, then dried and difficulty swallowing. She was started on prednisone and for counting. Results of inhibition testing were expressed as methotrexate, and 6 months after the rash had first appeared, the percent inhibition of the average activity seen with the she was referred to the Arthritis and Rheumatism Branch of normal serum included in that experiment, as follows: % in- the National Institute of Arthritis and Musculoskeletal and hibition ϭ [(average counts per minute with normal serum) Ϫ Skin Diseases, National Institutes of Health. There was a (cpm with test serum)] ϫ 100/(average cpm with normal widespread maculopapular rash of the trunk, extremities, and serum). Inhibition of Ͼ50% compared with the activity with head, and Gottron’s papules were observed. Proximal muscle normal serum was considered significant. In previous studies, weakness was present, and her CK level was 358 IU/liter. although nonspecific effects on aminoacylation reactions by Magnetic resonance imaging of the thighs showed both atro- serum were common, nonspecific inhibition was usually phy and probable inflammation on the STIR images. A biopsy Ͻ25%, and inhibition Ͼ50% reliably reflected specific anti- of the deltoid muscle showed changes of an active inflamma- body effects (6,7,12,13,26). tory myopathy. No malignancy was identified. She was treated DNA typing of the HLA class II (DRB1, DQA1, DQB1, with pulse methylprednisolone. However, her muscle weakness DPB1) alleles by polymerase chain reaction (PCR)–restriction and rash were not significantly improved, and infectious com- fragment length polymorphism (RFLP). Genomic DNA was plications limited the therapeutic options. Her disease course isolated by phenol extraction of SDS-lysed and proteinase was subsequently complicated by herpes zoster and the K–treated peripheral blood leukocytes, and then amplified by Ramsay-Hunt syndrome as well as by skin infections and the PCR procedure using an automated PCR thermal cycler cellulitis, mastoiditis, heart failure, and a cerebrovascular (PerkinElmer Cetus, Norwalk, CT). The primers used for accident. specific amplification of the polymorphic exon 2 domains of Patient 4. The patient, a 53-year-old Korean woman the DRB1, DQA1, DQB1, and DPB1 genes were previously with intermittent episodes of productive cough due to bron- described (27). Amplified DNA was digested by all-specific chiectasis, noticed easy fatigability and myalgia in 1994 and restriction endonucleases and subjected to electrophoresis later developed muscle weakness and was admitted to Seoul using a 12% polyacrylamide gel. Digested fragments were National University Hospital in February 1995. Proximal mus- detected by staining with ethidium bromide, and HLA geno- cle weakness in her extremities and a dark pigmentation over types were determined on the basis of the RFLP patterns the extensor surface of both knees were observed. The CK generated as previously described (27). level was elevated at 3,808 IU/liter. The findings on electro- Other. Ouchterlony double immunodiffusion was per- myogram and muscle biopsy were consistent with inflamma- formed as described previously, using HeLa cell extract as tory myopathy. A diagnosis of DM associated with ILD was antigen (10). made, and she was treated with prednisolone (60 mg/day). Her Cases. Patient 1. The patient, a 61-year-old Japanese muscle enzyme levels gradually normalized, and her muscle woman, noticed chest pain, followed 3 months later by dyspnea weakness improved. Her chest radiograph and high-resolution 1298 HIRAKATA ET AL

Figure 1. A, Immunoprecipitation (IP) for nucleic acids with anti-KS and control sera. Shown are patterns of transfer RNA (tRNA) resulting from 7M urea–10% polyacrylamide gel electrophoresis (PAGE) of phenol-extracted immunoprecipitates from HeLa cell extract, developed with silver stain. TNA ϭ total nucleic acids, with the 5.8S and 5S small ribosomal RNAs and the tRNA region indicated. Antisynthetase sera used for IP are indicated. Lane 1, Anti–histidyl–tRNA synthetase (a Jo-1); lane 2, anti–threonyl–tRNA synthetase (a PL-7); lane 3, anti–alanyl–tRNA synthetase (a PL-12); lane 4, anti–glycyl–tRNA synthetase (a EJ); lane 5, anti–isoleucyl–tRNA synthetase (a OJ/SS-A); lanes 6–11, anti-KS sera from patients KS, KN, and NI in the previous study (23) and from patients 1, 4, and 5 in the present study; lane 12, normal human serum (NHS) control. The tRNA pattern with anti-KS sera is easily distinguishable from that of other antisynthetases. B, IP for proteins with anti-KS and control sera. Autoradiogram of 10% sodium dodecyl sulfate–PAGE of immunoprecipitates from 35S-methionine–labeled HeLa cell extract. Mr. ϭ molecular weight markers. Antisynthetase sera used for IP are indicated as in A. Anti-KS sera immunoprecipitated a very strong protein band from 35S-methionine–labeled HeLa cell extracts (lanes 6–11), migrating at 65 kd, that was clearly different from the bands immunoprecipitated by sera with the described antisynthetases.

CT scan showed bilateral basilar interstitial fibrosis, and radiograph. Prednisolone was tapered and discontinued in pulmonary function tests showed a restrictive pattern with April 1998. He then developed polyarthritis and was treated decreased DLco. Her muscle weakness gradually improved, with a nonsteroidal antiinflammatory drug. No muscle weak- and the CK level normalized in January 1996. Prednisolone ness was found, and the CK level was normal (50 IU/liter at the was tapered and discontinued in March 1996. first visit) throughout his disease course. Patient 5. The patient, a 64-year-old Japanese man with a previous history of prostatic carcinoma, was admitted to the hospital due to bilateral infiltrates on chest radiography. He RESULTS did not notice cough or dyspnea at that time, but a chest CT Identification of anti-KS (anti-AsnRS) antibod- scan revealed bibasilar interstitial fibrosis. A transbronchial lung biopsy was performed, with histology showing usual ies. Sera from all 8 patients (the 3 patients with ILD interstitial pneumonia. He was started on prednisolone (40 and/or inflammatory arthritis without evidence of myo- mg/day), resulting in slight improvement seen on his chest sitis in our previous study [patients KS, KN, and NI; see CLINICAL SIGNIFICANCE OF ANTI-AsnRS AUTOANTIBODIES 1299

Table 1. Clinical features of 8 patients with anti-KS antibodies* Patient

KS KN NI 1 2 3 4 5 Age at onset, years/sex 36/F 44/F 61/F 60/F 51/F 72/F 53/F 65/M Ethnic background Japanese Japanese Japanese Japanese German US Korean Japanese ILD Yes Yes Yes Yes Yes No Yes Yes Myositis No No No No No Yes Yes No DM rash No No No No No Yes Yes No Arthritis Yes No No No Yes Yes No Yes Malignancy No No No No Ovarian cancer No No Prostate cancer Raynaud’s phenomenon No Yes No No No No No No Other autoantibodies No No No Anti-SSA/Ro No No No No Diagnosis ILD with arthritis Idiopathic ILD Idiopathic ILD Idiopathic ILD Idiopathic ILD DM DM ILD with arthritis * ILD ϭ interstitial lung disease; DM ϭ dermatomyositis. ref. 23] and the 5 additional patients described above) AsnRS, although sera with other antisynthetases inhib- were shown to immunoprecipitate a characteristic, iden- ited the expected enzymes. These results indicated that tical pattern of tRNA, with a strong predominant nucleic sera with anti-KS by IP showed specific inhibition of acid band of tRNA size, accompanied by a faster faint AsnRS, further supporting previous data indicating that band (Figure 1A). This gel pattern of tRNA was clearly anti-KS reacted with AsnRS. distinguishable from the pattern of tRNA precipitated Clinical findings. The clinical features of the 5 by the 5 other described antisynthetases (Figure 1A) and newly identified patients (patients 1–5) and the 3 pa- was identical in mobility and appearance to that of tients with anti-AsnRS reported previously (patients KS, serum KS, the originally reported anti-KS serum (23) KN, and NI) (23) are summarized in Table 1. All (Figure 1A). patients with anti-AsnRS antibodies were middle-aged A very strong band from 35S-methionine–labeled or elderly, and 7 of them were women. Five patients HeLa cell extracts (Figure 1B), migrating at 65 kd, that were Japanese, 1 was from the US, 1 was German, and was also identical in mobility to that of serum KS, was 1 was Korean. Seven of these 8 patients (88%) had ILD, found by IP for all 8 sera, with 5 representative sera documented in each case by both chest radiography and shown in Figure 1B. This was clearly different from the pulmonary function tests. In addition, 2 patients had characteristic bands immunoprecipitated by sera with myositis and a diagnosis of DM. Their clinical courses of the other described antisynthetases (Figure 1B). ILD were classified as the chronic type. Four patients Five of the newly recognized anti-KS antibody– (50%) had nonerosive arthritis or arthralgia. Raynaud’s positive sera were tested for their ability to inhibit the in phenomenon was seen in only 1 patient. None of the vitro enzymatic function of AsnRS (aminoacylation of patients had sclerodactyly or overlap syndromes with tRNAAsn). Four of the 5 new anti-KS sera significantly other connective tissue diseases. Malignant diseases inhibited (by Ͼ50% at 10 minutes) AsnRS activity (ovarian carcinoma and prostatic carcinoma) were ob- compared with normal serum or other controls (serum served in 2 patients. Regarding other autoantibodies, from patient KS by 87%, serum from patient KN by anti-SSA/Ro antibodies were detected in only 1 patient. 99%, serum from patient NI by 91%, serum from patient Anti-AsnRS was found in 0% of Japanese pa- 1 by 82%, serum from patient 2 by 100%, serum from tients with myositis, but was found in 3% of Japanese patient 3 by 18%, serum from patient 4 by 87%, and patients with “idiopathic” ILD. Thus, most patients with serum from patient 5 by 91%). This inhibition was strong anti-AsnRS antibodies had chronic ILD with or without and comparable with that seen with serum KS, for 4 of features of PM/DM or other connective tissue disease. the 5 new anti-KS sera. Purified IgG from the third new Immunogenetic features. The HLA class II gene serum (from patient 3) showed significant, but not was determined in 4 Japanese patients (Table 2). All 4 strong, inhibition (52%) that increased at 20 minutes (to patients had DR2 (DRB1*1501 or DRB1*1502) com- 84%). pared with 33% of healthy local controls. It should be There was no significant inhibition of other syn- noted that all patients with anti-AsnRS antibodies had thetases. Normal control serum and anti-KS–negative DR2, but the frequency of DR2 did not reach statistical myositis serum did not show significant inhibition of significance (P Ͼ 0.05). 1300 HIRAKATA ET AL

Table 2. HLA class II genes in Japanese patients with anti-KS previously found that the 3 original anti-KS (anti- autoantibodies AsnRS) sera did not immunoprecipitate any RNA from Patient deproteinized HeLa extracts (23). This suggests that Asn KS KN NI 1 anti-AsnRS antibodies can precipitate tRNA indi- rectly, through its affinity for AsnRS, although the DR 2/5 2/1 2/2 2/4 DRB1* 1502/1101 1501/0101 1502/1502 1501/0405 possibility of conformational epitopes on the tRNA has DQA1* 0103/0501 0102/0101 0103/0103 0102/0303 not been excluded (28). Further analysis will be neces- DQB1* 0601/0301 0602/0501 0601/0601 0602/0401 sary to determine the sequence and specificity of tRNA DPB1* 0901/1401 0201/0501 0901/0901 0201/0402 immunoprecipitated by anti-AsnRS. The specific inhibition of AsnRS function by most of the sera found to have anti-KS is consistent with DISCUSSION findings observed for other antisynthetases. It should be noted that our anti-KS sera also demonstrated inhibition We have identified anti-KS (anti-AsnRS) auto- of enzymatically active recombinant AsnRS (30). Most antibodies in 8 patients with ILD and DM, by IP of the sera with any of the 5 reported antisynthetases specifi- same distinctive set of tRNA and protein that differed cally inhibit the aminoacylation of the respective tRNA, from those precipitated by the other 5 antisynthetases. indicating inhibition of the enzymatic function of the Most of the anti-KS sera showed specific inhibition of synthetase (3,5–7,12). This functional inhibition may the enzyme target, AsnRS, without inhibiting other indicate that the autoantibodies are recognizing the synthetases. active sites of the synthetases. In contrast, it has been Several interesting characteristics of the previ- reported that animal antisera raised against synthetases ously studied antisynthetases have been described: 1) do not consistently show such inhibition, suggesting that they are associated with a distinctive clinical syndrome active sites tend not to be immunogenic for animals (31). referred to as the antisynthetase syndrome, 2) they are Hypothetically, this could relate to relative conservation directed at functionally related enzymes (performing the of the active site. However, there might be an alternative same function for different amino acids), 3) they do not mechanism for inhibition. For example, binding of anti- cross-react with other synthetases, and 4) they tend to be bodies outside the active site may alter the structure of mutually exclusive. Anti-AsnRS antibodies seem to have the enzyme or interfere with enzyme activity sterically. the same features. No serum with any other antisynthetase has had antibodies to AsnRS, and none of the 8 Further studies of the precise epitope on the aminoacyl– anti-AsnRS sera reported here showed signs of reaction tRNA synthetase might help to explain the development with other synthetases. The mechanism of this phenom- of these autoantibodies. enon remains unknown. Each of the 5 previous antisynthetases was first Multiple tRNA bands immunoprecipitated by identified in patients with myositis and then found to be anti-AsnRS were found on urea-PAGE. The patterns of associated with ILD. In previous studies, these autoan- tRNA for each of the 8 patients were very similar, highly tibodies were associated with myositis with a high fre- restricted compared with total tRNA, and distinctive quency of ILD (50–80%) and arthritis (50–90%) compared with the pattern of other anti–aminoacyl (1,2,17,18), as well as an increase in Raynaud’s phenom- tRNA synthetase autoantibodies. These bands are likely enon (60%), fever with exacerbations (80%), and the to represent different forms of tRNA for asparagine, skin lesion of the fingers referred to as mechanic’s hands which can include tRNA with different asparagine anti- (70%) when compared with the overall population of codons (uracil-uracil-adenine, uracil-uracil-guanine) or patients with myositis (9–11). The similarities between tRNA with the same anticodon but differences in other patients with different antisynthetases have been noted, parts of the sequence. Most sera with anti-HisRS, anti- whereas certain differences have been found, which ThrRS, anti-GlyRS, and anti-IleRS had not been de- must be considered preliminary due to the small re- scribed to react directly with tRNA, suggesting indirect ported number of patients with non-HisRS antisyntheta- precipitation of tRNA. However, approximately one- ses. Absence of significant myositis over the full disease third of anti-HisRS–positive sera were reported to con- course in patients with anti-HisRS is rare (Ͻ5%) (32), tain autoantibodies recognizing tRNAHis (28). Most whereas patients with anti-AlaRS are more likely than anti-AlaRS sera react directly with the sets of tRNAAla patients with anti-HisRS to have ILD and/or arthritis with the inosine-guanine-cytosine anticodon (29). We without clinical evidence of myositis (19). Anti-ThrRS CLINICAL SIGNIFICANCE OF ANTI-AsnRS AUTOANTIBODIES 1301

resembles anti-HisRS more than anti-AlaRS in Japa- (78%) with anti-HisRS tested had the HLA class II nese patients (33). DRB1*0405;DQA1*0302;DQB1*0401 haplotype, com- In the present study, 7 of 8 patients (88%) with pared with 22% of healthy controls (odds ratio [OR] 13, anti-AsnRS autoantibodies had ILD, some with other P ϭ 0.002), while 4 of 7 patients (57%) with anti-AlaRS associated features of connective tissue disease including had the DRB1*1501;DQA1*0102;DQB1*0602 haplo- arthritis and Raynaud’s phenomenon. In this respect, type, compared with 9% of healthy controls (OR 14, P ϭ anti-AsnRS appears to resemble anti-AlaRS more than 0.006) (35). Interestingly, all 4 Japanese patients tested anti-HisRS. It is noteworthy that the 2 patients with both had DR2 (DRB1*1501/1502), compared with 33% of anti-AsnRS and myositis were among the 3 patients healthy controls, although a definite statistical associa- from outside Japan, while none of 5 patients from Japan tion could not be established. These results suggest that had myositis. Thus, as with patients with anti-AlaRS, for the stronger association of anti-AlaRS and anti-KS with patients with anti-AsnRS, the frequency of ILD without ILD may be related to the DR2 phenotype. However, it myositis may be higher in Japanese patients. However, has been noted that different ethnic groups exhibit most of the group of patients with ILD without myositis different immunogenetic profiles that link with specific who were tested in this study were from Japan. autoantibodies (36). Therefore, further studies including The features of these 8 patients with anti-KS analysis of more patients with anti-KS antibodies in appeared to reside within the spectrum of the antisyn- different ethnic groups and major histocompatibility thetase syndrome that has been associated with other complex–restricted T cell responses could provide im- antisynthetases. ILD is one of the most important fea- portant clues for understanding the possible mechanisms tures of the antisynthetase syndrome, and Raynaud’s for the development of antisynthetase antibodies. phenomenon and arthritis, as seen in some patients with The mechanism for the association of antisyn- anti-AsnRS, are also likely to be part of the syndrome. thetases with ILD is unknown, but it seems to be related The syndrome associated with anti-AsnRS may be one to etiologic factors (37). Recently, a new association of end of the spectrum of patients with antisynthetase. This anti-HisRS–positive PM and ILD was reported in a highlights the clinical importance of looking for such patient with hepatitis C virus infection (38). It was antibodies in patients with ILD even if there are no signs hypothesized that viruses might interact with the syn- of myositis or connective tissue diseases. thetases and induce autoantibodies by molecular mim- The typical cutaneous features of DM were ob- icry or antiidiotype mechanisms in the anti-HisRS– served in 2 patients with anti-AsnRS antibodies. PM has positive patient with myositis associated with ILD (3,39). been reported to be much more common (60–80% or Another mechanism for generating autoantigenic more) than DM in patients with anti-HisRS in most epitopes of synthetase by granzyme B cleavage in apo- studies, whereas DM was most frequent with anti-GlyRS ptosis was also described recently (40,41). However, (15) and was also found to be common among patients these proposed mechanisms remain speculative, and with anti-AlaRS (13). Like anti-GlyRS and anti-AlaRS further studies could provide important clues for under- antibodies, anti-AsnRS antibodies were more associated standing the possible mechanisms for the development with DM in the small number of patients available. of these antibodies. Studies of these antibodies may Malignancy has been reported to be unusual in provide insight into the etiologic and pathogenetic patients with antisynthetases. In our studies, 2 patients mechanisms of ILD and myositis. were found to have malignancy during their disease course. However, malignancy in these patients may not be related to the DM or ILD, since these malignancies ACKNOWLEDGMENTS occurred separated in time from each other. We would like to thank Dr. Paul H. Plotz for providing Immunogenetic studies of connective tissue dis- the clinical information and serum, and Ms Mutsuko Ishida ease have been performed, but HLA associations pro- and Mr. Edward Trieu for expert technical assistance. We wish duced conflicting results. However, a strong correlation to thank Dr. John A. Hardin for helpful discussion and critical of HLA class II antigens with some autoantibodies has review of the manuscript. been reported (34). With regard to antisynthetase antibodies, HLA–DR3 (DRB1*0301), DQA1*0501, or AUTHOR CONTRIBUTIONS DQA1*0401 was found to be significantly increased in Dr. Hirakata had full access to all of the data in the study and myositis patients with antisynthetases (9,21). In Japa- takes responsibility for the integrity of the data and the accuracy of the nese patients, we have reported that 7 of 9 patients data analysis. 1302 HIRAKATA ET AL

Study design. Hirakata. the Jo-1, in connective tissue diseases: a marker for a subset of Acquisition of data. Hirakata, Nagai, Genth, Song, Targoff. polymyositis with interstitial pulmonary fibrosis. Arthritis Rheum Analysis and interpretation of data. Hirakata, Suwa, Takada, Sato, 1983;26:604–11. Mimori. 18. Bernstein RM, Morgan SH, Chapman J, Bunn CC, Mathews MB, Manuscript preparation. Hirakata, Takada, Targoff. Turner-Warwick M, et al. Anti-Jo-1 antibody: a marker for Statistical analysis. Hirakata, Suwa, Targoff. myositis with interstitial lung disease. Br Med J 1984;289:151–2. 19. Friedman AW, Targoff IN, Arnett FC. Interstitial lung disease with autoantibodies against aminoacyl-tRNA synthetase in the absence of clinically apparent myositis. Semin Arthritis Rheum REFERENCES 1996;26:459–67. 20. Hirakata M, Nakamura K, Okano Y, Suwa A, Inada S, Akizuki M, 1. Targoff IN. Laboratory testing in the diagnosis and management et al. Anti-alanyl tRNA synthetase (PL-12) antibodies are associ- of idiopathic inflammatory myopathies. Rheum Dis Clin North ated with interstitial lung disease in Japanese patients [abstract]. Am 2002;28:859–90. Arthritis Rheum 1995;38:S321. 2. Targoff IN. Myositis specific autoantibodies. Curr Rheumatol Rep 21. Arnett FC, Targoff IN, Mimori T, Goldstein R, Warner NB, 2006;8:196–203. Reveille JD. Interrelationship of major histocompatibility complex class II alleles and autoantibodies in four ethnic groups 3. Mathews MB, Bernstein RM. Myositis autoantibody inhibits his- with various forms of myositis. Arthritis Rheum 1996;39: tidyl-tRNA synthetase: a model for autoimmunity. Nature 1983; 1507–18. 304:177–9. 22. Hirakata M, Suwa A, Takeda Y, Matsuoka Y, Irimajiri S, Targoff 4. Mathews MB, Reichlin M, Hughes GR, Bernstein RM. Anti- IN, et al. Autoantibodies to glycyl–transfer RNA synthetase in threonyl-tRNA synthetase, a second myositis-related autoanti- myositis:associationwithdermatomyositisandimmunologichetero- body. J Exp Med 1984;160:420–34. geneity. Arthritis Rheum 1996;39:146–51. 5. Bunn CC, Bernstein RM, Mathews MB. Autoantibodies against Ala 23. Hirakata M, Suwa A, Nagai S, Kron MA, Trieu EP, Mimori T, et alanyl tRNA synthetase and tRNA coexist and are associated al. Anti-KS: identification of autoantibodies to asparaginyl-trans- with myositis. J Exp Med 1986;163:1281–91. fer RNA synthetase associated with interstitial lung disease. 6. Targoff IN. Autoantibodies to aminoacyl-transfer RNA syntheta- J Immunol 1999;162:2315–20. ses for isoleucine and glycine: two additional synthetases are 24. Bohan A, Peter JB. Polymyositis and dermatomyositis (first of two antigenic in myositis. J Immunol 1990;144:1737–43. parts). N Engl J Med 1975;292:344–7. 7. Targoff IN, Reichlin M. Measurement of antibody to Jo-1 by 25. Bohan A, Peter JB. Polymyositis and dermatomyositis (second of ELISA and comparison to enzyme inhibitory activity. J Immunol two parts). N Engl J Med 1975;292:403–7. 1987;138:2874–82. 26. Targoff IN, Arnett FC, Berman L, O’Brien CA, Reichlin M. 8. Oddis CV, Medsger TA Jr, Cooperstein LA. A subluxing arthro- Anti-KJ: a new antibody associated with the myositis/lung syn- pathy associated with the anti–Jo-1 antibody in polymyositis/ drome that reacts with translation-related protein. J Clin Invest dermatomyositis. Arthritis Rheum 1990;33:1640–5. 1989;84:162–72. 9. Love LA, Leff RL, Fraser DD, Targoff IN, Dalakas MC, Plotz PH, 27. Inoko H, Ota M. PCR-RFLP. In: Hui KM, Bidwell J, editors. et al. A new approach to the classification of idiopathic Handbook of HLA typing techniques. Boca Raton (FL): CRC inflammatory myopathy: myositis-specific autoantibodies de- Press; 1993. p. 9–70. fine useful homogeneous patient groups. Medicine (Baltimore) 28. Brouwer R, Vree Egberts W, Jongen PH, van Engelen BG, van 1991;70:360–74. Venrooij WJ. Frequent occurrence of anti-tRNAHis autoantibod- 10. Hirakata M, Mimori T, Akizuki A, Craft J, Hardin JA, Homma M. ies that recognize a conformational epitope in sera of patients with Autoantibodies to small nuclear and cytoplasmic ribonucleopro- myositis. Arthritis Rheum 1998;41:1428–37. teins in Japanese patients with inflammatory muscle disease. 29. Bunn CC, Mathews MB. Two human tRNA(Ala) families are Arthritis Rheum 1992;35:449–56. recognized by autoantibodies in polymyositis sera. Mol Biol Med 11. Marguerie C, Bunn CC, Beynon HL, Bernstein RM, Hughes 1987;4:21–36. JM, So AK, et al. Polymyositis, pulmonary fibrosis and autoan- 30. Beaulande M, Tarbouriech N, Hartlein M. Human cytosolic tibodies to aminoacyl-tRNA synthetase enzymes.QJMed asparaginyl-tRNA synthetase: cDNA sequence, functional expres- 1990;77:1019–38. sion in Escherichia coli and characterization as human autoanti- 12. Targoff IN, Arnett FC, Reichlin M. Antibody to threonyl–transfer gen. Nucleic Acids Res 1998;26:521–4. RNA synthetase in myositis sera. Arthritis Rheum 1988;31:515–24. 31. Miller FW, Waite KA, Biswas T, Plotz PH. The role of an 13. Targoff IN, Arnett FC. Clinical manifestations in patients with autoantigen, histidyl-tRNA synthetase, in the induction and main- antibody to PL-12 antigen (alanyl-tRNA synthetase). Am J Med tenance of autoimmunity. Proc Natl Acad SciUSA1990;87: 1990;88:241–51. 9933–7. 14. Targoff IN, Trieu EP, Miller FW. Reaction of anti-OJ autoanti- 32. Schmidt WA, Wetzel W, Friedlander R, Lange R, Sorensen HF, bodies with components of the multi-enzyme complex of amino- Lichey HJ, et al. Clinical and serological aspects of patients with acyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase. anti-Jo-1 antibodies: an evolving spectrum of disease manifesta- J Clin Invest 1993;91:2556–64. tions. Clin Rheumatol 2000;19:371–7. 15. Targoff IN, Trieu EP, Plotz PH, Miller FW. Antibodies to 33. Sato S, Hirakata M, Kuwana M, Nakamura K, Suwa A, Inada S, et glycyl–transfer RNA synthetase in patients with myositis and al. Clinical characteristics of Japanese patients with anti-PL-7 interstitial lung disease. Arthritis Rheum 1992;35:821–30. (anti-threonyl-tRNA synthetase) autoantibodies. Clin Exp Rheu- 16. Furuya T, Hakoda M, Tsuchiya N, Kotake S, Ichikawa N, Nanke matol 2005;23:609–15. Y, et al. Immunogenetic features in 120 Japanese patients with 34. Arnett FC. The genetics of human lupus. In: Wallace DL, Hahn idiopathic inflammatory myopathy. J Rheumatol 2004;31: BH, editors. Dubois’ lupus erythematosus. 5th ed. Baltimore: 1768–74. Williams & Wilkins; 1997. p.77–117. 17. Yoshida S, Akizuki M, Mimori T, Yamagata H, Inada S, Homma 35. Hirakata M, Satoh S, Suwa A, Nakamura K, Hama N, Ohsone Y, M. The precipitating antibody to an acidic nuclear protein antigen, et al. Clinical and immunogenetic features of anti-aminoacyl CLINICAL SIGNIFICANCE OF ANTI-AsnRS AUTOANTIBODIES 1303

tRNA synthetase autoantibodies in Japanese patients [abstract]. Polymyositis, pulmonary fibrosis, and hepatitis C. Arthritis Rheum Arthritis Rheum 1997;40 Suppl 9:S83. 1995;38:437–9. 36. Hirakata M, Suwa A, Kuwana M, Sato S, Mimori T, Hardin JA. 39. Plotz PH. Autoantibodies are anti-idiotype antibodies to antiviral Association between autoantibodies to the Ku protein and DPB1. antibodies. Lancet 1983;2:824–6. Arthritis Rheum 2005;52:668–9. 40. Casciola-Rosen L, Andrade F, Ulanet D, Wong WB, Rosen A. 37. Plotz PH, Dalakas M, Leff RL, Love LA, Miller FW, Cronin ME. Cleavage by granzyme B is strongly predictive of autoantigen Current concepts in the idiopathic inflammatory myopathies: status: implications for initiation of autoimmunity. J Exp Med polymyositis, dermatomyositis, and related disorders. Ann Intern 1999;190:815–26. Med 1989;111:143–57. 41. Plotz PH. The autoantibody repertoire: searching for order. Nat 38. Weidensaul D, Imam T, Holyst MM, King PD, McMurray RW. Rev Immunol 2003;3:73–8. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1304–1314 DOI 10.1002/art.22521 © 2007, American College of Rheumatology

A New Murine Model to Define the Critical Pathologic and Therapeutic Mediators of Polymyositis

Takahiko Sugihara,1 Chiyoko Sekine,2 Takashi Nakae,3 Kuniko Kohyama,4 Masayoshi Harigai,5 Yoichiro Iwakura,6 Yoh Matsumoto,4 Nobuyuki Miyasaka,5 and Hitoshi Kohsaka1

Objective. To establish a new murine model of in tumor necrosis factor ␣ (TNF␣)–null mutant mice. polymyositis (PM) for the understanding of its patho- Use of IVIG, a treatment with proven efficacy in PM, logic mechanisms and the development of new treat- suppressed CIM in the subgroup of treated mice. ment strategies. Conclusion. CIM mimics PM pathologically and Methods. C protein–induced myositis (CIM) was clinically. Infiltration of CD8 T cells is the most likely induced by a single immunization of recombinant hu- mechanism of muscle injury, and IL-1, but not B cells or man skeletal C protein in C57BL/6 mice, as well as in TNF␣, is crucial in the development of CIM. IVIG has CD4-depleted, CD8-depleted, and mutant mice as con- therapeutic effects in CIM, suggesting that the effects of trols. Some mice were treated with high-dose intra- IVIG are not mediated by suppression of antibody- venous immunoglobulin (IVIG) after disease induction. mediated tissue injury. This murine model provides a Muscle tissues were examined histologically. useful tool for understanding the pathologic mecha- Results. In mice with CIM, inflammation was nisms of PM and for developing new treatment strate- confined to the skeletal muscles. Histologic examination gies. revealed a common pathologic feature of CIM and PM, involving abundant infiltration of CD8 and perforin- Polymyositis (PM) is a chronic autoimmune in- expressing cells in the endomysial site of the injured flammatory myopathy affecting striated muscles (1). muscle. Suppression of myositis was achieved by deple- Damage of muscles results in varying degrees of muscle tion of both CD4 and CD8 T cells. Despite the develop- weakness. Dysphagia with choking episodes and respira- ment of serum anti–C protein antibodies in wild-type tory muscle weakness can occur in acute cases of PM. mice, severe myositis was induced in mice deficient in B Currently, the pathogenesis of PM is unknown, and cells. Induction of myositis was suppressed in patients are therefore treated with nonspecific immuno- interleukin-1␣/␤ (IL-1␣/␤)–null mutant mice, but not suppressants. High-dose corticosteroids are the first-line treatment but are not effective in all patients. Improve- Supported by grants-in-aid from the Japanese Ministry of ment of disease often depends on the dosage of cortico- Education, Culture, Sports, Science, and Technology and from the Japanese Ministry of Health, Labor and Welfare. steroids, making a dosage reduction difficult and thus, in 1Takahiko Sugihara, MD, Hitoshi Kohsaka, MD, PhD: Tokyo many cases, necessitating administration of methotrex- Medical and Dental University, Tokyo, Japan, and Research Center ate or other immunosuppressants as adjunctive treat- for Allergy and Immunology, RIKEN, Yokohama, Japan; 2Chiyoko Sekine, PhD: Research Center for Allergy and Immunology, RIKEN, ment. Because these medications can elicit a wide Yokohama, Japan; 3Takashi Nakae: Benesis Corporation, Osaka, variety of adverse drug reactions, new therapies to Japan; 4Kuniko Kohyama, MS, Yoh Matsumoto, MD, PhD: Tokyo address the specific pathologic features of PM are Metropolitan Institute for Neuroscience, Tokyo, Japan; 5Masayoshi Harigai, MD, PhD, Nobuyuki Miyasaka, MD, PhD: Tokyo Medical needed. and Dental University, Tokyo, Japan; 6Yoichiro Iwakura, DSc: Uni- In affected muscles of patients with PM, infiltra- versity of Tokyo, Tokyo, Japan. tion of mononuclear cells leads to muscle fiber necrosis. Address correspondence and reprint requests to Hitoshi Kohsaka, MD, PhD, Department of Medicine and Rheumatology, These cells are found in the endomysial site, where Graduate School, Tokyo Medical and Dental University, 1-5-45 non-necrotic muscle fibers are damaged, and also in the Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: kohsaka. perimysial and perivascular sites of the muscles. Immu- [email protected]. Submitted for publication March 31, 2006; accepted in revised nohistochemical studies have disclosed that CD8 T cells form January 16, 2007. are most abundant in the endomysial site and invade

1304 NEW MURINE MODEL OF POLYMYOSITIS 1305

non-necrotic muscle fibers (2,3). These CD8 T cells Susceptibility to CIM in B6 mice has facilitated express cytotoxic effector molecules, known as perforins, studies of the immunologic components required to that are oriented toward target muscle fibers (4). Sur- induce myositis. In the present study, we were able to face expression of class I major histocompatibility com- show that the effects of immunoglobulins are not neces- plex (MHC) molecules on muscle fibers is up-regulated sary for the development of CIM, despite the presence (5). In a study of T cells from patients with PM of B cells in the affected muscles and anti–C protein compared with normal donors, CD8 T cell clones ex- antibodies in the sera. Although both interleukin-1 panded more frequently in the peripheral blood of (IL-1)–positive and TNF␣-positive cells infiltrated the patients with PM (6,7). Moreover, some of these clones muscles of affected mice, only IL-1, not TNF␣, was could be found in muscle biopsy samples from the same crucial in the development of CIM. Interestingly, CIM patients (6,7). All of these observations support the view was suppressed by infusion of intravenous immuno- that PM is driven by cytotoxic CD8 T cells. globulins (IVIGs), which has been used as a last resort In rheumatoid arthritis (RA), another auto- for treatment of patients with PM who do not respond to immune disease, biologic agents that block tumor necro- or tolerate immunosuppressive reagents. Thus, we show ␣ ␣ sis factor (TNF ) have been introduced into clinical that our new model is useful in investigating the patho- ␣ use. TNF blockade has an enormous effect in modu- logic mechanisms of autoimmune myositis and in devel- lating the clinical course of RA (8). Animal models of oping new treatment strategies for this disease. arthritis serve to help identify therapeutic targets and to test the effect of these therapeutic reagents (9–11). In contrast, in PM, the lack of appropriate animal models MATERIALS AND METHODS has hampered basic research studies and delayed the Mice. B6, SJL/J, BALB/c, DBA/1, and C3H/He mice development of new treatments. were purchased from Charles River (Yokohama, Japan). NZB Experimental autoimmune myositis (EAM), es- and MRL/Mpϩ/ϩ mice were purchased from SLC (Shizuoka, tablished previously as an animal model of PM, is Japan). Mutant B6 mice rendered double-null for IL-1␣/␤ ␮ inducible specifically in SJL/J mice by repeated admin- were established previously (20), while Ig -null mutant B6 mice (21) and TNF␣-null mutant mice (22) were kindly istration of muscle homogenate or partially purified provided by Drs. Karasuyama (Tokyo Medical and Dental myosin (12,13). This model is a complex representation University) and Sekikawa (formerly at the National Institute of of disease, because SJL/J mice have a dysferlin gene Agrobiological Science), respectively. All experiments were mutation that causes spontaneous muscle necrosis and done under specific pathogen–free conditions in accordance secondary muscle inflammation (14). Immunohisto- with the ethics and safety guidelines for animal experiments of chemical studies have shown that infiltrating T cells in Tokyo Medical and Dental University and RIKEN. Recombinant human skeletal C protein. Four comple- the muscle are dominated by CD4 T cells, suggesting mentary DNA (cDNA) fragments encoding overlapping that the EAM disease model is mediated by CD4 T cells cDNA fragments 1, 2, 3, and 4 of human fast-type skeletal (15). muscle C protein were amplified from human skeletal muscle We established a new murine model that can be cDNA using polymerase chain reaction. Primers used were Ј Ј induced with a single injection of a recombinant skeletal 5 -GAGAGGTACCATGCCTGAGGCAAAACCAGCG-3 Ј muscle fast-type C protein. This myosin-binding protein and 5 -GAGAGTCGACTCAGAACCACTTGAGGGTCAG- GTC-3Ј for fragment 1, 5Ј-GAGAGGATCCGACCTGACC- is in the cross-bridge–bearing zone of A bands of CTCAAGTGGTTC-3Ј and 5Ј-GAGAAAGCTTTCACAGC- myofibrils (16,17). Biochemical purification studies CAGGTAGCGACGGGAGG-3Ј for fragment 2, 5Ј-AGA- showed that C protein appears to be the main immuno- GGATCCCCTCCCGTCGCTACCTGGCTG-3Ј and 5Ј-GA- pathogenic component of the crude skeletal-muscle my- GAAAGCTTTCACCGGGGCTTTCCCTGGAAGGG-3Ј for fragment 3, and 5Ј-AGAGGATCCCCCTTCAGGGAAAGC- osin preparation used for the induction of experimental Ј Ј myositis in Lewis rats (18,19). In this study, we used CCCGG-3 and 5 -GAGAAAGCTTTCACTGCGGCACTC- GGACCTC-3Ј for fragment 4 (Qiagen, Hilden, Germany) recombinant protein fragments to confirm the immuno- (underlining indicates the restriction enzyme recognition sites genicity of the C protein. This myositis, designated as C for subcloning into the pQE30 expression vector). These protein–induced myositis (CIM), can be induced in primers were introduced into the TOP10FЈ bacterial host C57BL/6 (B6) mice and in other strains of mice. Its (Invitrogen, Carlsbad, CA) and were used to prepare recom- histologic and immunohistochemical features mimic binant C protein fragments according to the manufacturer’s protocol. Soluble recombinant C proteins were dialyzed those of PM. Functional studies have indicated that against 0.5M arginine, 2 mM reduced glutathione, 0.2 mM cytotoxic CD8 T cells are primarily responsible for the oxydized glutathione in phosphate buffered saline (PBS), pH pathologic mechanisms of this disease. 7.4 (fragments 1 and 2) or 25 mM glycine HCl, pH 3.0 1306 SUGIHARA ET AL

