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Rheumatoid arthritis Studies of in other ...... autoimmune diseases demonstrate why the discovery of anti-CCP antibodies in patients before disease onset is of Unlocking the ‘‘PAD’’ lock on critical importance. Li and colleagues demonstrated in an elegant study that serum derived from family members of patients with pemphigus foliaceus con- P J Utz, M C Genovese, W H Robinson tained antibodies directed against syn- desmoglein.8 Antibodies in non-affected ...... family members recognised the carboxyl (COOH) portion of the molecule, in Perhaps a panel of antigens containing may explain RA: stark contrast with antibodies from many questions remain afflicted patients which recognised the amino (NH2) terminus. Over time, a heumatoid arthritis (RA) affects antibodies was a post-translationally subset of non-afflicted patients devel- about 1% of the world population, modified antigen (filaggrin) containing oped disease, and this correlated with Ryet the driving antigen(s) remain citrulline residues.23Other studies have the development of antibodies that unidentified. A number of different suggested that citrullinated antigens recognised both the NH2 and the antigens have been proposed to have a such as filaggrin, , and fibrin COOH terminus of syndesmoglein. As 1 fundamental role in RA, including anti- are physiological candidate targets in with the study of Rantapa¨a¨-Dahlqvist, bodies bound by rheumatoid factor RA (reviewed by van Boekel et al6). this study showed that serum antibodies (RF), collagen, BiP, Sa, and many others could serve as useful predictors of (reviewed by Rantapa¨a¨-Dahlqvist et al1). disease in people who lack symptoms SERUM ANTIBODIES AS The field has been revolutionised by the but are at risk of disease. Similar discovery that antigens containing argi- PREDICTORS OF RA findings have also been seen in systemic Three recent papers, including one in lupus erythematosus,9 multiple sclero- nine residues that have been deiminated 7 to form citrulline are prominent targets this issue of the Annals, have added sis,10 and insulin dependent diabetes of autoantibodies in RA.23 Their detec- further insights into the role played by mellitus.11 Taken together, studies in tion forms the basis of several assays PAD and in RA many, but not all, diseases quite clearly that are now standard diagnostic tests pathogenesis. Rantapa¨a¨-Dahlqvist and demonstrate that diagnostic autoanti- in rheumatology clinics world wide. The colleagues analysed the predictive value bodies precede disease manifestations goal of this editorial is to provide a brief of anti-cyclic citrullinated peptide (CCP) by years. An intriguing possibility is that history of the discovery of citrullinated antibodies in a cohort of patients with citrullinated antigen(s) represent an RA who had donated blood years early target of the immune response in antigens in RA, a review of what is 1 known about the enzymes (peptidyl- before the development of RA. In RA, and that arthritis only develops arginine deiminases, PADs) involved in addition to anti-CCP antibodies, IgG, when the T and B lymphocyte the catalysis of a reaction that forms IgM, and IgA RF were also measured in spreading has reached as yet unidenti- citrulline, and a roadmap of future areas 83 people who had donated blood before fied dominant . Work being for research. disease onset. The prevalence both of performed in the laboratory of one of anti-CCP autoantibodies and IgA RF the authors (WHR) employing large was 33.7%, with a lower prevalence HISTORY OF CITRULLINATED scale RA antigen arrays may answer observed for IgM and IgG RF. For this question.12–14 recogni- ANTIGENS donors who had provided serum (1.5 The history of citrullinated antigens is tion may ultimately lead to more accu- years before any symptoms of RA, the one that is particularly important for rate diagnostic assays, and perhaps sensitivity of the anti-CCP assay was students of rheumatology to review, more targeted therapeutic treatments 52%. It is interesting to note that serum because the initial discovery was largely for individual patients. autoantibodies were detectable in a few overlooked and rediscovered on several Although only a handful of antigens patients as long as 9–22 years (CCP and different occasions over the ensuing containing citrulline residues have been RF, respectively) before disease onset. four decades. In 1964, in the Annals of identified, the enzymes that catalyse the the Rheumatic Diseases, a search for cell deimination of arginine to form citrul- and tissue substrates containing anti- ‘‘Diagnostic autoantibodies precede line have been the subject of intense gens that could be bound by autoanti- the onset of RA by years’’ interest. The obvious hypothesis being bodies from patients with RA found that tested is that unidentified antigen(s) granules from differentiating buccal Antibody titres increased over time in present in the synovium are modified by mucosal cells expressed such an auto- almost all people. These studies clearly one or more PAD enzymes, generating antigen. The autoantibody system was demonstrate that anti-CCP antibodies an immune response that ultimately termed ‘‘antiperinuclear factor’’.4 A precede the onset of RA by over a year. leads to the clinical manifestations similar screen performed 15 years later These results further imply that this associated with RA. In this issue of the showed that rat oesophagus was an post-translational modification may Annals, Vossenaar and colleagues ideal and more easily studied substrate lead to the creation of a neoepitope that address some important aspects of this for detection of these serum autoanti- drives pathogenic autoreactive T and B hypothesis—namely, which PAD bodies, and these were named ‘‘anti- cells. Although this discovery will not enzymes might have a role in RA, and antibodies’’.5 This work went lead to large scale screening of patients which cells express these enzymes.7 largely unnoticed until nearly another outside rheumatology clinics, and is Four different PAD enzymes have been 20 years had passed, when two unlikely to lead to therapeutic interven- identified in humans. Of these, PAD2 European groups independently demon- tions in patients lacking symptoms, it and PAD4 are thought to be most strated that the target of both anti- may represent an important break- relevant to RA because both are perinuclear factor and antikeratin through in the pathogenesis of RA. expressed in haematopoietic cells such

