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. 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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. 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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

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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. 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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. 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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: [email protected] 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. 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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: [email protected] 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 auto- in 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. 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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. 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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. 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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. 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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

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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. 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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

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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. 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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]. 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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. 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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 subtract- ing 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 coeffi- cient (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.

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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. 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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. 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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). 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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. 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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, Ca