(fragments 3 and 4). Endotoxin was removed using Detoxi-Gel sites of the foci were enumerated separately. The frequency of Endotoxin Removal Gel (Pierce, Rockford, IL). each subset was calculated in relation to the sum of all subsets. Induction of CIM. Female mice, ages 8–10 weeks, were The CD4:CD8 ratios were calculated on the basis of these immunized intradermally with 200 ␮g of the C protein frag- calculations. The CD4:CD8 ratios in double immunofluores- ments emulsified in Freund’s complete adjuvant (CFA) con- cence staining were calculated in the same manner as in the taining 100 ␮g of heat-killed Mycobacterium butyricum (Difco, immunohistochemical analyses. CD68-positive and CD11b- Detroit, MI). The immunogens were injected at multiple sites positive cells in the serial sections were also enumerated in the of the back and foot pads, and 2 ␮g of pertussis toxin (PT) same manner, and the frequencies of CD68-positive cells were (Seikagaku Kogyo, Tokyo, Japan) in PBS was injected intra- calculated in relation to the number of CD11b-positive cells. peritoneally at the same time. Hematoxylin and eosin–stained Rotarod test. Muscle function was evaluated with a 10-␮m sections of the proximal muscles (hamstrings and MK-630 rotarod device (Muromachi Kikai, Tokyo, Japan) as quadriceps) were examined histologically for the presence of described previously (23). The rotarod test was performed on mononuclear cell infiltration and necrosis of muscle fibers. The each mouse by measuring the running time until the mouse fell histologic severity of inflammation in each muscle block was off the rod while the rod was turning at 20 revolutions per graded as follows (18,19): grade 1 ϭ involvement of a single minute for 200 seconds. The running ability of each mouse was muscle fiber or Ͻ5 muscle fibers; grade 2 ϭ a lesion involving scored in 5 categories of running time: score 1 ϭ 0–49 seconds, 5–30 muscle fibers; grade 3 ϭ a lesion involving a muscle score 2 ϭ 50–99 seconds, score 3 ϭ 100–149 seconds, score 4 ϭ fasciculus; and grade 4 ϭ diffuse, extensive lesions. When 150–200 seconds, score 5 ϭϾ200 seconds. Mice were initially multiple lesions with the same grade were found in a single trained to accommodate them to the task, and then tested 2 muscle block, 0.5 point was added to the grade. days thereafter. Immunohistochemical analysis. Cryostat-frozen sec- In vivo depletion of CD8 or CD4 T cells. For the tions (6 ␮m) fixed in cold acetone were stained with anti-CD8a depletion of CD8 or CD4 T cells in B6 mice, the mice were (53-6.7; BD Biosciences PharMingen, San Diego, CA), anti- injected intraperitoneally with 1 mg of purified anti-CD8 CD4 (H129.19; BD Biosciences PharMingen), anti-B220 (53.67.2) mAb (24), anti-CD4 (GK1.5) mAb (24), or purified (RA3-6B2; BD Biosciences PharMingen), anti-CD11b (M1/70; rat IgG (Sigma-Aldrich, St. Louis, MO) as a control, for 3 BD Biosciences PharMingen), anti-CD68 (FA-11; Serotec, consecutive days. This treatment started 10 days before the Oxford, UK), anti–IL-1␣ (40508; Genzyme/Techne, Minneap- immunization. Injection of 500 ␮g of the same mAb was olis, MN), or anti-TNF␣ (MP6-XT22; BioLegend, San Diego, repeated every other day for 14 days. Splenocytes and lymph CA) monoclonal antibodies (mAb). To stain perforin mole- node cells from the treated mice were stained with phyco- cules, air-dried sections were treated with 0.5% periodic acid erythrin (PE)–conjugated anti-CD8 mAb (53.67; BD Bio- solution and then stained with antiperforin mAb (CB5.4; sciences PharMingen) or PE-conjugated anti-CD4 mAb Alexis Biochemicals, San Diego, CA). Nonspecific staining was (H129.19; BD Biosciences PharMingen), together with FITC- blocked with 4% Blockace (Dainippon, Osaka, Japan). conjugated anti-CD3 mAb (145-2C11; BD Biosciences Phar- Bound antibodies were visualized with peroxidase- Mingen). The cells were then analyzed with FACSCalibur labeled anti-rat IgG antibodies and associated substrates (Hist- (Becton Dickinson, San Jose, CA). ofine Simple Stain Max PO; Nichirei, Tokyo, Japan). The IVIG treatment. A subgroup of the mice were treated sections were also stained with biotinylated mouse anti-mouse with IVIG. Human gamma immunoglobulins (Venoglobulin- b H-2K mAb (AF6-88.5; BD Biosciences PharMingen) and with IH; Benesis, Osaka, Japan) (400 mg/kg/day) were injected b biotinylated mouse anti-mouse I-A mAb (AF6-120.1; BD intravenously into the tail vein for 5 consecutive days, begin- Biosciences PharMingen). They were then incubated with ning 3 days after immunization. peroxidase-conjugated streptavidin and its substrates (Chemi- Statistical analysis. Histologic scores were compared con, Temecula, CA). For double immunofluorescence staining, with the Mann-Whitney U test. P values less than or equal to the sections were preincubated with 5% heat-inactivated rat 0.05 were considered significant. serum and 1% bovine serum albumin, and were stained with Alexa Fluor 647–conjugated anti-CD8a mAb (53-6.7; BD Biosciences PharMingen) and fluorescein isothiocyanate RESULTS (FITC)–conjugated anti-CD4 mAb (RM4-5; BD Biosciences PharMingen). Histologic features of CIM in immunized mice. The bound antibodies were visualized using SP2AOBS Recombinant human fast-type skeletal C protein frag- confocal laser microscopy (Leica, Heidelberg, Germany). ments were prepared using a prokaryotic expression Spleens or popliteal lymph nodes were stained as positive system. Because of the size of the C protein, 4 overlap- controls. Isotype controls were used as negative control. The stained sections were evaluated by 2 independent observers, ping protein fragments were generated: fragment 1 who reported results that were comparable. (amino acids 1–290), fragment 2 (amino acids 284–580), Quantification of mononuclear cell subsets. The fragment 3 (amino acids 567–877), and fragment 4 method used to quantify stained cells in the immunohisto- (amino acids 864–1142). B6 mice were immunized at chemical analysis was based on a previously published method multiple sites of the back and foot pads with each (2). Briefly, 5 inflammatory mononuclear cell foci in the serial sections from 4 mice with CIM were studied. At least 1 focus fragment emulsified in CFA. PT was also injected intra- from each mouse was evaluated. Stained mononuclear cells peritoneally. To compare the immunogenicity of the 4 infiltrating into the endomysial, perimysial, and perivascular fragments, 5 mice per group were immunized with each NEW MURINE MODEL OF POLYMYOSITIS 1307

Figure 1. Muscle inflammation in C protein–induced myositis (CIM). C57BL/6 mice were immunized once with recombinant human skeletal C protein fragment 2 to induce CIM. A and B, Mononuclear cell infiltration was found predominantly in the endomysial site (boxed area) but also in the perimysial site (solid arrows) and perivascular site (open arrows) (A). Many cells invaded non-necrotic muscle fibers (arrows), while necrotic fibers were also present (B). Bar in A ϭ 100 ␮m; bar in B ϭ 25 ␮m. C, Muscle function was evaluated with a rotarod test 21 days after immunization. Six mice with CIM and 5 control mice treated with adjuvant alone were examined. Running ability was scored as described in Materials and Methods. Values are the mean and SD score. .ϭ P Ͻ 0.01 ء fragment, and myositis was studied histologically 21 days perivascular sites of the muscle tissue (Figure 1A). Many after the immunizaton. mononuclear cells invaded non-necrotic muscle fibers None of the mice treated with adjuvant alone or (Figure 1B). No abnormality in cardiac muscle and other immunized without PT developed myositis. We found tissues, including lung and joint tissues, was observed. that a single immunization of fragments 2 or 4 induced Muscle function was assessed clinically with a rotarod myositis consistently, and the mean Ϯ SD histologic device on day 21. Consistent with the histologic findings scores were 2.8 Ϯ 0.2 and 1.0 Ϯ 0.3, respectively. in the muscle tissue, mice with CIM ran for a shorter Fragments 1 and 3 induced milder myositis at a lower time than did control mice, indicating a reduction in incidence, and the mean Ϯ SD histologic scores were motor function (Figure 1C). 0.2 Ϯ 0.3 and 0.1 Ϯ 0.2, respectively. Because fragment Inflammation is acute and regresses spontane- 2 induced the most severe myositis, it was used as an ously in most animal models of autoimmune diseases. To immunogen in subsequent experiments. study the course of CIM in mice, muscle sections from 4 Histologic analysis of the muscles affected by or 5 mice were examined at various time points after the CIM showed that mononuclear cells infiltrated predom- immunization. A small number of mononuclear cells inantly the endomysial site, but also the perimysial and appeared in 50% of the muscle samples on day 7 1308 SUGIHARA ET AL

Figure 2. Immunohistochemical findings in muscles of mice with C protein–induced myositis. A and B, Expression of CD4 (green, fluorescein isothiocyanate) and CD8 (red, Alexa Fluor 647) was examined. Infiltrating cells in the endomysial site (arrows) (A) and in the perimysial site (arrows) and perivascular site (arrowheads) (B) were individually enumerated for calculating CD4:CD8 ratios. C–H, Expression of CD11b (C and D), perforin (E), class I major histocompatibility complex (F), interleukin-1␣ (IL-1␣) (G), and tumor necrosis factor ␣ (TNF␣) (H) was examined with immunoperoxidase staining. CD11b-positive cells diffusely infiltrated the endomysial site (arrows) (C) and both the perimysial (arrows) and perivascular sites (arrowheads) (D). Perforin-positive cells infiltrated around non-necrotic muscle fibers at the endomysial sites (arrows) (E). Muscle fibers reacted to anti-H2Kb monoclonal antibodies (arrows) (F). IL-1␣ and TNF␣ were expressed on infiltrating mononuclear cells in muscles (G and H). Bars ϭ 50 ␮m. NEW MURINE MODEL OF POLYMYOSITIS 1309

Figure 3. Quantitative immunohistochemical analysis of mononuclear cells in muscles of mice with C protein–induced myositis. Frequencies of CD8, CD4, CD11b, and B220 cells in the infiltrating mononuclear cells at endomysial, perimysial, and perivascular sites are shown. Values are the mean Ϯ SD percentage of each cell subset and mean Ϯ SD CD4:CD8 ratio at 5 inflammatory mononuclear cell foci. The total mononuclear cell counts in the endomysial, perimysial, and perivascular sites were 1,385, 506, and 543, respectively.

(incidence 50%, mean Ϯ SD histologic score 0.6 Ϯ 0.7). ratios. Double immnunofluorescence staining of CD4 Inflammation of the muscle tissue peaked on days 14 and CD8 cells showed that the mean Ϯ SD CD4:CD8 and 21 after the immunization (incidence 100%, histo- ratios in the endomysium, perimysium, and perivascular logic scores 2.6 Ϯ 0.3 and 2.8 Ϯ 0.2, respectively) and site were 1.0 Ϯ 0.1, 3.3 Ϯ 0.3, and 3.5 Ϯ 0.4, respectively, started to resolve after day 28 (incidence 100%, histo- which was consistent with the ratios derived from immu- logic score 1.4 Ϯ 0.6). On day 49, mononuclear cell nohistochemical staining (Figure 3). The frequency of infiltration was absent from most skeletal muscles. On CD8-positive cells in endomysial sites was higher than days 35, 49, and 63, the histologic scores were 0.5 Ϯ 0.6, that in perimysial and perivascular sites, whereas the 0.1 Ϯ 0.2, and 0.0 Ϯ 0.0, respectively, and the incidences frequencies of CD4-positive T cells were similar among of CIM were 38%, 10%, and 0%, respectively. the 3 sites (Figure 3). Other strains of mice were immunized with frag- B cells and macrophages were identified by stain- ment 2 of the C protein in the same manner as in B6 ing with B220 and CD11b antibodies, respectively. mice, and the muscles were examined histologically. CD11b-positive cells were most abundant among the Studies of 4 or 5 mice per strain showed that myositis infiltrating cells in all 3 sites (Figures 2C and D and developed in NZB and SJL/J mice with an incidence Figure 3). Although natural killer cells are also CD11b similar to that in B6 mice. However, the mononuclear positive, our analysis of CD68 and CD11b expression in cell infiltration was less intense. The mean Ϯ SD histo- serial sections showed that 93.4 Ϯ 4.3% (mean Ϯ SD) of logic scores were 1.8 Ϯ 0.6 and 1.3 Ϯ 0.3 in NZB and CD11b-positive cells were CD68-positive cells, indicat- SJL/J mice, respectively. No inflammation was observed ing that the majority of CD11b-positive cells were in the muscles from BALB/c, DBA/1, C3H/He, or macrophages. MRL/Mpϩ/ϩ mice immunized with C protein frag- B cells were sparse in the muscle tissue, especially ments. in the endomysial and perimysial sites (Figure 3). Immunohistochemical findings in muscles of Perforin-positive cells were present mostly (82%) mice with CIM. Localization of CD4 and CD8 T cells around non-necrotic muscle fibers at the endomysial site was studied with double immnunofluorescence labeling (Figure 2E) and were sparse in the perimysial and or immunohistochemical staining of muscle sections. perivascular sites (results not shown). Thus, the distri- CD4 cells diffusely infiltrated the endomysial, perimy- bution of perforin-positive cells corresponded well to sial, and perivascular sites. In contrast, CD8 cells infil- that of CD8-positive cells. When muscle fibers were trated preferentially the endomysial site (Figures 2A and stained with anti-H2Kb (class I MHC) and anti–I-Ab B), which was reflected equally well in the CD4:CD8 cell (class II MHC) mAb, they reacted to the anti–class I 1310 SUGIHARA ET AL

MHC mAb (Figure 2F) but not to the anti–class II MHC Table 1. Studies of the pathologic features of C protein–induced mAb (results not shown). polymyositis (CIM) and effects of treatment with intravenous immu- Pathologic role of CD8 and CD4 T cells in CIM. noglobulin (IVIG)* Histologic studies have demonstrated that CD8 T cells Experiment, mouse Incidence of Muscle blocks Histologic score, Ϯ function as effector cells in injury of muscle fibers. To group CIM, % involved, %† mean SD establish the pathologic role of CD8 T cells, B6 mice CD8 depletion were pretreated by removal of circulating CD8 T cells CD8-depleted 33 25 0.6 Ϯ 1.0‡ Rat IgG–injected 100 100 1.9 Ϯ 0.6 using specific mAb. Ten days after injection of the CD4 depletion antibodies for 3 consecutive days, CD8 T cells in the CD4-depleted 60 50 0.7 Ϯ 0.7‡ spleens were depleted to fewer than 2%. The mice were Rat IgG–injected 100 100 2.0 Ϯ 0.4 Ig␮-null then immunized with C protein and treated with the Ig␮Ϫ/Ϫ 100 95 1.7 Ϯ 0.6 same antibodies every other day for 14 days. The WT 100 100 2.1 Ϯ 0.7 IL-1-null muscles were examined histologically 14 days after Ϫ Ϫ IL-1␣/␤ / 14 7 0.1 Ϯ 0.2§ the immunization, when the frequency of CD8 T cells in WT 100 100 2.1 Ϯ 0.5 the spleens and lymph nodes was still less than 2%. The TNF␣-null Ϫ/Ϫ number of CD4 T cells in the spleens and lymph nodes TNF␣ 80 80 1.9 Ϯ 1.0 WT 100 90 2.3 Ϯ 0.6 was maintained in the CD8-depleted mice. IVIG Significantly fewer CD8-depleted mice developed IVIG-injected 43 36 0.6 Ϯ 1.1‡ myositis compared with control mice, with a 33% inci- Saline-injected 100 100 2.3 Ϯ 1.0 dence of disease compared with 100% in controls (Table * Mice were immunized with 200 ␮g of the recombinant C protein 1). The histologic scores of the treated mice were fragments and then examined histologically 14 or 21 days after immunization. For in vivo depletion of CD8 or CD4 T cells, 5 or 6 B6 significantly lower than that of the controls. It is known mice were treated intraperitoneally with anti-CD8 or anti-CD4 mono- that CD4 T cells help CD8 T cells develop into mature clonal antibodies, while 5 or 6 control mice were treated with purified cytotoxic T lymphocytes. They also have the potential to rat IgG. To demonstrate the requirement for humoral immunity, the presence of interleukin-1 (IL-1), and the presence of tumor necrosis injure muscle fibers. Therefore, CD4 T cells were re- factor ␣ (TNF␣) for the development of CIM, 5 Ig␮-null mutant mice, moved with specific mAb in the same manner as de- 7 IL-1␣/␤–null mutant mice, 5 TNF␣-null mutant mice, and 5 wild- scribed above for CD8 T cells. The pretreated mice type (WT) B6 mice were studied. IVIG was administered at a dosage of 400 mg/kg/day intravenously into the tail vein for 5 consecutive days, exhibited fewer than 2% of circulating CD4 T cells, and from day 3 after immunization. Seven mice were treated with IVIG were then immunized for CIM induction. They also and 6 with control saline. developed a milder myositis compared with control mice † Calculated as the number of muscle blocks showing myositis divided by the total number of blocks. (Table 1). ‡ P Ͻ 0.05 versus control group. Investigation of essential immunologic mediators § P Ͻ 0.01 versus control group. in mutant mice. Mice with CIM developed serum antibodies directed to C protein. They also developed low-titer autoantibodies with a cytoplasmic pattern or homogeneous and speckled nuclear patterns on Hep-2 immunohistochemical analyses showed that these in- staining, which proved to be nonreactive to PM- flammatory cytokines were also found in mice with CIM associated autoantigens (results not shown). However, (Figures 2G and H). We then studied whether the the contribution of these autoantibodies to myositis was presence of IL-1 and TNF␣ is required for the develop- unclear. ment of CIM, using IL-1␣/␤ double-null mutant and The susceptibility of B6 mice to CIM allowed us TNF␣-null mutant B6 mice. Most IL-1␣/␤–null mutant to study the contribution of different immune mediators mice did not develop myositis, and those that did have to myositis using genetic mutant mice. Ig␮-null mutant myositis developed a mild form (Table 1). The rotarod mice developed CIM with features and a frequency score of 7 for the IL-1␣/␤–null mutant mice was signif- comparable with those in control wild-type (WT) mice icantly higher than that for the 6 WT mice (mean Ϯ SD (Table 1). These findings indicate that the functions of B score 4.7 Ϯ 0.5 versus 1.3 Ϯ 0.5; P Ͻ 0.01). In contrast, cells and immunoglobulins are not necessary for the TNF␣-null mutant mice were as susceptible to CIM as development of CIM. the control mice, and the TNF␣-null mice had a similar Inflammatory cytokines such as IL-1 and TNF␣ incidence and severity of myositis (Table 1). These are known to be expressed in mononuclear cells infil- results indicate the differential requirements for the trating the muscles of mice with PM (25,26). Our roles of IL-1 and TNF␣ in the development of CIM. NEW MURINE MODEL OF POLYMYOSITIS 1311

Effects of high-dose IVIG treatment. Infusion of Severe inflammation was found consistently in high-dose IVIG is effective treatment of inflammatory the proximal muscles of the lower extremities, but not in myopathies that are refractory to conventional immuno- other sites. Although injection of CFA alone did not suppressants (27–29). Although several mechanisms of induce myositis, we assume that activation of local action for IVIG have been proposed, they have not been innate immunity by injection of the foot pad with CFA fully characterized. One possibility is that IVIG acts via would contribute to induction of severe myositis. Unlike suppression of pathogenic immunoglobulins and B cells. inflammation in human PM, inflammation in other Thus, whether this treatment improves CIM, which does myositis models, such as EAM and cardiac myosin– not depend on humoral immunity for tissue injury, is of induced myocarditis, is transient (32,33). Because lipo- special interest. When mice with CIM were treated with polysaccharide injection in the recovery phase of exper- high-dose IVIG (400 mg/kg) for 5 consecutive days, imental myocarditis can induce a relapse of beginning 3 days after immunization, the incidence and inflammation (33), unknown factors might perpetuate histologic severity of CIM were suppressed significantly the chronic disease in humans. compared with that in control, saline-injected mice An elevation in the levels of creatine kinase (CK) (Table 1). was found in the mice with CIM. However, since some mice, including healthy animals, have unexpectedly high levels of CK, this elevation could be attributed to DISCUSSION uncontrollable hemolysis. Lung involvement in some CIM was established as a simple murine model of patients is characteristic of PM and also of dermatomy- PM. A single injection of mice with recombinant human ositis (DM). However, no abnormality in the lungs was muscle protein induced severe and clinically significant observed in the mice with CIM. Considering the fre- inflammation of the skeletal muscles. CD8 T cells were quency of lung disease in PM (34), we may have to enriched in the endomysial site (the site of muscle undertake extensive studies of this issue using a large injury) as compared with their distribution in other sites number of animals. of the mouse muscle. CD8 cells expressed perforins Recombinant C protein was used to confirm its preferentially at the endomysial site. Class I MHC immunogenicity, as suggested in a rat myositis model expression was up-regulated on the muscle fibers. Re- induced by biochemically purified C protein (19). Large- moval of CD8 T cells significantly suppressed myositis. scale production of recombinant C protein fragments in Thus, muscle injury in CIM appears to be driven, the prokaryotic expression system facilitated multiple primarily, by cytotoxic CD8 T cells, as is assumed in experiments to optimize an immunization protocol and human PM. In this regard, the new model provides a to analyze the pathologic features of myositis. Since at clear contrast to the previous EAM model, which ap- least 200 ␮g of the immunogen had to be injected to pears to be driven by CD4 T cells. Induction of EAM induce CIM consistently, we needed to use the back and requires repeated immunization with a specific mouse foot pads of the mice for immunization. strain having a dysferlin gene mutation that induces The rotarod test was useful to assess muscle spontaneous muscular degeneration and inflammation. function in the mice with CIM at a single time point. Sensitivity of B6 mice to CIM prompted the initiation of Analysis by Spearman’s rank correlation coefficient genetic studies of the pathologic mechanisms of auto- showed that the rotarod score correlated well with the immune myositis, which would provide information for histologic score (P Ͻ 0.001). However, this test was less the development of new treatments. useful in evaluating the disease course in these mice, Muscle tissues from mice with CIM contained because the mice could learn how to run for a longer more CD4 T cells and macrophages than are found in period of time when the test was repeated. We believe patients with PM, which may reflect the acute disease that the device should be improved so that muscle course of CIM. Although we observed critical participa- function or weakness can be evaluated in an easier way. tion by CD8 T cells, CD4 T cells were also important in Unlike in the EAM model, many strains of mice, the development of CIM. In this regard, it has been including SJL/J mice, were susceptible to CIM. This fact shown that the actions of CD4 T cells are essential for confirms the immunogenicity of the C protein and full differentiation of cytotoxic CD8 T cells (30,31). suggests that mouse strains may have their own hierar- Alternatively, CD4 T cells may also injure muscle tissues chy of susceptibility to myositis. directly. This issue should be addressed further in future Our observations of CIM induced in the B6 mice experiments. with mutations in inflammatory cytokine genes demon- 1312 SUGIHARA ET AL

strated the importance of IL-1, but not TNF␣,inthe been confirmed by a number of studies of patients with development of myositis. Previous histologic studies of PM and DM (27,28). Currently, IVIG therapy is the only PM muscle tissue showed that IL-1 and other proinflam- treatment that does not induce general immune suppres- matory cytokines, including TNF␣, IL-6, and sion. The same treatment exerted a minor therapeutic interferon-␥, are expressed by mononuclear cells in the effect in SJL/J mice in the myositis model (45). Although affected muscle tissue (25). IL-1 expression by mono- immunoglobulins are derived from human sera, the nuclear cells accompanies expression of class I MHC effect was not due to nonspecific immunomodulation by molecules on the muscle fibers and expression of adhe- a xenogenic protein. sion molecules, such as intercellular adhesion molecule We found that IVIG treatment was markedly 1 and vascular cell adhesion molecule 1, on endothelial effective in CIM, suggesting its relevance as a model for cells in muscle (35). Thus, we assume that IL-1 expres- human PM. When the treatment was started 8 days after sion of activated macrophages up-regulates expression immunization of the mice, the therapeutic effect ap- of the class I MHC molecules, as well as that of adhesion peared milder (results not shown). The mode of action molecules and chemokines, on muscle fibers, endothelial of IVIGs could vary, and all of the mechanisms have not cells, and inflammatory cells. All of these molecules can been fully characterized (46–48). Modulation of crystal- trigger CD8-mediated muscle damage. Other studies lizable fragment Fc␥ receptor IIb on phagocytes appears have shown that antigen-specific T cell responses are to be the primary mechanism for an increase in platelet ␣ ␤ impaired in IL-1 / double-null mutant mice (36,37). In counts in patients with immune thrombocytopenia (49). the autoimmune myocarditis model, IL-1 is important Theoretically, the treatment could also down-regulate for efficient activation of dendritic cells (DCs) and activating Fc␥ receptors, increase IgG catabolism, neu- priming of CD4 T cells (38). IL-1 may also contribute to tralize autoantibodies and inflammatory cytokines, at- activation of DCs and interacting T lymphocytes. tenuate complement-mediated tissue damage, and mod- Similar to IL-1, TNF␣ induces expression of class ulate cytokine production by B cells and monocytes. I MHC molecules on muscle fibers and expression of Because development of CIM does not depend on B adhesion molecules on endothelial cells (39,40). The cells or antibodies, the efficacy of IVIG treatment for results of one report suggested that TNF␣ released from this model suggests that down-regulation of B cells or infiltrating CD8 T cells in PM muscles was responsible autoantibody-mediated processes are not a prerequisite for the muscle damage (26). However, we found that to achieve improvement of PM. Further evaluation CD8 T cells can induce typical myositis without TNF␣. should lead to identification of key molecules for the In this regard, experimental myocarditis is suppressed in effect of IVIG and development of new treatments that both IL-1 receptor– and TNF␣ receptor–null mutant target defined molecules. mice (38,41), and this model is mediated by pathogenic Our new model mimics human PM and provides CD4 T cells (33,38). Thus, it is important to note the a useful tool to investigate its pathologic mechanisms. It differences between the 2 murine myositis models. holds promise for identification of specific targets that Our results do not necessarily refute the idea that will lead to the development of new therapeutic ap- TNF␣ is a therapeutic target in PM. Clinical findings proaches in the treatment of PM, and will also be useful from sporadic reports suggest that some patients with for confirming the efficacy of these treatments. PM respond to systemic delivery of anti-TNF␣ mAb (42). This fact and our results are similar to the findings in collagen-induced arthritis (CIA), which is an animal ACKNOWLEDGMENTS ␣ model of RA. TNF -null mutant mice are susceptible to We thank H. Karasuyama for providing the Ig␮-null CIA (43), but inhibition of TNF␣ can improve the mutant mice, K. Sekikawa for the TNF␣-null mutant mice, M. disease (9). Development of arthritis is suppressed in Azuma for the hybridoma-producing anti-CD8 (53.67.2) mAb, IL-1␣/␤ double-null mutant mice (37). Studies are in A. Suwa and T. Mimori for performing the detection of progress to investigate the therapeutic effects of IL-1 or autoantibodies, and E. Hirasawa for her critical advice. TNF␣ blockade in CIM. A high dose of IVIGs, pooled from the plasma of AUTHOR CONTRIBUTIONS healthy donors, is commonly administered as treatment Dr. Kohsaka had full access to all of the data in the study and in patients with autoimmune disorders (44). After an takes responsibility for the integrity of the data and the accuracy of the data analysis. initial study showing that a patient with refractory PM Study design. Sugihara, Kohsaka. was successfully treated with IVIG (29), the efficacy has Acquisition of data. Sugihara, Sekine, Nakae, Kohyama. NEW MURINE MODEL OF POLYMYOSITIS 1313

Analysis and interpretation of data. Sugihara, Harigai, Iwakura, tion of the A-band localization domain of myosin binding proteins Matsumoto, Miyasaka, Kohsaka. C and H (MyBP-C, MyBP-H) in skeletal muscle. J Cell Sci Manuscript preparation. Sugihara, Kohsaka. 1999;112:69–79. Statistical analysis. Sugihara, Kohsaka. 18. Kojima T, Tanuma N, Aikawa Y, Shin T, Sasaki A, Matsumoto Y. Myosin-induced autoimmune polymyositis in the rat. J Neurol Sci 1997;151:141–8. REFERENCES 19. Kohyama K, Matsumoto Y. C protein in the skeletal muscle induces severe autoimmune polymyositis in Lewis rats. J Neuro- 1. Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis. immunol 1999;98:130–5. Lancet 2003;362:971–82. 20. Horai R, Asano M, Sudo K, Kanuka H, Suzuki M, Nishihara M, et 2. Arahata K, Engel AG. Monoclonal antibody analysis of mono- al. Production of mice deficient in genes for interleukin (IL)-1␣, nuclear cells in myopathies. I. Quantitation of subsets according to IL-1␤, IL-1␣/␤, and IL-1 receptor antagonist shows that IL-1␤ is diagnosis and sites of accumulation and demonstration and counts crucial in turpentine-induced fever development and glucocorti- of muscle fibers invaded by T cells. Ann Neurol 1984;16:193–208. coid secretion. J Exp Med 1998;187:1463–75. 3. Engel AG, Arahata K. Monoclonal antibody analysis of mono- 21. Kubo S, Nakayama T, Matsuoka K, Yonekawa H, Karasuyama H. nuclear cells in myopathies. II. Phenotypes of autoinvasive cells in Long term maintenance of IgE-mediated memory in mast cells in polymyositis and inclusion body myositis. Ann Neurol 1984;16: the absence of detectable serum IgE. J Immunol 2003;170:775–80. 209–15. 22. Taniguchi T, Takata M, Ikeda A, Momotani E, Sekikawa K. 4. Goebels N, Michaelis D, Engelhardt M, Huber S, Bender A, Failure of germinal center formation and impairment of response Pongratz D, et al. Differential expression of perforin in muscle- to endotoxin in tumor necrosis factor ␣-deficient mice. Lab Invest infiltrating T cells in polymyositis and dermatomyositis. J Clin 1997;77:647–58. Invest 1996;97:2905–10. 23. Moll J, Barzaghi P, Lin S, Bezakova G, Lochmuller H, Engvall E, 5. Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibil- et al. An agrin minigene rescues dystrophic symptoms in a mouse ity complex class I antigen expression, immunolocalization of model for congenital muscular dystrophy. Nature 2001;413:302–7. interferon subtypes, and T cell-mediated cytotoxicity in myopa- 24. Mogi S, Sakurai J, Kohsaka T, Enomoto S, Yagita H, Okumura K, thies. Hum Pathol 1989;20:224–31. et al. Tumour rejection by gene transfer of 4-1BB ligand into a 6. Bender A, Ernst N, Iglesias A, Dornmair K, Wekerle H, Hohlfeld CD80ϩ murine squamous cell carcinoma and the requirements of R. T cell receptor repertoire in polymyositis: clonal expansion of co-stimulatory molecules on tumour and host cells. Immunology autoaggressive CD8ϩ T cells. J Exp Med 1995;181:1863–8. 2000;101:541–7. 7. Nishio J, Suzuki M, Miyasaka N, Kohsaka H. Clonal biases of 25. Lundberg I, Ulfgren AK, Nyberg P, Andersson U, Klareskog L. peripheral CD8 T cell repertoire directly reflect local inflamma- Cytokine production in muscle tissue of patients with idiopathic tion in polymyositis. J Immunol 2001;167:4051–8. inflammatory myopathies. Arthritis Rheum 1997;40:865–74. 8. Lipsky PE, van der Heijde DM, St.Clair EW, Furst DE, Breedveld 26. Kuru S, Inukai A, Liang Y, Doyu M, Takano A, Sobue G. Tumor FC, Kalden JR, et al, for the Anti–Tumor Necrosis Factor Trial in necrosis factor-␣ expression in muscles of polymyositis and der- Rheumatoid Arthritis with Concomitant Therapy Study Group. matomyositis. Acta Neuropathol (Berl) 2000;99:585–8. Infliximab and methotrexate in the treatment of rheumatoid 27. Cherin P, Pelletier S, Teixeira A, Laforet P, Genereau T, Simon A, arthritis. N Engl J Med 2000;343:1594–602. et al. Results and long-term followup of intravenous immunoglob- 9. Williams RO, Feldmann M, Maini RN. Anti-tumor necrosis factor ulin infusions in chronic, refractory polymyositis: an open study ameliorates joint disease in murine collagen-induced arthritis. with thirty-five adult patients. Arthritis Rheum 2002;46:467–74. Proc Natl Acad SciUSA1992;89:9784–8. 28. Dalakas MC, Illa I, Dambrosia JM, Soueidan SA, Stein DP, Otero 10. Takagi N, Mihara M, Moriya Y, Nishimoto N, Yoshizaki K, C, et al. A controlled trial of high-dose intravenous immune Kishimoto T, et al. Blockage of interleukin-6 receptor ameliorates globulin infusions as treatment for dermatomyositis. N Engl J Med joint disease in murine collagen-induced arthritis. Arthritis Rheum 1993;329:1993–2000. 1998;41:2117–21. 29. Cherin P, Herson S, Wechsler B, Piette JC, Bletry O, Coutellier A, 11. Taniguchi K, Kohsaka H, Inoue N, Terada Y, Ito H, Hirokawa K, et al. Efficacy of intravenous gammaglobulin therapy in chronic et al. Induction of the p16INK4a senescence gene as a new refractory polymyositis and dermatomyositis: an open study with therapeutic strategy for the treatment of rheumatoid arthritis. Nat 20 adult patients. Am J Med 1991;91:162–8. Med 1999;7:760–7. 30. Sun D, Whitaker JN, Huang Z, Liu D, Coleclough C, Wekerle H, 12. Rosenberg NL, Ringel SP, Kotzin BL. Experimental autoimmune et al. Myelin antigen-specific CD8ϩ T cells are encephalitogenic myositis in SJL/J mice. Clin Exp Immunol 1987;68:117–29. and produce severe disease in C57BL/6 mice. J Immunol 2001; 13. Matsubara S, Shima T, Takamori M. Experimental allergic myo- 166:7579–87. sitis in SJL/J mice immunized with rabbit myosin B fraction: 31. Edith M, Janssen EE, Lemmens TW, Urs C, Matthias GH, immunohistochemical analysis and transfer. Acta Neuropathol Stephen PS. CD4ϩ T cells are required for secondary expansion (Berl) 1993;85:138–44. and memory in CD8ϩ T lymphocytes. Nature 2003;421:852–6. 14. Bittner RE, Anderson LV, Burkhardt E, Bashir R, Vafiadaki E, 32. Matsubara S, Kitaguchi T, Kawata A, Miyamoto K, Yagi H, Hirai Ivanova S, et al. Dysferlin deletion in SJL mice (SJL-Dysf) defines S. Experimental allergic myositis in SJL/J mouse: reappraisal of a natural model for limb girdle muscular dystrophy 2B. Nat Genet immune reaction based on changes after single immunization. 1999;23:141–2. J Neuroimmunol 2001;119:223–30. 15. Rosenberg NL, Kotzin BL. Aberrant expression of class II MHC 33. Eriksson U, Ricci R, Hunziker L, Kurrer MO, Oudit GY, Watts antigens by skeletal muscle endothelial cells in experimental TH, et al. Dendritic cell-induced autoimmune heart failure re- autoimmune myositis. J Immunol 1989;142:4289–94. quires cooperation between adaptive and innate immunity. Nat 16. Weber FE, Vaughan KT, Reinach FC, Fischman DA. Complete Med 2003;9:1484–90. sequence of human fast-type and slow-type muscle myosin-bind- 34. Marie I, Hachulla E, Cherin P, Dominique S, Hatron PY, Hellot ing-protein C (MyBP-C): differential expression, conserved do- MF, et al. Interstitial lung disease in polymyositis and dermato- main structure and chromosome assignment. Eur J Biochem myositis. Arthritis Rheum 2002;47:614–22. 1993;216:661–9. 35. Lundberg I, Kratz AK, Alexanderson H, Patarroyo M. Decreased 17. Gilbert R, Cohen JA, Pardo S, Basu A, Fischman DA. Identifica- expression of interleukin-1␣, interleukin-1␤, and cell adhesion 1314 SUGIHARA ET AL

molecules in muscle tissue following corticosteroid treatment in matory arthritis and lymphadenopathy in the absence of TNF. patients with polymyositis and dermatomyositis. Arthritis Rheum J Clin Invest 2001;107:1519–27. 2000;43:336–48. 44. Sapir T, Blank M, Shoenfeld Y. Immunomodulatory effects of 36. Nakae S, Asano M, Horai R, Sakaguchi N, Iwakura Y. IL-1 intravenous immunoglobulins as a treatment for autoimmune enhances T cell-dependent antibody production through induction diseases, cancer, and recurrent pregnancy loss. Ann N Y Acad Sci of CD40 ligand and OX40 on T cells. J Immunol 2001;167:90–7. 2005;1051:743–78. 37. Saijo S, Asano M, Horai R, Yamamoto H, Iwakura Y. Suppression 45. Wada J, Shintani N, Kikutani K, Nakae T, Yamauchi T, Takechi of autoimmune arthritis in interleukin-1–deficient mice in which T K. Intravenous immunoglobulin prevents experimental auto- cell activation is impaired due to low levels of CD40 ligand and immune myositis in SJL mice by reducing anti-myosin antibody OX40 expression on T cells. Arthritis Rheum 2002;46:533–44. and by blocking complement deposition. Clin Exp Immunol 2001; 38. Eriksson U, Kurrer MO, Sonderegger I, Iezzi G, Tafuri A, Hunziker L, et al. Activation of dendritic cells through the 124:282–9. interleukin 1 receptor 1 is critical for the induction of autoimmune 46. De Grandmont MJ, Racine C, Roy A, Lemieux R, Neron S. myocarditis. J Exp Med 2003;197:323–31. Intravenous immunoglobulins induce the in vitro differentiation of 39. Nagaraju K, Raben N, Merritt G, Loeffler L, Kirk K, Plotz P. A human B lymphocytes and the secretion of IgG. Blood 2003;101: variety of cytokines and immunologically relevant surface mole- 3065–73. cules are expressed by normal human skeletal muscle cells under 47. Basta M, Dalakas MC. High-dose intravenous immunoglobulin proinflammatory stimuli. Clin Exp Immunol 1998;113:407–14. exerts its beneficial effect in patients with dermatomyositis by 40. Mantovani A, Bussolino F, Dejana E. Cytokine regulation of blocking endomysial deposition of activated complement frag- endothelial cell function. FASEB J 1992;6:2591–9. ments. J Clin Invest 1994;94:1729–35. 41. Wada H, Saito K, Kanda T, Kobayashi I, Fujii H, Fujigaki S, et al. 48. Kaveri S, Vassilev T, Hurez V, Lengagne R, Lefranc C, Cot S, et Tumor necrosis factor-␣ (TNF-␣) plays a protective role in acute al. Antibodies to a conserved region of HLA class I molecules, viral myocarditis in mice: a study using mice lacking TNF-␣. capable of modulating CD8 T cell-mediated function, are present Circulation 2001;103:743–9. in pooled normal immunoglobulin for therapeutic use. J Clin 42. Anandacoomarasamy A, Howe G, Manolios N. Advanced refrac- Invest 1996;97:865–9. tory polymyositis responding to infliximab. Rheumatology (Ox- 49. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory activ- ford) 2005;44:562–3. ity of IVIG mediated through the inhibitory Fc receptor. Science 43. Campbell IK, O’Donnell K, Lawlor KE, Wicks IP. Severe inflam- 2001;291:484–6. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1315–1324 DOI 10.1002/art.22456 © 2007, American College of Rheumatology

Role of Matrix Metalloproteinases, Proinflammatory Cytokines, and Oxidative Stress–Derived Molecules in Hepatitis C Virus–Associated Mixed Cryoglobulinemia Vasculitis Neuropathy

David Saadoun,1 Ivan Bieche,2 Franc¸ois-Je´rome Authier,3 Ingrid Laurendeau,2 Florence Jambou,1 Jean Charles Piette,1 Michel Vidaud,2 Thierry Maisonobe,4 and Patrice Cacoub1

Objective. Mixed cryoglobulinemia (MC) is a there was a >2-fold difference in mean expression levels systemic vasculitis, usually associated with hepatitis C between groups and the P value was less than 0.05. virus (HCV) infection. The molecular mechanisms re- Results. Expression levels of 8 genes were signif- sponsible for HCV-associated MC (HCV-MC) vasculitis icantly increased in HCV-MC patients versus control are largely unknown. This study was undertaken to patients with noninflammatory idiopathic neuropathy, assess the expression profile of selected genes involved with the highest increase for metallothionein 1 H in inflammatory vascular damage in patients with (MT1H), a hypoxic and oxidative stress protein. Com- HCV-MC vasculitis, patients with polyarteritis nodosa pared with PAN patients, HCV-MC patients had higher (PAN), and patients with noninflammatory idiopathic expression levels of genes encoding oxidative stress– neuropathy. derived molecules (MT1H, endothelial cell nitric oxide Methods. The quantitative expression levels of 42 synthase 3, Hsp70, and Hsp90) and tissue plasminogen selected genes involved in inflammatory vascular dam- activator and lower expression levels of matrix metallo- age were assessed in nerve lesions of patients with proteinase 7 (MMP-7). HCV-MC neuropathies were HCV-MC vasculitis, PAN (rheumatic disease controls), classified according to their morphologic pattern and and noninflammatory idiopathic neuropathy (nonin- the presence or absence of necrotizing arteritis. MMP-1, flammatory neuropathy controls), using real-time re- MMP-7, MMP-9, and interleukin-1␤ were up-regulated verse transcriptase–polymerase chain reaction. Genes in patients with necrotizing arteritis. were considered to be differentially expressed when Conclusion. This comprehensive molecular study of HCV-MC vasculitis provides strong evidence that MMPs, proinflammatory cytokines, and oxidative Dr. Saadoun’s work was supported by the Agence Nationale de Recherche sur le Sida et les He´patites. stress–derived molecules have a role in the pathogenesis 1David Saadoun, MD, Florence Jambou, MD, Jean Charles of HCV-MC vasculitis neuropathy. Piette, MD, Patrice Cacoub, MD: Universite´ Pierre et Marie Curie- Paris VI, CNRS UMR 7087, and Hoˆpital Pitie´-Salpe´trie`re, Paris, France; 2Ivan Bieche, MD, Ingrid Laurendeau, MD, Michel Vidaud, Mixed cryoglobulinemia (MC) is a systemic vas- MD: INSERM U 745, and Universite´ Paris V, Paris, France; culitis that affects mainly the small and, less frequently, 3Franc¸ois-Je´rome Authier, MD: Hoˆpital Henri-Mondor, Cre´teil, medium-sized vessels (1). MC is characterized by the France; 4Thierry Maisonobe, MD: Hoˆpital Pitie´-Salpe´trie`re, Paris, France. proliferation of B cell clones, which produce pathoge- Dr. Cacoub has received consulting fees and/or honoraria netic IgM with rheumatoid factor activity. Clinical man- (less than $10,000 each) from Sanofi-Aventis, Schering-Plough, Ser- ifestations associated with MC range from the so-called vier, and Roche. Address correspondence and reprint requests to Patrice Ca- “MC syndrome” (purpura, arthralgia, and asthenia) to coub, MD, AP-HP, Hoˆpital Pitie´-Salpeˆtrie`re, Service de Me´decine more serious lesions with neurologic and renal involve- Interne, 83 Boulevard de l’Hoˆpital, F-75013 Paris, France. E-mail: ment. Shortly after the discovery of hepatitis C virus [email protected]. Ͼ Submitted for publication July 25, 2006; accepted in revised (HCV) in 1989, evidence that 80% of cryoglob- form December 19, 2006. ulinemia vasculitis cases were associated with HCV