www.annrheumdis.com LEADER 331 as macrophages, whereas PAD1 and might account for the strong genetic an entirely new class of cytokine PAD3 are largely found in skin. association of haplotype 2 with RA. In inhibiting biological agents, as well as Vossenaar et al report that mRNAs light of the findings of Vossenaar et al in the discovery of a highly sensitive and encoding PAD2 and PAD4 can be easily this issue, it is also possible that the specific diagnostic assay for RA. As with identified by reverse transcriptase- PAD4 encoded by haplotype 2 is any important breakthrough, it is often polymerase chain reaction (RT-PCR) qualitatively different, rather than sim- the case that more questions arise than from CD14+ peripheral blood mono- ply being quantitatively different, from existed before the discovery. Let us hope nuclear cells. Upon differentiation into the protein encoded by haplotype 1. For that the interval between the initial macrophages in vitro, only PAD2 mRNA example, the calcium requirements, Annals of the Rheumatic Diseases paper in remained detectable. Analysis at the ability to interact with other regulatory 1964 and the repeated ‘‘rediscovery’’ of level of showed that PAD2 molecules or subunits, subcellular loca- citrullinated antigens over the ensuing was detectable only in macrophages, lisation, or substrate specificity might be 40 years far exceeds the period between whereas PAD4 was found in both uniquely different between these two the unlocking of the RA ‘‘PAD lock’’ and monocytes and macrophages. This result haplotypes. the opening of the door to a firm demonstrates that PAD2 is regulated understanding of RA pathogenesis. post-transcriptionally (supported by in QUESTIONS REMAIN vitro studies employing a luciferase Although the RA ‘‘PAD lock’’ seems to ACKNOWLEDGEMENTS regulated by the long 39 untranslated have been identified, the door to full PJU is supported by grants from the Dana (UTR) region derived from the PAD2 understanding of RA remains un- Foundation, the Northern California Chapter gene), and that PAD4 is likely to be a of the Arthritis Foundation, the Stanford opened, but unlocked. At least six relatively long lived protein. Similar Program in Molecular and Genetic Medicine questions remain unanswered and results were found when studying cells (PMGM), NIH grants DK61934, AI50854, should be the focus of future RA obtained from synovial fluid from AI50865, and AR49328, and NHLBI Proteomics research in the coming decade: (a) patients with RA. No differences were Contract N01-HV-28183. Dr Utz is a recipient of Which of the PAD enzymes are relevant an Arthritis Foundation Investigator award observed when comparing peripheral to autoantigen citrullination, and where and a Baxter Foundation Career Development blood mononuclear cells from normal are they expressed? For example, are award. MCG is supported by NIH Contract patients and healthy controls, either at there subsets of CD14+ cells, or other NO-AR-9-2241 and an Arthritis Foundation the level of mRNA or protein expression. relevant cell types, that might express Chapter Grant. WHR is supported by NIH K08 One of the most elegant aspects of this AR02133, an Arthritis Foundation Chapter PADs?; (b) Are PAD (s) dys- study was the observation that cells Grant and Investigator Award, and NIH regulated in patients at risk of develop- expressing PAD enzymes did not con- NHLBI contract N01 HV 28183. The authors ing RA? Careful analysis of the tain citrulline modified antigens unless declare no competing financial interests. biochemical and functional properties exogenous stimuli capable of markedly Ann Rheum Dis 2004;63:330–332. of the proteins encoded by PAD4 hap- increasing intracellular calcium (a doi: 10.1136/ard.2003.015990 lotypes 1 and 2 is a logical place to begin required cofactor for PAD activity) were such studies; (c) What role might micro- applied to the cells. In fact, one known ...... organisms play in the citrullination of autoantigen containing citrulline, Authors’ affiliations autoantigens, or PAD activation? Given vimentin, was detected within 15 min- P J Utz, M C Genovese, W H Robinson, the potential association of RA with utes of exposure to ionomycin. A unique Division of Immunology and Rheumatology, antecedent infections, one might spec- cadre of citrullinated proteins was Department of Medicine, Stanford University ulate that infectious agents could acti- observed when comparing lysates pre- School of Medicine, Stanford, California, USA vate endogenous cellular PAD enzymes, W H Robinson, GRECC, VA Palo Alto Health pared from monocytes and macro- could express biomolecules that are Care System, 3801 Miranda Ave, Palo Alto, phages, adding yet another level of molecular mimics of citrullinated anti- California, USA complexity to these already intriguing gens, or could encode their own PADs or results. PAD-like enzymes within their gen- Correspondence to: Dr P J Utz, Stanford omes; (d) Can overexpression of PADs, University, CCSR Building, Room 2215A, 269 ‘‘PAD2 and PAD4 are most likely to or exposure to citrullinated antigens in a Campus Drive, Stanford, CA 94305, USA; [email protected] have a role in RA’’ proinflammatory context, break toler- ance and induce a disease resembling The third recent paper shedding light RA?; (e) What are the physiological REFERENCES on citrullination and PAD enzymes took stimuli that activate PAD enzymes? 1 Rantapa¨a¨-Dahlqvist S, de Jong BA, Berglin E, a genetic approach to identify that Clearly, chemicals such as ionomycin Hallmans G, Wadell G, Stenlund H, et al. are linked to RA.15 The genes encoding and thapsigargin can activate PAD Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of PAD enzymes are encoded on human activity leading to production of citrulli- rheumatoid arthritis. Arthritis Rheum region 1p36, within a nated autoantigens such as vimentin. 2003;48:2741–9. previously identified RA susceptibility Perhaps other stimuli such as binding of 2 Schellekens G, de Jong B, van den Hoogen F, van de Putte L, van Venrooij WJ. Citrulline is an . Single nucleotide polymorphisms cytokines or chemokines to their respec- essential constituent of antigenic determinants in this region were used to identify a tive receptors might activate PADs, or recognized by rheumatoid arthritis-specific haplotype associated with RA in PAD4 might induce PAD expression in vivo; (f) autoantibodies. J Clin Invest 1998;101:273–281. but not in any of the other three PADs. Most importantly, what are the relevant 3 Girbal-Neuhauser E, Durieux J-J, Arnaud M, Dalbon P, Sebbag M, Vincent C, et al. The Sequencing of this gene in all patients citrullinated antigens that drive RA? The epitopes targeted by the rheumatoid arthritis identified two different haploytpes, an results of Vossenaar et al in this issue associated antifilaggrin autoantibodies are RA non-susceptible haplotype (haplo- strongly suggest that other citrullinated posttranslationally generated on various sites of antigens will soon be discovered. (pro)filaggrin by deimination of arginine residues. type 1) and an RA susceptible haplotype J Immunol 1999;162:585–94. (haplotype 2). In vitro experiments Perhaps there will be no single antigen 4 Nienhuis R, Mandena E. A new serum factor in performed using haplotype 2 mRNA to explain RA, but rather a panel of patients with rheumatoid arthritis. The perinuclear demonstrated significantly increased antigens containing citrulline. factor. Ann Rheum Dis 1964;23:302–5. 5 Young B, Mallya R, Leslie R, Clark C, Hamblin T. stability of the transcript, suggesting It has been an amazing decade for RA Antikeratin antibodies in rheumatoid arthritis. that enhanced stability of PAD4 mRNA research, one that saw the approval of BMJ 1979;ii:97–9.

