The Devil in the Details: The Emerging Role of Anticitrulline in Rheumatoid

This information is current as Floris van Gaalen, Andreea Ioan-Facsinay, Tom W. J. of September 23, 2021. Huizinga and René E. M. J Immunol 2005; 175:5575-5580; ; doi: 10.4049/jimmunol.175.9.5575 http://www.jimmunol.org/content/175/9/5575 Downloaded from

References This article cites 63 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/175/9/5575.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 23, 2021 *average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE

JOURNAL OF IMMUNOLOGY

BRIEF REVIEWS

The Devil in the Details: The Emerging Role of Anticitrulline Autoimmunity in Floris van Gaalen, Andreea Ioan-Facsinay, Tom W. J. Huizinga, and Rene´E. M. Toes1

Rheumatoid arthritis is a chronic inflammatory autoim- the inflamed . This leaves the third Witebsky postulate mune of unknown cause. The open, although recent data suggests that immunization with cit- against citrullinated Ags has recently become the prime rullinated peptides or proteins could lead to a significant suspect for disease pathogenesis. against citrul- response in rodents (our unpublished data and Ref. 4). linated Ags is thought to play a pivotal role in the disease As the response against citrullinated Ags is likely the first to for several reasons: 1) citrullinated Ags are expressed in the meet all criteria in Witebsky’s postulates, it is very tempting to target , the inflamed joint; 2) anti-citrullinated pro- speculate that the immunity to citrullinated Ags is intimately Downloaded from tein Abs are present before the disease becomes manifest; involved in the pathogenesis of RA. We propose here a model and 3) these Abs are highly specific for rheumatoid arthri- for the production of Abs against citrullinated Ags and the sub- tis. In this review, data from clinical, genetic, biochemi- sequent induction of chronic in the joint. cal, and animal studies is combined to create a profile of this remarkable response. Moreover, a

Pathogenic mechanisms of http://www.jimmunol.org/ model is proposed of how the anti-citrullinated proteins In autoimmune , autoantibodies provide diagnostic cri- response is generated and how it could eventually lead to teria, serve as surrogate markers for disease activity, and play a chronic inflammation. The Journal of Immunology, requisite role in pathogenesis. Although most autoantibodies 2005, 175: 5575–5580. are not known to be pathogenic and are mainly used for diag- nosis, several autoantibodies have been shown to be involved in utoimmune diseases are classified according to the or- development of autoimmune diseases. For instance, autoanti- gans and tissues that are damaged by the immune re- bodies against type IV in the glomerular basement A sponse. There is an directed membrane of the kidney and lung cause nephritis and lung by guest on September 23, 2021 against almost every organ in the body, involving, usually, an hemorrhage in Goodpasture’s syndrome, and autoantibodies to immune response to an Ag expressed in that organ (1). Auto- the acetylcholine cause muscle weakness in myasthenia immune diseases may be defined using Witebsky’s postulates. gravis (5, 6). Autoantibodies contribute to disease by directly These postulates require that 1) an autoimmune reaction is binding to end-organ tissue Ags, with triggering of immune- identified in the form of autoantibody or cell-mediated im- effector mechanisms, such as FcRs and the , mune reaction, 2) the corresponding Ag is known, and 3) an as a result (7). analogous response causes a similar disease in experimental an- RA is a chronic disease, with inflammation and deformities of imals (2, 3). the as the most striking features. The role of autoantibod- Rheumatoid arthritis (RA)2 is considered an autoimmune ies has been well established in several experimental arthritis disease, but specific immune responses against joint Ags have models, but their role is less clear in the human disease (8). For been difficult to demonstrate conclusively in the past. Recent one, most autoantibodies found in RA can also be detected in data indicate that the immune response against citrullinated individuals without RA. One such example is the IgM rheuma- Ags is an attractive candidate for the fulfillment of the three toid factor (RF). RFs are Abs directed to the Fc fragment of IgG Witebsky postulates. There has been considerable interest in re- molecules and are found in the majority of RA patients. How- cent years in the observation that a very high proportion of pa- ever, patients with other chronic inflammatory diseases, infec- tients with RA have IgG Abs to citrulline-containing proteins. tious diseases, as well as many healthy individuals, also produce Interestingly, these Abs appear early in RA and are rarely found RF, indicating that RF is not very specific for RA and that its in healthy people or patients with other diseases. Moreover, re- mere presence is insufficient for a chronic inflammatory re- cent data show that citrullinated Ags themselves are expressed in sponse (9).