1315 1316 SAADOUN ET AL

infection became available. The primary role of HCV in Table 1. Characteristics of patients with hepatitis C virus–associated the mechanism of cryoprecipitation is suggested mainly mixed cryoglobulinemia (HCV-MC) and patients with polyarteritis by its selective concentration in cryoglobulins (2,3). nodosa (PAN)* Neurologic complications in patients with HCV- HCV-MC vasculitis PAN ϭ ϭ associated MC (HCV-MC) predominantly affect the Characteristic (n 14) (n 7) peripheral nervous system (4). The prevalence of peri- Age, mean years 60.1 59.4 pheral nervous system involvement has varied in previ- Female sex 9 (64.2) 4 (57.1) Polyneuropathy 11 (78.6) – ous studies but has been as high as 50% of cases (5,6). Mononeuritis multiplex 3 (21.4) 7 (100) Neurologic manifestations range from pure sensory ax- Arthralgia/arthritis 9 (64.2) 5 (71.4) onopathy to mononeuritis multiplex, but the most fre- Skin involvement 6 (42.8) 3 (42.8) Kidney involvement 2 (14.2) 2 (28.5) quently described form is a distal sensory or sensorimo- Axonopathy tor polyneuropathy. Sensory 14 (100) 7 (100) The pathophysiology of HCV-MC vasculitis neu- Motor 9 (64.2) 7 (100) Necrotizing vasculitis 7 (50) 7 (100) ropathy is still unclear. Although HCV RNA has been HCV RNA positive 14 (100) – found in the nerve lesions of MC patients, HCV repli- Type II cryoglobulin positive 11 (78.6) – cation has never been evidenced in vasculitis lesions (7). * Except where indicated otherwise, values are the number (%) of These results support the hypothesis that an HCV- patients. driven cellular immune response, rather than HCV itself, plays an important role in HCV-MC. A leukocytoclastic reaction may be involved in vessel damage. The aim of this study was to assess the expression Leukocytoclastic reactions, however, are rare in the profile of 42 selected genes involved in inflammatory peripheral nerve tissue of MC patients. Instead, T cells vascular damage in nerve lesions of patients with and macrophages are the dominant infiltrating cells in HCV-MC vasculitis. The gene expression profiles of vascular walls, and a T cell–mediated process appears to patients with polyarteritis nodosa (PAN) and of controls be the primary mechanism of vessel injury (8,9). with noninflammatory idiopathic neuropathy were also In addition to the attraction and adhesion of assessed. inflammatory cells to the vessel wall, matrix metalloproteinases (MMPs) and oxidative stress–derived molecules may play a key role in the development of MC vasculitis PATIENTS AND METHODS neuropathy, by contributing to blood–nerve barrier dys- Patients. Characteristics of patients with HCV-MC function. MMPs are a family of endopeptidases with and idiopathic PAN are summarized in Table 1. The study overlapping substrate affinities against extracellular ma- population consisted of 14 patients with HCV-MC vasculitis trix components. Their expression is regulated by growth (mean age 60.1 years, range 33–73 years), 7 patients with idiopathic PAN who were studied as disease controls (mean factors, cytokines, and hormones, as well as by interac- age 59.4 years, range 45–76 years), and 3 control patients with tions with extracellular matrix proteins (10). MMPs are noninflammatory idiopathic axonopathy (mean age 58.8 years, able to degrade subendothelial basement membrane and range 52–68 years). Informed consent was obtained from each may therefore act as effector molecules of blood–nerve patient, and the study conformed to the ethical guidelines of barrier disruption and of penetration of neural tissue by the Declaration of Helsinki (1975 version). HCV-MC patients had serum cryoglobulin levels of Ն0.05 gm/liter on at least 2 blood-derived immune cells. Endogenous inhibitors, occasions and were positive for serum HCV RNA. Patients such as tissue inhibitors of metalloproteinases (TIMPs), with PAN or noninflammatory neuropathy were negative for also exist to counterbalance MMP activity. HCV infection. All patients and controls were negative for the Oxidative stress–derived molecules are important human immunodeficiency virus and hepatitis B virus. Patients in the vascular inflammatory response. The induction with HCV-MC and patients with PAN were untreated and had clinically active disease. The type of vasculitis was defined and activation of oxidant-generating enzymes upon stim- according to the Chapel Hill criteria (12). All patients had ulation of inflammatory cells provide the first line of severe axonal degeneration and biopsy evidence of an inflam- defense against pathogens (11). Based on their cytolytic matory process involving nerves. Necrotizing arteritis was activities, oxidants are expected to be a key factor in present in all PAN patients and in 7 of 14 patients with tissue injuries in vasculitis. Vascular endothelium, one of HCV-MC neuropathy (Table 1). Nerve specimens. All patients and controls underwent the main sites where inflammatory lesions can develop full-thickness open biopsy of the superficial peroneal nerve in systemic autoimmune diseases, is easily exposed to and peroneous brevis muscle in the most affected limb. reactive oxygen species. Specimens were immediately snap-frozen in liquid nitrogen MOLECULAR PROFILE OF HCV-MC NEUROPATHY 1317

Table 2. The 42 genes selected for the gene expression study of hepatitis C virus–associated mixed cryoglobulinemia vasculitis neuropathy Gene Alternate term Gene definition Extracellular matrix (n ϭ 16) MMP1 Matrix metalloproteinase 1 MMP2 Matrix metalloproteinase 2 MMP7 Matrix metalloproteinase 7 MMP9 Matrix metalloproteinase 9 MMP13 Matrix metalloproteinase 13 MMP14 Matrix metalloproteinase 14 TIMP1 Tissue inhibitor of metalloproteinases 1 TIMP2 Tissue inhibitor of metalloproteinases 2 TIMP3 Tissue inhibitor of metalloproteinases 3 TIMP4 Tissue inhibitor of metalloproteinases 4 BSG Emmprin, CD147 Basigin PLAT tPA Tissue plasminogen activator PLAU uPA Urokinase plasminogen activator PLAUR uPAR Urokinase-type plasminogen activator SERPINB2 PAI-2 Plasminogen activator inhibitor 2 SERPINE1 PAI-1 Plasminogen activator inhibitor 1 Oxidative stress (n ϭ 15) HIF1A Hypoxia-inducible factor 1␣ SLC2A1 GLUT-1 Solute carrier family 2 member 1 gene (facilitated glucose transporter 1) CA9 Carbonic anhydrase IX NOS1 nNOS Neuronal nitric oxide synthase NOS2A iNOS Inducible nitric oxide synthase NOS3 eNOS Endothelial cell nitric oxide synthase HSPA1A Hsp70 70-kd heat-shock protein HSPB1 Hsp27 27-kd heat-shock protein HSPCA Hsp90 90-kd heat-shock protein MT1H Metallothionein 1 H TRA1 GRP94 Tumor rejection antigen (gp96) 1 PP1R15A GADD34 Protein phosphatase 1 regulatory (inhibitor) subunit 15A EGR1 KROX24 Early growth response 1 (KROX-24) GFAP Glial fibrillary acidic protein DUSP1 MKP-1, CL100 Dual specificity phosphatase 1 Inflammation (n ϭ 11) NKFB1 NF-␬B Nuclear factor of kappa light polypeptide gene (p105) RelA v-rel reticuloendotheliosis viral oncogene homolog A, p65 gadd45B Growth arrest and DNA damage– induced TNFAIP3 Tumor necrosis factor ␣–induced protein 3 IER3S Immediate early-response 3 PTGS2 COX-2 Prostaglandin endoperoxide synthetase 2 CD40L CD40 ligand (tumor necrosis factor superfamily member 5, hyper-IgM syndrome) CD40 CD40 antigen (tumor necrosis factor receptor superfamily member 5) IL1B Interleukin-1␤ jun Jun oncogene fos Fos oncogene

and stored at Ϫ80°C. Part of the specimen was paraffin- samples by examination of paraffin-embedded specimens on embedded, and both transversal and longitudinal sections were serial sections. Only the superficial peroneal nerve samples stained with hematoxylin and eosin (H&E), periodic acid– were analyzed for messenger RNA (mRNA) quantification. Schiff, or Congo red. In each case, the presence of inflamma- HCV-MC neuropathies were separated into 2 groups (with tory vascular lesions was evaluated in both muscle and nerve necrotizing arteritis and without necrotizing arteritis), accord- 1318 SAADOUN ET AL

Table 3. Genes with significant differential levels of expression in patients with HCV-MC versus controls, patients with PAN versus controls, and patients with HCV-MC versus patients with PAN* P, HCV-MC P, PAN versus P, HCV-MC Gene HCV-MC PAN versus controls controls versus PAN MMP7 5.3 Ϯ 2.3 17.4 Ϯ 8.0 0.04 0.01 0.01 MMP9 13.7 Ϯ 7.6 8.1 Ϯ 3.2 0.01 0.02 NS PLAT 4.5 Ϯ 1.1 2.4 Ϯ 0.4 0.008 0.02 0.04 PAI1 5.4 Ϯ 1.9 4.6 Ϯ 1.6 0.008 0.01 NS PAI2 3.5 Ϯ 1.6 2.2 Ϯ 0.5 NS 0.02 NS PLAUR 5.6 Ϯ 2.3 2.8 Ϯ 0.4 0.04 0.02 NS IL1B 6.5 Ϯ 1.3 5.0 Ϯ 2.1 0.01 0.03 NS GLUT1 22.1 Ϯ 4.9 10.2 Ϯ 2.1 0.008 0.01 0.03 MT1H 41.3 Ϯ 18.7 0.5 Ϯ 0.2 0.02 NS 0.02 HIF1A 2.0 Ϯ 0.3 2.4 Ϯ 0.3 NS 0.02 NS NOS3 2.7 Ϯ 0.8 0.4 Ϯ 0.1 NS NS 0.02 PP1R15A 3.6 Ϯ 0.8 1.3 Ϯ 0.2 NS NS 0.01 hsp70 2.1 Ϯ 0.6 0.3 Ϯ 0.1 NS NS Ͻ0.001 hsp90 2.7 Ϯ 1.2 0.5 Ϯ 0.1 NS NS 0.01 * Values are the mean Ϯ SEM fold difference compared with nerve samples from controls with noninflammatory neuropathy, after normalization of each measurement. Genes were considered to be differentially expressed when there was a Ͼ2-fold difference in mean expression levels between groups and the P value was less than 0.05. HCV-MC ϭ hepatitis C virus–associated mixed cryoglobulinemia; PAN ϭ polyarteritis nodosa; NS ϭ not significant.

ing to the presence or absence of fibrinoid necrosis in the nerve value of the sample was determined by subtracting the Ct value vessel wall. HCV and control nerve samples did not overlap of the target gene from the Ct value of the TBP gene. The with those investigated in our previous study (9). For immu- Ntarget values of the samples were subsequently normalized to nohistochemistry studies, all consecutive serial sections with the median value in samples from control patients with noninflammatory vascular lesions were analyzed by the same inflammatory neuropathies; the Ntarget values for the nonin- pathologist (TM). Paraffin-embedded longitudinal nerve sec- flammatory control nerves were set at 1. Primers for TBP and tions were incubated at room temperature for 30 minutes with the 42 target genes were chosen using Oligo, version 5.0 primary anti–MMP-9 antibody (ab2167) and anti– ␤ ␤ (National Biosciences, Plymouth, MN). To avoid amplification interleukin-1 (anti–IL-1 ) antibody (ab8320) (both from of contaminating genomic DNA, 1 of the 2 primers was placed Abcam, Cambridge, UK), and revealed with the Novolink kit at the junction between 2 exons. The thermal cycling condi- (Novocastra, Newcastle-upon-Tyne, UK) using the immuno- tions comprised an initial denaturation step at 95°C for 10 peroxidase method. minutes, followed by 50 cycles at 95°C for 15 seconds and 65°C Gene quantification with mRNA. A total of 42 genes, for 1 minute. Duplicate experiments were performed for each selected based on their supposed role in inflammatory nerve vascular damage, were quantified (Table 2). The theoretical up-regulated gene. and practical aspects of real-time quantitative reverse Statistical analysis. Statistical analyses were performed using StatView software (SAS Institute, Cary, NC). All transcriptase–polymerase chain reaction (RT-PCR) using the Ϯ ABI Prism 7700 Sequence Detection System (PerkinElmer data are expressed as the mean SEM. Comparison of the Applied Biosystems, Foster City, CA) have been described in gene expression level between different groups was performed detail elsewhere (13). Briefly, total RNA was reverse- using the nonparametric Mann-Whitney U test. Genes were transcribed before real-time PCR amplification. Quantitative considered to be differentially expressed when there was a Ͼ2-fold difference in mean expression levels between groups values were obtained from the threshold cycle (Ct) number at which the increase in the signal associated with exponential and the P value was less than 0.05 (14). Hierarchical clustering growth of PCR products began to be detected using analysis was performed using GeneANOVA software (15). software, according to the recommendations of the manufacturer (PerkinElmer Applied Biosystems). The precise amount of total RNA added to each reaction mixture (based on optical RESULTS density) and its quality (i.e., lack of extensive degradation) Differential gene expression in nerve samples were both difficult to assess. We therefore also quantified transcripts of the TBP gene, which encodes the TATA box– from patients with HCV-MC vasculitis and patients binding protein (a component of the DNA binding protein with PAN versus controls with noninflammatory idio- complex transcription factor IID), as the endogenous RNA pathic neuropathy. Relative RNA expression levels of control, and each sample was normalized on the basis of its the 42 selected genes were quantified by real-time TBP content. Results, expressed as fold differences in target gene RT-PCR and compared between the HCV-MC vasculi- expression relative to the TBP gene (termed Ntarget), were tis group and controls with noninflammatory idiopathic ⌬ ϭ Ct ⌬ determined using the formula Ntarget 2 sample. The Ct neuropathy. Results were normalized to the level of MOLECULAR PROFILE OF HCV-MC NEUROPATHY 1319

expression of each gene in the group of control patients with noninflammatory disease. Significant differential gene expression in nerve specimens from patients with HCV-MC, at least 2 times the value observed in the control group and with a P value of less than 0.05 (14), was observed for 8 of the 42 genes tested. The highest increase in the gene expression level was observed for the metallothionein 1 H gene (MT1H), which belongs to the metallothionein family of stress-induced proteins. Significant differential expression in nerve specimens from patients with PAN, at least 2 times the value observed in nerve specimens from control patients with noninflammatory disease and with a P value of less than 0.05 (14), was observed for 9 of the 42 genes tested, with up-regulation of all of them in the PAN group. The highest increase in gene expression level was observed for the MMP-7 gene (Table 3). Differential gene expression in nerve samples from patients with HCV-MC vasculitis versus patients with PAN (disease control patients). PAN is the prototypic disorder of the systemic necrotizing arteritis group. In order to assess whether specific regulation pathways may be involved in the vasculitic process, differential gene expression was investigated in patients with HCV-MC versus patients with PAN (Table 3). This analysis showed significant differential expression of 8 genes, with higher expression in the HCV-MC group compared with the PAN group for 7 of the 8. Most of Figure 1. a, Hierarchical clustering of patients with hepatitis C virus– them encoded oxidative stress–derived molecules (glu- associated mixed cryoglobulinemia (HCV-MC; n ϭ 14) and patients cose transporter 1 [GLUT-1], MT1H, endothelial cell with polyarteritis nodosa (PAN; n ϭ 7) according to the expression nitric oxide synthase [eNOS], protein phosphatase 1 profile of the 8 differentially expressed genes. On the basis of similarity in gene expression profiles, 2 main branches, one with an HCV-MC regulatory subunit 15A, Hsp70, and Hsp90) and tissue cluster and the other with a PAN cluster, were observed. b, Hierar- plasminogen activator (tPA). The expression level of the chical clustering of the HCV-MC patients with necrotizing arteritis extracellular matrix endopeptidase MMP-7 gene was (HCV-MC NA; n ϭ 7) and patients without necrotizing arteritis lower in HCV-MC patients compared with PAN pa- (HCV-MC; n ϭ 7) according to the expression profile of the 5 tients. differentially expressed genes. Clustering clearly subdivided the HCV-MC population into 2 groups according to the presence or A hierarchical clustering of the samples from absence of necrotizing arteritis. patients with HCV-MC and patients with PAN, based on expression of the 8 differentially expressed genes (Table 3), distinguished these 2 groups of vasculitis patients. A patients versus control patients with noninflammatory representative dendrogram is shown in Figure 1a. On idiopathic neuropathy (Table 4). Among 42 selected the basis of the similarity of gene expression profiles, 2 genes, significant differential expression was observed main branches, one with an HCV-MC cluster, and the for 7 genes, with 6 being up-regulated in the necrotizing other with a PAN cluster, were observed. arteritis group. These genes involved MMPs (MMP-1, Differential gene expression in HCV-MC vascu- MMP-7, and MMP-9), oxidative stress–derived mole- litis according to the presence or absence of necrotizing cules (GLUT-1 and MT1H), and IL-1␤. The down- vasculitis. It is well known that there are pathologic regulated gene in HCV-MC patients with necrotizing variations in HCV-MC neuropathy, ranging from arteritis was related to an extracellular matrix endopep- perivascular infiltration to necrotizing arteritis. To ad- tidase, MMP-14. dress this issue, we analyzed the pattern of gene expres- Expression levels of the 42 selected genes in sion according to the clinical features of HCV-MC HCV-MC patients with necrotizing arteritis were com- 1320 SAADOUN ET AL

Table 4. Genes with significant differential levels of expression in patients with HCV-MC with necrotizing arteritis versus controls, patients with HCV-MC without necrotizing arteritis versus controls, and patients with HCV-MC with necrotizing arteritis versus patients with HCV-MC without necrotizing arteritis* HCV-MC P, HCV-MC P, HCV-MC with HCV-MC with without P, HCV-MC with without necrotizing necrotizing arteritis versus necrotizing necrotizing necrotizing arteritis arteritis versus HCV-MC without Gene arteritis arteritis versus controls controls necrotizing arteritis MMP1 6.0 Ϯ 2.0 0.5 Ϯ 0.3 0.01 NS 0.007 MMP7 10.6 Ϯ 3.8 1.1 Ϯ 0.4 0.01 NS 0.002 MMP9 24.6 Ϯ 14.5 2.8 Ϯ 0.7 0.01 NS 0.001 MMP14 1.6 Ϯ 0.2 6.0 Ϯ 2.1 NS 0.03 0.03 IL1B 8.6 Ϯ 2.5 3.6 Ϯ 0.8 0.02 NS NS GLUT1 22.9 Ϯ 8.5 21.2 Ϯ 5.9 0.01 0.01 NS MT1H 67.0 Ϯ 35.2 16.2 Ϯ 9.4 0.02 NS NS hsp90 4.4 Ϯ 2.3 0.9 Ϯ 0.3 NS NS 0.03 * Values are the mean Ϯ SEM fold difference compared with nerve samples from controls with noninflammatory neuropathy, after normalization of each measurement. Genes were considered to be differentially expressed when there was a Ͼ2-fold difference in mean expression levels between groups and the P value was less than 0.05. HCV-MC ϭ hepatitis C virus–associated mixed cryoglobulinemia; NS ϭ not significant. pared with those in HCV-MC patients without necrotiz- the epineurium of patients with HCV-MC with necro- ing arteritis (Table 4). This analysis showed differential tizing arteritis and patients with PAN compared with expression for 5 genes (MMP1, MMP7, MMP9, MMP14, that in patients with HCV-MC without necrotizing ar- and hsp90), with 4 up-regulated in the necrotizing arteri- teritis and those with noninflammatory idiopathic neu- tis group. In contrast, MMP14 was significantly down- ropathy. regulated in HCV-MC patients with necrotizing arteritis IL-1␤ was not expressed in the nerve samples compared with those without necrotizing arteritis. from controls with noninflammatory idiopathic neurop- A hierarchical clustering of the samples from athy. In contrast, it was expressed in the perivascular HCV-MC patients with and those without necrotizing mononuclear cell infiltrates in patients with HCV-MC, arteritis, based on the expression of the 5 differentially HCV-MC with necrotizing arteritis, and PAN. IL-1␤ expressed genes (Table 4), clearly subdivided the expression was higher in patients with HCV-MC with HCV-MC population into 2 groups according to the necrotizing arteritis and PAN than in those with presence or absence of necrotizing arteritis. A represen- HCV-MC without necrotizing arteritis. tative dendrogram is shown in Figure 1b. HCV-MC neuropathy could be readily distinguished not only by its DISCUSSION pathologic features, but also by its molecular genetic profile. On the basis of similarity in gene expression In this study, we addressed the pathogenesis of profiles, 2 main branches, one with a cluster of nerve vasculitis related to HCV-MC, using a molecular HCV-MC patients with necrotizing arteritis and the approach. Factors that have been associated with axonal other with a cluster of HCV-MC patients without necro- damage include cytokines, nitric oxide (NO), proteases, tizing arteritis, were observed. superoxides, and T cells (16–18). This gene expression Immunohistochemical analysis. Immunohisto- investigation provided further insight into the molecular chemical analyses of nerve tissue specimens from pa- mechanisms involved in the development of vasculitic tients with noninflammatory neuropathy, patients with lesions associated with HCV-MC. The results of the HCV-MC, patients with HCV-MC neuropathy with present study provide strong evidence for a role of necrotizing arteritis, and patients with PAN were used to MMPs, proinflammatory cytokines, and oxidative stress– investigate the pattern of expression of MMP-9 and derived molecules in the pathogenesis of HCV-MC IL-1␤ (Figure 2). H&E staining showed a perivascular vasculitis neuropathy. mononuclear cell infiltrate in samples from patients with Among the MMPs, MMP-7 and MMP-9 were HCV-MC vasculitis neuropathy, and transmural inflam- up-regulated in nerve samples from patients with mation with concurrent fibrinoid necrosis in patients HCV-MC vasculitis, whereas the level of TIMPs re- with HCV-MC with necrotizing arteritis and in patients mained unchanged in comparison with levels in control with PAN. Higher expression of MMP-9 was observed in patients with noninflammatory disease. The ratio of MOLECULAR PROFILE OF HCV-MC NEUROPATHY 1321

Figure 2. Paraffin-embedded longitudinal nerve sections from control patients with noninflammatory neuropathy (Ctrl), patients with HCV-MC, patients with HCV-MC with necrotizing arteritis, and patients with PAN. Top, Hematoxylin and eosin (HE) staining. Perivascular mononuclear cell infiltration was observed in patients with HCV-MC, and transmural inflammation with concurrent fibrinoid necrosis was observed in patients with HCV-MC with necrotizing arteritis and in patients with PAN. Middle, Staining for matrix metalloproteinase 9 (MMP-9). Low-level baseline expression of MMP-9 was observed in endothelial cells of blood vessels from control patients with noninflammatory idiopathic neuropathy. Higher expression of MMP-9 was observed in the epineurium of patients with HCV-MC with necrotizing arteritis and patients with PAN compared with that in patients with HCV-MC without necrotizing arteritis. MMP-9 colocalized with perivascular mononuclear cell infiltrates. Bottom, Staining for interleukin-1␤ (IL-1␤). IL-1␤ was not expressed in nerve samples from control patients with noninflammatory idiopathic neuropathy but was expressed in mononuclear cells within the perivascular infiltrates in patients with HCV-MC, patients with HCV-MC with necrotizing arteritis, and patients with PAN. Higher expression of IL-1␤ was noted in patients with HCV-MC with necrotizing arteritis and patients with PAN as compared with patients with HCV-MC without necrotizing arteritis. (Original magnification ϫ 270 for control and PAN specimens; ϫ 540 for HCV-MC and HCV-MC with necrotizing arteritis specimens.) See Figure 1 for other definitions.

MMPs to TIMPs determines overall MMP activity. III collagen, and thus may have an important role in the Thus, maintenance of this equilibrium is essential, and connective tissue turnover or remodeling associated with any disturbance in this balance likely results in tissue inflammation and wound healing (21). MMP-1 has been damage by increased proteolysis, as observed in this reported to be up-regulated by HCV core protein (22). study. MMP1, MMP7, and MMP9 were clearly overex- MMP-7 displays broad proteolytic activity against a pressed in the subgroup of HCV-MC patients with variety of extracellular matrix substrates, including col- necrotizing arteritis. In contrast, MMP14 appears to lagens, proteoglycans, elastin, laminin, fibronectin, and provide protection against necrotizing arteritis. casein. An increase in MMP-7 expression has been The MMP genes MMP1, MMP7, and MMP9 linked to IL-8 stimulation (23). Interestingly, up- harbor an activator protein 1 (AP-1) binding site in the regulation of both molecules has been demonstrated in proximal promoter, whereas the MMP-14 gene does not our HCV-MC vasculitis patients, particularly those with contain AP-1 elements (19,20). Whether the necrotizing necrotizing arteritis (9). arteritis process involves an AP-1–dependent signal MMP-9 is known to digest gelatins and types III, transduction pathway needs further substantiation. IV, and V collagen. Moreover, findings support a central MMP-1 cleaves fibrillar collagens, such as types I, II, and role of MMP-9 in T cell migration (16) and in the 1322 SAADOUN ET AL

disruption of vascular basement membranes (24). Steroids The plasminogen activator system seems to play a (24) and type I interferon (25) suppress the production of key role in the pathogenesis of HCV-MC neuropathy. In MMP-9, which may be one of the factors in their beneficial the present study, urokinase-type plasminogen activator effect in HCV-MC vasculitis neuropathy. MMP-9 is ex- receptor (uPAR), tPA, and plasminogen activator inhib- pressed at increased levels in the peripheral blood cells itor 1 (PAI-1) mRNA expression were ϳ5 times higher of patients infected with HCV (26). in HCV-MC vasculitis lesions than in nerve specimens Unbalanced MMP expression has been demon- from controls with noninflammatory idiopathic neurop- strated in several other inflammatory disorders (18,27– athy. Several studies have demonstrated that infectious 30). Immunostaining for MMP-1, MMP-2, and MMP-9 agents can alter endothelial cells by induction of pheno- has been shown in sural biopsy specimens from patients typic procoagulant changes (39–41). Serine protease with vasculitis neuropathy (18,28). Selective up- activity is considered to be a factor in the disturbance of regulation of MMP-2 has been evidenced in peripheral the blood–brain barrier and subsequent leukocyte entry blood mononuclear cells from patients with active We- leading to inflammation and causing myelin breakdown gener’s granulomatosis compared with healthy controls (42). Significantly up-regulated levels of uPAR and (29). PAI-1 are among the earliest detectable signs of inflam- Inflammatory cytokines are potent inducers of matory demyelination (43). MMPs. In the present study, IL-1␤ was significantly Tissue-type plasminogen activator is constitu- up-regulated in the nerve lesions of patients with HCV- tively expressed in the central nervous system by neurons MC, particularly in the subgroup of patients with necro- and microglia, and it is involved in the regulation of tizing arteritis. These findings are consistent with the synaptic remodeling and neuronal activity. In multiple previously reported increased levels of expression of sclerosis lesions, tPA is colocalized on demyelinated, tumor necrosis factor ␣ (TNF␣) mRNA in the nerves of damaged axons with nonphosphorylated neurofilament patients with HCV-MC vasculitis (9). This involvement and fibrin, suggesting a role in axonal fibrin dissolution of proinflammatory cytokines in HCV-MC neuropathies (44). Leukocytes, including monocytes, macrophages, may have several pathogenetic consequences. Both cy- and activated T cells, all constitutively express uPAR, tokines may exert a cytotoxic effect on nerve tissue which is an important mediator of adhesion and migra- (31–35). TNF␣ may induce neurotoxicity and neuronal tion to sites of inflammation via interactions with vitro- damage through caspase-dependent cascades and silenc- nectin and integrins (45). ing of cell survival signals (31). A direct myocytotoxic As compared with the levels in PAN patients, effect of IL-1␤ and TNF␣ has been observed in human levels of Hsp70, Hsp90, and eNOS mRNA expression myotubes in vitro (32,33), and TNF␣ may also partici- were higher in the nerve lesions of HCV-MC vasculitis pate in the initiation of myocytotoxicity in vivo in patients. The expression of heat-shock proteins (HSPs) polymyositis patients (36). can be markedly up-regulated by all cells under condi- Interestingly, the highest degree of mRNA up- tions of stress, e.g., fever, exposure to proinflammatory regulation in HCV-MC patients was observed for mediators such as TNF␣, oxidative stress, and viral MT1H, a hypoxic and oxidative stress protein that infection (46). The vascular wall is the object of various contributes to the resistance of mammalian cells to stresses that induce HSP expression. Injury to the vessel reactive oxygen intermediates (37). HCV core protein wall and the associated inflammatory response to injury has been shown to alter mitochondrial function and are now generally recognized as the essential compo- contribute to oxidative stress (38). MT1H may also nents of vasculitis. HSPs are believed to trigger auto- regulate both cellular proliferation and apoptotic path- immune reactions by means of molecular mimicry. An- ways (37). Up-regulation of MT1H in nerve samples tibodies to these proteins also develop, and although from HCV-MC patients in the present study, especially controversial, some studies suggest that these antibodies in the subgroup displaying necrotizing arteritis, may may play a role in autoimmunity-induced damage to the account for an elevated hypoxic stress response. The vessel wall (47,48). clinical distinction between HCV-MC patients with and Alternatively, HSPs can prevent or arrest inflam- those without necrotizing arteritis may also be reflected matory damage. In initial clinical trials in patients with at the molecular level. Indeed, a molecular signature of chronic inflammatory disease (such as rheumatoid ar- 5 differentially expressed genes clearly subdivided the thritis or type 1 diabetes mellitus), HSP-derived peptides HCV-MC population into 2 groups, according to the were shown to promote the production of antiinflamma- presence or absence of necrotizing arteritis. tory cytokines, indicating that HSPs have immunoregu- MOLECULAR PROFILE OF HCV-MC NEUROPATHY 1323

latory potential (49). Levels of Hsp70 may also be Chinaglia G, et al. T-cell-mediated epineurial vasculitis and hu- increased in the more susceptible small nociceptive moral-mediated microangiopathy in cryoglobulinemic neuropathy. J Neuroimmunol 1997;73:145–54. neurons in diabetes (50). Its protective effect against 9. Saadoun D, Bieche I, Maisonobe T, Asselah T, Laurendeau I, neuronal death has been repeatedly reported (51,52). Piette JC, et al. Involvement of chemokines and type 1 cytokines NO contributes to protecting against ischemic injury, in the pathogenesis of hepatitis C virus–associated mixed cryoglobulinemia vasculitis neuropathy. Arthritis Rheum 2005;52: and chronic hypoxia increases eNOS production of NO 2917–25. in response to growth factor stimulation. Recent studies 10. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate suggest that Hsp90 modulates eNOS function (53–55). cell behavior. Annu Rev Cell Dev Biol 2001;17:463–516. 11. Bogdan C. Nitric oxide and the immune response. Nat Immunol The association of Hsp90 with eNOS appears to be a 2001;2:907–16. critical factor in increasing NO production and limiting 12. Jennette JC, Falk RJ. Small-vessel vasculitis. N Engl J Med eNOS-dependent superoxide anion generation (55). 1997;337:1512–23. 13. Bieche I, Parfait B, Le Doussal V, Olivi M, Rio MC, Lidereau R, In conclusion, this study is the first comprehen- et al. Identification of CGA as a novel estrogen receptor-responsive screen of metalloproteinase, proinflammatory cyto- sive gene in breast cancer: an outstanding candidate marker to kine, and oxidative stress–derived molecules in HCV- predict the response to endocrine therapy. Cancer Res 2001;61: MC vasculitis, and provides a comparison between non- 1652–8. 14. Paradis V, Bieche I, Dargere D, Cazals-Hatem D, Laurendeau I, inflammatory, PAN, and HCV-MC neuropathies in Saada V, et al. Quantitative gene expression in Budd-Chiari terms of genetic expression of these molecules. The data syndrome: a molecular approach to the pathogenesis of the provide a basis for further study of the role of the genes disease. Gut 2005;54:1776–81. 15. Didier G, Brezellec P, Remy E, Henaut A. GeneANOVA: gene that underlie the pathologic processes in vasculitis. expression analysis of variance. Bioinformatics 2002;18:490–1. 16. Leppert D, Waubant E, Galardy R, Bunnett NW, Hauser SL. T cell gelatinases mediate basement membrane transmigration in AUTHOR CONTRIBUTIONS vitro. J Immunol 1995;154:4379–89. Dr. Cacoub had full access to all of the data in the study and 17. Hu W, Mathey E, Hartung HP, Kieseier BC. Cyclo-oxygenases takes responsibility for the integrity of the data and the accuracy of the and prostaglandins in acute inflammatory demyelination of the data analysis. peripheral nerve. Neurology 2003;61:1774–9. Study design. Saadoun, Bieche, Authier, Piette, Vidaud, Maisonobe, 18. Satoi H, Oka N, Kawasaki T, Miyamoto K, Akiguchi I, Kimura J. Cacoub. Mechanisms of tissue injury in vasculitic neuropathies. Neurology Acquisition of data. Saadoun, Bieche, Laurendeau, Jambou, Mai- 1998;50:492–6. sonobe. 19. Onodera S, Nishihira J, Iwabuchi K, Koyama Y, Yoshida K, Analysis and interpretation of data. Saadoun, Bieche, Maisonobe, Tanaka S, et al. Macrophage migration inhibitory factor up- Cacoub. regulates matrix metalloproteinase-9 and -13 in rat osteoblasts: Manuscript preparation. Saadoun, Bieche, Piette, Cacoub. relevance to intracellular signaling pathways. J Biol Chem 2002; Statistical analysis. Saadoun, Bieche. 277:7865–74. 20. Vihinen P, Kahari VM. Matrix metalloproteinases in cancer: prognostic markers and therapeutic targets. Int J Cancer 2002;99: REFERENCES 157–66. 21. Fields GB, Netzel-Arnett SJ, Windsor LJ, Engler JA, Birkedal- 1. Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N. Hansen H, Van Wart HE. Proteolytic activities of human fibro- Cryoglobulinemia: a clinical and laboratory study. II. Cryoglobu- blast collagenase: hydrolysis of a broad range of substrates at a lins with rheumatoid factor activity. Am J Med 1966;40:837–56. single active site. Biochemistry 1990;29:6670–7. 2. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus 22. Li K, Prow T, Lemon SM, Beard MR. Cellular response to infection in type II cryoglobulinemia. N Engl J Med 1992;327: conditional expression of hepatitis C virus core protein in Huh7 1490–5. cultured human hepatoma cells. Hepatology 2002;35:1237–46. 3. Cacoub P, Fabiani FL, Musset L, Perrin M, Frangeul L, Leger JM, 23. Wroblewski LE, Noble PJ, Pagliocca A, Pritchard DM, Hart CA, et al. Mixed cryoglobulinemia and hepatitis C virus. Am J Med Campbell F, et al. Stimulation of MMP-7 (matrilysin) by Helico- 1994;96:124–32. bacter pylori in human gastric epithelial cells: role in epithelial cell 4. Cacoub P, Saadoun D, Limal N, Leger JM, Maisonobe T. Hepa- migration. J Cell Sci 2003;116(Pt 14):3017–26. titis C virus infection and mixed cryoglobulinaemia vasculitis: a 24. Rosenberg GA, Dencoff JE, Correa N Jr, Reiners M, Ford CC. review of neurological complications. AIDS 2005;19 Suppl Effect of steroids on CSF matrix metalloproteinases in multiple 3:S128–34. sclerosis: relation to blood-brain barrier injury. Neurology 1996; 5. Cacoub P, Poynard T, Ghillani P, Charlotte F, Olivi M, Piette JC, 46:1626–32. et al, for the MULTIVIRC Group. Extrahepatic manifestations of 25. Stuve O, Dooley NP, Uhm JH, Antel JP, Francis GS, Williams G, chronic hepatitis C. Arthritis Rheum 1999;42:2204–12. et al. Interferon ␤-1b decreases the migration of T lymphocytes in 6. Ferri C, La Civita L, Cirafisi C, Siciliano G, Longombardo G, vitro: effects on matrix metalloproteinase-9. Ann Neurol 1996;40: Bombardieri S, et al. Peripheral neuropathy in mixed cryo- 853–63. globulinemia: clinical and electrophysiologic investigations. 26. Lichtinghagen R, Huegel O, Seifert T, Haberkorn CI, Michels D, J Rheumatol 1992;19:889–95. Flemming P, et al. Expression of matrix metalloproteinase-2 and 7. Authier FJ, Bassez G, Payan C, Guillevin L, Pawlotsky JM, Degos -9 and their inhibitors in peripheral blood cells of patients with JD, et al. Detection of genomic viral RNA in nerve and muscle of chronic hepatitis C. Clin Chem 2000;46:183–92. patients with HCV neuropathy. Neurology 2003;60:808–12. 27. Perez P, Kwon YJ, Alliende C, Leyton L, Aguilera S, Molina C, et 8. Bonetti B, Invernizzi F, Rizzuto N, Bonazzi ML, Zanusso GL, al. Increased acinar damage of salivary glands of patients with 1324 SAADOUN ET AL

Sjo¨gren’s syndrome is paralleled by simultaneous imbalance of 42. Lo EH, Wang X, Cuzner ML. Extracellular proteolysis in brain matrix metalloproteinase 3/tissue inhibitor of metalloproteinases 1 injury and inflammation: role for plasminogen activators and and matrix metalloproteinase 9/tissue inhibitor of metalloprotein- matrix metalloproteinases. J Neurosci Res 2002;69:1–9. ases 1 ratios. Arthritis Rheum 2005;52:2751–60. 43. Cuzner ML, Gveric D, Strand C, Loughlin AJ, Paemen L, 28. Leppert D, Hughes P, Huber S, Erne B, Grygar C, Said G, et al. Opdenakker G, et al. The expression of tissue-type plasminogen Matrix metalloproteinase upregulation in chronic inflammatory activator, matrix metalloproteases and endogenous inhibitors in demyelinating polyneuropathy and nonsystemic vasculitic neurop- the central nervous system in multiple sclerosis: comparison of athy. Neurology 1999;53:62–70. stages in lesion evolution. J Neuropathol Exp Neurol 1996;55: 29. Bjerkeli V, Halvorsen B, Damas JK, Nordoy I, Yndestad A, 1194–204. Aukrust P, et al. Expression of matrix metalloproteinases in 44. Gveric D, Hanemaaijer R, Newcombe J, van Lent NA, Sier CF, patients with Wegener’s granulomatosis. Ann Rheum Dis 2004;63: Cuzner ML. Plasminogen activators in multiple sclerosis lesions: 1659–63. implications for the inflammatory response and axonal damage. 30. Choi YC, Dalakas MC. Expression of matrix metalloproteinases in Brain 2001;124(Pt 10):1978–88. the muscle of patients with inflammatory myopathies. Neurology 45. Tarui T, Mazar AP, Cines DB, Takada Y. Urokinase-type plas- 2000;54:65–71. minogen activator receptor (CD87) is a ligand for integrins and 31. Takeuchi H, Jin S, Wang J, Zhang G, Kawanokuchi J, Kuno R, et mediates cell-cell interaction. J Biol Chem 2001;276:3983–90. al. Tumor necrosis factor-␣ induces neurotoxicity via glutamate 46. Kaufmann SH, Schoel B, van Embden JD, Koga T, Wand- release from hemichannels of activated microglia in an autocrine Wurttenberger A, Munk ME, et al. Heat-shock protein 60: impli- manner. J Biol Chem 2006;281:21362–8. cations for pathogenesis of and protection against bacterial infec- 32. Kalovidouris AE, Plotkin Z. Synergistic cytotoxic effect of inter- tions. Immunol Rev 1991;121:67–90. feron-␥ and tumor necrosis factor-␣ on cultured human muscle 47. Xu Q, Wick G. The role of heat shock proteins in protection and cells. J Rheumatol 1995;22:1698–703. pathophysiology of the arterial wall. Mol Med Today 1996;2: 33. Dalakas MC. The molecular and cellular pathology of inflamma- 372–9. tory muscle diseases. Curr Opin Pharmacol 2001;1:300–6. 48. Schett G, Xu Q, Amberger A, van der Zee R, Recheis H, Willeit 34. Sherwin C, Fern R. Acute lipopolysaccharide-mediated injury in J, et al. Autoantibodies against heat shock protein 60 mediate neonatal white matter glia: role of TNF-␣, IL-1␤, and calcium. endothelial cytotoxicity. J Clin Invest 1995;96:2569–77. J Immunol 2005;175:155–61. 49. Van Eden W, van der Zee R, Prakken B. Heat-shock proteins 35. Taylor DL, Jones F, Kubota ES, Pocock JM. Stimulation of induce T-cell regulation of chronic inflammation. Nat Rev Immu- microglial metabotropic glutamate receptor mGlu2 triggers tumor nol 2005;5:318–30. necrosis factor ␣-induced neurotoxicity in concert with microglial- 50. Kamiya H, Zhangm W, Sima AA. Apoptotic stress is counterbal- derived Fas ligand. J Neurosci 2005;25:2952–64. anced by survival elements preventing programmed cell death of 36. Kuru S, Inukai A, Liang Y, Doyu M, Takano A, Sobue G. Tumor dorsal root ganglions in subacute type 1 diabetic BB/Wor rats. necrosis factor-␣ expression in muscles of polymyositis and der- Diabetes 2005;54:3288–95. matomyositis. Acta Neuropathol (Berl) 2000;99:585–8. 51. Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, 37. Murphy BJ, Andrews GK, Bittel D, Discher DJ, McCue J, Green Zamzami N, et al. Heat-shock protein 70 antagonizes apoptosis- CJ, et al. Activation of metallothionein gene expression by hypoxia inducing factor. Nat Cell Biol 2001;3:839–43. involves metal response elements and metal transcription factor-1. 52. Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana Cancer Res 1999;59:1315–22. T, et al. Heat-shock protein 70 inhibits apoptosis by preventing 38. Okuda M, Li K, Beard MR, Showalter LA, Scholle F, Lemon SM, recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell et al. Mitochondrial injury, oxidative stress, and antioxidant gene Biol 2000;2:469–75. expression are induced by hepatitis C virus core protein. Gastro- 53. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, enterology 2002;122:366–75. Papapetropoulos A, et al. Dynamic activation of endothelial nitric 39. Vercellotti GM. Effects of viral activation of the vessel wall on oxide synthase by Hsp90. Nature 1998;392:821–4. inflammation and thrombosis. Blood Coagul Fibrinolysis 1998;9 54. Pritchard KA Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Suppl 2:S3–6. Fontana JT, et al. Heat shock protein 90 mediates the balance of 40. East E, Baker D, Pryce G, Lijnen HR, Cuzner ML, Gveric D. A nitric oxide and superoxide anion from endothelial nitric-oxide role for the plasminogen activator system in inflammation and synthase. J Biol Chem 2001;276:17621–4. neurodegeneration in the central nervous system during experi- 55. Shi Y, Baker JE, Zhang C, Tweddell JS, Su J, Pritchard KA Jr. mental allergic encephalomyelitis. Am J Pathol 2005;167:545–54. Chronic hypoxia increases endothelial nitric oxide synthase gener- 41. Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: ation of nitric oxide by increasing heat shock protein 90 association emerging mechanistic paradigms. Circulation 1999;100:e20–8. and serine phosphorylation. Circ Res 2002;91:300–6. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1325–1335 DOI 10.1002/art.22441 © 2007, American College of Rheumatology