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6 van Boekel M, Vossenaar E, van den Hoogen F, autoantibodies before the clinical onset of microarrays for multiplex characterization of van Venrooij WJ. Autoantibody systems in systemic lupus erythematosus. N Engl J Med autoantibody responses. Nat Med rheumatoid arthritis: specificity, sensitivity and 2003;349:1526–33. 2002;8:295–301. diagnostic value. Arthritis Res 2002;4:87–93. 10 Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, 13 Robinson W, Fontoura P, Lee B, deVegvar H, 7 Vossenaar ER, Radstake TRD, van der Heijden A, Mayringer I, et al. Antimyelin antibodies as a Tom J, Pedotti R, et al. Protein microarrays van Mansum MAM, Dieteren C, de Rooij D-J, et predictor of clinically definite multiple sclerosis guide tolerizing DNA vaccine treatment of al. Expression and activity of citrullinating after a first demyelinating event. N Engl J Med autoimmune encephalomyelitis. Nat Biotech peptidylarginine deiminase enzymes in 2003;349:139–45. 2003;21:1033–9. monocytes and macrophages. Ann Rheum Dis 11 Hagopian W, Sanjeevi C, Kockum I, Landin- 14 Robinson W, Steinman L, Utz PJ. Protein arrays 2004;63:373–81. Olsson M, Karlsen A, Sundkvist G, et al. for autoantibody profiling and fine specificity 8 Li N, Aoki V, Hans-Filho G, Rivitti E, Diaz L. The Glutamate decarboxylase, insulin, and islet mapping. Proteomics 2003;3:2077–84. role of intramolecular epitope spreading in the cell autoantibodies and HLA typing to detect 15 Suzuki A, Yamada R, Chang X, Tokuhiro S, pathogenesis of endemic pemphigus foliaceus diabetes in a general population-based study of Sawada T, Suzuki M, et al. Functional haplotype (Fogo Selvagem). J Exp Med Swedish children. J Clin Invest of PADI4, encoding citrullinating enzyme peptidyl 2003;197:1501–10. 1995;95:1505–11. arginine deiminase 4, are associated with 9 Arbuckle M, McClain M, Rubertone M, Scofield R, 12 Robinson W, DiGennaro C, Hueber W, Haab B, rheumatoid arthritis. Nat Gene Dennis G, James J, et al. Development of Kamachi M, Dean E, et al. Autoantigen 2003;34:395–402.