Department of , Leiden University Medical Centre, Leiden, The Netherlands 2 Abbreviations used in this paper: RA, rheumatoid arthritis; RF, ; APF, antiperinuclear factor; AKA, anti-keratin Ab; ACPA, anti-citrullinated protein/ Received for publication June 2, 2005. Accepted for publication September 8, 2005. peptide Ab; PAD, peptidylarginine deiminase; SE, shared ; GPI, glucose-6- The costs of publication of this article were defrayed in part by the payment of page phosphate isomerase. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Rene´E. M. Toes, Department of Rheumatology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Lei- den, The Netherlands. E-mail address: [email protected]

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5576 BRIEF REVIEW: THE EMERGING ROLE OF ANTICITRULLINE AUTOIMMUNITY IN RA

Autoantibodies against the unusual amino acid citrulline PAD enzymes were found in (PAD4) and macro- In 1964, Nienhuis and Mandema (10) discovered a novel Ab, phages (PAD2 and PAD4) in (27), indicating the antiperinuclear factor (APF), while investigating the occur- that they could be involved in of synovial proteins rence of autoantibodies in patients with various rheumatic dis- once they become activated. Indeed, it has been shown that cit- eases. Later on, also anti-keratin Abs (AKAs) (11) were identi- rullination of synovial proteins is an active process during in- fied by using rat or human esophagus cryostat sections for flammation (28, 29) and that several citrullinated proteins, detection. Despite the fact that several research groups have rec- such as fibrin (30), can be found in the RA synovium. Together ognized the clinical and diagnostical value of these Abs, espe- with citrullinated proteins in the inflamed joint, B cells actively cially because of their high specificity for RA (12–15), APFs and secreting ACPA have been detected in synovial fluid and syno- AKA never became very popular. This was mainly a conse- vium from RA patients (31, 32) but not in peripheral blood or quence of the fact that testing their presence was more laborious in healthy controls. The presence of IgM ACPA-secreting B than the RF test, which soon became the standard laboratory cells in synovial fluid is indicative of a continuous activation of test for RA. The first step toward identifying the Ag recognized B cells specific for citrullinated Ags from naive precursors, sug- by these Abs was done by the group of Serre (16) in 1993 when gesting an Ag-driven proliferation and maybe local differentia- it became clear that the Ag recognized by the so-called AKAs is tion of these cells. actually . The ACPA response became prime suspect in the - More than 30 years after its initial description, a major break- esis of RA once it became clear that, while being found in 60– through was made through the pioneering work of the groups 70% of patients with RA, ACPAs display a unique specificity Downloaded from of Serre and Van Venrooij (17, 18). They showed that the APF for RA and are rarely detected in other diseases or in healthy is an Ab directed against proteins containing the unusual amino controls (13, 33–36). Remarkably, using recently developed acid citrulline. In addition, they demonstrated that the APF and ELISAs containing cyclic citrullinated peptides, ACPAs of the AKA are directed to the same Ag and that other RA-specific IgG have been detected up to nine years before symp- autoantibodies such as anti-Sa Abs are also directed against cit- toms of RA occurred (37, 38). A similar observation was made rulline-containing proteins (19, 20). Although the fine-speci- in patients with undifferentiated arthritis (or unclassified arthri- http://www.jimmunol.org/ ϳ ficity of these Abs might differ and is