High Levels of Inflammatory Chemokines and Cytokines in Joint Fluid and Synovial Tissue Throughout the Course of Antibiotic-Refractory Lyme Arthritis

Junghee J. Shin, Lisa J. Glickstein, and Allen C. Steere

Objective. To investigate the possible role of che- patients with antibiotic-refractory arthritis continued to mokines and cytokines in the pathogenesis of Lyme exhibit high SF and ST levels of these chemokines and arthritis. cytokines. In addition, synovial samples showed marked Methods. Using cytometric bead array and flow expression of the receptors for T cell or macrophage cytometry techniques, chemokine and cytokine levels chemokines, CXCR3 and CCR5. were determined in 65 synovial fluid (SF) samples and 7 Conclusion. Patients with antibiotic-refractory synovial tissue (ST) samples from 17 patients with Lyme arthritis have high synovial fluid levels of proin- antibiotic-responsive Lyme arthritis and 35 patients flammatory chemokines and cytokines, especially with antibiotic-refractory Lyme arthritis seen during CXCL9 and IFN␥, throughout the illness. Thus, even the past 18 years. In the ST samples, expression of when antibiotic treatment reduces or completely clears chemokine receptors was measured using immunohis- the infection in these patients, the inflammatory re- tochemistry. sponse in synovium persists. Results. Before or during antibiotic therapy, when the majority of patients had positive polymerase chain Lyme arthritis, which is caused by the tick-borne reaction (PCR) results for Borrelia burgdorferi DNA, SF spirochete Borrelia burgdorferi, is characterized by inter- from patients with antibiotic-refractory arthritis con- mittent or chronic arthritis in a few large joints, espe- tained exceptionally high levels of Th1 chemoattractants cially the knee, over a period of several years (1). In most and cytokines, particularly CXCL9 and interferon-␥ patients, the infection can be treated successfully with a (IFN␥). Compared with the patients whose arthritis was 1-month course of oral antibiotic therapy (2). However, responsive to antibiotic treatment, those with antibiotic- in a small percentage of patients the arthritis persists refractory arthritis had significantly higher levels of despite treatment with oral antibiotics for Ն2 months or CXCL9 and CXCL10 (both P < 0.001) and CCL3, intravenous (IV) antibiotics for Ն1 month or both; the CCL4, CXCL8, IFN␥, tumor necrosis factor ␣, persistent disease in these patients has been termed interleukin-1␤ (IL-1␤), and IL-6 (all P < 0.01). During antibiotic-refractory Lyme arthritis (2). After such ther- the post-antibiotic period, when the results of PCR for B apy, polymerase chain reaction (PCR) results for B burgdorferi DNA in SF and ST were uniformly negative, burgdorferi DNA in joint fluid are usually negative, suggesting that the symptoms of Lyme arthritis may Supported in part by the NIH (grant AR-20358), the English, persist after the near or total eradication of the spiro- Bonter, Mitchell Foundation, the Lyme/Arthritis Research Fund, and chete from the joint with antibiotic therapy (3). the Eshe Fund. Dr. Shin is recipient of a Walter J. and Lille A. Antibiotic-refractory Lyme arthritis is associated Berbecker Foundation scholarship for the study of Lyme disease. Junghee J. Shin, PhD, Lisa J. Glickstein, PhD, Allen C. with DRB1 molecules (primarily rheumatoid arthritis Steere, MD: Massachusetts General Hospital, and Harvard Medical [RA] alleles) that bind an epitope of B burgdorferi OspA School, Boston, Massachussets. (OspA ) (4), and with T cell recognition of this Dr. Steere has received an honorarium (more than $10,000) 163–175 from Novartis. epitope (5–8). The synovial lesions in antibiotic- Address correspondence and reprint requests to Junghee J. refractory Lyme arthritis, which exhibit synovial hyper- Shin, PhD, Massachusetts General Hospital, 55 Fruit Street, CNY trophy, fibrin deposition, infiltration of lymphocytes and 149/8301, Boston, MA 02114. E-mail: [email protected]. Submitted for publication July 28, 2006; accepted in revised plasma cells, and up-regulation of adhesion molecules, form December 6, 2006. are similar to those in other forms of chronic inflamma-

1325 1326 SHIN ET AL

tory arthritis, including RA (9,10). In Lyme arthritis, as (or the parents of patients who were minors) provided written in RA, interferon-␥ (IFN␥)–producing Th1 cells domi- informed consent. All patients met the Centers for Disease nate the T cell profile in synovial fluid (SF) (11,12). Control and Prevention (CDC) criteria for the diagnosis of Lyme arthritis (18). They had monarticular or oligoarticular However, a more thorough analysis of cytokine and arthritis in a few large joints, especially the knee, and a positive chemokine expression in the joints of patients with Lyme antibody response to B burgdorferi by enzyme-linked immu- arthritis has not yet been performed. nosorbent assay (ELISA) and Western blot, interpreted ac- In RA, characteristic features of the cytokine and cording to the CDC criteria (18). The clinical characteristics and HLA profiles of the patients seen through May 2004 have chemokine response in SF and synovial tissue (ST) been reported previously (4,19). include high levels of potent, proinflammatory cyto- For the present study, 65 stored SF samples and 7 kines, particularly tumor necrosis factor ␣ (TNF␣) and stored ST samples, obtained from 52 of the 134 patients (39%), interleukin-1␤ (IL-1␤) (13,14). High concentrations of were available. As in previous studies (4), antibiotic-responsive IL-6, a pleomorphic cytokine, play a role in the induc- Lyme arthritis was defined as the resolution of arthritis within 3 months after treatment with IV antibiotics for Յ4 weeks or tion of these proinflammatory cytokines (15). RA syno- oral antibiotics for Յ8 weeks, and antibiotic-refractory Lyme via exhibit a predominant Th1 cytokine pattern (higher arthritis was defined as persistent joint swelling for Ն3 months levels of IFN␥ than IL-4) and elevated levels of the after the start of treatment with IV antibiotics for Ն4 weeks or Ն IFN␥-inducible chemokines CXCL9 and CXCL10, oral antibiotics for 8 weeks, or both. At each visit, the degree of knee swelling in each patient was determined by joint which are chemoattractants for Th1 cells, effector aspiration or estimated by physical examination. PCR testing CD8ϩ T cells, and natural killer (NK) T cells (13,14,16). for B burgdorferi DNA in each SF sample was performed as In addition, there is marked expression of CXCR3, the previously described (3), and if enough SF remained, the white receptor for CXCL9 and CXCL10 (16). Furthermore, blood cell count was determined. moderate levels of chemoattractants for macrophages Chemokine and cytokine determinations. Protein levels of 6 chemokines and 9 cytokines in serum, SF, or ST were (CCL3 and CCL4) are found, as is high expression of measured by cytometric bead array according to the instruc- their receptor, CCR5 (16). tions of the manufacturer (BD Biosciences, San Diego, CA). In Lyme arthritis, unlike in RA, the triggering The array included tests for proinflammatory cytokines (IFN␥, event for synovial inflammation is known (B burgdorferi TNF␣, TNF␤, IL-1␤, and IL-12 p70), anti-inflammatory cyto- infection of the joint). Therefore, it is possible to kines (IL-4, IL-5, and IL-10), a pleomorphic cytokine (IL-6), a neutrophil chemokine (CXCL8), macrophage chemokines compare chemokine and cytokine levels in patients with (CCL2, CCL3, and CCL4), and T cell chemokines (CXCL9 B burgdorferi–infected joints who do and those who do and CXCL10). not develop antibiotic-refractory arthritis, and in those First, the precoated microbeads with capture antibod- with refractory arthritis, it is possible to compare the ies specific for each cytokine or chemokine were mixed to- infectious and post-antibiotic period. In the present gether in groups and incubated with SF or serum diluted in sample buffer (BD Biosciences). Based on preliminary exper- study we found that the degree of joint swelling was iments, beads detecting IFN␥, TNF␣, TNF␤, IL-1␤, IL-12 p70, similar in patients with antibiotic-responsive arthritis IL-4, IL-5, IL-10, and CCL3 were mixed together and incu- and those with antibiotic-refractory arthritis, but levels bated with 2-fold dilutions of SF or serum, beads detecting of Th1 chemoattractants and cytokines, especially IL-6, CXCL8, CCL2, and CCL4 were mixed together and CXCL9 and IFN␥, in joint fluid were significantly higher incubated with a 10-fold dilution of SF or a 5-fold dilution of serum, and beads detecting CXCL9 and CXCL10 were mixed in patients with antibiotic-refractory than in those with together and incubated with a 100-fold or 1,000-fold dilution of antibiotic-responsive arthritis. In the post-antibiotic pe- SF or a 10-fold dilution of serum. Following incubation with a riod in patients with refractory arthritis, when PCR mixture of phycoerythrin-conjugated antibodies to the corre- results for B burgdorferi DNA were uniformly negative, sponding chemokines or cytokines, the samples were analyzed the proinflammatory response remained high. by flow cytometry with a FACSCalibur (BD Biosciences). Levels of cytokines and chemokines in the same mixture were determined simultaneously, since beads that detect a given PATIENTS AND METHODS cytokine or chemokine have a distinct fluorescence intensity. The amount of each cytokine or chemokine in the SF or serum Patients. From November 1987 through July 2005, 134 was interpolated from a standard curve, which was generated consecutive patients (ages 12–79 years), who were treated with with each recombinant cytokine or chemokine, using FCAP antibiotics according to the guidelines of the Infectious Dis- Array software (BD Biosciences). Levels of CXCL13, a B cell eases Society of America (17), met criteria for inclusion in a chemoattractant, were measured separately in SF samples by study called Immunity in Lyme Arthritis (18). The study was Quantikine Human CXCL13/BLC/BCA-1 Immunoassay, ac- approved by the Human Investigations Committees at Tufts– cording to the instructions of the manufacturer (R&D Systems, New England Medical Center (Boston, MA) (1987–2002) and Minneapolis, MN). Massachusetts General Hospital (2002–2004), and all patients For chemokine and cytokine determinations in ST, CHEMOKINES AND CYTOKINES IN LYME ARTHRITIS 1327

Table 1. Clinical and demographic characteristics of the patients with Lyme arthritis Group 1 (antibiotic-responsive Group 2 (antibiotic-refractory arthritis) (n ϭ 17) arthritis) (n ϭ 35) Age, median (range) years 49 (16–67) 38 (12–79) No. male/no. female 12/5 27/8 Duration of arthritis prior to diagnosis, 1 (0–7) 1 (0–12) median (range) months Total WBCs in synovial fluid, median 4,167 (540–33,000) 10,100 (84–21,500) (range) per mm3*

OspA163–175 peptide–binding HLA–DR 5/15 (33) 26/33 (80)‡ molecules, no. of patients positive/ no. tested (%)† * White blood cell (WBC) counts were available in 13 patients in group 1 and 24 patients in group 2. † DRB1*0401, 0101, 0404, or 0405, DRB5*0101, DRB1*0402 or 0102. ‡ P ϭ 0.006 versus group 1.

20–30 mg of the tissue, which had been snap-frozen and stored a scale of 0–3 (0 ϭ absent; 1 ϭ slight; 2 ϭ moderate; 3 ϭ in liquid nitrogen, was suspended in 600 ␮l of CellLytic marked). Mammalian tissue lysis/extraction reagent, with protease inhib- Statistical analysis. Cytokine and chemokine levels in itors (Sigma, St. Louis, MO). The tissue was transferred to a patients with antibiotic-refractory arthritis and those with prechilled Tissue Gri-Tube (Fisher Scientific, Pittsburgh, PA) antibiotic-responsive arthritis were compared by Mann- and homogenized. The lysed sample was centrifuged at 12,000g Whitney rank sum test. Among patients who had Ͼ1 sample for 10 minutes, the protein-containing supernatant was col- available from the post-antibiotic period, the values were lected, and the concentration of protein was measured by averaged and a single value was included for each patient. absorbance at 280 nm. The difference in total protein concen- Because of multiple comparisons, P values less than or equal to tration in the supernatants was corrected by diluting all 0.01 were considered significant. samples to the same protein concentration as the sample with the lowest concentration. All samples were then diluted 2-fold and incubated with microbeads for each of the chemokines and RESULTS cytokines, as was done with serum and SF samples. Immunohistochemistry. ST specimens were snap- Clinical characteristics of the patients. Clinical frozen in TissueTek OTC (Miles, Elkhart, IN) and stored in and demographic characteristics of the Lyme arthritis ␮ liquid nitrogen. For this analysis, serial 6- m–thick cryostat patients are shown in Table 1. The majority of patients sections from each sample were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA); the sections were air in both the antibiotic-responsive group and the dried and fixed in cold acetone for 10 minutes (9). The slides antibiotic-refractory group were male. The median du- were then stored at Ϫ20°C until ready for use. ration of joint swelling prior to diagnosis (1 month), the Immunoperoxidase staining with a Vectastain Elite amount of swelling of 1 or both knees, and the SF white ABC kit (Vector, Burlingame, CA) was performed according to a modification of a previously described method (9). The blood cell count were similar in the 2 groups. HLA–DR slides were brought to room temperature, rinsed with phos- molecules that bound the OspA163–175 peptide of B phate buffered saline, and blocked for 20 minutes with normal burgdorferi were present with significantly greater fre- horse serum. Subsequent incubations were carried out with quency in patients with antibiotic-refractory arthritis ␧ purified mouse anti-human monoclonal antibodies to CD3 than in those with antibiotic-responsive arthritis (P ϭ (clone UCHT1; 1.25 ␮g/ml), CD14 (clone M5E2; 1.25 ␮g/ml), CXCR3 (clone 1C6/CXCR3; 2.5 ␮g/ml), or CCR5 (clone 0.006) (Table 1). 2D7/CCR5; 2.5 ␮g/ml) (all from BD Biosciences), followed by The 17 patients with antibiotic-responsive arthri- incubation with Vectastain biotinylated horse anti-mouse sec- tis (designated group 1) were, in most cases, treated ondary antibody, Vectastain ABC reagent, and the chromogen successfully with a 1-month course of oral doxycycline. substrate 3,3Ј-diaminobenzidine (Vector). After a final rinse with distilled water, the slides were counterstained with hema- In 14 of these patients, it was no longer possible to toxylin (Fisher Scientific, Middletown, VA) for 20 seconds and obtain SF samples after the start of antibiotic therapy mounted with VectaMount (Vector). A negative control sec- (Table 2). The 35 patients with antibiotic-refractory tion, stained with an irrelevant primary antibody (anti-OspA arthritis were treated with a 2–3-month course of oral [30 ␮g/ml]) was included on each slide. Anti-OspA was a gift from Dr. Alan G. Barbour (University of California, Irvine). doxycycline, a 1-month course of IV ceftriaxone, or (in For synovial sections from each of the 7 patients, expression of most cases) with both medications. However, only 2 of each cell surface marker or chemokine receptor was scored on these patients were seen at our institution prior to 1328 SHIN ET AL

Table 2. Clinical and laboratory findings at the time the SF or ST sample was obtained Group 2 (antibiotic-refractory arthritis)*

Group 1 (antibiotic-responsive 2A, SF obtained 2B, SF obtained 2C, ST obtained arthritis), SF obtained during during antibiotic during post-antibiotic during post-antibiotic antibiotic period (n ϭ 17) period (n ϭ 17) period (n ϭ 23) period (n ϭ 7) Duration from the start of 0 (0–2) 2 (0–4) 9 (4–33) 11 (7–38) antibiotics to sample date, median (range) months Degree of joint swelling, median 25 (5–130) 30 (5–130) 22.5 (2–60) 20 (15–20) (range) ml PCR results for Borrelia burgdorferi 6/14 10/17 0/23 0/6 DNA in SF, no. positive/no. tested† * In the antibiotic-refractory group, 17 patients had a single synovial fluid (SF) sample obtained during the period of antibiotic therapy (group 2A). In 9 of these patients, 1–3 additional samples were available from the post-antibiotic period, and in 14 other patients, 1 sample was obtained during this period only (group 2B). Synovial tissue (ST) obtained during arthroscopic synovectomy was available from 7 patients with antibiotic-refractory arthritis (group 2C), 3 of whom had SF samples obtained concurrently. † PCR ϭ polymerase chain reaction. initiation of antibiotic treatment; the remaining 33 were cytokines (IFN␥, TNF␣, and IL-1␤), and a pleomorphic referred because of lack of response to Ն1 course of cytokine (IL-6) compared with patients who had antibiotics. Therefore, in patients with antibiotic- antibiotic-responsive arthritis (group 1) (Figure 1). In refractory arthritis, from whom Ͼ1 sample was some- both the antibiotic-refractory and the antibiotic- times available, data were stratified according to responsive groups, moderate levels of CXCL13, a B cell whether the sample was obtained during the 1–4-month chemoattractant, were found (Figure 1), and levels of period of antibiotic therapy (when PCR results for B IL-12 p70 and TNF␤ were minimal (data not shown). burgdorferi DNA in SF were often positive) (group 2A), The median IFN␥:IL-4 ratio was 10 in the antibiotic- during the post-antibiotic period (when PCR results refractory group and 4 in the antibiotic-responsive were uniformly negative) (group 2B), or at the time of group, but this difference was not statistically significant. arthroscopic synovectomy (ST samples) (group 2C). Chemokine and cytokine levels in the 3 patients Joint swelling was usually greatest prior to anti- with antibiotic-responsive arthritis whose SF samples biotic treatment, and even in patients with refractory were obtained 1 or 2 months after the start of antibiotic arthritis, joint effusions often diminished as the disease therapy were similar to those in the 14 patients who were progressed, but synovial hypertrophy became more ap- seen prior to the initiation of antibiotic treatment. parent. Among these 35 patients, joint swelling persisted Similarly, the levels in the 2 patients with antibiotic- for a median of 13 months after the start of antibiotic refractory arthritis whose SF was obtained prior to treatment (range 4–53 months). The clinical character- antibiotic therapy were similar to those in the 15 patients istics and HLA profiles of the 52 patients with antibiotic- who were first seen several months after the start of responsive or antibiotic-refractory arthritis were repre- antibiotic therapy (data not shown). sentative of the complete group of 134 Lyme arthritis The levels of each chemokine and cytokine were patients who were seen during this time period (4,19). significantly higher in SF than in serum, where they were Chemokine and cytokine levels in SF and serum detectable at negligible levels if at all (in each instance, during the period of antibiotic therapy. During the P Ͻ 0.001) (data not shown). In addition, to confirm that period of antibiotic therapy (0–4 months after the start the SF from patients with antibiotic-responsive arthritis of antibiotics), when PCR results for B burgdorferi DNA was not inhibitory in the assay, SF samples with very low in SF were often positive, patients with antibiotic- levels of cytokines from 3 patients in the antibiotic- refractory arthritis (group 2A) had exceptionally high SF responsive group and 3 in the antibiotic-refractory group levels of Th1 chemoattractants and cytokines, particu- were spiked with 1,000 pg/ml of recombinant TNF␣, larly CXCL9 and IFN␥. Patients in this group had IL-1␤,orIFN␥. Levels of these cytokines were ϳ1,000 significantly higher levels of CXCL9 and CXCL10, pg/ml higher in the spiked samples compared with macrophage chemoattractants (CCL3 and CCL4), a unspiked samples from both patient groups (data not neutrophil chemoattractant (CXCL8), proinflammatory shown). Thus, early in the course of Lyme arthritis, a CHEMOKINES AND CYTOKINES IN LYME ARTHRITIS 1329

Figure 1. Chemokine and cytokine levels in synovial fluid from patients with antibiotic-responsive Lyme arthritis during the period of antibiotic therapy (the infectious period) and in patients with antibiotic-refractory Lyme arthritis during the period of antibiotic therapy and during the post-antibiotic period. Data are shown as box plots, where the boxes represent the first through third quartiles, the lines within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values (excluding outliers). P values are versus patients with antibiotic-responsive arthritis. IFN␥ ϭ interferon-␥; TNF␣ ϭ tumor necrosis factor ␣; IL-1␤ ϭ interteukin-1␤.

proinflammatory chemokine and cytokine response was to or during antibiotic treatment in patients with observed in SF. However, even after several months of antibiotic-responsive arthritis. antibiotic therapy, patients with antibiotic-refractory ar- Chemokine and cytokine levels in SF during the thritis had significantly higher proinflammatory chemo- post-antibiotic period. A median of 9 months after the kine and cytokine responses in SF than were seen prior start of antibiotic therapy, when test results for B 1330 SHIN ET AL

Figure 2. Serial determinations of chemokine and cytokine levels in joint fluid, degree of joint swelling, and polymerase chain reaction (PCR) results for Borrelia burgdorferi DNA in joint fluid from 2 representative patients with antibiotic-refractory Lyme arthritis. Patient 1 (A) was treated with oral doxycycline for 3 months and intravenous (IV) ceftriaxone for 1 month, after which PCR results became negative. Although his arthritis resolved 10 months after the start of antibiotic treatment, levels of proinflammatory chemokines and cytokines remained high or increased in the post-antibiotic period. Similarly, patient 2 (B) received a 2-month course of oral doxycycline and a 1-month course of IV ceftriaxone, after which PCR results for B burgdorferi DNA were negative. During the post-antibiotic period, levels of proinflammatory chemokines and cytokines increased, especially CXCL9 and IFN␥. After persistence of arthritis for 24 months, he underwent arthroscopic synovectomy, with resolution of the arthritis. See Figure 1 for other definitions.

burgdorferi DNA in SF were uniformly negative, high SF and cytokine responses in 2 patients from whom 4 SF levels of CXCL9 and CXCL10, intermediate levels of samples were available are shown in Figure 2. In both CCL4, and high levels of the proinflammatory cytokine patients, joint swelling was greatest when they were first IFN␥ were found. Although the levels of certain chemo- seen during the period of antibiotic therapy, when test kines and cytokines were lower in the post-antibiotic results for B burgdorferi DNA in SF were positive. period than during the infectious period, the median However, during the post-antibiotic period, proinflam- levels of the Th1 and macrophage chemoattractants and matory chemokine and cytokine responses remained IFN␥ remained significantly higher in the antibiotic- high, and the levels of CXCL9 and IFN␥ increased, even refractory group (group 2B) than in the antibiotic- as PCR results for B burgdorferi DNA became negative responsive group (group 1) (Figure 1), even though and joint effusions decreased. samples from the antibiotic-responsive group were usu- Chemokine and cytokine levels in synovial tissue ally obtained prior to initiation of antibiotic therapy. during the post-antibiotic period. A median of 11 To demonstrate these responses throughout the months after the start of antibiotic treatment, 7 patients course of the illness, the clinical courses and chemokine with antibiotic-refractory Lyme arthritis underwent CHEMOKINES AND CYTOKINES IN LYME ARTHRITIS 1331

Figure 3. Chemokine and cytokine levels in 23 joint fluid samples and 7 synovial tissue samples collected during the post-antibiotic period in patients with antibiotic-refractory Lyme arthritis. Bars show the median values; vertical lines above the bars show the third-quartile values. The general pattern of chemokine and cytokine levels was similar in the synovial tissue and joint fluid samples. See Figure 1 for definitions.

arthroscopic synovectomy. Although the concentration cytes and cells of monocyte lineage (16,22). Since these of total protein extracted from synovial tissue was at chemokine ligands were commonly found in SF and ST, least 50–100 fold lower than that obtained from joint we examined synovial samples for the receptors for these fluid, the general pattern of cytokine and chemokine ligands, using immunohistologic techniques. levels in these patients (group 2C) was similar to that in In ST, CD3ϩ T cells were found in a patchy or the 23 patients in whom SF samples were obtained in the diffuse distribution in sublining areas (mean Ϯ SD score post-antibiotic period (group 2B) (Figure 3). In both ST 2.0 Ϯ 0.8 on a scale of 0–3), and CXCR3, a receptor on and SF samples, the levels of the Th1 chemoattractants these cells, was also markedly expressed on infiltrating CXCL9 and CXCL10 were high. However, consistent and resident cells in these areas (mean Ϯ SD score 2.3 Ϯ with the fact that neutrophils are found primarily in joint 0.9) (Figures 4A–D). Moreover, the levels of CXCL9 fluid rather than in ST, the level of the neutrophil and CXCL10 correlated directly with the amount of chemoattractant CXCL8 was higher than that of the staining for CD3 (r ϭ 0.9, P ϭ 0.01 and r ϭ 0.8, P ϭ 0.05, macrophage chemoattractants CCL2, CCL3, and CCL4 respectively) and CXCR3 (r ϭ 0.93, P ϭ 0.007 and r ϭ in SF, whereas the opposite was generally the case in ST. 0.8, P ϭ 0.05, respectively) in the tissue. In addition to In addition, in SF, levels of IFN␥ were high, whereas T cells, CD14ϩ macrophages and synoviocytes were IFN␥ was barely detectable in ST. Conversely, levels of scattered throughout the sublining areas (score 2.4 Ϯ TNF␣ and, particularly, IL-1␤, were high in ST. 0.8), and CCR5, a receptor on these cells, was also Expression of chemokine receptors in ST. The widely expressed in the tissue (score 2.6 Ϯ 0.5) (Figures receptor for CXCL9 and CXCL10 is CXCR3, which is 4E–H). There was a trend toward increased levels of expressed primarily on activated T cells, but also on NK CCL3 and CCL4 and increased staining for CD14 and cells and some B cells (20,21). The receptor for CCL3 CCR5, but the correlations did not reach statistical and CCL4 is CCR5, which is expressed on Th1 lympho- significance. 1332 SHIN ET AL

start of antibiotic treatment had an antibiotic-responsive course, and because their arthritis usually resolved quickly, it was possible to obtain samples only prior to the start of antibiotic therapy. In contrast, most of the patients with antibiotic-refractory arthritis were referred several months later in their illness because of lack of response to 1 or more courses of antibiotics. Therefore, in most instances, samples from this group were first obtained several months after the start of antibiotics. Despite this difference, the duration of arthritis prior to diagnosis, the degree of joint swelling when the samples were obtained, and the percentage with positive PCR results for B burgdorferi DNA were similar in patients in the antibiotic-responsive and antibiotic- refractory groups. Moreover, there was no relationship in either group between the amount of time after antibiotic treatment and chemokine and cytokine levels. Although the levels of some chemokines and cytokines decreased during the post-antibiotic period in patients with refractory arthritis, the values were still significantly higher at that time than in samples obtained prior to antibiotic therapy in patients with antibiotic-responsive arthritis. Thus, despite the differences in timing, we believe the comparisons made here between patients with refractory arthritis and patients with responsive arthritis are robust. Except for CXCL13, chemokine and cytokine levels in this study were determined by cytometric bead array and flow cytometry. The advantage of this method is that the protein levels of a large number of chemo- Figure 4. Infiltrating cells and expression of chemokine receptors in kines or cytokines can be measured simultaneously using sections of synovial tissue from the knee joint of a representative a small volume of SF, with comparable sensitivity, patient with antibiotic-refractory Lyme arthritis. Sections were stained accuracy, and reproducibility to individual ELISAs (23). with monoclonal antibodies to cell surface markers (brown) and In addition, this method measures the actual protein ϩ counterstained with hematoxylin (purple). CD3 T cells are seen levels rather than messenger RNA expression, which diffusely in synovial sublining areas and in clusters (A and C). CD14ϩ synovial cells of macrophage lineage and infiltrating macrophages are may not accurately reflect protein concentrations at the seen throughout the synovial sublining areas (E and G). There is site of inflammation. marked CXCR3 expression on infiltrating and resident cells, especially Genetically determined differences in spirochetal in lymphocyte clusters in the synovial sublining and perivascular areas factors or host immune responses may account for (B and D). CCR5 expression is widespread in synoviocytes and different levels of chemokines and cytokines in patients lymphocytes (F and H). (Original magnification ϫ 100 in A, B, E, and F; ϫ 400 in C, D, G, and H.) with antibiotic-responsive or antibiotic-refractory arthritis. Prior to and during antibiotic treatment, the number of spirochetes in the SF was small in both groups, and ϳ50% of the patients in both groups had positive PCR DISCUSSION results for B burgdorferi DNA. After antibiotic treat- We determined cytokine and chemokine levels in ment, patients with antibiotic-refractory arthritis had synovial fluid samples from a unique cohort of patients uniformly negative PCR results. Thus, if persistent sy- with antibiotic-refractory and antibiotic-responsive novitis after antibiotic therapy is driven by a few remain- Lyme arthritis seen during an 18-year period. As one ing spirochetes, the number of organisms is below the would expect in a study of patients from the community level detectable by PCR. Alternatively, the negative (2), most of the patients whom we evaluated prior to the PCR results during the post-antibiotic period suggest CHEMOKINES AND CYTOKINES IN LYME ARTHRITIS 1333

that refractory Lyme arthritis is not caused by persistent nonhuman primates, the level of CXCL13 in skeletal infection, but rather by a host inflammatory response muscle was significantly higher than that in uninfected that does not decline appropriately with spirochetal animals (39). Although some patients with refractory killing. arthritis have germinal center–like B cell clusters in An animal model of antibiotic-refractory Lyme synovial tissue (9,10), this is an inconsistent finding. In arthritis is not available. However, B burgdorferi infec- the present study, moderate levels of CXCL13 were tion of inbred strains of mice has demonstrated the found in joint fluid, but the levels were similar in importance of host immune factors in the expression of patients with responsive and those with refractory Lyme acute Lyme arthritis. At one end of the spectrum, B arthritis, suggesting that T cells, rather than B cells, are burgdorferi–infected C57BL/6 mice have spirochetes in important in the pathogenesis of refractory Lyme arthritis. their joints but develop minimal or no arthritis. Appar- Antibiotic-refractory Lyme arthritis fits into a ently, the secretion of macrophage-derived antiinflam- pathogenetic framework with similarities to that of RA matory cytokines, particularly IL-10 and IL-6, protects (39–43). In Lyme arthritis, however, it is known that the these mice against severe arthritis (24,25). stimulus for synovial inflammation is an infectious agent. In the middle of the spectrum, B burgdorferi– We postulate that, when B burgdorferi invades synovium, infected BALB/c mice develop mild to severe joint resident fibroblast- and macrophage-derived synovio- inflammation, depending on the infectious dose. In cytes secrete proinflammatory cytokines (particularly these mice, a prominent, early Th1 response participates TNF␣ and IL-1␤) and chemokines (for example, CCL4, in B cell–dependent reduction of spirochetes (26,27). CXCL9, and CXCL10) that are biased toward a Th1 After spirochetal killing, arthritis resolves and an IL-4 phenotype. CXCL9 and CXCL10 are strong chemoat- response predominates. tractants for Th1 CD4ϩ T cells and effector CD8ϩ T At the severe end of the spectrum, B burgdorferi– cells. Through the chemokine receptors CXCR3 and infected C3H/He mice develop marked joint swelling CCR5, these cells, as well as NKT cells and B cells, home and inflammation (26,28,29). In these mice, an early Th1 to SF and ST. These T cells secrete IFN␥, which response is minimal or lacking, resulting in a greater up-regulates class II major histocompatibility complex spirochetal burden. Although a Th1 response develops molecules and adhesion molecules on synoviocytes and later, it does not result in spirochetal eradication, even endothelial cells, and in a feedback loop, would lead to though the joint inflammation lessens over a period of the continued ingress of inflammatory cells. These early weeks. When stimulated with B burgdorferi ex vivo, stages of inflammation probably occur in all patients macrophages from C3H/He and C57BL/6 mice differed significantly in levels of production of proinflammatory with Lyme arthritis, and are down-regulated appropri- cytokines (30). ately in most patients when spirochetes are killed by Variation in arthritis severity has been noted in antibiotics. human Lyme disease. In the late 1970s, before the cause However, in the presence of certain HLA–DR of Lyme disease was known, 55 patients with erythema molecules (primarily RA alleles) (44), a T cell response migrans were followed up for at least 4 years without to a particular epitope of B burgdorferi OspA (OspA163–175 antibiotic treatment (1). Of the 55 patients, 11 (20%) or other unidentified borrelial epitopes) may lead to had no later symptoms, 10 (18%) subsequently had especially high levels of proinflammatory cytokines in transient arthralgias, 28 (51%) developed intermittent the joint. In these patients, retained OspA antigens attacks of arthritis, and only 6 (11%) had persistent, might continue to elicit synovial inflammation after chronic arthritis. Since the institution of antibiotic treat- antibiotic treatment. However, in a recent study, the ment for Lyme arthritis, ϳ90% of patients have been frequencies of OspA163–175–specific T cells declined found to respond to a 1-month course of antibiotics, and both in patients with responsive arthritis and in patients the remaining 10% have not (2). with refractory arthritis within 2–3 months after the start Studies of patients with dermatologic or neuro- of antibiotics, months before the joint swelling resolved logic manifestations of B burgdorferi infection have also in patients with refractory arthritis (45), suggesting that shown elevated levels of proinflammatory chemokines retained spirochetal antigens are not the cause of per- and cytokines at sites of inflammation (31–36). In addi- sistent synovial inflammation. Thus, either this proin- tion, in cerebrospinal fluid of patients with neuroborre- flammatory response is not down-regulated appropri- liosis, high levels of the B cell chemoattractant CXCL13 ately after spirochetal killing, or the response could lead were found (37,38). Moreover, in B burgdorferi–infected to the breaking of tolerance to autoantigens, which 1334 SHIN ET AL

continue to induce synovial inflammation after apparent Study design. Shin, Glickstein, Steere. eradication of spirochetes from the joint. Acquisition of data. Shin, Steere. Analysis and interpretation of data. Shin, Glickstein, Steere. Inflamed ST in antibiotic-refractory Lyme arthri- Manuscript preparation. Shin, Glickstein, Steere. tis appears similar to that in all of the chronic inflam- Statistical analysis. Shin, Steere. matory arthritides (9,10). As in RA, the ST from patients with Lyme arthritis in the present study showed a REFERENCES predominance of macrophage-derived proinflammatory ␤ 1. Steere AC, Schoen RT, Taylor E. The clinical evolution of Lyme cytokines, particularly IL-1 . After antibiotic therapy, arthritis. Ann Intern Med 1987;107:725–31. Lyme arthritis patients were usually treated with antiin- 2. Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JH III, Liu flammatory agents, often including disease-modifying NY, et al. Treatment of Lyme arthritis. Arthritis Rheum 1994;37: 878–88. antirheumatic drugs (DMARDs), especially hydroxy- 3. Nocton JJ, Dressler F, Rutledge BJ, Rys PN, Persing DH, Steere chloroquine. In RA, patients treated with methotrexate AC. Detection of Borrelia burgdorferi DNA by polymerase chain alone or in combination with TNF␣ blockers have shown reaction in synovial fluid from patients with Lyme arthritis. N Engl J Med 1994;330:229–34. a decline in SF levels of infiltrating cells and proinflam- 4. Steere AC, Klitz W, Drouin EE, Falk BA, Kwok WW, Nepom GT, matory cytokines and chemokines (46–49). Our study et al. Antibiotic-refractory Lyme arthritis is associated with was not designed to test the response to DMARD HLA-DR molecules that bind a Borrelia burgdorferi peptide. J Exp Med 2006;203:961–71. therapy, but this treatment may have been a factor in the 5. Chen J, Field JA, Glickstein L, Molloy PJ, Huber BT, Steere AC. decline in cytokine responses in some Lyme arthritis Association of antibiotic treatment–resistant Lyme arthritis with T patients. cell responses to dominant epitopes of outer surface protein A of In conclusion, marked differences in proinflam- Borrelia burgdorferi. Arthritis Rheum 1999;42:1813–22. 6. Drouin EE, Glickstein LJ, Steere AC. Molecular characterization matory chemokine and cytokine responses, especially of the OspA (161-175) T cell epitope associated with treatment- CXCL9 and IFN␥ levels, were seen between patients resistant Lyme arthritis: differences among the three pathogenic with antibiotic-refractory Lyme arthritis and patients species of Borrelia burgdorferi sensu lato. J Autoimmun 2004;23: 281–92. with antibiotic-responsive Lyme arthritis in the present 7. Gross DM, Forsthuber T, Tary-Lehmann M, Etling C, Ito K, Nagy study. Moreover, even when antibiotic therapy reduced ZA, et al. Identification of LFA-1 as a candidate autoantigen in or completely cleared the infection, the inflammatory treatment-resistant Lyme arthritis. Science 1998;281:703–6. 8. Trollmo C, Meyer AL, Steere AC, Hafler DA, Huber BT. Molec- response in synovium from patients with refractory ular mimicry in Lyme arthritis demonstrated at the single cell arthritis persisted for months or even several years. In level: LFA-1 ␣ L is a partial agonist for outer surface protein these patients, either the proinflammatory response is A-reactive T cells. J Immunol 2001;166:5286–91. 9. Akin E, Aversa J, Steere AC. Expression of adhesion molecules in not down-regulated appropriately after spirochetal kill- synovia of patients with treatment-resistant Lyme arthritis. Infect ing or something else, such as an autoantigen, drives the Immun 2001;69:1774–80. proinflammatory response after spirochetal eradication. 10. Steere AC, Duray PH, Butcher EC. Spirochetal antigens and lymphoid cell surface markers in Lyme synovitis: comparison with In contrast to RA, but similar to findings in reactive rheumatoid synovium and tonsillar lymphoid tissue. Arthritis arthritis, the inflammatory process in the synovium of Rheum 1988;31:487–95. patients with Lyme disease recedes and disappears 11. Gross DM, Steere AC, Huber BT. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. within months to several years. Thus, without the stim- J Immunol 1998;160:1022–8. ulus of the infectious agent, even a putative infection- 12. Yssel H, Shanafelt MC, Soderberg C, Schneider PV, Anzola J, induced autoimmune response seems to right itself Peltz G. Borrelia burgdorferi activates a T helper type 1-like T cell subset in Lyme arthritis. J Exp Med 1991;174:593–601. within months to several years, and synovial inflamma- 13. Arend WP. Physiology of cytokine pathways in rheumatoid arthri- tion resolves. tis. Arthritis Rheum 2001;45:101–6. 14. Canete JD, Martinez SE, Farres J, Sanmarti R, Blay M, Gomez A, et al. Differential Th1/Th2 cytokine patterns in chronic arthritis: ACKNOWLEDGMENTS interferon ␥ is highly expressed in synovium of rheumatoid arthritis compared with seronegative spondyloarthropathies. Ann The authors thank Gail McHugh for PCR testing, Dr. Rheum Dis 2000;59:263–8. Elise Drouin for help with immunohistochemistry, and Colleen 15. Nishimoto N. Interleukin-6 in rheumatoid arthritis. Curr Opin Squires for help in gathering patient data. Rheumatol 2006;18:277–81. 16. Patel DD, Zachariah JP, Whichard LP. CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 2001;98:39–45. AUTHOR CONTRIBUTIONS 17. Wormser GP, Nadelman RB, Dattwyler RJ, Dennis DT, Shapiro ED, Steere AC, et al, for the Infectious Diseases Society of Dr. Shin had full access to all of the data in the study and America. Practice guidelines for the treatment of Lyme disease. takes responsibility for the integrity of the data and the accuracy of the Clin Infect Dis 2000;31 Suppl 1:1–14. data analysis. 18. Centers for Disease Control. Recommendation for test perfor- CHEMOKINES AND CYTOKINES IN LYME ARTHRITIS 1335

mance and interpretation from the Second National Conference specimens from patients with erythema migrans or acrodermatitis on Serologic Diagnosis of Lyme disease. MMWR Morb Mortal chronica atrophicans. J Invest Dermatol 2000;115:1115–23. Wkly Rep 1995;44:590–1. 34. Pashenkov M, Teleshova N, Kouwenhoven M, Smirnova T, Jin 19. Steere AC, Angelis SM. Therapy for Lyme arthritis: strategies for YP, Kostulas V, et al. Recruitment of dendritic cells to the the treatment of antibiotic-refractory arthritis [review]. Arthritis cerebrospinal fluid in bacterial neuroinfections. J Neuroimmunol Rheum 2006;54:3079–86. 2002;122:106–16. 20. Muehlinghaus G, Cigliano L, Huehn S, Peddinghaus A, Leyen- 35. Rupprecht TA, Koedel U, Muhlberger B, Wilske B, Fontana A, deckers H, Hauser AE, et al. Regulation of CXCR3 and CXCR4 Pfister HW. CXCL11 is involved in leukocyte recruitment to the expression during terminal differentiation of memory B cells into central nervous system in neuroborreliosis. J Neurol 2005;252: plasma cells. Blood 2005;105:3965–71. 820–3. 21. Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher 36. Salazar JC, Pope CD, Sellati TJ, Feder HM Jr, Kiely TG, Dardick M, et al. The chemokine receptors CXCR3 and CCR5 mark KR, et al. Coevolution of markers of innate and adaptive immu- subsets of T cells associated with certain inflammatory reactions. nity in skin and peripheral blood of patients with erythema J Clin Invest 1998;101:746–54. migrans. J Immunol 2003;171:2660–70. 22. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. 37. Pachner AR, Dail D, Narayan K, Dutta K, Cadavid D. Increased Nat Rev Immunol 2005;5:953–64. expression of B-lymphocyte chemoattractant, but not pro-inflam- 23. De Jager W, te Velthuis H, Prakken BJ, Kuis W, Rijkers GT. matory cytokines, in muscle tissue in rhesus chronic Lyme borre- Simultaneous detection of 15 human cytokines in a single sample liosis. Cytokine 2002;19:297–307. of stimulated peripheral blood mononuclear cells. Clin Diagn Lab 38. Rupprecht TA, Pfister HW, Angele B, Kastenbauer S, Wilske B, Immunol 2003;10:133–9. Koedel U. The chemokine CXCL13 (BLC): a putative diagnostic 24. Anguita J, Rincon M, Samanta S, Barthold SW, Flavell RA, Fikrig E. Borrelia burgdorferi-infected, interleukin-6-deficient mice have marker for neuroborreliosis. Neurology 2005;65:448–50. decreased Th2 responses and increased Lyme arthritis. J Infect Dis 39. Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 1998;178:1512–5. 2003;423:356–61. 25. Brown JP, Zachary JF, Teuscher C, Weis JJ, Wooten RM. Dual 40. Miossec P. Pro- and antiinflammatory cytokine balance in rheu- role of interleukin-10 in murine Lyme disease: regulation of matoid arthritis. Clin Exp Rheumatol 1995;13 Suppl 12:S13–6. arthritis severity and host defense. Infect Immun 1999;67:5142–50. 41. Nishimoto N. Cytokine signal regulation and autoimmune disor- 26. Kang I, Barthold SW, Persing DH, Bockenstedt LK. T-helper-cell ders. Autoimmunity 2005;38:359–67. cytokines in the early evolution of murine Lyme arthritis. Infect 42. Steere AC, Glickstein L. Elucidation of Lyme arthritis. Nat Rev Immun 1997;65:3107–11. Immunol 2004;4:143–52. 27. Zeidner N, Mbow ML, Dolan M, Massung R, Baca E, Piesman J. 43. Steiner G, Tohidast-Akrad M, Witzmann G, Vesely M, Studnicka- Effects of Ixodes scapularis and Borrelia burgdorferi on modula- Benke A, Gal A, et al. Cytokine production by synovial T cells in tion of the host immune response: induction of a TH2 cytokine rheumatoid arthritis. Rheumatology (Oxford) 1999;38:202–13. response in Lyme disease-susceptible (C3H/HeJ) mice but not in 44. Winchester RJ, Gregersen PK. The molecular basis of susceptibil- disease-resistant (BALB/c) mice. Infect Immun 1997;65:3100–6. itytorheumatoidarthritis:theconformationalequivalencehypothe- 28. Barthold SW, Beck DS, Hansen GM, Terwilliger GA, Moody KD. sis. Springer Semin Immunopathol 1988;10:119–39. Lyme borreliosis in selected strains and ages of laboratory mice. 45. Kannian P, Glickstein LJ, Drouin EE, Kwok WW, Nepom GT,