Clinical Evidence—Call for contributors

Clinical Evidence is a regularly updated evidence based journal available worldwide both as a paper version and on the internet. Clinical Evidence needs to recruit a number of new contributors. Contributors are health care professionals or epidemiologists with experience in evidence based medicine and the ability to write in a concise and structured way. Currently, we are interested in finding contributors with an interest in the following clinical areas: Altitude sickness; Autism; Basal cell carcinoma; Breast feeding; Carbon monoxide poisoning; Cervical cancer; Cystic fibrosis; Ectopic pregnancy; Grief/bereavement; Halitosis; Hodgkins disease; Infectious mononucleosis (glandular fever); Kidney stones; Malignant melanoma (metastatic); Mesothelioma; Myeloma; Ovarian cyst; Pancreatitis (acute); Pancreatitis (chronic); Polymyalgia rheumatica; Post-partum haemorrhage; Pulmonary embolism; Recurrent miscarriage; Repetitive strain injury; Scoliosis; Seasonal affective disorder; Squint; Systemic lupus erythematosus; Testicular cancer; Varicocele; Viral meningitis; Vitiligo However, we are always looking for others, so do not let this list discourage you. Being a contributor involves: N Appraising the results of literature searches (performed by our Information Specialists) to identify high quality evidence for inclusion in the journal. N Writing to a highly structured template (about 2000–3000 words), using evidence from selected studies, within 6–8 weeks of receiving the literature search results. N Working with Clinical Evidence Editors to ensure that the text meets rigorous epidemiological and style standards. N Updating the text every eight months to incorporate new evidence. N Expanding the topic to include new questions once every 12–18 months. If you would like to become a contributor for Clinical Evidence or require more information about what this involves please send your contact details and a copy of your CV, clearly stating the clinical area you are interested in, to Claire Folkes ([email protected]).

Call for peer reviewers

Clinical Evidence also needs to recruit a number of new peer reviewers specifically with an interest in the clinical areas stated above, and also others related to general practice. Peer reviewers are health care professionals or epidemiologists with experience in evidence based medicine. As a peer reviewer you would be asked for your views on the clinical relevance, validity, and accessibility of specific topics within the journal, and their usefulness to the intended audience (international generalists and health care professionals, possibly with limited statistical knowledge). Topics are usually 2000–3000 words in length and we would ask you to review between 2–5 topics per year. The peer review process takes place throughout the year, and our turnaround time for each review is ideally 10–14 days. If you are interested in becoming a peer reviewer for Clinical Evidence,please complete the peer review questionnaire at www.clinicalevidence.com or contact Claire Folkes([email protected]).

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