most probably influenced tis). Although 40% of these patients will eventually progress by the citrulline-flanking residues, citrulline is the critical con- to RA, the remaining patients experience remission or develop stituent of the antigenic determinant recognized by these Abs other rheumatic diseases. Unfortunately, physicians are cur- because its absence leads to lack of recognition (17, 18, 21). rently not able to predict which patients will progress to RA and Thus, although the nomenclature (APF, AKA, anti-filaggrin, which patients will have a more favorable outcome of disease. and anti-cyclic citrullinated peptide Abs) of the several Ab re- We have recently reported that patients with undifferentiated Ͼ sponses might differ as a result of the different Ags used for de- arthritis with ACPA have a chance of 90% to progress to full- tection, they all recognize citrulline as common antigenic entity blown RA within 3 years (39). This not only shows that detec- by guest on September 23, 2021 and will, therefore, be named in this review as anti-citrullinated tion of ACPAs is a powerful tool in predicting RA, but again protein/peptide Abs (ACPAs). points to the highly specific nature of the ACPA response. Citrullination is the posttranslational modification of pro- The second observation, indicating that the immune reaction tein-bound arginine into the nonstandard amino acid citrulline against citrullinated Ags is involved in the pathogenesis of RA, and results in a small change in molecular mass and the loss of a comes from genetic association studies. A haplotype of the gene positive charge in the modified proteins (Fig. 1). Although the encoding one of the citrullinating enzymes, PADI4, was shown physiological role of citrullination remains to be elucidated, it to be associated with susceptibility to RA (Ref. 40 and our un- has been proposed that citrullination plays an important role in published observations). Although it could not be confirmed in preparing intracellular proteins for degradation during apopto- two other studies in Caucasians (41, 42), possibly as a result of sis (22, 23), as well as in regulation of transcription through differences in the haplotype structure between different ethnic citrullination of histones (24, 25). populations (43), this haplotype was shown to be associated Five mammalian peptidylarginine deiminases (PADs), with an enhanced stability of PADI4 transcripts. Therefore, it PAD1–4 and PAD6, each with a defined tissue distribution, was postulated that the more “stabile haplotype” leads to an in- mediate citrullination of arginine in the presence of sufficient creased production of PAD4 protein and thereby to a higher ϩ concentrations of Ca2 (reviewed in Ref. 26). Interestingly, citrullinating enzyme activity in the joint. This will result in a higher concentration of citrullinated proteins that could serve as a target for the immune response against citrullinated Ags. A third line of evidence is found in the strong association be- tween production of ACPA and the presence of RA susceptibil- ity HLA-DRB1 genes (44). It has long been observed in many different populations that specific HLA-DR gene variants in the MHC region are highly associated with RA. The association has been mapped to the third hypervariable region of DR␤-chains, especially aa 70–74, encoding a conserved amino acid sequence (QKRAA, QRRAA, or RRRAA) that forms the fourth anchoring pocket in the HLA groove. This susceptibility epitope, called the shared epitope FIGURE 1. Enzymatic conversion of arginine to citrulline is catalyzed (SE), is found in multiple RA-associated DR molecules, includ- by PAD enzymes. ing DR4, DR1, and DR14 (e.g., DRB*0401, DRB*0404, The Journal of Immunology 5577