J Infect Dis 1990;162:133–8. Steere AC. Frequencies of Borrelia burgdorferi OspA161-175- 29. Keane-Myers A, Nickell SP. T cell subset-dependent modulation specific T cells in HLA-DRB1*0401-positive patients with Lyme of immunity to Borrelia burgdorferi in mice. J Immunol 1995;154: arthritis [abstract]. Arthritis Rheum 2006;54 Suppl 9:S546–7. 1770–6. 46. Gao IK, Leins C, Bohlen H, Heilig B, Lemmel EM. Inhibition of 30. Glickstein LJ, Coburn JL. Macrophage inflammatory response interleukin-8 synthesis by intraarticular methotrexate therapy in and cell death following Borrelia burgdorferi infection in vitro patients with rheumatoid arthritis. Z Rheumatol 1998;57:95–100. correlates with arthritis resistance. Am J Trop Med Hyg 2006;75: 47. Kopp S, Alstergren P, Ernestam S, Nordahl S, Morin P, Bratt J. 964–7. Reduction of temporomandibular joint pain after treatment with a 31. Grygorczuk S, Pancewicz S, Zajkowska J, Kondrusik M, Rwier- combination of methotrexate and infliximab is associated with zbinska R, Hermanowska-Szpakowicz T. Concentrations of mac- changes in synovial fluid and plasma cytokines in rheumatoid rophage inflammatory proteins MIP-1␣ and MIP-1␤ and interleu- arthritis. Cells Tissues Organs 2005;180:22–30. kin 8 (IL-8) in Lyme borreliosis. Infection 2004;32:350–5. 48. Thomas R, Carroll GJ. Reduction of leukocyte and interleukin-1␤ 32. Lepej SZ, Rode OD, Jeren T, Vince A, Remenar A, Barsic B. concentrations in the synovial fluid of rheumatoid arthritis patients Increased expression of CXCR3 and CCR5 on memory CD4ϩ treated with methotrexate. Arthritis Rheum 1993;36:1244–52. T-cells migrating into the cerebrospinal fluid of patients with 49. Van Oosterhout M, Levarht EW, Sont JK, Huizinga TW, Toes neuroborreliosis: the role of CXCL10 and CXCL11. J Neuroim- RE, van Laar JM. Clinical efficacy of infliximab plus methotrexate munol 2005;163:128–34. in DMARD naive and DMARD refractory rheumatoid arthritis is 33. Muellegger RR, McHugh G, Ruthazer R, Binder B, Kerl H, associated with decreased synovial expression of TNF ␣ and IL-18 Steere AC. Differential expression of cytokine mRNA in skin but not CXCL12. Ann Rheum Dis 2005;64:537–43. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1336–1344 DOI 10.1002/art.22457 © 2007, American College of Rheumatology

Gabapentin in the Treatment of Fibromyalgia

A Randomized, Double-Blind, Placebo-Controlled, Multicenter Trial

Lesley M. Arnold,1 Don L. Goldenberg,2 Sharon B. Stanford,1 Justine K. Lalonde,3 H. S. Sandhu,2 Paul E. Keck, Jr.,1 Jeffrey A. Welge,1 Fred Bishop,1 Kevin E. Stanford,1 Evelyn V. Hess,1 and James I. Hudson3

Objective. To assess the efficacy and safety of bad as you can imagine). Response to treatment was gabapentin in patients with fibromyalgia. defined as a reduction of >30% in this score. The Methods. A 12-week, randomized, double-blind primary analysis of efficacy for continuous variables study was designed to compare gabapentin (1,200–2,400 was a longitudinal analysis of the intent-to-treat sample, pa- with treatment-by-time interaction as the measure of 75 ؍ patients) with placebo (n 75 ؍ mg/day) (n tients) for efficacy and safety in treating pain associated effect. with fibromyalgia. The primary outcome measure was Results. Gabapentin-treated patients displayed a the Brief Pain Inventory (BPI) average pain severity significantly greater improvement in the BPI average ؍ ؍ estimated difference ;0.015 ؍ score (range 0–10, where 0 no pain and 10 pain as pain severity score (P ؊0.92 [95% confidence؍ between groups at week 12 -Supported by NIH grant N01-AR-2-2264 from the National interval ؊1.75, ؊0.71]). A significantly greater propor Institute of Arthritis and Musculoskeletal and Skin Diseases (Dr. Arnold, Principal Investigator). tion of gabapentin-treated patients compared with 1Lesley M. Arnold, MD, Sharon B. Stanford, MD, Paul E. placebo-treated patients achieved response at end point Gabapentin compared .(0.014 ؍ Keck, Jr., MD, Jeffrey A. Welge, PhD, Fred Bishop, BS, Kevin E. (51% versus 31%; P Stanford, MPH, Evelyn V. Hess, MD: University of Cincinnati College of Medicine, Cincinnati, Ohio; 2Don L. Goldenberg, MD, H. S. with placebo also significantly improved the BPI aver- Sandhu, MD: Newton-Wellesley Hospital, Newton, Massachusetts, age pain interference score, the Fibromyalgia Impact and Tufts University School of Medicine, Boston, Massachusetts; 3Justine K. Lalonde, MD (current address: AstraZeneca Pharmaceu- Questionnaire total score, the Clinical Global Impres- ticals, Zug, Switzerland), James I. Hudson, MD, ScD: McLean Hos- sion of Severity, the Patient Global Impression of pital, Belmont, Massachusetts, and Harvard Medical School, Boston, Improvement, the Medical Outcomes Study (MOS) Massachusetts. Dr. Arnold has received consulting fees from Eli Lilly (more Sleep Problems Index, and the MOS Short Form 36 than $10,000) and from Pfizer, Cypress Bioscience, Wyeth Pharma- vitality score, but not the mean tender point pain ceuticals, Sanofi-Aventis, Boehringer Ingelheim, Sepracor, Forest threshold or the Montgomery Asberg Depression Rating Laboratories, Allergan, and Vivus (less than $10,000 each). She also has received research support from Eli Lilly, Pfizer, Cypress Bio- Scale. Gabapentin was generally well tolerated. science, Wyeth Pharmaceuticals, Sanofi-Aventis, and Boehringer In- Conclusion. Gabapentin (1,200–2,400 mg/day) is gelheim. Dr. Keck has received consulting fees (less than $10,000) safe and efficacious for the treatment of pain and other from or is a member of the scientific advisory boards of Abbott, AstraZeneca Pharmaceuticals, Bristol-Myers Squibb, GlaxoSmith- symptoms associated with fibromyalgia. Kline, Eli Lilly, and Pfizer. He is a principal or coinvestigator on research studies sponsored by Abbott, the American Diabetes Asso- Fibromyalgia is a common, chronic musculoskel- ciation, AstraZeneca Pharmaceuticals, Bristol-Myers Squibb, Glaxo- SmithKline, Eli Lilly, Janssen Pharmaceutica, the National Institute of etal pain disorder that is characterized by widespread Mental Health, the National Institute of Drug Abuse, Pfizer, the pain and tenderness and is frequently accompanied by Stanley Medical Research Institute, and UCB. Address correspondence and reprint requests to Lesley M. fatigue, insomnia, depression, and anxiety (1,2). Fibro- Arnold, MD, University of Cincinnati Medical Arts Building, 222 myalgia occurs in ϳ2% of the US general population, is Piedmont Avenue, Suite 8200, Cincinnati, OH 45219. E-mail: more common in women (3.4% of women and 0.5% of [email protected]. Submitted for publication August 29, 2006; accepted in men) (3), and is associated with substantial morbidity revised form December 19, 2006. and disability.

1336 GABAPENTIN IN FIBROMYALGIA TREATMENT 1337

The pathophysiology of fibromyalgia is unknown, randomized, controlled study of gabapentin in the treat- but evidence suggests that fibromyalgia is associated ment of fibromyalgia. with aberrant central nervous system (CNS) processing of pain (4–7). As frequently observed in patients with PATIENTS AND METHODS neuropathic or inflammatory pain conditions, fibromyalgia patients often develop an increased response to Overview. The study was conducted in 3 outpatient painful stimuli (hyperalgesia) and experience pain from research centers in the US. Enrollment began in September 2003, and the study was completed in January 2006. The stimuli that are not usually noxious (allodynia) (6), various Institutional Review Boards approved the protocol, which may reflect enhanced CNS processing of both and all patients provided written informed consent after the painful and other stimuli that is characteristic of central study was explained and their questions were answered but sensitization (8). Unlike neuropathic or inflammatory before study procedures were initiated. Patients were identi- pain disorders, fibromyalgia is not associated with dam- fied by physician referral or response to an advertisement for a fibromyalgia medication trial. age to or a lesion of the peripheral nervous system or Entry criteria. Female or male patients were eligible CNS (9). However, fibromyalgia may share pathogenic for the study if they were Ն18 years of age and met the ACR mechanisms with neuropathic or inflammatory pain criteria for fibromyalgia (1). Patients with other rheumatic or conditions (10,11). medical disorders that contributed to the symptoms of fibro- Ն In preclinical pain models, gabapentin, a struc- myalgia were excluded. Patients were required to score 4on ␥ the average pain severity item of the Brief Pain Inventory tural analog of the neurotransmitter -aminobutyric acid (BPI) (26) at screening and randomization. Exclusion criteria (GABA), exerted robust analgesic and anti-allodynic consisted of the following: pain from traumatic injury or effects in syndromes secondary to sensitization of pain structural or regional rheumatic disease; rheumatoid arthritis, responses (12,13), but had minimal effects in models of inflammatory arthritis, or autoimmune disease; unstable med- acute, transient pain (14). Taylor et al (15) suggested ical or psychiatric illness; lifetime history of psychosis, hypo- mania or mania, epilepsy, or dementia; substance abuse in the that gabapentin did not appear to reduce immediate last 6 months; serious risk of suicide; pregnancy or breastfeed- pain from injury, but appeared to be effective in reducing; unacceptable contraception in those of childbearing po- ing abnormal hypersensitivity (allodynia and hyperalge- tential; patients who, in the opinion of the investigator, were sia) induced by inflammatory responses or nerve injury. treatment refractory; prior treatment with gabapentin or pre- The antinociceptive effects of gabapentin are hypothe- gabalin; and treatment with an investigational drug within 30 days of screening. Concomitant medication exclusions con- sized to be mediated by modulation of calcium channels ␣ ␦ sisted of medications or herbal agents with CNS effects, with via 2 binding, modulation of transmission of GABA, the exception of episodic use of sedating antihistamines (anti- and possibly other additional unidentified mechanisms depressants required a 14-day washout period prior to begin- (16). ning study medication except for fluoxetine, which required a Gabapentin has been found to have substantial 30-day washout period); analgesics, with the exception of analgesic effects in randomized, controlled clinical trials acetaminophen or over-the-counter nonsteroidal antiinflammatory drugs; and unconventional or alternative therapies. in diabetic neuropathy (17,18), postherpetic neuralgia Study design. Patients who met the entry criteria (19,20), migraine prophylaxis (21), and other neuro- following the 7–60-day screening phase were randomly as- pathic pain conditions (22). In addition to its antinoci- signed to 1 of 2 treatment groups, gabapentin or placebo, in a ceptive properties, data from placebo-controlled, ran- 1:1 ratio. Treatment was double-blind for 12 weeks. Patients domized trials indicate that gabapentin also has an were seen weekly for the first 2 weeks of the 12-week therapy phase; thereafter, study visits were scheduled at 2-week inter- anxiolytic effect and beneficial effects on sleep (17,23– vals. Patients then entered into a 1-week study-drug tapering 25). phase. Based on these preclinical and clinical findings, Gabapentin or matching placebo was titrated in the we hypothesized that gabapentin would be safe and following manner: 300 mg once a day at bedtime for 1 week, efficacious in reducing pain severity in patients with 300 mg twice a day for 1 week, 300 mg twice a day and 600 mg once a day at bedtime for 2 weeks, 600 mg 3 times a day for 2 fibromyalgia. To test this hypothesis, we conducted a weeks, and 600 mg twice a day and 1,200 mg once a day at randomized, double-blind, placebo-controlled, parallel- bedtime (2,400 mg/day) for the remainder of the study begin- group, flexible-dose study to assess the safety and effi- ning at week 6. If a patient could not tolerate 2,400 mg/day, the cacy of gabapentin (dosage range 1,200–2,400 mg/day, dosage was reduced to a minimum of 1,200 mg/day, administered 3 times a day. The study medication dose was stable for administered in 3 doses) in 150 outpatients who met the at least the last 4 weeks of the therapy phase. During the American College of Rheumatology (ACR) criteria for tapering phase, the dosage was decreased by 300 mg/day until fibromyalgia (1). To our knowledge, this is the first discontinuation. This study used a true intent-to-treat (ITT) 1338 ARNOLD ET AL

design, whereby patients were assessed regardless of adherence to study medication treatment (27,28). Outcome measures. The protocol-defined primary outcome measure was pain severity as measured by the self- reported BPI (short form) average pain severity score (26), which assesses average pain severity during the past 24 hours (0–10 scale, where 0 ϭ no pain and 10 ϭ pain as bad as you can imagine). There were several secondary outcome measures. Interference of pain with general activity, mood, walking ability, normal work, relationships with other people, sleep, and enjoyment of life was assessed using the BPI average pain interference score (0–10 scale, where 0 ϭ does not interfere and 10 ϭ completely interferes). Response to treatment was defined as a Ն30% reduction in the BPI average pain severity score. The overall impact of fibromyalgia was measured using the Fibromyalgia Impact Questionnaire (FIQ) (29), a self- administered questionnaire that is used to measure components of health status that are affected by fibromyalgia over the previous week. The total score ranges from 0 to 80; a higher score indicates a more negative impact. For the tender point assessment, the Fischer dolorimeter with a 1-cm2 rubber disk (30) was applied to the 18 tender point sites defined by the ACR (1), and the pressure was increased at a rate of 1 kg/cm2/second until the patient indicated verbally that he/she first felt discomfort or pain. The mean tender point pain threshold was calculated from the 18 points and recorded in Figure 1. Disposition of study patients from screening to completion kg/cm2. of the trial. Other measures included the Clinical Global Impres- sion of Severity scale (1–7 scale, where 1 ϭ normal, not at all ill, and 7 ϭ among the most extremely ill patients) (31), the Weight and height were measured at randomization, Patient Global Impression of Improvement scale (1–7 scale, and weight was measured again at the end of the therapy where 1 ϭ very much better and 7 ϭ very much worse), the phase. The mean tender point pain threshold, the Montgomery Medical Outcomes Study (MOS) sleep measure (32), which Asberg Depression Rating Scale, and the MOS sleep measure consists of 12 items that assess key constructs of sleep and were conducted at randomization and at weeks 4, 8, and at the generates a Sleep Problems Index that measures sleep ade- end of the therapy phase or week 12. The Patient Global quacy and disturbance, and the Montgomery Asberg Depres- Impression of Improvement scale was completed at week 1 and sion Rating Scale (33), a clinician-rated scale with 10 items that at all subsequent visits. The SF-36 was performed at random- measure apparent sadness, reported sadness, inner tension, ization and at the end of the therapy phase. Laboratory tests reduced sleep, reduced appetite, concentration difficulties, (hematologic and chemistry studies) and the EKG were re- lassitude, inability to feel, pessimistic thoughts, and suicidal peated at week 8 (urine pregnancy test conducted at weeks 4 thoughts. Additional patient-reported health outcomes were and 8), and a physical examination, EKG, and a urine preg- measured using the MOS Short Form 36 (SF-36) health survey nancy test were conducted at the end of the therapy phase. (34), which consists of 36 items in 8 health domains (sub- Statistical analysis. This study required the enrollment scales): bodily pain, general health, mental health, physical of 150 patients to have at least 90% power to detect a functioning, role–physical, role–emotional, social function, and moderately large effect size (0.60) for gabapentin using point vitality. and variance estimates based on the results of the Arnold et al Schedule of assessments. The screening protocol in- study comparing fluoxetine with placebo (37). The BPI average cluded the medical history and the Mini-International Neuro- pain severity score was chosen a priori as the primary outcome psychiatric Interview (35), and the Diagnostic and Statistical measure to test the efficacy of gabapentin in the treatment of Manual of Mental Disorders, Fourth Edition, to identify axis I pain associated with fibromyalgia. Type I error was controlled psychiatric disorders (36). Patients also underwent a physical at a significance level of 0.05 for the analysis of this primary examination, electrocardiography (EKG), and laboratory tests variable. Several secondary efficacy measures were included to (hematologic studies, chemistry panel, urinalysis, serum preg- confirm the findings of the primary measure. A multiplicity nancy test, urine drug screening, thyroid-stimulating hormone, adjustment was not performed for the secondary measures antinuclear antibody level, erythrocyte sedimentation rate, and because it was not the intent of the study to assess the rheumatoid factor), and completed the BPI. At the random- secondary measures at the same experimental significance ization visit, and at each subsequent visit until the end of the level as was established for the primary outcome variable. therapy phase, the BPI, FIQ, and Clinical Global Impression For the primary analysis of continuous variables col- of Severity scale were completed, vital signs were checked, and lected at more than 2 time points, we used a longitudinal adverse events and concomitant medication were reviewed. analysis that compared the rate of change of the outcome GABAPENTIN IN FIBROMYALGIA TREATMENT 1339

Table 1. Patient characteristics and scores on efficacy measures at baseline* Treatment group

Gabapentin Placebo (n ϭ 75) (n ϭ 75) Age, years 49.2 Ϯ 10.6 47.3 Ϯ 11.8 Women, no. (%) 70 (93.3) 65 (86.7) Race, no. (%) White 73 (97.3) 73 (97.3) African American 1 (1.3) 1 (1.3) Asian 1 (1.3) 0 Other 0 1 (1.3) Patients with current major depressive disorder, no. (%) 14 (18.7) 15 (20.0) Patients with current anxiety disorder, no. (%)† 8 (10.7) 6 (8.0) Brief Pain Inventory average pain severity score, range 0–10 5.7 Ϯ 1.4 6.0 Ϯ 1.5 Brief Pain Inventory average pain interference score, range 0–10 4.7 Ϯ 2.0‡ 5.3 Ϯ 1.9 FIQ total score, range 0–80 46.3 Ϯ 11.5 47.7 Ϯ 10.3 CGI Severity scale score, range 1–7 4.4 Ϯ 0.6 4.5 Ϯ 0.6 Mean tender point pain threshold, kg/cm2 1.8 Ϯ 0.7 1.7 Ϯ 0.7 Medical Outcomes Study Sleep Problems Index score, range 0–100 56.0 Ϯ 16.3 55.8 Ϯ 18.5 Montgomery Asberg Depression Rating Scale score, range 0–60 15.9 Ϯ 7.2 17.1 Ϯ 7.6 SF-36 score, range 0–100 Physical functioning 47.6 Ϯ 22.6 46.1 Ϯ 21.2 Role–physical 19.0 Ϯ 28.4 11.3 Ϯ 20.3 Social functioning 61.7 Ϯ 25.7 57.8 Ϯ 23.1 Bodily pain 37.0 Ϯ 13.1‡ 32.3 Ϯ 14.2 Mental health 67.6 Ϯ 17.1 64.3 Ϯ 20.5 Role–emotional 60.9 Ϯ 42.2 54.2 Ϯ 42.7 Vitality 21.7 Ϯ 15.1 20.1 Ϯ 16.7 General health 52.6 Ϯ 22.3 51.3 Ϯ 24.7 * Except where indicated otherwise, values are the mean Ϯ SD. FIQ ϭ Fibromyalgia Impact Question- naire; CGI Severity ϭ Clinical Global Impression of Severity; SF-36 ϭ Medical Outcomes Study Short Form 36. † Generalized anxiety disorder, panic disorder, agoraphobia, posttraumatic stress disorder, or obsessive- compulsive disorder. ‡ P Ͻ 0.05 versus placebo.

during the treatment period between groups. The difference in ITT sample, which included observations of participants re- rate of change was estimated by random regression methods, gardless of whether they were adherent to study medication as described elsewhere (38,39). We used a model for the mean treatment. We also performed a secondary analysis using only of the outcome variable that included terms for treatment, observations from visits while patients were adherent to study time, treatment-by-time interaction, and center. Time was medication treatment. modeled as a continuous variable. To account for the correla- We evaluated the differences between groups in the tion of observations among participants, we used the SAS incidence of treatment-emergent adverse events using Fisher’s procedure MIXED (SAS Institute, Cary, NC) with the best exact test. We compared the baseline characteristics of each fitting of the following covariance structures: unstructured, group using Fisher’s exact test for categorical variables, and the first-order heterogeneous autoregressive, and first-order auto- 2-sample t-test for continuous variables. Treatment effects regressive. The longitudinal analyses used all available obser- were tested at a 2-sided significance level of 0.05. vations from all time points from all patients who completed a baseline evaluation. As a secondary analysis, changes from baseline to end point (the last observation carried forward RESULTS [LOCF] method) were analyzed using an analysis of variance model, with a term for center. We also used this analysis as the Patient disposition. A total of 252 patients were primary analysis for the SF-36, which was obtained only at screened to identify 150 who met the entry criteria. They baseline and end point. were randomly assigned to either the gabapentin (n ϭ The primary analysis for response to treatment and for 75) or the placebo (n ϭ 75) group. Thirty-one patients participant ratings of global improvement was the Cochran- (21%) withdrew during the 12-week therapy phase, 18 Mantel-Haenszel test for end point values, using LOCF. All analyses employing LOCF used all available observations of (24%) from the gabapentin group and 13 (17%) from subjects who had at least one postbaseline assessment. the placebo group (P ϭ 0.42 by Fisher’s exact test) The primary analysis for all variables was based on the (Figure 1). Of 1,200 possible study visits, the number of 1340 ARNOLD ET AL

Efficacy. The median dosage at the end point for patients treated with gabapentin was 1,800 mg/day (interquartile range 1,200–2,400 mg/day). The mean BPI average pain severity scores decreased over time in both treatment groups, but more so in the gabapentin group (Figure 2). In the primary longitudinal analysis, compared with the placebo group, the gabapentin group had a significantly greater improvement in the BPI average pain severity score (Table 2). Gabapentin was also significantly superior to placebo in all secondary efficacy measures except for the mean tender point pain threshold and the Montgomery Asberg Depression Rating Scale (Table 2). Analysis of the BPI average pain severity score response rates (defined as Ն30% reduction from baseline to end point) revealed a significant difference between patients treated with gabapentin (38 Figure 2. Mean observed and estimated Brief Pain Inventory average of 75 [51%]) compared with patients treated with pla- pain severity scores in the gabapentin and placebo groups during the cebo (23 of 75 [31%]) (P ϭ 0.014). Compared with 12 weeks of treatment. Estimates were obtained by longitudinal placebo, gabapentin was associated with a significantly analysis. higher level of global improvement in patient ratings at the end point (P Ͻ 0.001) (Figure 3). The vitality domain of the SF-36 was the only domain that improved signif- observed visits was 1,077 (90.0%), of which 989 (82.4% icantly more in the gabapentin group compared with the of total possible) were obtained while participants were placebo group (P ϭ 0.032) (data not shown). adherent to study medication treatment. In the secondary end point analysis of the pri- Baseline clinical and demographic characteris- mary outcome measure, the gabapentin group had sig- tics. The majority of the patients were women (90%) nificantly greater improvement in the BPI average pain and white (97%). There were no significant differences severity score (mean Ϯ SD score at week 12 using LOCF between the treatment groups in demographic or clinical 3.8 Ϯ 2.2 for the gabapentin group versus 5.0 Ϯ 2.6 for variables (Table 1). For most outcome variables, there the placebo group). The estimated mean difference in were no significant differences between the groups at scores from baseline to week 12 was Ϫ0.95 (95% confi- baseline. However, the groups had significantly different dence interval [95% CI] Ϫ1.68, Ϫ0.23) (P ϭ 0.010). The ratings in the BPI average pain interference score and results of the end point analysis for the secondary the bodily pain domain of the SF-36 (Table 1). outcome measures were consistent with the findings

Table 2. Observed values and model-based estimates of differences in outcome measures between groups after 12 weeks of treatment with gabapentin or placebo* Difference between groups Gabapentin Placebo (n ϭ 57) (n ϭ 62) Estimate (95% CI)† P Brief Pain Inventory average pain severity score, range 0–10 3.2 Ϯ 2.0 4.6 Ϯ 2.6 Ϫ0.92 (Ϫ1.75, Ϫ0.71) 0.015 Brief Pain Inventory average pain interference score, range 0–10 2.2 Ϯ 2.2 3.6 Ϯ 2.8 Ϫ0.81 (Ϫ1.56, Ϫ0.07) 0.032 FIQ total score, range 0–80 26.2 Ϯ 15.1 37.3 Ϯ 18.1 Ϫ8.4 (Ϫ13.0, Ϫ3.3) 0.001 CGI Severity scale score, range 1–7 3.1 Ϯ 1.0 3.8 Ϯ 1.3 Ϫ0.66 (Ϫ1.08, Ϫ0.24) 0.002 Mean tender point pain threshold, kg/cm2 2.0 Ϯ 0.9 1.8 Ϯ 1.0 0.17 (Ϫ0.04, 0.39) 0.11 Medical Outcomes Study Sleep Problems Index score, range 0–100 33.4 Ϯ 19.5 47.8 Ϯ 20.9 Ϫ11.5 (Ϫ18.6, Ϫ4.4) 0.001 Montgomery Asberg Depression Rating Scale score, range 0–60 9.1 Ϯ 9.4 13.9 Ϯ 8.9 Ϫ2.79 (Ϫ6.13, 0.56) 0.067 * Values are the mean Ϯ SD. FIQ ϭ Fibromyalgia Impact Questionnaire; CGI Severity ϭ Clinical Global Impression of Severity. † Estimate is the mean (week 12 minus baseline) for gabapentin minus the mean (week 12 minus baseline) for placebo. The test statistic is the treatment-by-time interaction term, which represents the mean difference in rate of change between the gabapentin and placebo groups, with time modeled as weeks since baseline. The estimate and 95% confidence interval (95% CI) were obtained by multiplying the treatment-by-time interaction and 95% CI by 12. GABAPENTIN IN FIBROMYALGIA TREATMENT 1341

longitudinal and end point analyses were consistent with the findings obtained in the ITT analysis. Safety. Of the 150 randomized patients, a total of 19 patients discontinued the study during the therapy phase due to adverse events, with no significant differences between treatment groups (12 in the gabapentin group [16%] and 7 in the placebo group [9%]; P ϭ 0.34, by Fisher’s exact test) (Figure 1). Gabapentin-treated patients reported dizziness, sedation, lightheadedness, and weight gain significantly more frequently than did placebo-treated patients (Table 3). Notably, there were no significant differences in weight change between gabapentin- and placebo-treated patients from baseline Figure 3. Participant ratings of global improvement at week 12 (last to end point, as measured in the clinic (mean Ϯ SD observation carried forward) in the gabapentin and placebo groups. change 1.7 Ϯ 6.2 kg increase in the gabapentin group versus 1.1 Ϯ 5.8 kg increase in the placebo group) (P ϭ 0.56). Most treatment-emergent adverse events were obtained in the primary longitudinal analysis. The ana- mild to moderate in severity, and there were no signifi- lyses using only observations from visits at which partic- cant group differences in the percentage of serious ipants remained adherent to study medication treatment treatment-emergent adverse events. There were no clin- also showed significant improvement in the BPI average ically important findings in the laboratory results, phys- pain severity score (at week 12, estimated mean differ- ical examinations, or EKGs. ence between groups Ϫ0.86 [95% CI Ϫ1.69, Ϫ0.04], P ϭ 0.039, for the longitudinal analysis; Ϫ0.87 [95% CI Ϫ1.63, Ϫ0.11], P ϭ 0.025, for the end point analysis). DISCUSSION The results of the secondary outcomes in both the In this 12-week, randomized, double-blind, flexible-dose trial, gabapentin (1,200–2,400 mg/day), Table 3. Most frequently reported treatment-emergent adverse compared with placebo, significantly reduced pain asso- events* ciated with fibromyalgia, as measured by the BPI aver- Gabapentin Placebo age pain severity score, which was the primary efficacy (n ϭ 75) (n ϭ 75) measure. In addition, patients taking gabapentin compared with those taking placebo experienced a signifi- Headache 20 (26.7) 16 (21.3) Dizziness 19 (25.3)† 7 (9.3) cant reduction in their total level of pain interference on Sedation 18 (24.0)‡ 3 (4.0) the BPI. A significantly greater proportion of Nausea 16 (21.3) 16 (21.3) gabapentin-treated patients compared with placebo- Somnolence 14 (18.7) 6 (8.0) Edema 12 (16.0) 6 (8.0) treated patients achieved response at end point, defined Lightheadedness 11 (14.7)§ 1 (1.3) as Ն30% reduction in the BPI average pain severity Insomnia 9 (12.0) 6 (8.0) score from baseline to end point, which is considered to Diarrhea 8 (10.7) 5 (6.7) Pharyngitis 7 (9.3) 11 (14.7) be a clinically meaningful change in pain intensity (40). Asthenia 6 (8.0) 5 (6.7) Although fibromyalgia is defined by the ACR Depression 6 (8.0) 3 (4.0) criteria as a chronic, widespread condition that is asso- Flatulence 6 (8.0) 4 (5.3) Nervousness 6 (8.0) 1 (1.3) ciated with pain at Ն11 of 18 specific tender point sites Weight gain 6 (8.0)† 0 on the body (1), 75–80% of patients with fibromyalgia Amblyopia 5 (6.7) 1 (1.3) also experience fatigue and sleep disturbance (1). In the Anxiety 5 (6.7) 2 (2.7) Cold virus 5 (6.7) 11 (14.7) analysis of secondary outcomes, gabapentin, compared Dry mouth 5 (6.7) 3 (4.0) with placebo, significantly improved sleep on the MOS * Values are the number (%) of affected patients. Adverse events Sleep Problems Index and the vitality domain of the shown are those reported by at least 5% of the patients in the SF-36. Thus, treatment with gabapentin may result in gabapentin group. broad relief of important symptom domains associated † P Ͻ 0.05 versus placebo. ‡ P Ͻ 0.001 versus placebo. with fibromyalgia. Indeed, both clinicians and patients § P Ͻ 0.01 versus placebo. rated significantly greater global improvement with 1342 ARNOLD ET AL

gabapentin compared with placebo, and gabapentin- study medication. The advantage of this design is that it treated patients reported significant reduction in the preserves the validity of comparisons between treatment total impact of fibromyalgia. groups established by randomization (27,28). However, Other secondary outcomes, including depressive there continues to be debate about the advantages and symptoms and tender point pressure pain thresholds, did disadvantages of this design compared with one in which not significantly improve in patients taking gabapentin data are included only from time points at which partic- compared with those taking placebo. The mean Mont- ipants remain adherent to assigned treatment (27). gomery Asberg Depressive Rating Scale scores at base- Therefore, we included secondary analyses that used a line were mild, which may have limited the possibility for modified ITT design in which only outcomes from the significant change in depressive symptoms, although the visits during which participants remained adherent to gabapentin-treated patients showed numerically supe- medication treatment were included in the analyses. rior improvement in depressive symptoms compared Importantly, the results of these secondary analyses with patients taking placebo. Tender points have been were consistent with the primary analysis. unresponsive in some previous clinical trials of fibromy- Several limitations of this study should be consid- algia (41), suggesting that they may be less responsive to ered. First, because the trial was 12 weeks in duration, treatment than other symptoms of fibromyalgia (42) or the results may not generalize to longer treatment that gabapentin may not affect the underlying mecha- periods, and the long-term efficacy of gabapentin should nism that causes tender points. be explored in future clinical trials. Second, the treat- The results of the present study are consistent ment groups were relatively small, and the study may with the pregabalin trial of fibromyalgia (43) in which have lacked the power to detect potentially relevant pain, but not tender points, significantly improved in differences between groups, particularly on tender patients taking 450 mg/day pregabalin compared with points. Third, the trial used a flexible-dose design, which placebo. In addition, pregabalin was associated with limited our ability to establish a single effective dose of significant improvement in other important symptom gabapentin, although the median dose of gabapentin domains, including sleep and fatigue, and other mea- used in the present study is within the range recom- sures of health status. Pregabalin, like gabapentin, is mended for the treatment of other chronic pain disor- thought to exert its antinociceptive effects primarily by ders (22). Finally, the results of the trial may not modulation of calcium channels via ␣ ␦ binding, which 2 generalize to patients with some comorbid psychiatric reduces the release of several neurotransmitters involved in pain processing, such as glutamate, noradren- disorders, such as bipolar disorder or psychosis, patients aline, and substance P (43). The results of the pregabalin with comorbid rheumatologic or other painful musculo- and gabapentin trials provide substantial evidence that skeletal disorders, or those with unstable psychiatric or ␣ ␦ medical disorders because patients with these conditions 2 ligands have the potential to benefit patients with fibromyalgia. were excluded from the trial. Gabapentin was generally well tolerated. Signifi- In summary, this is the first randomized, placebo- cantly more gabapentin-treated patients than placebo- controlled study to evaluate gabapentin in the treatment treated patients reported dizziness, sedation, lighthead- of fibromyalgia. The results demonstrated that gabapen- edness, and weight gain, although upon clinical tin, taken for up to 12 weeks, is effective and safe in the measurement, there were no significant differences in treatment of pain and other symptoms associated with weight gain between patient groups. Most gabapentin- fibromyalgia. treated patients who reported weight gain also reported edema, which may explain some of the patients’ percep- ACKNOWLEDGMENTS tion of weight gain. There were no significant differences The authors would like to thank the following mem- between treatment groups in the number of patients who bers of the Data Safety and Monitoring Board who provided discontinued participation in the study due to treatment- valuable advice during the trial: Lyle Sensenbrenner, MD emergent adverse events. The safety findings are gener- (Chair), Nancy Olsen, MD, Janet Holbrook, PhD, Daniel ally consistent with the findings in studies of gabapentin Clauw, MD, and Theresa O’Lonergan, MA. We also thank the in patients with other pain disorders (22). staff at the NIH/National Institute of Arthritis and Musculo- skeletal and Skin Diseases for their support. We appreciate the This clinical trial was designed to allow for a true logistical help provided by KAI Research Inc. We would like to ITT analysis. Thus, patient outcomes were collected at acknowledge our research staff at each of the investigator sites: all patient visits, regardless of the patient’s adherence to Carrie Gibson, Catherine Brooks, and Jennifer Hoff (Univer- GABAPENTIN IN FIBROMYALGIA TREATMENT 1343