DRB*0101, and DRB*1402) (reviewed in Ref. 45). Due to the of the RA patients do not harbor these Abs. Likewise, the emer- presence of mainly positively charged amino acid residues in gence of these Abs does not appear to have an immediate effect, this sequence, it is hypothesized that HLA molecules contain- suggesting the requirement for an additional factor involved in ing the SE would preferentially bind peptides containing a neg- disease onset. atively charged or nonpolar amino acid in anchor position 4. Therefore, we propose a “multiple hit model” for RA (Fig. 2) Intriguingly, when the patient cohort in our studies was strati- in which environmental and genetic factors have to come to- fied for ACPA status, it was found that the SE-encoding HLA gether within one individual for induction and progression of alleles only associate with ACPA-positive disease but not with the disease and in which ACPA are associated both with an in- ACPA-negative RA (Ref. 44 and our article in press). These creased risk for developing RA and with progression to erosive data indicate that these HLA alleles do not associate with RA as disease. Considering the clinical association data and the im- such, but rather with a defined disease phenotype and suggest munological data published thus far, we have elaborated a two- that the SE alleles predispose for ACPA positivity rather than step model attempting to explain the development of RA. for RA. Studies in mice transgenic for the HLA allele DRB1*0401 Step one: induction of ACPA indicate how SE alleles may be involved in initiating an auto- For the induction of a response against citrullinated Ags, acti- immune response to citrullinated self-Ags. Conversion of argi- vation of both B cells specific for citrullinated Ags and, most nine to citrulline in a selected model peptide, in the position likely, Th cells has to occur. Citrullination has been described as interacting with the SE, increased peptide-MHC binding affin- a physiological process occurring during apoptosis at multiple Downloaded from ϩ ity, and led to the efficient activation of CD4 T cells in these sites in the body (22, 47, 48). This process is believed to involve mice. Although the general relevance of these findings needs mainly intracellular proteins, which need to be unfolded to be- further confirmation, these data indicate that citrullination come more accessible to degradation by proteases (23) and most could influence and antigenicity of pro- probably does not lead to an immune response against citrulli- teins (4). nated Ags. Citrullination can, in contrast, occur also during in-

These observations, combined with the finding that SE al- flammation (28, 29). Considering that citrullinated proteins http://www.jimmunol.org/ leles are highly correlated with production of ACPAs in RA pa- are attractive targets for the when presented in tients, strongly support the hypothesis that these HLA SE-con- a proinflammatory environment, this could lead to the induc- ϩ taining molecules play a role in the activation of CD4 T cells tion of effector T cells providing help to B cells specific for cit- through preferential presentation of citrullinated Ags. These rullinated Ags. Moreover, inflammation could also result in the “citrulline”-specific T cells may provide the help required for generation of citrullinated neoepitopes, which, in contrast to the IgG Ab response to citrullinated Ags. the physiologically generated citrullinated proteins, occur ex- tracellularly, and are thus “visible” for B cells specific for citrul- A two-step model of ACPA in the pathogenesis of RA

linated Ags. Fibrin is the main extracellular citrullinated protein by guest on September 23, 2021 A remarkable feature of ACPAs is that they may appear before identified thus far in the synovial extract from the inflamed the onset of clinical disease. Once they appear, the probability joint and is, as such, a prominent synovial candidate Ag in of developing RA is very high, suggesting an intimate relation- ACPA-positive RA (30). However, the presence of citrullinated ship between the immunity against citrullinated Ags and dis- fibrin is not specific for RA, but rather a result of inflammation ease. However, considering that these Abs can persist for years (28, 29, 49). Nevertheless, despite the fact that inflammation is without apparent arthritis, it is likely that the production of relatively abundant in everyone’s daily life, only Ͻ1% of the ACPA and development of disease are two distinct events (29, population develops ACPA. Therefore, it is conceivable that an 46). Indeed, while the presence of ACPAs is intimately associ- accumulation of genetic and environmental factors is necessary ated with RA, it is not required for disease induction, as ϳ20% for a response to citrullinated Ags to develop.

FIGURE 2. Multiple hit model for RA. 5578 BRIEF REVIEW: THE EMERGING ROLE OF ANTICITRULLINE AUTOIMMUNITY IN RA