sity of Cincinnati, Cincinnati, Ohio), Mary Rogers (Newton- 15. Taylor CP, Gee NS, Su TZ, Kocsis JD, Welty DF, Brown JP, et al. Wellesley Hospital, Newton, Massachusetts), and Judy Berry, A summary of mechanistic hypotheses of gabapentin pharmacol- Kate Fogarty, Yael Nillni, Lindsay Pindyck, Rachel Placidi, ogy [review]. Epilepsy Res 1998;29:233–49. and Micheala Vine (McLean Hospital, Belmont, Massachu- 16. Urban MO, Ren K, Park KT, Campbell B, Anker N, Stearns B, et setts). Finally, we thank the patients for their participation in al. Comparison of the antinociceptive profiles of gabapentin and 3-methylgabapentin in rat models of acute and persistent pain: this clinical trial. implications for mechanism of action. J Pharmacol Exp Ther 2005;313:1209–16. AUTHOR CONTRIBUTIONS 17. Backonja M, Beydoun A, Edwards KR, Schwartz SL, Fonseca V, Hes M, et al. Gabapentin for the symptomatic treatment of painful Dr. Arnold had full access to all of the data in the study and neuropathy in patients with diabetes mellitus: a randomized takes responsibility for the integrity of the data and the accuracy of the controlled trial. JAMA 1998;280:1831–6. data analysis. 18. Morello CM, Leckband SG, Stoner CP, Moorhouse DF, Sahagian Study design. Arnold, Goldenberg, Sandhu, Keck, Hess, Hudson. GA. Randomized double-blind study comparing the efficacy of Acquisition of data. Arnold, Goldenberg, Sharon Stanford, Lalonde, gabapentin with amitriptyline on diabetic peripheral neuropathy Sandhu, Hess, Hudson. pain. Arch Intern Med 1999;159:1931–7. Analysis and interpretation of data. Arnold, Lalonde, Keck, Welge, 19. Rowbotham M, Harden N, Stacey B, Bernstein P, Magnus-Miller Kevin Stanford, Hess, Hudson. L. Gabapentin for the treatment of postherpetic neuralgia: a Manuscript preparation. Arnold, Goldenberg, Sandhu, Keck, Hess, randomized controlled trial. JAMA 1998;280:1837–42. Hudson. 20. Rice AS, Maton S, Postherpetic Neuralgia Study Group. Gabap- Statistical analysis. Welge, Bishop, Kevin Stanford, Hudson. entin in postherpetic neuralgia: a randomised, double blind, Database design. Bishop. placebo controlled study. Pain 2001;94:215–24. 21. Mathew NT, Rapoport A, Saper J, Magnus L, Klapper J, Ra- madan N, et al. Efficacy of gabapentin in migraine prophylaxis. REFERENCES Headache 2001;41:119–28. 1. Wolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, 22. Backonja M, Glanzman RL. Gabapentin dosing for neuropathic Goldenberg DL, et al. The American College of Rheumatology pain: evidence from randomized, placebo-controlled clinical trials 1990 criteria for the classification of fibromyalgia: report of the [review]. Clin Ther 2003;25:81–104. Multicenter Criteria Committee. Arthritis Rheum 1990;33:160–72. 23. Pande AC, Davidson JR, Jefferson JW, Janney CA, Katzelnick DJ, 2. Hudson JI, Pope HG Jr. The relationship between fibromyalgia Weisler RH, et al. Treatment of social phobia with gabapentin: a and major depressive disorder [review]. Rheum Dis Clin North placebo-controlled study. J Clin Psychopharmacol 1999;19:341–8. Am 1996;22:285–303. 24. Pande AC, Pollack MH, Crockatt J, Greiner M, Chouinard G, 3. Wolfe F, Ross K, Anderson J, Russell IJ, Hebert L. The preva- Lydiard RB, et al. Placebo-controlled study of gabapentin treat- lence and characteristics of fibromyalgia in the general population. ment of panic disorder. J Clin Psychopharmacol 2000;20:467–71. Arthritis Rheum 1995;38:19–28. 25. Foldvary-Schaefer N, De Leon Sanchez I, Karafa M, Mascha E, 4. Pillemer SR, Bradley LA, Crofford LJ, Moldofsky H, Chrousos Dinner D, Morris HH. Gabapentin increases slow-wave sleep in GP. The neuroscience and endocrinology of fibromyalgia. Arthri- normal adults. Epilepsia 2002;43:1493–7. tis Rheum 1997;40:1928–39. 26. Cleeland CS, Ryan KM. Pain assessment: global use of the Brief Pain 5. Lautenbacher S, Rollman GB. Possible deficiencies of pain mod- Inventory [review]. Ann Acad Med Singapore 1994;23:129–38. ulation in fibromyalgia. Clin J Pain 1997;13:189–96. 27. Fisher LD, Dixon DO, Herson J, Frankowski RK, Hearron MS, 6. Bennett RM. Emerging concepts in the neurobiology of chronic Peace KE. Intention to treat in clinical trials. In: Peace KE, editor. pain: evidence of abnormal sensory processing in fibromyalgia Statistical issues in drug research and development. New York: [review]. Mayo Clin Proc 1999;74:385–98. Marcel Dekker; 1990. p. 331–50. 7. Staud R. Evidence of involvement of central neural mechanisms in 28. Ware JH. Interpreting incomplete data in studies of diet and generating fibromyalgia pain [review]. Curr Rheumatol Rep 2002; weight loss. N Engl J Med 2003;348:2136–7. 4:299–305. 29. Burckhardt CS, Clark SR, Bennett RM. The Fibromyalgia Impact 8. Baranauskas G, Nistri A. Sensitization of pain pathways in the Questionnaire: development and validation. J Rheumatol 1991;18: spinal cord: cellular mechanisms [review]. Prog Neurobiol 1998; 728–33. 54:349–65. 30. Fischer AA. Pressure threshold meter: its use for quantification of 9. Rowbotham MC. Is fibromyalgia a neuropathic pain syndrome? tender spots. Arch Phys Med Rehabil 1986;67:836–8. [review]. J Rheumatol Suppl 2005;75:38–40. 31. Guy W. ECDEU assessment manual for psychopharmacology, 10. Woolf CJ. Pain: moving from symptom control toward mecha- revised. US Department of Health, Education, and Welfare nism-specific pharmacologic management [review]. Ann Intern publication (ADM). Rockville (MD): National Institute of Mental Med 2004;140:441–51. Health; 1976. p. 76–338. 11. Crofford LJ. The relationship of fibromyalgia to neuropathic pain 32. Hays RD, Stewart AL. Sleep measures. In: Stewart AL, Ware JE, syndromes [review]. J Rheumatol Suppl 2005;75:41–5. editors. Measuring functioning and well-being. Durham (NC): 12. Pan HL, Eisenach JC, Chen SR. Gabapentin suppresses ectopic Duke University Press; 1992. p. 232–59. nerve discharges and reverses allodynia in neuropathic rats. 33. Montgomery SA, Asberg M. A new depression scale designed to J Pharmacol Exp Ther 1999;288:1026–30. be sensitive to change. Br J Psychiatry 1979;134:382–9. 13. Hao JX, Xu XJ, Urban L, Wiesenfeld-Hallin Z. Repeated admin- 34. Ware JE, Snow KK, Kosinski M, Gandek B. SF-36 health survey istration of systemic gabapentin alleviates allodynia-like behaviors manual and interpretation guide. Boston: The Health Institute, in spinally injured rats. Neurosci Lett 2000;280:211–4. New England Medical Center; 1993. 14. Abdi S, Lee DH, Chung JM. The anti-allodynic effects of amitrip- 35. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, tyline, gabapentin, and lidocaine in a rat model of neuropathic Weiller E, et al. The Mini-International Neuropsychiatric Inter- pain. Anesth Analg 1998;87:1360–6. view (M.I.N.I.): the development and validation of a structured 1344 ARNOLD ET AL

diagnostic psychiatric interview for DSM-IV and ICD-10 [review]. 40. Farrar JT, Young JP Jr, LaMoreaux L, Werth JL, Poole RM. Clinical J Clin Psychiatry 1998;59 Suppl 20:22–33. importance of changes in chronic pain intensity measured on an 36. American Psychiatric Association. Diagnostic and statistical man- 11-point numerical pain rating scale. Pain 2001;94:149–58. ual of mental disorders, 4th edition. Washington, DC: American 41. Arnold LM, Keck PE Jr, Welge JA. Antidepressant treatment of Psychiatric Association; 1994. fibromyalgia: a meta-analysis and review. Psychosomatics 2000;41: 37. Arnold LM, Hess EV, Hudson JI, Welge JA, Berno SE, Keck PE 104–13. Jr. A randomized, placebo-controlled, double-blind, flexible-dose 42. Arnold LM, Rosen A, Pritchett YL, D’Souza DN, Goldstein DJ, study of fluoxetine in the treatment of women with fibromyalgia. Iyengar S, et al. A randomized, double-blind, placebo-con- Am J Med 2002;112:191–7. trolled trial of duloxetine in the treatment of women with 38. Gibbons RD, Hedeker D, Elkin I, Waternaux C, Kraemer HC, fibromyalgia with or without major depressive disorder. Pain Greenhouse JB, et al. Some conceptual and statistical issues in 2005;119:5–15. analysis of longitudinal psychiatric data: application to the NIMH 43. Crofford LJ, Rowbotham MC, Mease PJ, Russell IJ, Dworkin RH, Treatment of Depression Collaborative Research Program data- Corbin AE, et al, for the Pregabalin 1008-105 Study Group. set. Arch Gen Psychiatry 1993;50:739–50. Pregabalin for the treatment of fibromyalgia syndrome: results of 39. Fitzmaurice GM, Laird NM, Ware JH. Applied longitudinal a randomized, double-blind, placebo-controlled trial. Arthritis analysis. Hoboken (NJ): John Wiley & Sons; 2004. Rheum 2005;52:1264–73. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1345–1354 DOI 10.1002/art.22460 © 2007, American College of Rheumatology

Arthritic Pain Is Processed in Brain Areas Concerned With Emotions and Fear

B. Kulkarni,1 D. E. Bentley,1 R. Elliott,2 P. J. Julyan,3 E. Boger,1 A. Watson,1 Y. Boyle,1 W. El-Deredy,1 and A. K. P. Jones1

Objective. Functional neuroimaging studies have pain. The search for new analgesics for arthritis that act shown that experimentally induced acute pain is pro- on the brain should focus on drugs that modify this cessed within at least 2 parallel networks of brain circuitry. structures collectively known as the pain matrix. The relevance of this finding to clinical pain is not known, It is now well established that pain is processed because no direct comparisons of experimental and within a network of brain structures collectively known clinical pain have been performed in the same group of as the pain matrix (1–4). Within this matrix, 2 parallel patients. The aim of this study was to compare directly systems have been identified: the medial pain system and the brain areas involved in processing arthritic pain and the lateral pain system, each of which subserves different experimental pain in a group of patients with osteoar- functions. The medial pain system comprises structures thritis (OA). including the medial thalamus and the perigenual, Methods. Twelve patients with knee OA under- midcingulate, and insular cortices (5) and is involved in went positron emission tomography of the brain, using processing the affective (emotional) aspects of pain. The 18 F-fluorodeoxyglucose (FDG). Scanning was performed lateral pain system is involved in processing the sensory- during 3 different pain states: arthritic knee pain, discriminative (intensity, location, and duration) aspects experimental knee pain, and pain-free. Significant dif- of pain and comprises the lateral thalamus and its ferences in the neuronal uptake of FDG between differ- projections to the primary and secondary somatosensory ent pain states were investigated using statistical para- cortices in addition to thalamoinsular projections from metric mapping software. the ventromedial posterior nucleus of the thalamus (6). Results. Both pain conditions activated the pain This division of function between the medial and lateral matrix, but arthritic pain was associated with increased pain systems was first suggested by Bowsher in 1957 (1) activity in the cingulate cortex, the thalamus, and the and has now been demonstrated directly and noninva- amygdala; these areas are involved in the processing of sively in humans, using functional brain imaging tech- fear, emotions, and in aversive conditioning. niques (2,3,7,8). Conclusion. Our results suggest that studies of We recently showed that when healthy volunteers experimental pain provide a relevant but quantitatively received painful experimental heat stimuli, selectively incomplete picture of brain activity during arthritic attending to the affective aspects of the pain resulted in increased regional cerebral blood flow in areas of the Supported by the Arthritis Research Campaign, UK. medial pain system (the perigenual anterior cingulate 1B. Kulkarni, MRCP, PhD, D. E. Bentley, PhD, E. Boger, cortex, prefrontal cortex, orbitofrontal cortex, and amyg- BSc, A. Watson, BSc, Y. Boyle, PhD, W. El-Deredy, PhD, A. K. P. Jones, FRCP, MD: University of Manchester Rheumatic Diseases dala). However, selectively attending to the location of Centre, Hope Hospital, Salford, UK; 2R. Elliott, PhD: University of the pain was associated with increased regional cerebral Manchester, Manchester, UK; 3P. J. Julyan, PhD: Christie Hospital blood flow in the contralateral primary somatosensory National Health Service Trust, Withington, Manchester, UK. Address correspondence and reprint requests to B. Kulkarni, cortex and the inferior parietal cortex of the lateral pain MRCP, PhD, Human Pain Research Group, University of Manchester system. Rheumatic Diseases Centre, Hope Hospital, Eccles Old Road, Salford It remains unclear whether these brain responses M6 8HD, UK. E-mail: [email protected]. Submitted for publication November 23, 2006; accepted in to experimental pain are relevant to the processing of revised form December 19, 2006. ongoing clinical pain. Several groups of investigators

1345 1346 KULKARNI ET AL

have compared brain responses to acute experimental Participants. Twelve patients with knee OA (6 women pain in healthy volunteers and patients with chronic pain and 6 men; mean age 59 years [range 52–67 years]) participated in this study. All patients fulfilled the American College (9–12) and have shown some differences, predominantly of Rheumatology criteria for the classification of OA (15). Six within areas of the medial pain system. For example, in patients had predominant left knee arthritis, and the other 6 patients with atypical facial pain, a type of chronic had predominant right knee arthritis. All patients were right- regional pain syndrome, anterior cingulate responses to handed and had no psychiatric condition and no other medical painful stimulation were exaggerated compared with condition. None of the patients had received opiates or antidepressants for at least 1 year prior to the study. The these responses in healthy control subjects (11). In arthritic pain was treated mainly by nonpharmacologic mea- contrast, patients with rheumatoid arthritis showed di- sures and with simple analgesics such as acetaminophen. All minished responses to pain compared with those dem- analgesics were discontinued at least 12 hours prior to PET onstrated by age- and sex-matched healthy control sub- scanning and were restarted after the scanning session. All patients were instructed to continue their routine activities jects, particularly in the anterior cingulate and prefrontal prior to undergoing the arthritic pain condition and were asked cortices (12). These differences between patients and not to engage in unaccustomed physical activities prior to healthy control subjects in the response of the medial undergoing the pain-free and experimental pain conditions. pain system to experimental pain are likely to be asso- The order of the 3 conditions was randomized between patients. ciated with greater emotional salience of pain in patients PET protocol. We used the radioisotope FDG, which, with clinical pain and possible differential recruitment of as a glucose analog, provides an image of the rate of regional the limbic and medial pain circuitry in different clinical glucose metabolism (13,14). The metabolic product of FDG, pain conditions. However, no studies to date have FDG-6-phosphate, remains trapped within neuronal tissue for a longer duration (half-life 6.5 hours) compared with radioac- compared brain responses to experimental and clinical tive water. This unique metabolic behavior makes FDG an pain in the same group of patients, and therefore, the excellent marker for mapping regional function in the brain in relevance of these studies to ongoing clinical pain is response to various stimuli. unclear. All patients underwent PET scanning under 3 condi- We compared the brain activity of 12 patients tions, in randomized order, at least 24 hours apart: arthritic pain, pain-free, and experimental pain. The arthritic pain with osteoarthritis (OA) between 3 different pain states: condition assessed ongoing knee pain experienced during arthritic knee pain, experimental knee pain (experimen- patients’ accustomed physical activities (patients reported this tal pain applied on an occasion when patients were not pain as being moderately strong). The pain-free and experi- experiencing arthritic pain), and pain-free. Arthritic mental pain conditions were assessed during a period when patients were free from arthritic pain. In the absence of pain, by its nature, is usually acute and recurrent, in arthritic pain, acute experimental heat pain stimuli were contrast to many chronic pain conditions (e.g., neuro- delivered to the skin over the arthritic knee, using a Marstock pathic pain) in which pain is more often continuous. thermal threshold stimulator (Thermotest type 1; Somedic, Arthritic pain therefore provides an ideal model for Stockholm, Sweden) (16). This device delivers reproducible comparing experimental and common clinical pain. We intermittent ramps of increasing heat via a water-cooled probe (2.5 cm ϫ 5 cm). used positron emission tomography (PET) to measure In a training session prior to PET scanning, patients 18F-fluorodeoxyglucose (FDG) uptake in the brain as a were given a standard explanation of the study procedure. For correlate of neuronal activity (13,14). Our hypothesis each patient, the temperature that was reproducibly experi- was that both arthritic pain and acute experimental pain enced as moderately painful was carefully established using a 0–100-point scale (mean temperature 46.3°C). This tempera- would be processed within the same areas of the pain ture was used during scanning to induce acute experimental matrix, but that the arthritic pain state would be associ- pain. The standard instruction given to patients was to rate the ated with increased activation within the medial pain perceived intensity and unpleasantness of pain using a 0–100- system. point scale. All patients completed the Hospital Anxiety Depression Scale (17), the Center for Epidemiologic Studies Depression Scale (18), the Spielberger State Anxiety Inventory (19), and the Pain Catastrophizing Scale (20) during the PATIENTS AND METHODS training session and before each scanning session. These questionnaires were used to screen for current symptoms of The study protocol was approved by the Salford and mood and behavioral disorders, because altered emotional Trafford Research Ethics Committee, and permission to ad- states might influence the processing of pain in the brain minister radioactive substances was obtained from the Admin- (Table 1). istration of Radioactive Substances Advisory Committee of the All patients were asked to fast for 4 hours prior to Department of Health, UK. All patients gave informed written scanning, to ensure a relatively stable basal blood glucose level. consent. To ensure that the patients were experiencing the condition of PROCESSING OF ARTHRITIC PAIN AND EXPERIMENTAL PAIN IN OA 1347

Table 1. Demographic and clinical characteristics of the patients* Characteristic Baseline Session 1 Session 2 Session 3 Age, mean (range) years 59.16 (52–67) – – – No. men/no. women 6/6 – – – Arthritis, no. left knee/no. right knee 6/6 – – – HADS depression score 3.36 Ϯ 3.83 3.25 Ϯ 3.79 3.66 Ϯ 4.61 3.16 Ϯ 3.29 HADS anxiety score 4.86 Ϯ 3.69 4.83 Ϯ 3.61 5.25 Ϯ 3.72 4.50 Ϯ 4.03 PCS 12.78 Ϯ 13.7 14.09 Ϯ 15.65 11.63 Ϯ 12.80 12.63 Ϯ 13.77 CES-D score 12.66 Ϯ 2.16 13.09 Ϯ 2.42 12.66 Ϯ 2.18 12.09 Ϯ 2.11 STAI score 47.66 Ϯ 3.66 47.5 Ϯ 2.11 48.00 Ϯ 4.28 47.54 Ϯ 4.45 * Except where indicated otherwise, values are the mean Ϯ SD. HADS ϭ Hospital Anxiety Depression Scale; PCS ϭ Pain Catastrophizing Scale; CES-D ϭ Center for Epidemiologic Studies Depression Scale; STAI ϭ Spielberger State Anxiety Inventory. interest, pain ratings were obtained prior to the injection of to the space described by Talairach and Tournoux in their atlas FDG and every 10 minutes during the FDG uptake period of the human brain (23). (activation condition), which lasted for 30 minutes following To increase the signal-to-noise ratio and to accommo- the injection of FDG. The patients lay down on the scanning date variability in functional anatomy, each image was table, outside the scanner, in a quiet, dimly lit room. Rating smoothed with a Gaussian filter with a width of 16 mm full scales were suspended in the patient’s field of view to allow width at half maximum. A correction was made for global verbal reporting of perceived pain intensity and unpleasant- changes in rCMRGlu between scans. Differences between one ness. The thermode was in place on the skin 5 cm above the condition and another were assessed in the appropriate con- arthritic knee during all conditions, but heat stimuli were trast, using t statistics. This analysis was performed for each delivered only during the experimental pain condition. Follow- pixel, and the resulting set of t values constituted a statistical ing the activation condition, each patient was positioned in the parametric map (t). A further analysis was performed with scanner with his or her head secured firmly, using a molded subjective unpleasantness ratings as covariates, and differences headrest and a head-restraining Velcro band to reduce motion between arthritic pain and experimental pain were assessed. artifact. Only results that were significant (P Ͻ 0.05) after correction Data acquisition. Imaging was performed using a GE for multiple comparisons across the brain were reported. Advance PET scanner (General Electric Medical Systems, A second-step analysis was performed in 2 subgroups Milwaukee, WI). An initial 30-second scout scanning was (6 patients with right knee arthritis and 6 patients with left performed to ensure correct positioning of the patients within knee arthritis), to identify any laterality effects. Direct statis- the scanner, before the commencement of the main scanning tical comparisons were made between the 2 subgroups to (40 minutes after injection of the FDG). Scanning consisted of demonstrate brain activity in the arthritic pain condition versus a 20-minute 3-dimensional (3-D) emission scan preceded by a the pain-free condition and the experimental pain condition 3-minute 2-D transmission scan (to correct for tissue attenua- versus the pain-free condition. tion) and a 2-minute 2-D emission scan (to correct for emission contamination of the transmission scan). Thirty-five slices at 4.25-mm intervals were obtained to cover the whole brain. RESULTS From the acquired data, images of FDG distribution were produced in 2 15-minute blocks during the 3-D emission scan. Questionnaires for current symptoms of depres- Images were reconstructed by fully 3-D filtered back projec- sion, anxiety, or catastrophizing pain (see Table 1) were ϫ ϫ tion, with reprojection into 128 128 35 image matrices administered before each scanning session, and the (voxel size 1.95 ϫ 1.95 ϫ 4.25 mm3), using measured attenuation correction. results did not vary significantly from those obtained at Statistical analysis. The goal of this study was to baseline (training session), suggesting no significant compare pain-related responses in the brain during different change in mood states during different conditions. pain conditions. Statistical parametric mapping software im- Behavioral data. All patients rated their per- plemented in MatLab (MathWorks, Natick, MA; online at ceived pain intensity and unpleasantness on separate http://www.fil.ion.ucl.ac.uk/spm) was used to compare alter- 0–100-point scales at 10-minute intervals, from 10 min- ations in regional cerebral glucose metabolism (rCMRGlu) between conditions. Individual data were preprocessed, fol- utes before the FDG injection to the end of the scan. In lowed by statistical analysis of the group data (see the following the arthritic pain condition, the mean Ϯ SD intensity web site: www.fil.ion.ucl.ac.uk). To correct for head movement rating was 62.9 Ϯ 11.4, and the mean Ϯ SD unpleasant- between scans, all images were realigned to the first one to ness rating was 57.7 Ϯ 13.8. In the experimental pain create a mean PET image. Each realigned set of images was Ϯ Ϯ then normalized to enable data from an individual patient to condition, the mean SD intensity rating was 62.4 be transformed onto a standardized stereotaxic anatomic space 15.3, and the mean Ϯ SD unpleasantness rating was using an FDG template (21,22). This standard space conforms 49.2 Ϯ 12.4. In the pain-free condition, the mean Ϯ SD 1348 KULKARNI ET AL

Table 2. Brain areas that were significantly more active during arthritic pain compared with the pain-free condition* MNI coordinates

Brain region xyzBA Z score KE P Right posterior cingulate cortex 10 Ϫ36 36 31 4.21 626 0.01 Left posterior cingulate cortex Ϫ10 Ϫ36 36 31 4.21 626 0.01 Right anterior midcingulate cortex 10 24 20 24/32 4.01 256 0.02 Left anterior midcingulate cortex Ϫ10 24 20 24/32 4.01 256 0.02 Left perigenual anterior cingulate cortex Ϫ8 40 12 32/24 3.92 243 0.03 Right orbitofrontal cortex 27 40 Ϫ17 11 4.25 235 0.01 Left orbitofrontal cortex Ϫ22 40 Ϫ17 11 4.25 235 0.01 Right prefrontal cortex 18 40 20 10 4.42 648 0.01 Left prefrontal cortex Ϫ16 42 20 10 4.42 648 0.01 Right primary motor cortex 8 Ϫ2 76 4, 6 3.94 395 0.03 Left primary motor cortex Ϫ8 Ϫ2 76 4, 6 3.94 395 0.03 Right primary somatosensory cortex 55 Ϫ22 15 3, 1, 2 3.94 325 0.01 Left primary somatosensory cortex Ϫ51 Ϫ22 15 3, 1, 2 3.94 325 0.01 Left amygdala Ϫ20 Ϫ8 Ϫ14 – 4.36 226 0.01 Right insula/secondary somatosensory cortex 45 2 6 – 4.45 325 0.02 Left insula/secondary somatosensory cortex Ϫ35 23 6 – 4.32 325 0.02 Right inferior parietal cortex 54 Ϫ38 30 40 5.02 650 0.01 Left inferior parietal cortex Ϫ60 Ϫ45 28 40 5.02 650 0.01 Left thalamus Ϫ13 Ϫ14 Ϫ3 – 4.36 235 0.01 Right supplementary motor area 4 Ϫ2 50 6 3.26 235 0.03 Ͻ ϭ ϭ ϭ * For all brain areas reported, corrected P 0.05. MNI Montreal Neurological Institute; BA Brodmann’s area; KE cluster size. intensity rating was 4.0 Ϯ 10, and the mean Ϯ SD than those during the pain-free condition (P ϭ 0.00 [to unpleasantness rating was 3.6 Ϯ 6.4. These differences in 2 decimal places] by paired t-test for both comparisons). the mean intensity and unpleasantness ratings between Alterations in rCMRGlu. The reported results the arthritic and experimental pain conditions were not represent the pooled data for all 12 patients. However, 6 statistically significant (P ϭ 0.9 and P ϭ 0.08 by paired patients had predominant left knee arthritis, and the t-test, respectively). Both the mean intensity and the other 6 had predominant right knee arthritis. In the mean unpleasantness ratings during the arthritic and experimental pain condition, when patients were free experimental pain conditions were significantly higher from arthritic pain, the experimental pain stimulus was

Table 3. Brain areas that were significantly more active during experimental pain compared with the pain-free condition* MNI coordinates

Brain region xyz BA Z score KE P Right perigenual cingulate cortex 10 44 8 32 5.83 850 0.01 Left perigenual cingulate cortex Ϫ830Ϫ8 32 3.52 393 0.03 Right orbitofrontal cortex 4 55 Ϫ16 11 3.28 228 0.04 Left orbitofrontal cortex Ϫ150Ϫ16 11 3.33 393 0.01 Right prefrontal cortex 45 40 12 10 5.23 850 0.01 Left primary motor cortex Ϫ60 0 20 4, 6 5.32 850 0.01 Right primary motor cortex 34 Ϫ26 62 4, 6 5.23 383 0.01 Right primary somatosensory cortex 25 Ϫ28 60 3, 1, 2 4.26 1,054 0.01 Left secondary somatosensory cortex Ϫ40 Ϫ32 56 3, 1, 2 5.23 1,054 0.03 Right putamen 25 10 1 – 3.83 343 0.01 Left putamen Ϫ22 10 1 – 3.42 363 0.01 Right insula/secondary somatosensory cortex 4646– 5.85 1,250 0.01 Left insula/secondary somatosensory cortex Ϫ40 20 8 – 3.65 1,106 0.01 Right inferior parietal cortex 50 Ϫ28 20 40 5.83 850 0.01 Left inferior parietal cortex Ϫ60 Ϫ30 20 40 5.83 850 0.01 Left thalamus Ϫ16 Ϫ25 2 – 3.84 243 0.03 Left supplementary motor area Ϫ24 1 54 6 4.25 154 0.00 Ͻ ϭ ϭ ϭ * For all reported brain areas, corrected P 0.05. MNI Montreal Neurological Institute; BA Brodmann’s area; KE cluster size. PROCESSING OF ARTHRITIC PAIN AND EXPERIMENTAL PAIN IN OA 1349

Figure 1. Brain areas of increased activation in the arthritic pain (AP) condition and the experimental pain (EP) condition, both relative to the pain-free (PF) condition. The images show statistical parametric maps (SPMs) of each data comparison, with color coding reflecting the Z scores. The SPMs are displayed on the sagittal (top row), coronal (middle row), and axial (bottom row) sections of a standard Montreal Neurological Institute (MNI) template. The MNI coordinates (in millimeters) are indicated for the sagittal sections on the x-axis, the coronal sections on the y-axis, and the axial sections on the z-axis. The key areas for each comparison are labeled. For descriptive purposes only, the threshold for SPM images was uncorrected P Ͻ 0.01. PCC ϭ posterior cingulate cortex; aMCC ϭ anterior midcingulate cortex; pACC ϭ perigenual anterior cingulate cortex; SII ϭ secondary somatosensory cortex. applied to the skin of the arthritic knee. Therefore, no P Ͻ 0.05) within areas of the pain matrix (based on a inferences can be made from the pooled results for priori hypotheses) are reported. either the experimental pain or arthritic pain condition Arthritic pain versus pain-free condition. During regarding the laterality of any effects seen. However, this the arthritic pain condition (compared with the pain-free issue was addressed in further analyses comparing the 2 condition), rCMRGlu was enhanced in all areas of the subgroups of patients (see Patients and Methods). Those pain matrix. Most areas were activated bilaterally, in- analyses revealed no consistently lateralized activations cluding the posterior cingulate cortex (Brodmann’s area within any areas of the pain matrix. [BA] 31), anterior midcingulate cortex (BA 24/32), The pooled data for all 12 patients were sub- prefrontal cortex (BA 10), orbitofrontal cortex (BA 11), jected to 4 statistical comparisons to investigate signifi- inferior parietal cortex, as well as the primary motor cant differences between the arthritic pain and experi- cortex, insula/secondary somatosensory cortex, and pri- mental pain conditions, as described below and shown in mary somatosensory cortex (Table 2 and Figure 1, left). Tables 2 and 3 and Figures 1 and 2. The threshold for Unilateral activations were seen in the left thalamus activations was uncorrected P Ͻ 0.01 (extent thresh- (extending to the midline), left perigenual cingulate old ϭ 25 voxels). Only significant activations (corrected cortex (BA 32/24), left amygdala, and right supplemen- 1350 KULKARNI ET AL

Figure 2. Brain areas of increased activation in the arthritic pain versus the experimental pain condition and in the experimental pain condition versus the arthritic pain condition. The key areas for each comparison are labeled. SGC ϭ subgenual cingulate cortex (see Figure 1 for other definitions). tary motor area (SMA). Activations in these areas were of several areas of the pain matrix and associated limbic significant at P Ͻ 0.05 (corrected for multiple compari- and motor areas. Bilateral activations were seen in the sons). perigenual cingulate (BA 32/24), anterior midcingulate Experimental pain versus pain-free condition. (BA 24b), posterior midcingulate (BA 23b), subgenual The experience of experimental heat pain, compared cingulate (BA 25) and posterior cingulate (BA 31), the with the pain-free condition, was associated with in- amygdala, and the orbitofrontal cortex (Table 4 and creased brain activation in most areas of the pain matrix, Figure 2, left). Left-sided activations were seen in the including the bilateral perigenual cingulate cortex, posterior insula/secondary somatosensory cortex, pre- insula/secondary somatosensory cortex, primary so- frontal cortex, inferior parietal cortex, and the thalamus, matosensory cortex, primary motor cortex, orbitofrontal whereas the SMA, primary motor cortex, caudate nu- cortex, inferior parietal cortex, putamen and left thala- cleus, and putamen were activated only in the right mus, SMA, and right prefrontal cortex (P Ͻ 0.01) (Table hemisphere. All of these areas were significant at P Ͻ 3 and Figure 1, right). Activations in the right-sided 0.01. The most statistically significant activations were in regions were generally more extensive than those in the the anterior cingulate cortex, where the cluster extended left hemisphere. from the anterior midcingulate to the posterior midcin- Arthritic pain versus experimental pain. When gulate. compared with experimental knee pain, arthritic pain in The correlation analysis with the unpleasantness the same knee was associated with increased activation ratings during this comparison also demonstrated in- PROCESSING OF ARTHRITIC PAIN AND EXPERIMENTAL PAIN IN OA 1351

Table 4. Brain areas that were significantly more active during arthritic pain compared with experimental pain* MNI coordinates

Brain region xyzBA Z score KE P Right posterior cingulate cortex 6 Ϫ36 40 31 5.25 543 0.01 Left posterior cingulate cortex Ϫ10 Ϫ36 36 31 5.02 543 0.01 Right anterior midcingulate perigenual anterior cingulate cortex 1 40 12 32/24 5.34 1,054 0.01 Left anterior midcingulate perigenual anterior cingulate cortex Ϫ2 40 5 32/24 5.45 1,054 0.01 Right posterior midcingulate cortex 2 Ϫ2 38 24 3.65 245 0.01 Left posterior midcingulate cortex Ϫ4 Ϫ2 38 24 4.95 343 0.03 Right subgenual cingulate cortex 8 10 Ϫ16 25 5.02 245 0.01 Left subgenual cingulate cortex Ϫ910Ϫ16 25 5.02 245 0.01 Right orbitofrontal cortex 6 28 Ϫ27 11 5.25 645 0.01 Left orbitofrontal cortex Ϫ816Ϫ25 11 5.02 645 0.01 Left prefrontal cortex Ϫ45 Ϫ45 2 10 5.45 845 0.01 Right primary motor area 32 Ϫ14 47 4, 6 5.25 543 0.02 Right amygdala 26 Ϫ2 Ϫ21 – 5.25 645 0.01 Left amygdala Ϫ20 Ϫ6 Ϫ18 – 5.25 645 0.01 Left insula/secondary somatosensory cortex Ϫ40 Ϫ23 12 – 4.02 645 0.03 Left inferior parietal cortex Ϫ50 Ϫ32 40 40 4.95 543 0.01 Right putamen 27 Ϫ11 Ϫ6 – 3.65 245 0.03 Left thalamus Ϫ20 Ϫ13 1 – 3.65 245 0.04 Right caudate nucleus 20 Ϫ12 21 – 3.85 245 0.04 Right supplementary motor area 32 0 50 6 3.45 245 0.04 Ͻ ϭ ϭ ϭ * For all reported brain areas, corrected P 0.01. MNI Montreal Neurological Institute; BA Brodmann’s area; KE cluster size. creased activity in the bilateral subgenual (BA 25) and DISCUSSION perigenual cingulate (BA 32) cortices, bilateral posterior The results of this study show that both the medial cingulate cortices (BA 31), amygdalae, and left-sided and lateral pain systems are activated during arthritic and insula. Experimental pain versus arthritic pain. Exper- experimental pain, but that the medial system is more imental pain, compared with arthritic pain, was associ- active during arthritic pain. To our knowledge, this study is the first to document the cerebral substrates of the percep- ated with bilateral increases in rCMRGlu in the posterior/inferior parietal cortices, primary motor cor- tion of arthritic pain. It is also the first study to directly tex, and SMA and with right-sided increases in the compare experimental pain with ongoing clinical pain in insula/secondary somatosensory cortex (extending from the same group of patients at the same level of perceived the anterior insula to the posterior insula), primary pain intensity. The finding that both experimental pain and somatosensory cortex, subgenual cingulate (BA 25), and arthritic pain activate the medial and lateral pain systems orbitofrontal cortex. All of these areas were significant suggests that there is no unique brain network for process- at P Ͻ 0.01 (Table 5 and Figure 2, right). ing arthritic pain.