For example, it is possible that citrullination of proteins as a In many of the small animal models of human RA, such as in result of inflammation or inflicted by environmental factors will the K/BxN and the collagen-induced arthritis mouse model, initiate an HLA class II-restricted T cell response (Fig. 3; nos. 1 immune complexes play an important role in autoantibody- and 2 uptake and presentation) only in a genetically predis- mediated autoimmunity (8). In addition, in both models, joint- posed (e.g., SE-positive) individual. It is, in this respect, intrigu- specific Ag expression is not a requirement for joint-specific dis- ing that the environmental factor smoking is associated with an ease. Type II collagen, the autoantibody target in collagen- increased risk to develop ACPA-positive RA only in subjects induced arthritis, is not only present in the joint but also in that are SE positive, pointing to a clear gene-environment in- tracheal and bronchial , the vitreous humor of the eye, teraction involved in the development of ACPA-positive disease and the cartilage of the ear. Nevertheless, anti-type II collagen (50). Other factors, such as an altered negative selection and/or Abs cause disease in the joints (54). This is even more pro- activation of B or T cells could also play a role in the emergence nounced in the K/BxN model where autoantibodies to glucose- of an immune response against citrullinated Ags. An altered re- 6-phosphate isomerase (GPI) cause arthritis, despite the fact sponsiveness could be a consequence of a mutation in the pro- that GPI is present in virtually every organ (55). tein tyrosine phosphatase PTPN22, a negative regulator of lym- After injection in healthy animals, anti-GPI Abs rapidly ac- phocyte activation, which predisposes to multiple autoimmune cumulate in the joints (56). This process is dependent on the diseases, including RA (51). presence of mast cells, , complement receptors, and The help provided by T cells allows for maturation and class FcRs and is related to the fact that anti-GPI Abs form immune switching by B cells, which results in a further maturation of the complexes with GPI in serum (57). Similarly, anti-type II col- Downloaded from ACPA IgG Ab response (Fig. 3; no. 3 autoantibody produc- lagen Abs do not accumulate in the distal joints, but do so after tion). This response specific for citrullinated Ags, induced coinjection of irrelevant, preformed immune complexes. Inter- either in the lymph nodes draining the joints or other sites of estingly, control autoantibodies localize to the joint in a similar inflammation, can become pathogenic once citrullinated Ags manner, but their presence is of limited duration. Only Abs are generated in the joint and ACPA are able to enter the joint. against Ags expressed in the joint, such as GPI and type II col-

lagen persist in the joint, and cause arthritis. These observations http://www.jimmunol.org/ Immune complexes allow autoantibodies access to the joints are intriguing, as they indicate that 1) the presence of circulat- Several clinical observations indicate that the joint is an organ ing immune complexes mediates Ab access specifically to the sensitive to systemic inflammatory reactions. For instance, ar- joints and 2) the presence of Ag ensures persistence of Ab in the thritis is one of the presenting features of - joint. Moreover, they could explain why ACPAs are present associated complications of meningococcal disease in children sometimes years before clinical signs of disease, as arthritis will (52). Moreover, in , immune complex deposi- not develop until articular Ags will be expressed and Abs will tion and the subsequent inflammatory response not only cause have access to the joints. The latter could occur when circulat-

skin lesions but often also (transient) arthritis (53). ing (irrelevant) immune complexes such as RF are produced, by guest on September 23, 2021