Table 5. Brain areas that were significantly more active during experimental pain compared with arthritic pain* MNI coordinates

Brain region xyzBA Z score KE P Right subgenual cingulate 5 20 Ϫ11 25 3.35 343 0.03 Right orbitofrontal cortex 6 64 Ϫ13 11 4.28 546 0.01 Right primary motor cortex 50 Ϫ7 55 4, 6 3.65 565 0.03 Left primary motor cortex Ϫ48 Ϫ13 55 4, 6 3.45 484 0.03 Right primary somatosensory cortex 50 Ϫ23 55 3, 1, 2 3.82 343 0.02 Right insula/secondary somatosensory cortex 46 5 Ϫ6 – 4.85 865 0.01 Right inferior parietal cortex 50 Ϫ40 55 40 3.65 546 0.02 Left inferior parietal cortex Ϫ46 Ϫ45 40 40 3.48 485 0.02 Right supplementary motor area 4 14 50 6 3.45 243 0.04 Left supplementary motor area Ϫ5 0 50 6 5.14 485 0.00 Ͻ ϭ ϭ ϭ * For all reported brain areas, corrected P 0.01. MNI Montreal Neurological Institute; BA Brodmann’s area; KE cluster size. 1352 KULKARNI ET AL

The present study confirms that we are justified arthritic pain were accompanied by greater activity in in using experimental pain in humans as a tool for the amygdala, orbitofrontal cortex, and putamen. These investigating some of the generalized mechanisms of areas are associated with aversive conditioning, reward, pain perception. However, it also demonstrates the and fear (32–36), suggesting that processing the fear of possibility of using FDG-PET to measure subtle changes further injury and disability has a possible role in in brain activity in patients experiencing different types arthritic pain. This circuitry is less commonly revealed of clinical pain, as has been achieved for mood disorders during experimental pain (2,3,5,37) and therefore may (24). Because arthritic pain is rarely continuous and be important in the development of pain chronicity. generally is characterized by recurrent acute pain, it is In the present study, arthritic pain (compared particularly suited to this experimental approach. with experimental pain) was also associated with in- The present study showed that arthritic pain, creased activation of the prefrontal cortex and the inferior posterior parietal cortex. Coactivation of these when compared with experimental pain, was associated areas is consistent with the finding of dense intercon- with increased activity within the medial pain system of nections between them and their projections to common the brain, including most of the cingulate, insula/ cortical and subcortical areas (38). Evidence suggests a secondary somatosensory cortex, and thalamus. It has role of these regions in the supervision of attention (39). been previously shown that activity within the medial Therefore, their activation during the experience of pain system and related structures is increased during arthritic pain may reflect the recruitment of appropriate selective attention to the unpleasantness of an experi- coping strategies in these patients (40). This is also mental pain stimulus (8,25). Therefore, the present consistent with reduced responses to painful experimen- findings are consistent with the idea that, for these tal stimuli within BA 10 of the dorsolateral prefrontal patients, arthritic pain has more emotional salience than cortex in patients with chronic regional pain and depres- does experimental pain. This is also consistent with the sion, who tend to have poor coping strategies (11). trend toward higher scores for the unpleasantness of The greater activation of the primary motor arthritic pain compared with the unpleasantness of cortex that was observed in the present study during experimental pain observed in this study, although the arthritic pain compared with experimental pain, even in difference was not significant. Although both mood and the absence of actual movement, may be attributable to pain are processed within the orbitofrontal, perigenual, an increased motivational response or the inhibition of a and midcingulate cortices (25,26), patients’ mood states desire to move the painful knee during arthritic pain. did not differ between scans, and therefore, the changes Increased activation of the primary motor cortex was in brain activation observed are likely to be specific to also seen in our previous study, when healthy volunteers changes in the pain state. selectively attended to the unpleasantness of acute ex- During arthritic pain, activation of the cingulate perimental pain stimuli in the absence of actual move- cortex (including the perigenual, midcingulate, and pos- ment (25). The close association between motivational terior cingulate cortices) was much more extensive. The and affective responses, particularly within the midcin- greater perigenual and midcingulate activity is consis- gulate cortex (5), is likely to explain the more extensive tent with increased affective processing (5,7,25). The activations within this cortical area during arthritic pain posterior cingulate cortex is traditionally thought to be compared with experimental pain. involved in visuospatial processing (27), although there The present study demonstrates the importance is increasing evidence for a role in processing affect and of the medial pain system during the experience of pain memory (28,29). This area has been shown to have arthritic pain and suggests that it is a likely target for increased blood flow during ongoing neuropathic pain both pharmacologic and nonpharmacologic interven- (compared with a pain-free condition) following re- tions. The endogenous opioid system provides one pos- gional nerve block in patients with neuropathic pain sible candidate for modulation (41,42). High concentra- (30), supporting a possible role in the affective process- tions of opiate receptors have been identified in the ing of pain. The adjacent retrosplenial cortex (BAs 29 medial pain system compared with the concentrations in and 30) has been shown to be activated in response to the lateral pain system (43). Changes in the occupation unpleasant stimuli such as fearful and sad words and of opioid receptors, reflecting their occupation by en- images (31). Activity in this region might therefore be dogenous opioid peptides such as enkephalins, have also related to the emotional salience during arthritic pain. been demonstrated during chronic arthritic pain and Increased activations in the cingulate cortex during neuropathic pain (44,45). The identification of brain- PROCESSING OF ARTHRITIC PAIN AND EXPERIMENTAL PAIN IN OA 1353

specific inhibitors of enkephalin breakdown (46) raises AUTHOR CONTRIBUTIONS the possibility of enhancing such endogenous responses Dr. Kulkarni had full access to all of the data in the study and without the gastrointestinal side effects associated with takes responsibility for the integrity of the data and the accuracy of the data analysis. most of the currently available analgesics. Considering Study design. Kulkarni, Bentley, Elliott, Julyan, Watson, Jones. the recent concerns about the long-term safety of cyclo- Acquisition of data. Kulkarni, Julyan, Boger, Watson, Boyle. oxygenase inhibitors (47), we hope that our current Analysis and interpretation of data. Kulkarni, Elliott, Julyan, El- Deredy, Jones. findings will stimulate partnerships between academia Manuscript preparation. Kulkarni, Bentley, Elliott, Julyan, El-Deredy, and the pharmaceutical industry to develop a new class Jones. of analgesics for arthritic pain that specifically target the Statistical analysis. Kulkarni, Elliott. medial pain system. The main limitation of the present study is the REFERENCES small number of subjects. Previous functional imaging 1. Bowsher D. Termination of the central pain pathway in man: the studies in patients and healthy volunteers have been conscious appreciation of pain. Brain 1957;80:606–22. performed in as few as 6 subjects and have reproducibly 2. Jones AK, Kulkarni B, Derbyshire SW. Pain mechanisms and their demonstrated the cortical areas involved in nociceptive disorders [review]. Br Med Bull 2003;65:83–93. 3. Peyron R, Laurent B, Garcia-Larrea L. Functional imaging of processing. Larger studies need to be performed, partic- brain responses to pain: a review and meta-analysis [review]. ularly to look at further details of nociceptive processing Neurophysiol Clin 2000;30:263–88. with variables such as depression, anxiety, and coping 4. Melzack R, Casey KL. Sensory, motivational and central control determinants of pain: a new conceptual model. In: Kenshalo DR, strategies, which can affect chronic pain conditions. editor. The skin senses. Springfield (IL): Charles C. Thomas; 1968. One of the possible criticisms of the study is that p. 423–43. we are comparing 2 different types of pain stimulations, 5. Vogt BA. Pain and emotion interactions in subregions of the cingulate gyrus [review]. Nat Rev Neurosci 2005;6:533–44. such as heat pain and arthritic pain. Most of the 6. Craig AD. Pain mechanisms: labeled lines versus convergence in evidence for cortical nociceptive processing comes from central processing [review]. Annu Rev Neurosci 2003;26:1–30. studies that have used experimentally induced heat pain 7. Rainville P, Duncan GH, Price DD, Carrier B, Bushnell MC. Pain affect encoded in human anterior cingulate but not somatosensory (3,7,11,12,25,48). Those studies reproducibly activated cortex. Science 1997;277:968–71. the structures of the pain matrix and demonstrated a 8. Tolle TR, Kaufmann T, Siessmeier T, Lautenbacher S, Berthele A, division of function within this pain matrix. This suggests Munz F, et al. Region-specific encoding of sensory and affective that heat pain is sensitive enough to activate both the components of pain in the human brain: a positron emission tomography correlation analysis. Ann Neurol 1999;45:40–7. medial and lateral pain systems. Thus far, however, there 9. Giesecke T, Gracely RH, Grant MA, Nachemson A, Petzke F, has not been a direct comparison between clinical pain Williams DA, et al. Evidence of augmented central pain process- and artificially induced pain in the same group of ing in idiopathic chronic low back pain. Arthritis Rheum 2004;50: 613–23. individuals. Our studies aimed to examine possible 10. Derbyshire SW, Jones AK, Creed F, Starz T, Meltzer CC, differences in cortical processing between artificial pain Townsend DW, et al. Cerebral responses to noxious thermal and clinical pain, and there is no other way to compare stimulation in chronic low back pain patients and normal controls. Neuroimage 2002;16:158–68. them directly. 11. Derbyshire SW, Jones AK, Devani P, Friston KJ, Feinmann C, In summary, the present study demonstrates that Harris M, et al. Cerebral responses to pain in patients with atypical although both experimental pain and arthritic pain facial pain measured by positron emission tomography. J Neurol predominantly activate the same areas of the brain Neurosurg Psychiatry 1994;57:1166–72. 12. Jones AK, Derbyshire SW. Reduced cortical responses to noxious (confirming the relevance of experimental pain studies), heat in patients with rheumatoid arthritis. Ann Rheum Dis arthritic pain is also associated with increased activity in 1997;56:601–7. areas of the brain that play a role in affect, aversive 13. Phelps ME. Positron computed tomography studies of cerebral glucose metabolism in man: theory and application in nuclear conditioning, and motivational responses. This suggests medicine. Semin Nucl Med 1981;11:32–49. that researchers should be moving toward more natural- 14. Ginsberg MD, Chang JY, Kelley RE, Yoshii F, Barker WW, istic studies in patients, in order to fully understand the Ingenito G, et al. Increases in both cerebral glucose utilization and blood flow during execution of a somatosensory task. Ann Neurol perception of different types of clinical pain. 1988;23:152–60. 15. Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of criteria for the classification and reporting of ACKNOWLEDGMENTS osteoarthritis: classification of osteoarthritis of the knee. Arthritis Rheum 1986;29:1039–49. We are grateful to all of the radiographers and tech- 16. Fruhstorfer H, Lindblom U, Schmidt WC. Method for quantitative nicians at the Manchester PET Centre, North Western Med- estimation of thermal thresholds in patients. J Neurol Neurosurg ical Physics, Christie Hospitals NHS Trust, Manchester. Psychiatry 1976;39:1071–5. 1354 KULKARNI ET AL

17. Zigmond AS, Snaith RP. The Hospital Anxiety and Depression pocampal involvement in human aversive trace conditioning re- Scale. Acta Psychiatr Scand 1983;67:361–70. vealed through event-related functional magnetic resonance imag- 18. Radloff LS. The CES-D Scale: a self report depression scale for ing. J Neurosci 1999;19:10869–76. research in the general population. Appl Psychol Meas 1977;1: 35. Chudler EH, Dong WK. The role of the basal ganglia in nocicep- 385–401. tion and pain [review]. Pain 1995;60:3–38. 19. Spielberger CD. Assessment of state and trait anxiety. South 36. Anderson AK, Phelps EA. Lesions of the human amygdala impair Psychologist 1985;1:6–16. enhanced perception of emotionally salient events. Nature 2001; 20. Sullivan M, Bishop S, Pivik J. The Pain Catastrophizing Scale: 411:305–9. development and validation. Psychol Assess 1995;7:524–32. 37. Petrovic P, Carlsson K, Petersson KM, Hansson P, Ingvar M. 21. Gispert JD, Pascau J, Reig S, Martinez-Lazaro R, Molina V, Context-dependent deactivation of the amygdala during pain. J Garcia-Barreno P, et al. Influence of the normalization template Cogn Neurosci 2004;16:1289–301. on the outcome of statistical parametric mapping of PET scans. 38. Kupfermann I. Localisation of higher cognitive and affective Neuroimage 2003;19:601–12. functions: the association cortices. In: Kandel ER, Schwartz JH, 22. Gispert JD, Pascau J, Reig S, Garcia-Barreno P, Desco M. Jessell TM, editors. Principles of neural science. Amsterdam: Statistical parametric mapping (SPM) in nuclear medicine. Rev Elsevier; 1991. p. 823–38. Esp Med Nucl 2003;22:43–53. In Spanish. 39. Posner MI, Petersen SE. The attention system of the human brain 23. Talairach J, Tournoux P. Co-planar stereotaxic atlas of the human [review]. Annu Rev Neurosci 1990;13:25–42. brain. New York: Georg Thieme Verlag; 1998. 40. Lorenz J, Minoshima S, Casey KL. Keeping pain out of mind: the 24. Mayberg HS, Liotti M, Brannan SK, McGinnis S, Mahurin RK, role of the dorsolateral prefrontal cortex in pain modulation. Brain Jerabek PA, et al. Reciprocal limbic-cortical function and negative 2003;126(Pt 5):1079–91. mood: converging PET findings in depression and normal sadness. 41. Jones AK, Watabe H, Cunningham VJ, Jones T. Cerebral de- Am J Psychiatry 1999;156:675–82. creases in opioid receptor binding in patients with central neuro- 25. Kulkarni B, Bentley DE, Elliott R, Youell P, Watson A, Derby- pathic pain measured by 11C diprenorphine binding in PET. Eur J shire SW, et al. Attention to pain localization and unpleasantness Pain 2004;8:479–85. discriminates the functions of the medial and lateral pain systems. 42. Jones A, Pike V, Luthra S, Turton D, Brady F, Jones T, et al. Eur J Neurosci 2005;21:3133–42. 11C-diprenorphine and 11C-meptazinol: new specific and non- 26. Mayberg HS. Modulating limbic-cortical circuits in depression: specific probes for studying opioid binding sites using positron targets of antidepressant treatments [review]. Semin Clin Neuro- tomography. J Cereb Blood Flow Metab 1985;5(S1):603–4. psychiatry 2002;7:255–68. 43. Jones AK, Qi LY, Fujirawa T, Luthra SK, Ashburner J, Bloom- 27. Bentley DE, Derbyshire SW, Youell PD, Jones AK. Caudal field P, et al. In vivo distribution of opioid receptors in man in cingulate cortex involvement in pain processing: an inter-individ- relation to the cortical projections of the medial and lateral pain ual laser evoked potential source localisation study using realistic systems measured with positron emission tomography. Neurosci head models. Pain 2003;102:265–71. Lett 1991;126:25–8. 28. Maddock RJ, Garrett AS, Buonocore MH. Remembering familiar 44. Jones AK, Cunningham VJ, Ha-Kawa S, Fujiwara T, Luthra SK, people: the posterior cingulate cortex and autobiographical mem- Silva S, et al. Changes in central opioid receptor binding in relation ory retrieval. Neuroscience 2001;104:667–76. to inflammation and pain in patients with rheumatoid arthritis. 29. Maddock RJ, Garrett AS, Buonocore MH. Posterior cingulate Br J Rheumatol 1994;33:909–16. cortex activation by emotional words: fMRI evidence from a 45. Jones AK, Kitchen ND, Watabe H, Cunningham VJ, Jones T, valence decision task. Hum Brain Mapp 2003;18:30–41. Luthra SK, et al. Measurement of changes in opioid receptor 30. Hsieh JC, Belfrage M, Stone-Elander S, Hansson P, Ingvar M. binding in vivo during trigeminal neuralgic pain using 11Cdi- Central representation of chronic ongoing neuropathic pain stud- prenorphine and positron emission tomography. J Cereb Blood ied by positron emission tomography. Pain 1995;63:225–36. Flow Metab 1999;19:803–8. 31. Maddock RJ. The retrosplenial cortex and emotion: new insights 46. Le Guen S, Mas NM, Canestrelli C, Chen H, Fournie-Zaluski MC, from functional neuroimaging of the human brain [review]. Trends Cupo A, et al. Pain management by a new series of dual inhibitors Neurosci 1999;22:310–6. of enkephalin degrading enzymes: long lasting antinociceptive 32. Bohus B, Koolhaas JM, Luiten PG, Korte SM, Roozendaal B, properties and potentiation by CCK2 antagonist or methadone. Wiersma A. The neurobiology of the central nucleus of the Pain 2003;104:139–48. amygdala in relation to neuroendocrine and autonomic outflow. 47. Hippisley-Cox J, Coupland C. Risk of myocardial infarction in Prog Brain Res 1996;107:447–60. patients taking cyclo-oxygenase-2 inhibitors or conventional non- 33. Bornhovd K, Quante M, Glauche V, Bromm B, Weiller C, Buchel steroidal anti-inflammatory drugs: population based nested case- C. Painful stimuli evoke different stimulus-response functions in control analysis. BMJ 2005;330:1366. the amygdala, prefrontal, insula and somatosensory cortex: a 48. Derbyshire SW, Jones AK. Cerebral responses to a continual tonic single-trial fMRI study. Brain 2002;125(Pt 6):1326–36. pain stimulus measured using positron emission tomography. Pain 34. Buchel C, Dolan RJ, Armony JL, Friston KJ. Amygdala-hip- 1998;76:127–35. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1355–1364 DOI 10.1002/art.22513 © 2007, American College of Rheumatology

Risk Factors for More Severe Regional Musculoskeletal Symptoms

A Two-Year Prospective Study of a General Working Population

Johan H. Andersen,1 Jens P. Haahr,1 and Poul Frost2

Objective. To quantify the relative contribution of high body mass index was highly associated with lower work-related physical and psychosocial factors, individ- limb pain. ual factors, and health-related factors to the develop- Conclusion. Very few workers are totally free of ment of more severe musculoskeletal pain in the neck pain in musculoskeletal regions, and we question the and upper limbs and the back and lower limbs. concept of incidence of musculoskeletal pain. The tran- Methods. In this cohort study of 5,604 workers sition from no or minor pain to more severe pain was from industrial and service companies, we collected influenced by physical and psychosocial work place information on work-related physical and psychosocial factors together with individual and health-related fac- exposures and on individual and health-related factors. tors. Questionnaires were completed at baseline by 4,006 participants (71.5%) and after 24 months by 3,276 Regional musculoskeletal pain is common in (82%). At followup, participants with no or minor pain working populations and in the general population were included in Cox regression analyses to determine (1–4). Within the last 30 years, much effort has been put which factors predicted more severe regional pain. into research to identify risk factors for primary preven- Results. Of the 4,006 baseline respondents, only tion, first by focusing on physical hazards in the work 7.7% were free of regional pain. A total of 1,513 partic- place (e.g., heavy lifting, awkward postures, repetitive ipants were free of severe pain at baseline and com- movements) and second by also introducing psychoso- pleted the 24-month followup. Highly repetitive work cial work place factors (e.g., job demands, job control, predicted arm pain, heavy lifting and prolonged stand- social support, and job satisfaction). Many risk factors ing predicted low back pain, and heavy pushing or have been identified in various studies, but no great pulling predicted lower limb pain. Low job satisfaction success has been seen in intervention studies (5,6). It predicted neck/shoulder pain and lower limb pain, looks as if regional musculoskeletal pain problems are whereas other psychosocial work place factors were only here to stay, and are perhaps a ubiquitous part of of marginal importance. High levels of fear avoidance modern working life (7,8). were associated with arm pain and lower limb pain. A It has recently been proposed in a European guideline on back pain that the general nature and course of commonly experienced low back pain means Supported by grants from the Danish Working Environment that there is limited scope for preventing its incidence, Authority. 1Johan H. Andersen, PhD, Jens P. Haahr, MD: Herning and that prevention should be focused on reduction of Hospital, Herning, Denmark; 2Poul Frost, PhD: Herning Hospital, the impact and consequences of back pain (ref. 9 and Herning, Denmark, and Aarhus University Hospital, Aarhus, Den- www.backpaineurope.org). This message could possibly mark. Address correspondence and reprint requests to Johan H. be extended to other regional pain, such as arm pain, Andersen, PhD, Department of Occupational Medicine, Herning neck/shoulder pain, and lower limb pain. Hospital, Gammel Landevej 61, DK-7400 Herning, Denmark. E-mail: The aim of the present study was to examine the [email protected]. Submitted for publication July 6, 2006; accepted in revised effect of work-related factors and individual and health- form January 4, 2007. related factors on the onset of more severe musculoskel-

1355 1356 ANDERSEN ET AL

etal pain at 24 months of followup among participants for Ͻ10 minutes per hour, a medium repetition group per- with no or minor pain at baseline in 4 regions (the forming such work for 10–44 minutes per hour, and a high Ͼ neck/shoulders; the elbow, forearm, and hand; the low repetition group performing such work for 45 minutes per hour. Posture variables were dichotomized into sitting for Ͼ30 back; the hip, knee, and foot) as well as in any of the 4 minutes per hour or not, standing for Ͼ30 minutes per hour or regions. not, and squatting for Ͼ5 minutes per hour or not. Psychosocial risk factors. Psychosocial risk factors were assessed using a standardized questionnaire developed by SUBJECTS AND METHODS the Danish National Institute of Occupational Health (13,14). The questionnaire included 10 items about job demands (4 on Study design. This was a prospective cohort study of a work load, 3 on sensory demands, and 3 on cognitive de- general working population in western Denmark. At baseline mands), 7 items about job control (4 on decision latitude and and at 24 months, subjects completed a questionnaire, which 3 on freedom at work), and 9 items about social support (4 on ascertained regional pain status, work-related physical and social support from supervisors and 5 on social support from psychosocial factors, and health-related factors. colleagues). The questionnaire items had 5 alternative re- Study subjects. The study population consisted of sponses for each question (always, often, sometimes, seldom, workers from 39 different work places, of which 19 were in the and never/almost never); these responses were dichotomized service sector and 20 were in different kinds of industry. Work and given a raw score of 1 or 0. Four scales were constructed places were recruited through the 3 local occupational health as raw score summations for job demands, job control, and services with the aim of composing a study population of social support from supervisors or colleagues. High scale approximately equal proportions of workers from the service values indicated a high level of job demands, a low level of job sector and workers from industrial companies. The purpose control, and low levels of social support. We also included was to include a general working population with the intention single-item questions on quality of leadership (“All in all, are of subsequently performing a randomized controlled study of you satisfied with the way your workplace is managed?”; the effect of information and ergonomic interventions versus responses on a 6-point Likert scale ranged from “Hardly at all” no intervention. This subsequent part of the study will be to “To a very great extent”) and job satisfaction (“How reported separately. satisfied are you with your work, all in all?”; responses on a Baseline information. A self-administered question- 6-point Likert scale ranged from “Very dissatisfied” to “Very naire was mailed to all the workers on the basis of information satisfied”). received from the companies. The questionnaire included Individual and health-related risk factors. Education information across the following domains: physical risk factors level was categorized as 1 of 3 groups: low (7–9 years of school in the work place, psychosocial factors at work, health-related education), medium (10 years), and high (college education factors, and other individual factors. The occupations of the and higher). Leisure time physical activity was categorized as participants were based on information received from the work low (almost none or light activity for Ͻ2 hours/week or light places and were classified according to International Standard activity for 2–4 hours/week) and high (light activity Ͼ4 hours/ Classification of Occupations (ISCO-88), developed by the week or 2–4 hours of hard physical activity or hard physical International Labour Organisation. activity for Ͼ4 hours/week). On the basis of self reports of Physical risk factors. The participants were asked height and weight, the body mass index (BMI) was categorized about several manual handling activities, posture, and repeti- as low or normal (index Ͻ25), overweight (index 25–29), or tive movements. These included lifting with the hands, lifting severely overweight (index Ն30). weight at or above shoulder height, pulling or pushing weights, Fear avoidance was assessed by the Fear-Avoidance squatting, standing, and repetitive movements with the hands. Beliefs Questionnaire (FABQ) (15). On the basis of factor Participants were asked to estimate the weights they had lifted, analysis, 2 scales were used, one for fear avoidance of work how many lifts per hour, and the duration of each task. (Cronbach’s ␣ ϭ 0.86) and the other for fear avoidance of Postures and repetitive movements were assessed by questions physical activity (Cronbach’s ␣ ϭ 0.84). Cut points for low, about the amount of time spent in these postures and perform- medium, and high levels of fear avoidance were made according these repetitive movements in a typical work hour during ing to quartiles. the last day of work. These questions have been validated by Health anxiety was assessed by the 7-item version of comparing the responses according to a direct observation the Whiteley Index (16). The responses were dichotomized as technique and have been used in studies of different outcomes a little bit and quite a bit, and were summed to produce a scale (10–12). with 3 categories (Cronbach’s ␣ ϭ 0.83). From questions about The physical exposure variables were categorized as persistent diseases lasting more than 6 months (diabetes, follows. For lifting with the hands, lifting at or above shoulder depression, migraine, hypertension, chronic bronchitis, rheu- level, and pushing or pulling, we combined the questions on matic diseases, and other somatic diseases), a variable was weight with the number of activities per hour to form a created for having a disease or not. cumulative exposure measure, where the referent group con- Pain status assessment. Regional pain status was sisted of participants who did not perform these activities. The assessed for each of the 4 regions by asking, “How much have remaining participants were dichotomized according to the you been bothered by pain during the past 12 months?” There distribution level to obtain contrasts between groups. Repeti- were 7 categories of answers, which are shown here with their tive movements were divided into a reference group perform- percentages of distribution at baseline: not at all (7.7%), very ing repetitive work (repetitive movements of the hand or arm) little (16.7%), little (22.3%), some (20.0%), quite a lot RISK FACTORS FOR SEVERE REGIONAL MUSCULOSKELETAL SYMPTOMS 1357

Figure 1. Distribution of the study participants with onset of severe pain over the 2-year followup. Participants were asked to complete a questionnaire at baseline and at 24 months (followup 1). Intervention group 1 represents controls, group 2 received information, and group 3 received information and a work place visit.

(17.5%), much (9.8%), and very much (6.1%) (17,18). More bothered not at all, very little, and little according to the severe pain was operationalized as some pain to very much 7-point Likert scale used for pain assessment. Pain status at 24 pain, and none or minor pain was operationalized as not at all, months was dichotomized at the same level as at baseline, as very little, and little pain. more severe pain (from some to very much pain) versus no or Followup assessment. All subjects who answered the minor pain. baseline questionnaire received a questionnaire at 24 months The analysis was performed for each of the 4 regions as in followup. Up to 2 reminders were mailed to the participants. well as for any of the 4 regions. First, partially adjusted HRs Pain status and other health status, including fear-avoidance were obtained for each potential risk factor, adjusting for age, beliefs, were assessed in the same way as at baseline. The sex, occupational group, and intervention group for the body participants were also asked about job status at followup, if region in question. Second, a multivariate model within each they had changed jobs, and if so, whether this change was domain was constructed, and factors with a P value of less than related to musculoskeletal pain. 0.10 were included in the final multivariate model. When there Absence because of sickness. Episodes of absences were high correlations between variables, the one with the because of sickness for Ͼ2 weeks were collected from the highest point estimate in the partially adjusted model was used. DREAM register in the Danish Ministry of Labor, where all We tested for interaction between all risk factors if they were sickness absences for Ͼ2 weeks are centrally collected. The considered to be plausible (e.g., interaction between job de- DREAM database includes information on all public transfer mands, job control, and social support), but no interaction payments administered by Danish ministries and municipali- term contributed to the final model (e.g., with a P value of less ties and Statistics Denmark for all Danish citizens on a weekly than 0.10). basis since 1991. The data for our cohort were merged with the All analyses were performed with the Stata statistical DREAM register data in order to analyze sickness absences package (version 9.0; Stata, College Station, TX). among respondents and nonrespondents. Statistical analysis. The analysis of risk factors for more severe pain at 24 months of followup was conducted with RESULTS Cox proportional hazards analysis, yielding hazard ratios (HRs) with 95% confidence intervals (95% CIs). The analysis A flow chart showing the distribution of the was conducted among those who had no or minor pain at participants, from the 5,604 eligible participants to the baseline, which were those indicating that they had been 4,006 who answered the baseline questionnaire, and to 1358 ANDERSEN ET AL

Table 1. Study participation and prevalence rates of severe musculoskeletal pain in the 4 assessment regions at baseline and at the 24-month followup* Baseline 24-month followup

No. of Neck/ Elbow, Neck/ Elbow, eligible Response shoulder forearm, Low back Hip, knee, Response shoulder forearm, Low back Hip, knee, Study group subjects rate pain hand pain pain foot pain rate pain hand pain pain foot pain Production Administrative work 676 72 32 12 16 12 82 22 11 15 13 Skilled workers 141 75 22 17 17 12 79 16 8 19 17 Unskilled workers 1,874 67 38 22 25 22 76 32 21 27 24 Service Administrative 582 80 43 17 26 14 83 31 16 26 19 Nurses 825 76 29 11 22 14 84 23 9 21 13 Nurse assistants 1,042 71 44 21 31 23 87 34 20 30 26 Cleaning and kitchen 303 67 49 28 29 27 83 37 23 33 23 Technical staff 161 78 32 13 23 17 86 35 24 30 35 Total 5,604 72 37 18 25 18 82 30 17 25 21 * Except where indicated otherwise, values are percentages. the 3,276 who answered the followup questionnaire at 24 in the service sector (24%). Low back pain ranged from months, is shown in Figure 1. The 1,513 participants who 15% among administrative workers in industry to 33% were free of severe pain at baseline and completed the among cleaning and kitchen workers and 30% among followup were the main group used for further analysis nurse assistants. Lower limb pain was highest among in this study. As shown in Figure 1, there was no cleaning and technical staff in the service sector (35%) selection based on the intervention group or on pain and among the nurse assistants (26%). The differences from baseline to the 24-month followup assessment. between occupational groups were significant (at the Table 1 shows the distribution of participants and 0.05 level) for all 4 assessment regions. the prevalence rates of severe pain in the 4 assessment Partially adjusted associations. Table 2 shows regions at baseline and at 24 months. At baseline, the the associations between physical risk factors and the prevalence rates of regional pain varied among the onset of more severe regional pain, adjusted for sex, age, different occupational groups, where skilled workers in occupational group, and intervention group. Highly re- industrial work places had the lowest rate of neck/ petitive work was significantly associated with neck/ shoulder pain (22%), and were at the lowest end on the shoulder pain (HR 1.5 [95% CI 1.0–2.1]), arm pain (HR other 3 outcomes, whereas cleaning and kitchen workers 1.9 [95% CI 1.2–3.1]), low back pain (HR 1.7 [95% CI had high levels of neck/shoulder pain (49%), arm pain 1.2–2.6]), and any pain (HR 1.4 [95% CI 1.1–1.8]). (28%), low back pain (29%), and lower leg pain (27%). Lifting and pushing/pulling was associated in an Nurse assistants had the highest level of low back pain exposure-response pattern with all 5 outcomes, with (31%). The 4 regional pain outcomes were highly cor- 30–60% elevated risk in those with medium exposure related with each other: r ϭ 0.45 for the correlation and 50–100% elevated risk in those with the highest between neck/shoulder pain and arm pain; r ϭ 0.42 for exposure. Lifting Ն50 kg per hour at or above shoulder neck/shoulder pain and low back pain; and r ϭ 0.38 for level was associated with neck/shoulder pain (HR 2.1 low back pain and lower leg pain. [95% CI 1.3–3.5]), arm pain (HR 2.2 [95% CI 1.1–4.3]), At followup, the response rates ranged from 76% lower limb pain (HR 2.0 [95% CI 1.1–3.5]), and any among unskilled industrial workers to 87% among nurse regional pain (HR 1.6 [95% CI 1.1–2.3]). Sitting for Ͼ30 assistants (Table 1). The transition of no or minor pain minutes per hour was not associated with any of the to some or more severe pain showed that more severe outcomes, whereas standing for Ͼ30 minutes per hour pain in the neck/shoulder region occurred in 16% was associated with all outcomes (risk estimates elevated (skilled industrial workers) to 37% (cleaning and kitchen from 1.7 to 2.1). Squatting for Ͼ5 minutes per hour was workers) of subjects at 24 months of followup. Arm pain associated with lower leg pain (HR 1.6 [95% CI 1.1–2.3]) was lowest among skilled industrial workers (8%), and and neck/shoulder pain (HR 1.6 [95% CI 1.1–2.2]) and highest among unskilled industrial workers (21%), was marginally associated with low back pain and any cleaning and kitchen workers (23%), and technical staff regional pain. RISK FACTORS FOR SEVERE REGIONAL MUSCULOSKELETAL SYMPTOMS 1359

Table 2. Work-related physical risk factors and new onset of severe regional musculoskeletal pain, partially adjusted* Hazard ratio (95% confidence interval)

Neck/ Elbow, Any No. of shoulder forearm, Low back Hip, knee, regional Physical risk factor subjects pain hand pain pain foot pain pain Repetitive work 0–9 minutes per hour 893 1.0 1.0 1.0 1.0 1.0 10–44 minutes per hour 256 1.0 (0.7–1.5) 1.2 (0.7–2.1) 1.3 (0.8–1.9) 1.4 (0.9–2.1) 1.1 (0.8–1.4) 45–60 minutes per hour 260 1.5 (1.0–2.1) 1.9 (1.2–3.1) 1.7 (1.2–2.6) 1.1 (0.7–1.8) 1.4 (1.1–1.8) Lifting, cumulative Never 684 1.0 1.0 1.0 1.0 1.0 1–99 kg per hour 479 1.4 (0.9–1.9) 1.3 (0.8–2.1) 1.4 (0.9–2.0) 1.6 (1.1–2.3) 1.3 (1.0–1.7) Ն100 kg per hour 290 1.9 (1.3–2.7) 1.6 (0.9–2.7) 1.9 (1.3–2.8) 1.8 (1.2–2.8) 1.6 (1.2–2.0) Pushing, cumulative Never 824 1.0 1.0 1.0 1.0 1.0 1–354 kg per hour 327 1.3 (0.9–1.9) 1.6 (0.9–2.7) 1.9 (1.3–2.8) 1.6 (1.1–2.5) 1.5 (1.1–1.9) Ն355 kg per hour 305 1.5 (1.0–2.2) 1.8 (1.1–3.1) 1.7 (1.1–2.5) 2.0 (1.4–3.0) 1.5 (1.1–1.9) Lifting at or above shoulder level Never 1,307 1.0 1.0 1.0 1.0 1.0 1–49 kg per hour 90 1.2 (0.7–2.2) 0.9 (0.4–2.2) 1.2 (0.6–2.2) 1.4 (0.8–2.7) 1.3 (0.9–1.9) Ն50 kg per hour 78 2.1 (1.3–3.5) 2.2 (1.1–4.3) 1.0 (0.5–2.0) 2.0 (1.1–3.5) 1.6 (1.1–2.3) Sitting Ͼ30 minutes per hour No 1,084 1.0 1.0 1.0 1.0 1.0 Yes 286 0.7 (0.5–1.1) 1.0 (0.6–1.7) 0.9 (0.6–1.4) 1.0 (0.6–1.5) 0.9 (0.7–1.3) Standing Ͼ30 minutes per hour No 1,384 1.0 1.0 1.0 1.0 1.0 Yes 114 1.8 (1.2–2.9) 2.0 (1.1–3.7) 2.1 (1.3–3.3) 1.7 (1.0–2.9) 1.7 (1.1–2.3) Squatting Ͼ5 minutes per hour No 1,082 1.0 1.0 1.0 1.0 1.0 Yes 283 1.6 (1.1–2.2) 1.2 (0.7–2.0) 1.5 (1.0–2.1) 1.6 (1.1–2.3) 1.3 (1.0–1.7) * Adjusted for sex, age, occupational group, and intervention group.

Table 3. Work-related psychosocial risk factors and new onset of severe regional musculoskeletal pain, partially adjusted* Hazard ratio (95% confidence interval)

Neck/ Elbow, Any No. of shoulder forearm, Low back Hip, knee, regional Psychosocial risk factor subjects pain hand pain pain foot pain pain Job demands Low 940 1.0 1.0 1.0 1.0 1.0 High 565 0.9 (0.7–1.3) 0.8 (0.5–1.2) 1.2 (0.9–1.7) 0.9 (0.7–1.4) 0.9 (0.7–1.1) Job control High 1,030 1.0 1.0 1.0 1.0 1.0 Low 475 1.3 (0.9–1.8) 1.5 (0.9–2.2) 1.7 (1.2–2.3) 1.6 (1.1–2.2) 1.2 (1.0–1.5) Social support from supervisors High 1,030 1.0 1.0 1.0 1.0 1.0 Low 472 1.2 (0.8–1.6) 1.3 (0.8–1.9) 1.1 (0.8–1.6) 1.4 (1.0–2.0) 1.2 (0.9–1.5) Social support from colleagues High 1,234 1.0 1.0 1.0 1.0 1.0 Low 270 1.3 (0.9–1.8) 1.5 (0.9–2.4) 1.1 (0.8–1.6) 1.9 (1.3–2.7) 1.3 (1.1–1.7) Management quality High 1,050 1.0 1.0 1.0 1.0 1.0 Low 436 1.2 (0.9–1.6) 1.3 (0.9–2.0) 1.3 (0.9–1.9) 1.3 (0.9–1.8) 1.2 (1.0–1.5) Job satisfaction High 1,422 1.0 1.0 1.0 1.0 1.0 Low 78 1.9 (1.1–3.2) 1.3 (0.5–2.9) 1.2 (0.6–2.2) 2.2 (1.3–3.8) 1.6 (1.1–2.3) * Adjusted for sex, age, occupational group, and intervention group. 1360 ANDERSEN ET AL

Table 4. Demographic and health-related risk factors and new onset of severe regional musculoskeletal pain, partially adjusted* Hazard ratio (95% confidence interval)

No. of Neck/shoulder Elbow, forearm, Low back Hip, knee, Any regional Risk factor subjects pain hand pain pain foot pain pain Education level High 353 1.0 1.0 1.0 1.0 1.0 Medium 668 1.2 (0.8–1.9) 2.0 (1.1–3.9) 0.9 (0.6–1.4) 1.3 (0.8–2.2) 1.1 (0.9–1.5) Low 454 1.8 (1.1–3.0) 1.9 (0.9–4.0) 1.1 (0.7–1.8) 1.4 (0.8–2.5) 1.2 (0.9–1.7) Smoking No 1,120 1.0 1.0 1.0 1.0 1.0 Yes 382 1.3 (0.9–1.7) 1.3 (0.8–2.0) 1.0 (0.7–1.4) 1.1 (1.0–1.1) 1.1 (0.9–1.4) Leisure time physical activity Low 820 1.0 1.0 1.0 1.0 1.0 High 672 0.8 (0.6–1.1) 0.7 (0.4–1.0) 1.0 (0.7–1.3) 1.2 (0.9–1.7) 0.9 (0.7–1.1) Body mass index Ͻ25 896 1.0 1.0 1.0 1.0 1.0 25–29 480 1.1 (0.8–1.5) 0.9 (0.5–1.4) 1.2 (0.8–1.7) 1.5 (1.0–2.1) 1.1 (0.9–1.4) Ն30 116 1.8 (1.1–2.8) 1.5 (0.8–2.8) 1.8 (1.1–2.9) 2.8 (1.7–4.5) 1.5 (1.1–2.2) Fear avoidance, work Low 382 1.0 1.0 1.0 1.0 1.0 Medium 611 0.8 (0.5–1.1) 0.9 (0.5–1.4) 1.0 (0.7–1.4) 1.3 (0.9–2.0) 0.9 (0.7–1.2) High 363 0.9 (0.6–1.3) 1.1 (0.6–1.8) 0.9 (0.6–1.5) 0.8 (0.5–1.3) 0.9 (0.6–1.2) Fear avoidance, physical activity Low 382 1.0 1.0 1.0 1.0 1.0 Medium 664 1.1 (0.7–1.6) 1.9 (1.1–3.4) 1.0 (0.7–1.5) 1.3 (0.9–2.1) 1.1 (0.9–1.5) High 319 1.2 (0.8–1.8) 1.3 (0.7–2.7) 1.2 (0.7–1.8) 1.4 (0.9–2.4) 1.0 (0.8–1.4) Whiteley Index for health anxiety Low 1,286 1.0 1.0 1.0 1.0 1.0 Medium 149 1.3 (0.8–2.1) 1.3 (0.7–2.4) 1.4 (0.9–2.2) 1.1 (0.7–1.9) 1.1 (0.8–1.6) High 66 1.3 (0.7–2.6) 1.0 (0.4–2.7) 0.9 (0.4–2.0) 1.1 (0.5–2.3) 1.1 (0.6–1.7) Other chronic disease No 1,190 1.0 1.0 1.0 1.0 1.0 Yes 315 1.4 (1.0–1.9) 1.6 (1.0–2.4) 1.1 (0.7–1.6) 1.7 (1.2–2.4) 1.3 (1.0–1.6) * Adjusted for sex, age, occupational group, and intervention group.

Of the psychosocial risk factors (Table 3), high for physical activity showed minor positive associations job demands were not associated with worsening of pain. with all outcomes, except for any regional pain. Health Low job control was significantly associated with low anxiety was only marginally associated with neck/ back pain (HR 1.7 [95% CI 1.2–2.3]) and lower limb shoulder pain. Other chronic diseases at baseline were pain (HR 1.6 [95% CI 1.1–2.2]). Social support from significantly associated with pain worsening for all re- supervisors was marginally associated with lower limb gions, most pronounced for arm pain (HR 1.6 [95% CI pain (HR 1.4 [95% CI 1.0–2.0]), and social support from 1.0–2.4]) and lower leg pain (HR 1.7 [95% CI 1.2–2.4]). colleagues was associated with lower limb pain (HR 1.9 Final model. In the final multivariate model [95% CI 1.3–2.7]), and any regional pain (HR 1.3 [95% (Table 5), subjects with high levels of repetitive work CI 1.1–1.7]). Management quality was only marginally had an increased risk of arm pain (HR 1.7 [95% CI associated with the outcomes. Low job satisfaction was 1.0–2.9]). Lifting Ն100 kg per hour predicted worsening significantly associated with neck/shoulder pain, lower of low back pain (HR 1.5 [95% CI 1.0–2.3]). Lifting Ն50 limb pain, and any regional pain, with risk estimates kg at or above shoulder level remained significant for from 1.6 for any regional pain to 2.2 for lower limb pain. neck/shoulder pain (HR 1.9 [95% CI 1.1–3.3]) and Many of the individual and health-related factors pushing/pulling for lower limb pain (HR 1.6 [95% CI conferred an increased risk of worsening pain, including 1.0–2.5]). Standing for Ͼ30 minutes per hour was asso- low education level for neck/shoulder pain (HR 1.8 ciated with low back pain (HR 1.9 [95% CI 1.2–3.0]) and [95% CI 1.1–3.0]) and BMI Ն30 for neck/shoulder pain, any regional pain (HR 1.6 [95% CI 1.2–2.3]). low back pain, and lower leg pain (HR 2.8 [95% CI The effects of the psychosocial risk factors at 1.7–4.5]) (Table 4). Fear avoidance for work was not work were modest, aside from the association of low job associated with pain worsening, whereas fear avoidance satisfaction with increased risk of neck/shoulder pain RISK FACTORS FOR SEVERE REGIONAL MUSCULOSKELETAL SYMPTOMS 1361

Table 5. Final model of risk factors for more severe regional pain, multivariate associations* Hazard ratio (95% confidence interval)

Neck/shoulder Elbow, forearm, Low back Hip, knee, Any regional Risk factor pain hand pain pain foot pain pain Repetitive work 0–9 minutes per hour 1.0 10–44 minutes per hour 1.1 (0.6–2.0) 45–60 minutes per hour 1.7 (1.0–2.9) Lifting, cumulative Never 1.0 1–99 kg per hour 1.2 (0.8–1.8) Ն100 kg per hour 1.5 (1.0–2.3) Lifting at or above shoulder level Never 1.0 1–49 kg per hour 1.1 (0.6–2.0) Ն50 kg per hour 1.9 (1.1–3.3) Pushing, cumulative Never 1.0 1–354 kg per hour 1.4 (0.9–2.3) Ն355 kg per hour 1.6 (1.0–2.5) Squatting Ͼ5 minutes per hour No 1.0 1.0 Yes 1.4 (1.0–2.0) 1.2 (0.8–1.8) Standing Ͼ30 minutes per hour No 1.0 1.0 Yes 1.9 (1.2–3.0) 1.6 (1.2–2.3) Job control High 1.0 Low 1.5 (1.1–2.2) Social support from colleagues High 1.0 Low 1.6 (1.0–2.4) Job satisfaction High 1.0 1.0 Low 2.1 (1.2–3.6) 1.6 (0.9–3.0) Body mass index Ͻ25 1.0 1.0 25–29 1.4 (0.9–2.1) 1.1 (0.8–1.4) Ն30 2.3 (1.3–3.9) 1.4 (1.0–2.0) Fear avoidance, physical activity Low 1.0 1.0 Medium 1.9 (1.1–3.5) 1.6 (0.9–2.6) High 1.3 (0.6–2.7) 1.8 (1.1–3.2) Education level High 1.0 1.0 Medium 1.2 (0.7–1.8) 1.9 (0.9–3.7) Low 1.6 (0.9–2.7) 1.8 (0.8–3.9) Other chronic disease No 1.0 1.0 1.0 Yes 1.3 (0.9–1.9) 1.9 (1.2–3.1) 1.7 (1.1–2.5) * Adjusted for sex, age, occupational group, intervention group, and all other factors in each column.

(HR 2.1 [95% CI 1.2–3.6]). BMI Ն30 increased the risk 1.2–3.1]) and for lower limb pain (HR 1.7 [95% CI of lower leg pain (HR 2.3 [95% CI 1.3–3.9]) and, to a 1.1–2.5]). In the final model, there was only a tendency lesser extent, the risk of any regional pain (HR 1.4 [95% for the education level to have an impact on neck/ CI 1.0–2.0]). Fear avoidance for physical activity was a shoulder and arm pain. risk factor for arm pain and for lower limb pain. Sex was Respondents versus nonrespondents. Table 6 not significantly associated with any of the 5 outcomes shows the distribution of mean age, sex (% female), and for pain worsening (data not shown). Other chronic sickness-related absences for Ͼ2 weeks during the 2 diseases were significant for arm pain (HR 1.9 [95% CI years preceding the study for all eligible study partici- 1362 ANDERSEN ET AL

Table 6. Demographic characteristics and prevalence of sickness-related absence for Ͼ2 weeks during the 2 years preceding the study Nonrespondents Dropouts Followup Total Variable (n ϭ 1,598) (n ϭ 730) (n ϭ 3,276) (n ϭ 5,604) Age, mean Ϯ SD years 41.1 Ϯ 10.9 39.6 Ϯ 10.4 44.9 Ϯ 9.9 43.9 Ϯ 10.5 No. (%) female 893 (55.9) 397 (54.4) 2,087 (63.7) 3,377 (60.3) No. (%) with sickness-related absence for Ͼ2 weeks During 2000 252 (15.8) 119 (16.3) 368 (11.2) 739 (13.2) During 2001 257 (16.1) 102 (14.0) 359 (11.0) 718 (12.8) pants, grouped according to nonrespondents at baseline, pain, and low social support from colleagues was asso- dropouts from baseline to 24 months, those who partic- ciated with lower limb pain. These associations indicate ipated in both assessments (baseline and followup), and nonspecific effects of psychosocial factors at work. the total population. Both the nonrespondents and the Fear avoidance of physical activity, but not work, dropouts seemed to be younger, included more males, was of some importance for lower limb pain and arm and had more episodes of sick leave before the study was pain, but it is possible that fear avoidance is of minor initiated. A comparison between the 730 who dropped importance in this rather healthy working population out and the 3,276 who were followed up at 24 months than in a population in the later stages of more severe showed no differences in pain or other health-related and disabling pain. Other chronic disease should be outcomes at baseline, but significantly more of the acknowledged in the secondary prevention of musculo- dropouts lived alone (22.5% versus 12.5%), and there skeletal pain. were more smokers among dropouts (42.1% versus Nonrespondents and dropouts in this study were 29.1%). There were no significant differences for the characterized as younger males who smoked, lived variables that were included in the final multivariate alone, and had more lengthy periods of sick leave during model. the 2 years before the study. The same findings have been noted in other studies (19,20), and this group’s DISCUSSION unwillingness to participate is well known. The dropouts were no different in terms of pain status or exposures at In this study, we examined the effect of work- baseline, so we do not think that this selection would related physical and psychosocial factors in combination with individual and health-related factors in relation to severely interfere with our risk estimates. more severe regional pain in a prospective cohort of In this study, we chose to include all participants 4,006 industrial and service workers. The study benefits from baseline with no or only minor pain, instead of from investigating risk factors from several domains restricting the study to a followup of pain-free partici- together and from investigating different regional pain pants, which has been the method of choice in other symptoms in the same population, rather than analyzing studies (12,21). We did that deliberately. When it comes different specific outcomes in separate studies. The to musculoskeletal pain incidence, it seems difficult to us main findings were that physical work place factors, to define a pain-free population. In our population, only psychosocial factors, and factors related to health and 7.7% had not been bothered by pain in 1 of the 4 regions beliefs about health were all associated with more severe within the previous year. The occurrence of regional regional pain. Physical factors at work predicted pain pain is normally distributed in the population. Further- worsening in specific regions, rather than just any re- more, pain in one region is strongly correlated with pain gional pain. Highly repetitive work predicted arm pain, in other regions and is strongly correlated with pain heavy lifting predicted low back pain, and pushing/ status in subsequent years. We would postulate that pulling heavy weights predicted lower limb pain. These there is no such thing as a pain-free population, and effects favor a specific effect of physical factors on pain even if there were, the characteristics of such a small worsening. Low job satisfaction was associated with all group would be so special that it would hamper its use as outcomes, but in the final model was significantly asso- a reference group in epidemiologic studies. ciated only with neck/shoulder pain and lower limb pain, The exposures in the current study were mea- whereas low job control was associated with low back sured 2 years prior to the measurement of pain status. RISK FACTORS FOR SEVERE REGIONAL MUSCULOSKELETAL SYMPTOMS 1363

Changes in exposure may well have occurred during the regions was also correlated with mental health and followup period, and we have no later measures. But we vitality (according to the Short Form 36 health survey) know that 80% of the participants in the followup cohort (data not shown), which would further confirm that still had the same job and 10% had another similar job, some pain symptoms belong to this large pool of func- so we have no reason to think that the exposures were tional symptoms. We do not know anything about the radically changed. This study was a part of an interven- pathology behind the regional pain symptoms in our tion study with 3 arms, and all the analyses were adjusted cohort, but from other studies of general working pop- for the intervention group, which did not contribute ulations, we have found very few specific disorders significantly to any of the models (data not shown). among the study participants with pain complaints The most serious drawback to exposure assess- (19,20). In a recent study of arm pain, evidence was ments in this study is that the physical exposures were found for a higher importance of physical exposures for derived from self reports. Even though the temporal more specific disorders, which calls for better descrip- relationship between measures fulfills an acceptable tions of case definitions in epidemiologic studies based standard procedure, the self reports could easily be not only on self reports, but also on clinical examination flawed by the subjects’ pain status, given the discussion findings (23). Because of the low prevalence and inci- above concerning the very few pain-free individuals in dence of specific disorders, very large studies have to be such a population. Furthermore, common beliefs in conducted or, next best, one has to rely on well- different occupational groups about the occupational conducted case reference studies. hazards experienced by their group could also influence We found some effects of work-related psycho- self reports of exposure. In Denmark, nurse assistants social factors. A review of psychosocial risk factors and have received a great deal of attention with regard to low back pain in prospective studies found no effect their musculoskeletal pain problems in recent years, and (27). A more recent 6-month longitudinal study found in our study, they scored highest on pushing and pulling that high job strain (the combination of high job de- and next highest on lifting, which could not be confirmed mands and low job control) was associated with the by observation in 2 nursing homes for 2 hours on typical occurrence of low back pain and neck/upper extremity working days (data not shown). We do not know in what symptoms as well as with the taking of sick leave because other way the results of this study could be distorted of musculoskeletal pain (21). In that study, pain was because of self reports. It is possible that any such defined as any pain within the last 12 months, and distortion could affect the results both positively and participants with pain at baseline were excluded from negatively. But, from the results, it nevertheless seems the analyses. In our study, we found no effect of job plausible that repetitive movements could be a risk strain, but low job control alone was associated to a factor for arm pain, which also has been found by others minor degree with low back pain. In order to resolve (22,23). Lifting is a well-known risk factor for worsening these discrepancies of the importance of psychosocial of low back pain, and squatting as a risk factor for lower factors at work, there is a need to develop more person- leg pain also seems possible, since squatting has been independent measures of the psychosocial work environ- found to be a risk factor for knee arthritis (24). ment (28). Pain status was ascertained only at 2 time points, In summary, we have demonstrated that the and even if we know that pain status at baseline was reporting of more severe regional musculoskeletal pain strongly associated with pain status at followup, we do is multifactorial, as has been demonstrated in other not know the exact time frame for pain worsening in the studies. We have questioned the notion of incidence of study. Fluctuations in pain status have been confirmed in regional musculoskeletal pain, and we agree that inter- a previous study (25). ventions should address efforts to reduce the conse- Beliefs about pain and health predicted pain quences of regional pain (9,29). In addition, the con- status at followup, but this association was small after certed action of all stakeholders in the work place is adjustment for other factors. Several studies have indi- necessary to provide work places that support workers cated the importance of health beliefs with regard to with regional pain complaints so that they can keep up back pain, but the association is of the same magnitude with their jobs and to make work places comfortable and as for other regional musculoskeletal pain. A part of the accommodating so that workers with regional pain can burden of regional musculoskeletal pain could well fit cope with their regional pain complaints, which seems to under the umbrella of medically unexplained symptoms be a ubiquitous part of modern working life (30). (26), and in this study, the regional pain from all 4 Doctors and other health care professionals should 1364 ANDERSEN ET AL

refrain from upgrading unexplained pain complaints to 14. Kristensen TS, Hannerz H, Hogh A, Borg V. The Copenhagen somatic or psychiatric diseases, should provide reassur- Psychosocial Questionnaire: a tool for the assessment and improvement of the psychosocial work environment. Scand J Work ance about the good prognosis of most musculoskeletal Environ Health 2005;31:438–49. pain, and should support the necessary changes in the 15. Waddell G, Newton M, Henderson I, Somerville D, Main CJ. A work place (31). Fear-Avoidance Beliefs Questionnaire (FABQ) and the role of fear-avoidance beliefs in chronic low back pain and disability. Pain 1993;52:157–68. 16. Fink P, Ewald H, Jensen J, Sorensen L, Engberg M, Holm M, et AUTHOR CONTRIBUTIONS al. Screening for somatization and hypochondriasis in primary care Dr. Andersen had full access to all of the data in the study and and neurological in-patients: a seven-item scale for hypochondri- takes responsibility for the integrity of the data and the accuracy of the asis and somatization. J Psychosom Res 1999;46:261–73. data analysis. 17. Lassen CF, Mikkelsen S, Kryger AI, Brandt LP, Overgaard E, Study design. Andersen, Frost. Thomsen JF, et al. Elbow and wrist/hand symptoms among 6,943 Acquisition of data. Andersen, Haahr, Frost. computer operators: a 1-year follow-up study (the NUDATA Analysis and interpretation of data. Andersen, Haahr, Frost. study). Am J Ind Med 2004;46:521–33. Manuscript preparation. Andersen, Haahr, Frost. 18. Lassen CF, Mikkelsen S, Kryger AI, Andersen JH. Risk factors for Statistical analysis. Andersen. persistent elbow, forearm and hand pain among computer workers. Scand J Work Environ Health 2005;31:122–31. 19. Andersen JH, Kaergaard A, Frost P, Thomsen JF, Bonde JP, REFERENCES Fallentin N, et al. Physical, psychosocial, and individual risk factors for neck/shoulder pain with pressure tenderness in the muscles 1. Hagberg M, Silverstein BA, Wells RP, Smith R, Carayon P, among workers performing monotonous, repetitive work. Spine Hendrick H, et al, editors. Work-related musculoskeletal disorders 2002;27:660–7. (WMSD): a handbook for prevention. London: Taylor & Francis; 20. Andersen JH, Kaergaard A, Mikkelsen S, Jensen UF, Frost P, 1995. Bonde JP, et al. Risk factors in the onset of neck/shoulder pain in 2. Panel on Musculoskeletal Disorders and the Workplace, Commis- a prospective study of workers in industrial and service companies. sion on Behavioral and Social Sciences and Education, National Occup Environ Med 2003;60:649–54. Research Council, Institute of Medicine. Musculoskeletal disor- 21. Ijzelenberg W, Burdorf A. Risk factors for musculoskeletal symp- ders and the workplace: low back and upper extremities. Wash- toms and ensuing health care use and sick leave. Spine 2005;30: ington: National Academy Press; 2001. 1550–6. 3. Silman AJ, Hochberg MC, editors. Epidemiology of the rheumatic 22. Macfarlane GJ, Hunt IM, Silman AJ. Role of mechanical and diseases. 2nd ed. New York: Oxford University Press; 2001. psychosocial factors in the onset of forearm pain: prospective 4. Nachemson AL, Jonsson E. Neck and back pain: the scientific population based study. BMJ 2000;321:676–9. evidence of causes, diagnosis and treatment. Philadelphia: Lippin- 23. Ryall C, Coggon D, Peveler R, Reading I, Palmer KT. A case- cott Williams & Wilkins; 2000. control study of risk factors for arm pain presenting to primary 5. Linton SJ, van Tulder MW. Preventive interventions for back and care services. Occup Med (Lond) 2006;56:137–43. neck pain problems: what is the evidence? Spine 2001;26:778–87. 24. Coggon D, Croft P, Kellingray S, Barrett D, McLaren M, Cooper 6. Van Poppel MN, Hooftman WE, Koes BW. An update of a C. Occupational physical activities and osteoarthritis of the knee. systematic review of controlled clinical trials on the primary Arthritis Rheum 2000;43:1443–9. prevention of back pain at the workplace. Occup Med (Lond) 25. Papageorgiou AC, Croft PR, Thomas E, Ferry S, Jayson MI, 2004;54:345–52. Silman AJ. Influence of previous pain experience on the episode 7. Macfarlane GJ, McBeth J, Garrow A, Silman AJ. Life is as much incidence of low back pain: results from the South Manchester a pain as it ever was [letter]. BMJ 2000;321:897. Back Pain Study. Pain 1996;66:181–5. 8. Hadler NM, Carey TS. Low back pain: an intermittent and 26. Wessely S, Nimnuan C, Sharpe M. Functional somatic syndromes: remittent predicament of life. Ann Rheum Dis 1998;57:1–2. one or many? Lancet 1999;354:936–9. 9. Burton AK, Balague F, Cardon G, Eriksen HR, Henrotin Y, 27. Hartvigsen J, Lings S, Leboeuf-Yde C, Bakketeig L. Psychosocial Lahad A, et al. How to prevent low back pain. Best Pract Res Clin factors at work in relation to low back pain and consequences of Rheumatol 2005;19:541–55. low back pain: a systematic, critical review of prospective cohort 10. Pope DP, Silman AJ, Cherry NM, Pritchard C, Macfarlane GJ. studies. Occup Environ Med 2004;61:e2. Validity of a self-completed questionnaire measuring the physical 28. Walker-Bone K, Cooper C. Hard work never hurt anyone: or did demands of work. Scand J Work Environ Health 1998;24:376–85. it? A review of occupational associations with soft tissue muscu- 11. Pope DP, Silman AJ, Cherry NM, Pritchard C, Macfarlane GJ. loskeletal disorders of the neck and upper limb. Ann Rheum Dis Association of occupational physical demands and psychosocial 2005;64:1391–6. working environment with disabling shoulder pain. Ann Rheum 29. Burton AK, Balague F, Cardon G, Eriksen HR, Henrotin Y, Dis 2001;60:852–8. Lahad A, et al. Chapter 2. European guidelines for prevention in 12. Harkness EF, Macfarlane GJ, Nahit E, Silman AJ, McBeth J. low back pain: November 2004. Eur Spine J 2006;15 Suppl Mechanical injury and psychosocial factors in the work place 2:S136–68. predict the onset of widespread body pain: a two-year prospective 30. Hadler NM. Back pain in the workplace: what you lift or how you study among cohorts of newly employed workers. Arthritis Rheum lift matters far less than whether you lift or when. Spine 1997;22: 2004;50:1655–64. 935–40. 13. Kristensen TS, Borg V, Hannerz H. Socioeconomic status and 31. Waddell G, Burton AK. Occupational health guidelines for the psychosocial work environment: results from a Danish national management of low back pain at work: evidence review. Occup study. Scand J Public Health Suppl 2002;59:41–8. Med (Lond) 2001;51:124–35. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1365–1367 © 2007, American College of Rheumatology

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DOI 10.1002/art.22528 infiltrate, lymphocytes and plasma cells also showed positive Liver X receptor is a therapeutic target in staining for LXR␣ (Figure 1D). Immunostaining of normal ␤ ϭ collagen-induced arthritis synovium with LXR -specific antibody (SC 1001; Santa Cruz Biotechnology, Santa Cruz, CA) showed negative to Liver X receptors (LXR␣/NR1H3 and LXR␤/NR1H2) occasionally faintly positive staining of synoviocytes (Figure exert their atheroprotective effects by modulating the expres- 1E). In synovial samples from RA patients, superficial reactive sion of genes involved in lipid metabolism and inflammation in synoviocytes showed faint to patchy areas of moderately posi- macrophages (1). Ligand-activated LXRs induce the expres- tive staining for LXR␤ (Figure 1F), and subsynovial histiocytes sion of genes involved in cholesterol efflux (ABCA1, ABCG1 showed faintly to moderately positive staining for LXR␤ and ApoE) in macrophages, and inhibit the expression of (Figure 1G). Plasma cells within the inflammatory infiltrate mediators of inflammation (inducible nitric oxide synthase and also showed moderately positive staining for LXR␤ (Figure cyclooxygenase 2), cytokines (interleukin-6 [IL-6], IL-1␤, gran- 1H). Therefore, both LXR␣ and LXR␤ proteins are overex- ulocyte colony-stimulating factor, monocyte chemotactic pro- pressed in RA. tein 1 [MCP-1], MCP-3, macrophage inflammatory protein 1␤, Since tumor necrosis factor ␣ (TNF␣) blockade ame- and interferon-␥–inducible 10-kd protein), and matrix metal- liorates RA symptoms in humans (4,5) and in murine models loproteinase 9 in lipopolysaccharide (LPS)–treated mouse of arthritis, we examined the effect of a synthetic LXR ligand, macrophages (1,2). To extend the potential use of LXR ligands T0901317 (6), on LPS-induced TNF␣ elaboration in vivo in a in inflammatory and autoimmune conditions beyond athero- mouse model of RA. Female BALB/c mice 7–8 weeks old (n ϭ sclerosis (1) and atopic dermatitis (3), we explored the possi- 10 mice per group) were administered different doses of bility of using LXR as a therapeutic target in rheumatoid T0901317 or vehicle. One hour after dosing, mice were chal- arthritis (RA). We demonstrated that a synthetic LXR ligand lenged with LPS (400 ␮g/kg; E coli serotype 0111:B4). Animals inhibits the progression of arthritis symptoms in a murine were killed 90 minutes after challenge with LPS, and serum collagen-induced arthritis (CIA) model. was collected. TNF␣, IL-1␤, and IL-6 were measured by Immunostaining with an LXR␣-specific antibody (Pa1- enzyme-linked immunosorbent assay. LPS treatment of mice 330; Affinity Bioreagents, Golden, CO) showed negative to increased serum TNF␣ protein levels to 6,400 pg/ml. Oral faintly positive staining of normal synoviocytes (Figure 1A), administration of T0901317 to LPS-treated animals resulted and moderately to strongly positive staining of synovio- in a dose-dependent inhibition of TNF␣ protein levels cytes (Figure 1B), subsynovial histiocytes, and macrophages (Figure 2A). We also analyzed the effect of the LXR ligand on (Figure 1C) from RA synovium. Within the inflammatory IL-1␤ and IL-6, which are involved in the pathophysiology of

Figure 1. Expression of liver X receptor ␣ (LXR␣) and LXR␤ proteins in rheumatoid arthritis (RA) synovial tissue. Synovia obtained at surgery from either normal subjects (A and E) or RA subjects (B, C, D, F, G, and H) were immunostained with either LXR␣-specific antibody or LXR␤-specific antibody. Normal synoviocytes and subsynovial fibroblasts (A and E) show negative or faintly positive staining for LXR␣ and LXR␤. Superficial reactive synoviocytes (B and F), subsynovial histiocytes (C and G), and inflammatory infiltrate cells (D and H) all show positive staining for both LXR␣ and LXR␤.

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Figure 2. T0901317 inhibition of proinflammatory cytokine production and collagen-induced arthritis in vivo. A, T0901317 inhibition of lipopolysaccharide-induced tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), and IL-6 production in vivo in female BALB/c mice. Values are the mean Ϯ SEM from 3 independent experiments. P values are versus untreated controls. B, T0901317 inhibition of collagen-induced arthritis in male DBA/1 mice. Mice were treated from the time of onset of arthritis (day 28) with vehicle or T0901317 (50 mg/kg) once daily for 7 days. Mice were examined visually for the appearance of arthritis in the peripheral joints, and the severity of arthritis was graded on a scale of 0–4 for each paw. Values are the mean Ϯ SEM of a total of 6 mice per group. C, Significant reduction of joint pathology in mice with collagen-induced arthritis by T0901317. Values are the ϭ P Ͻ 0.05 versus vehicle-treated mice, by Dunnett’s ء .(mean histologic scores of 4 joints from all mice (n ϭ 6 t-test.

RA. T0901317 significantly inhibited IL-1␤ and IL-6 produc- The macroscopic clinical score for each mouse was the sum of tion in LPS-treated animals, in a dose-dependent manner the scores for all 4 paws (maximum possible score 16). (Figure 2A). Compared with vehicle-treated arthritic mice, T0901317- Given the ability of T0901317 to block LPS-induced treated animals showed a marked reduction in mean clinical TNF␣, we hypothesized that the LXR ligand might be capable scores. Three days after treatment, there was a statistically of inhibiting T cell–mediated autoimmunity. To test this pronounced suppression of the mean clinical score in the hypothesis, we used a murine CIA model, a widely used T0901317-treated group. The mean maximum clinical score experimental model of polyarthritis that shares many patho- was 6.0 in vehicle-treated mice and 2.0 in T0901317-treated logic and histologic features with human RA (7). Male DBA/1 mice (Figure 2B). mice immunized with chicken type II collagen in Freund’s Histologic examination of the paws and ankles on day complete adjuvant for 21 days were challenged with the 7 of treatment showed significant differences between autoantigen in Freund’s incomplete adjuvant. Starting on day T0901317-treated and vehicle-treated mice. Scores for inflam- 28, mice were administered either T0901317 (50 mg/kg) or mation, pannus formation, cartilage damage, and bone de- vehicle daily for 7 days, by oral gavage. struction (ranging from 0 to 5 for each parameter) were Mice were examined visually for the appearance of determined by a pathologist who was blinded with regard to arthritis in the peripheral joints, and each paw was assigned a treatment group (7,8). Vehicle-treated mice developed severe score of 0–4, where 0 ϭ normal, 0.5 ϭ swelling and redness of arthritis, which was characterized by marked inflammation of 1 digit, 1.0 ϭ swelling and redness of Ն2 digits, 2.0 ϭ mild synovium and joint tissue, with resultant erosion of articular swelling and redness of ankle or tarsal joint, 2.5 ϭ mild cartilage and bone. However, T0901317-treated animals swelling and redness of ankle to tarsal area, 3.0 ϭ gross showed significantly reduced lymphocyte infiltration, pannus swelling and redness of the entire paw, and 4.0 ϭ ankylosis. formation, and cartilage and bone damage (Figure 2C). Ac- CONCISE COMMUNICATION 1367

cordingly, the total number of affected joints per mouse (data Subba R. Chintalacharuvu, PhD not shown), the macroscopic arthritis score, and the histologic George E. Sandusky, PhD severity score were substantially lower in the LXR agonist– Thomas P. Burris, PhD treated group. Eli Lilly and Company These results suggest that LXR agonists may be a new Indianapolis, IN class of therapeutic agents for the treatment of autoimmune Glenna C. Burmer, MD, PhD diseases, in particular, RA. The major side effect of this class LifeSpan Biosciences of ligands is hypertriglyceridemia, which occurs as a result of Seattle, WA their action on the liver via induction of sterol regulatory Sunil Nagpal, PhD element binding protein 1c and fatty acid synthase gene (current address: Wyeth Research, Collegeville, PA) expression (1). Since LXR␣ is the major receptor type ex- Eli Lilly and Company pressed in the liver (1), the presence of both LXR forms in the Indianapolis, IN cellular rheumatoid synovial tissue suggests that either tissue- selective LXR modulators, LXR␤-selective ligands, or LXR 1. Kalaany NY, Mangelsdorf DJ. LXRS AND FXR: the yin and yang ligands dissociated for transactivation and transrepression may of cholesterol and fat metabolism. Annu Rev Physiol 2006;68: be potential candidates for use in the treatment of RA. 159–191. Dr. Burris has received consulting fees and honoraria from Orphagen 2. Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Pharmaceuticals, Invitrogen, GTx, Inc. (less than $10,000 each), Eli Lilly and Reciprocal regulation of inflammation and lipid metabolism by liver Company, and Phenex Pharmaceuticals (more than $10,000 each). Dr. Burris holds X receptors. Nat Med 2003;9:213–9. stock or stock options in Eli Lilly and Company and Orphagen Pharmaceuticals. Dr. 3. Fowler AJ, Sheu MY, Schmuth M, Kao J, Fluhr JW, Rhein L, et al. Nagpal holds stock or stock options in Eli Lilly and Company, Allergan, and Wyeth, Liver X receptor activators display anti-inflammatory activity in and holds patents on retinoids and vitamin D analogs. irritant and allergic contact dermatitis models: liver-X-receptor- specific inhibition of inflammation and primary cytokine produc- AUTHOR CONTRIBUTIONS tion. J Invest Dermatol 2003;120:246–55. 4. Maini RN. Current and new antitumor necrosis factor agents in Dr. Nagpal had full access to all of the data in the study and perspective. Arthritis Res Ther 2004;6 Suppl 2:S1–2. takes responsibility for the integrity of the data and the accuracy of the 5. Nash PT, Florin TH. Tumour necrosis factor inhibitors. Med J Aust data analysis. 2005;183:205–8. Study design. Chintalacharuvu, Burris, Nagpal. 6. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, et al. Role of Acquisition of data. Chintalacharuvu, Burmer. LXRs in control of lipogenesis. Genes Dev 2000;14:2831–8. Analysis and interpretation of data. Chintalacharuvu, Sandusky, 7. Chintalacharuvu SR, Wang JX, Giaconia JM, Venkataraman C. An Burris, Burmer, Nagpal. essential role for CCL3 in the development of collagen antibody- Manuscript preparation. Chintalacharuvu, Sandusky, Burris, Burmer, induced arthritis. Immunol Lett 2005;100:202–4. Nagpal. 8. Bendele A, McComb J, Gould T, McAbee T, Senello G, Chlipala E, Statistical analysis. Chintalacharuvu. et al. Animal models of arthritis: relevance to human disease. Study conceptualization. Nagpal. Toxicol Pathol 1999;27:134–42. ARTHRITIS & RHEUMATISM Vol. 56, No. 4, April 2007, pp 1368–1370 © 2007, American College of Rheumatology LETTERS

DOI 10.1002/art.22519 tures leads to the identification of a homogeneous disease What is juvenile psoriatic arthritis? Comment on the group. article by Stoll et al Alberto Martini, MD Universita` di Genova To the Editor: and Istituto G Gaslini A few years ago, in an editorial (1) challenging some Genoa, Italy aspects of the current classification criteria for juvenile idiopathic arthritis (JIA) (2), I suggested that a presumably 1. Martini A. Are the number of joints involved or the presence of homogeneous disease category is currently being classified into psoriasis still useful tools to identify homogeneous disease entities 4 different types of JIA. The features of this disease entity in juvenile idiopathic arthritis? [editorial]. J Rheumatol 2003;30: include early onset, female predominance, HLA–DRB1*0801 1900–3. positivity, antinuclear antibody positivity, asymmetric arth- 2. Petty RE, Southwood TR, Baum J, Bhettay E, Glass DN, Manners P, et al. Revision of the proposed classification criteria for juvenile ritis, and high risk of chronic iridocyolitis. Patients with these idiopathic arthritis: Durban, 1977. J Rheumatol 1998;25:1991–4. features are classified as having either persistent oligoarth- 3. Stoll ML, Zurakowski D, Nigrovic LE, Nichols DP, Sundel RP, ritis, extended oligoarthritis, rheumatoid factor–negative poly- Nigrovic PA. Patients with juvenile psoriatic arthritis comprise two arthritis, or psoriatic arthritis (PsA). With regard to juvenile distinct populations. Arthritis Rheum 2006;54:3564–72. PsA, the available literature described 2 distinct types of 4. Fitzgerald O, Dougados M. Psoriatic arthritis: one or more dis- disease (1). One is very similar to early-onset oligoarthritis, eases? Best Pract Res Clin Rheumatol 2006;20:435–50. although with a higher frequency of dactylitis and a more rapid 5. Southwood TR, Petty RE, Malleson PN, Delgado EA, Hunt DW, Wood B, et al. Psoriatic arthritis in children. Arthritis Rheum spread of arthritis, and the other shares the features of 1989;32:1007–13. enthesitis-related arthritis and therefore belongs to the group of spondylarthritides. I therefore read with much interest the article by Stoll et al (3), which confirms that the current classification criteria for PsA identify at least 2 distinct groups of patients with the same above-mentioned features. In their interpretation of the DOI 10.1002/art.22517 results, the authors favor the hypothesis that there are 2 broad The irreversible component of the disability index of types of PsA in children. However, I find it difficult to believe the Health Assessment Questionnaire: comment on that a single disease could have clinical features that are as different as those of oligoarticular JIA are from those of the article by Aletaha et al enthesitis-associated arthritis. Moreover, it would be expected To the Editor: that a childhood disease would exhibit strong similarities with Aletaha et al (1) have usefully called attention to a its adult counterpart. For example, rheumatoid factor–positive neglected observation: instruments that assess the latent trait polyarthritis has, in children, features that are identical to of disability, such as the disability index (DI) of the Health those observed in the disease in adults and differs only in some Assessment Questionnaire (HAQ) (2) in diseases such as aspects related to skeletal growth. rheumatoid arthritis (RA), must include a component related In adults the concept of PsA itself is not universally to the inflammatory activity of the disease and a component accepted, and there is no universally agreed-upon defini- related to irreversible joint damage. They have proposed that tion. Various hypotheses have been considered that are not one may determine the magnitude of the “irreversible” com- mutually exclusive. These include the notion that PsA is a ponent by inspecting the residual HAQ DI scores after a unique, although not yet well-characterized, disease entity, seemingly excellent clinical response (equivalent to that that psoriasis enhances susceptibility to arthritis and/or modi- achieved in the top 10% of responders to a treatment). fies the disease phenotype, conferring some features (such as The model suggested, however, is incomplete, and the higher frequency of dactylitis) on an underlying rheumatic results are thereby biased. A more complete model for HAQ disease, and that the association of psoriasis and arthritis is DI scores would include as a minimum 1) a disease activity coincidental. component, 2) a physical comorbidity component (disability The current available evidence from studies of adults due to increased age, acute or chronic diseases, or trauma), 3) suggests that PsA could be viewed as a member of the a mental health component (to assess mainly depression), 4) spondylarthritides family (4). If this is true, it could be assumed an error term component, and 5) an irreversible RA compo- that in the group identified by Stoll et al according to a nent. In addition to these omissions, the model presented previous definition of PsA (5), only those patients with the assumes that no future more powerful new antiinflammatory features of enthesitis-related arthritis could possibly have the drug treatment could have any additional effect on the residual childhood equivalent of adult PsA. Patients with early-onset HAQ DI score since this is the irreversible component; it oligoarthritis and psoriasis may simply be children with oligo- tacitly assumes that present treatments are ideal. Further, the articular JIA in whom the concurrent presence of psoriasis has definition of remission used does not require total remission, slightly modified the phenotype of the disease (higher fre- so additional improvement obviously remains possible. quency of dactylitis and more rapid spread of arthritis). There The omissions from the model each contribute to a is no convincing evidence to date that grouping together bias whereby the irreversible component is overestimated. children with arthritis and psoriasis or definite psoriatic fea- There are likely to be better drugs in the future, and the

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suggested model would then imply that the new drugs had chose radiographic damage and the duration of RA as inde- reversed the irreversible component. Some patients will have pendent variables to study this. Our findings demonstrate that had the flu or a bad back at the time of the last HAQ DI the clinical irreversible portion of the HAQ DI (as estimated assessment. Some will have had irreversibility due to osteoar- during remission) increases with the duration of RA and the thritis. Some values will be in error, and the correct value will degree of radiographic damage, while the overall HAQ DI at generally lie closer to the mean than the observed value. baseline is not related to either measure. Increasing age and increasing disease duration are linearly Dr. Fries’ main criticism of our study relates to the fact related, and recent observations indicate that much residual that assessment of the irreversible component might be biased. disability is related to age effects rather than disease effects First, he argues that apart from functional impairment due to (3). Data from a 20-year longitudinal study of normal senior RA activity and joint damage, the HAQ DI is affected by adult populations show similarly large effects of age (4,5), with comorbid conditions, age, and mental health. Second, he an increase in the HAQ DI of 0.016 units per year of age after points out that the criteria for remission are not completely 50 years. These data also demonstrate that exercise is a specific and residual disease activity might have continued to dominant factor in reducing the frailty and disability associated influence function. We agree with Dr. Fries on both points and with aging. would like to reemphasize these issues. Thus, the estimates of irreversible disease effects by Regarding the first point, we stated that our analysis Aletaha et al are systematically high. As we work to further estimated the upper limit of the latent trait irreversible disabil- improve RA outcomes, we will need better treatments for RA, ity, and that the true irreversible component may be lower. greater attention to comorbidities, lifestyle risk factors, and This is, in fact, supported by another analysis, in which we depressive reactions, and increased attention to reduction of showed that HAQ DI scores improved slightly during sus- the error terms (6). tained clinical remission (2). Moreover, we have hesitated to attribute the HAQ DI during remission to any specific cause. James F. Fries, MD Although the level of HAQ DI during remission was associated Stanford University School of Medicine with the degree of radiographic joint damage, other factors Stanford, CA including age and comorbid conditions may certainly contribute. However, we adjusted for age in our analyses, and major 1. Aletaha D, Smolen J, Ward MM. Measuring function in rheuma- comorbidity was an exclusion criterion in the clinical trials toid arthritis: identifying reversible and irreversible components. analyzed. With regard to a potential mental health component Arthritis Rheum 2006;54:2784–92. of irreversible functional limitations, we have observed in 2. Fries JF, Spitz P, Kraines RG, Holman HR. Measurement of ongoing studies in this area of research that the reversibility of patient outcome in arthritis. Arthritis Rheum 1980;23:137–45. the mental health component of the Short Form 36 (SF-36) in 3. Sokka T, Kautiainen H, Hannonen P, Pincus T. Changes in Health patients with RA is not affected by disease duration, in Assessment Questionnaire disability scores over five years in patients with rheumatoid arthritis compared with the general popula- contrast to the physical health component of the SF-36 (3). tion. Arthritis Rheum 2006;54:3113–18. As for the second point, the specificity of the definition 4. Chakravarty EF, Hubert HB, Lingala B, Fries JF. Reduced disabil- of remission was an important consideration when we designed ity and mortality among older runners: a 21-year longitudinal study the study. We based the definition of remission on that used in [abstract]. Arthritis Rheum 2006;54 Suppl 9:S338. the Simplified Disease Activity Index because it was highly 5. Hubert HB, Lingala B, Fries JF. Lifestyle risk factors predict specific (4). We also employed sensitivity analyses using more disability and death in an aging cohort [abstract]. Arthritis Rheum stringent criteria, which did not alter the results. 2006;54 Suppl 9:S499–500. It is important to recognize that the “irreversible” 6. Fries JF, Bruce B, Cella D. The promise of PROMIS: using item functional limitations we demonstrated should be considered response theory to improve assessment of patient-reported outcomes. Clin Exp Rheumatol 2005;5 Suppl 39:S53–7. only within the context and time frame of the clinical trials we analyzed. For example, a joint replacement may make previously impossible tasks possible, and thereby reverse some previously irreversible functional impairment. We agree with Dr. Fries that future therapies may be more efficacious than current ones, and may be able to reverse structural damage DOI 10.1002/art.22518 and the irreversible component of disability. The prospect of Reply such therapies provides additional rationale for recognizing and studying activity-related and damage-related components To the Editor: of functional measures such as the HAQ DI. We appreciate that Dr. Fries agrees with the major As noted by Dr. Fries, it is difficult to derive a precise hypothesis and evidence presented in our study, that in pa- estimate of the irreversible component of damage, and it will tients with RA, the HAQ DI (1) encompasses a component differ across patient samples, trials, interventions, and obser- related to irreversible joint damage. Our primary goal was to vational cohorts. We believe that recognition of the dual investigate the concept of reversibility and irreversibility. For contributions of activity and damage to functional limitations is this purpose, it was important to know if the latent trait of important, and that appreciation of the role of these 2 factors irreversibility was greater among patients who would be ex- will be helpful in any study that uses functional impairment pected to have more irreversible functional limitations. We either as an outcome or as a predictor of outcome. 1370 LETTERS

Daniel Aletaha, MD, MHS 1. Fries JF, Spitz P, Kraines RG, Holman HR. Measurement of NIH patient outcome in arthritis. Arthritis Rheum 1980;23:137–45. Medical University of Vienna 2. Aletaha D, Segurado OG, Breedveld F, Smolen JS. Physical Vienna, Austria function keeps improving during sustained remission in early RA and NIH [abstract]. Arthritis Rheum 2006;54 Suppl 9:S198. Bethesda, MD 3. Aletaha D, Smolen JS, Ward MM. Measurement of health status in Josef S. Smolen, MD rheumatoid arthritis: accumulation of irreversible limitations over Medical University of Vienna time [abstract]. Arthritis Rheum 2006;54 Suppl 9:S198. Vienna, Austria 4. Aletaha D, Ward MM, Machold KP, Nell VP, Stamm T, Smolen JS. Remission and active disease in rheumatoid arthritis: defining Michael M. Ward, MD, MPH criteria for disease activity states. Arthritis Rheum 2005;52: NIH 2625–36. Bethesda, MD

DOI 10.1002/art.22648 Erratum

In the article by Sundy et al published in the March 2007 issue of Arthritis & Rheumatism (pp. 1021–1028), the half-life of intravenous PEGylated recombinant mammalian urate oxidase in plasma, though correct in the abstract and the Results section, was incorrectly stated in the second paragraph of the Discussion section. The sentence in the Discussion should have read, “The half-life of IV PEG-uricase in plasma ranged from 6.4 days to 13.8 days.” This has been corrected in the online version of the article (http://www3.interscience. wiley.com/cgi-bin/jissue/114130641). We regret the error. ACR ANNOUNCEMENTS AMERICAN COLLEGE OF RHEUMATOLOGY 1800 Century Place, Suite 250, Atlanta, Georgia 30345-4300 www.rheumatology.org

ACR Meetings Meeting in November. Positions are available on the following committees: Communications and Marketing, Corporate Re- Annual Meetings lations, Education, Ethics and Conflict of Interest, Finance, November 6–11, 2007, Boston Government Affairs, Journal Publications, Nominations and October 24–29, 2008, San Francisco Appointments, Quality Measures, Research, Rheumatologic Care, and Rheumatology Training and Workforce Issues. State-of-the-Art Clinical Symposium Positions are also available on the ACR Board of Directors April 13–15, 2007, Chicago and on the Research and Education Foundation Board of Directors, Scientific Advisory Council, Development Advisory For additional information, contact the ACR office. Council, and Study Sections. Two ACR officer positions—Vice President and Secretary/Treasurer—and one Research and Nominations for ACR Awards of Distinction and Education Foundation officer position—Vice President—are Masters Due May 15 available as well. Members are invited to submit names of Nominations for the 2007 ACR Awards of Distinction candidates to serve as officers, on committees, or on the Board and ACR Masters are due May 15. To nominate someone for of Directors. For more information, contact the Director of one of the Awards of Distinction (Paulding Phelps Award, Membership by phone at (404) 633-3777 or by e-mail at Distinguished Service Award, Distinguished Clinician Scholar [email protected]. Volunteer forms and com- Award, Distinguished Basic Investigator Award, Distinguished mittee descriptions are available from the Membership De- Clinical Investigator Award, Henry Kunkel Young Investigator partment at the ACR office and in the Membership section of Award, Excellence in Investigative Mentoring, and Presiden- the ACR Web site. Nominations should be sumbitted by June tial Gold Medal), the sponsor must submit a nomination form, 1 to Chair, Committee on Nominations and Appointments, c/o a letter of nomination, two additional letters of recommenda- the ACR office. tion, and a copy of the nominee’s curriculum vitae. Recognition as a Master of the American College of Education Programs Rheumatology is one of the highest honors the College bestows. The designation of Master is conferred on ACR Rheumatic Diseases in the Elderly. April 27, 2007, members, age 65 or older, who have made outstanding contri- Indianapolis, IN. Presented by the Division of Rheumatology, butions to the field of rheumatology through scholarly achieve- Indiana University School of Medicine. For complete program ment and/or service to their patients, students, and profession. information or to register online, visit the Web site http:// This honor is usually bestowed upon no more than 15 members cme.medicine.iu.edu. For more conference information, con- per year. To nominate someone for Master status, the sponsor tact Deborah Jenkins, [email protected]. must submit a nomination form and two supporting letters 8th International Congress on Systemic Lupus Ery- from voting members of the ACR. Nominees for ACR Master thematosus. May 23–27, 2007, Shanghai, China. Sponsored by must have reached the age of 65 before October 1, 2007. the Chinese Medical Association, the Shanghai Jiao Tong Nomination forms for Awards of Distinction and Mas- University School of Medicine, and the Shanghai Medical ters are available from the Membership Department at the Center for Rheumatology. Official language will be English. ACR office and in the Membership section of the ACR Web Registration fees, if received before April 15, 2007, are as site. Completed nomination packets should be sent to Chair, follows: $450 for participants, $100 for guests, $150 for train- Committee on Nominations and Appointments, c/o the ACR ees, $100 for patients, and $550 for corporate representatives. office. The 2007 award recipients will be honored at the ACR For registration on-site or after April 15, fees will be increased. Annual Meeting in Boston in November. For information on hotel reservations, contact the Shanghai International Conference Management Organization by e-mail ACR Invites Nominations for Leadership Positions at [email protected]. To register for the congress or for additional information, contact the Chinese Med- The ACR encourages all members to participate in ical Association, 42 Dongsi Xidajie, Beijing 100710, China, forming policy and conducting activities of the College by phone 86-10-8515-8148, fax 86-10-6512-3754, e-mail lupus2007 assuming positions of leadership in the organization. Appoint- @cma.org.cn, or visit the Web site www.chinamed.com.cn/ ments will become effective at the 2007 Annual Scientific lupus2007.