FIGURE 3. Schematic representation of the potential contri- bution of ACPA to inflammation in the RA joint. A, Citrullinated proteins (C), produced in the joint or elsewhere, are taken up by APCs (1) and, if generated in an inflammatory environment, are presented to T cells (T) by activated dendritic cells in the drain- ing lymph node. This will promote the induction of a T cell response (2). T cells provide help to B cells specific for citrul- linated Ags (B) that will subsequently produce high-affinity IgG ACPAs (red Abs) (3). The Abs are allowed to enter the joint when a local inflammation occurs or as a result of immune com- plex-facilitated vascular leakage (4). Gra, ; M, mac- rophage/; f, irrelevant Ag; black Ab, Ab to irrelevant Ag. B, During inflammation or after trauma, monocytes (M), and (Gra) migrate into the synovium of the joint. During cell death (5), the membrane integrity of these cells is lost, and PAD enzymes become activated due to extracellular calcium in- flux, leading to citrullination of synovial proteins (6). In the joint, ACPAs, bound to citrullinated Ags (7), activate complement and attract and trigger granulocytes, monocytes, and mast cells (8). These cells (mainly granulocytes and ) exert their function and die. As a consequence, more PAD becomes acti- vated. The generation of more Ags will drive the inflammation into a vicious circle, contributing to the development of chronic disease. The Journal of Immunology 5579 thereby providing an explanation for the presence of both Table I. Outstanding questions ACPAs and RF Abs in many RA patients (our unpublished data). 1) Using a combination of citrullinated peptides or one citrullinated protein, ACPAs are detected in 60–70% of patients Step two: citrullinated Ags in the target organ with RA (34, 63, 64). Does this imply that a considerable proportion of patients with RA do not produce ACPAs or are The joints are not only sensitive to immune complex-mediated current assays simply not able to detect them? influx of Abs, but are also a site where small occur 2) Current assays generally detect ACPAs of the IgG isotype. Does this indicate the presence of an underlying T cell response, regularly. Bleedings in the joints in patients with hemophilia are and does this imply that this T cell response is specific for daily events and are believed to be due to the extensive vascu- citrullinated Ags? larization of the synovium and to the mechanical stress applied 3) Is blocking PAD enzymes a way to treat RA? Selectively to the joints with every movement (reviewed in Ref. 58). Al- blocking PAD enzymes involved in RA may be beneficial, as this would prevent production of citrullinated proteins. though coagulation will occur in healthy persons, these findings 4) What is the Ag responsible for the generation of the immune do indicate that the joint is a site where spontaneous bleedings response against citrullinated Ags? Identifying the relevant Ag is occur regularly. These could result in hypoxia-induced cell important and could be the basis of a therapeutic intervention, e.g., tolerizing vaccine. death and release of endogenous danger signals, such as uric 5) How, when, and where is the immune response against acid (59) and high-mobility group box 1 (60), which will alarm citrullinated proteins generated. Or, how, when and where is the immune system and attract at least a few immune cells (e.g., tolerance broken? neutrophils). Especially granulocytes, which express PAD4, are 6) Does immunity against citrullinated Ags cause arthritis in Downloaded from animals? known to have a high turnover under inflammatory conditions (Fig. 3; no. 4 cell death) (61). PAD enzymes are normally present in an inactive state, as they require high concentrations of calcium for activation. Although in living cells the intracel- Valuable information may come from follow-up studies in lular concentration of calcium is ϳ100 times lower than the healthy individuals harboring ACPAs. Analyses of their B and T threshold concentration for activation of PAD (62), the situa- cell responses against citrullinated Ags might give important in- http://www.jimmunol.org/ tion in dying cells is likely to be completely different. During sights into the events resulting in full-blown arthritis. At the cell death, the integrity of the cell membrane is disrupted, re- same time, these individuals are the ones most likely to benefit sulting in an influx of extracellular calcium and subsequent from Ag-specific intervention strategies. If ACPAs truly are in- PAD activation within the cell (22). Likewise, PAD enzymes volved in the pathogenesis of RA, Ag-specific interventions may may leak out of the cell and become activated (the normal ex- prevent chronic arthritis and long-term joint destruction with- tracellular calcium concentration is above the threshold), lead- out the side effects associated with today’s treatment regiments. ing to citrullination of the extracellular matrix proteins and thereby generating the target Ag for ACPAs. References by guest on September 23, 2021 1. Marrack, P., J. Kappler, and B. L. Kotzin. 2001. Autoimmune disease: why and where PAD4 and PAD2 are expressed by granulocytes, monocytes, it occurs. Nat. Med. 7: 899–905. and macrophages. Recruitment of granulocytes and monocytes 2. Rose, N. R., and C. Bona. 1993. Defining criteria for autoimmune diseases (Witeb- sky’s postulates revisited). Immunol. Today 14: 426–430. to an inflamed joint, followed by their demise will most prob- 3. Witebsky, E., N. R. Rose, K. Terplan, J. R. Paine, and R. W. Egan. 1957. Chronic ably result in the activation of these two enzymes, allowing the thyroiditis and autoimmunization. J. Am. Med. Assoc. 164: 1439–1447. citrullination of intra- and extracellular proteins including ex- 4. Hill, J. A., S. Southwood, A. Sette, A. M. Jevnikar, D. A. Bell, and E. Cairns. 2003. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide tracellular fibrin (Fig. 3; no. 5 citrullination), which is gener- interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II ated upon activation of the coagulation system. Recognition of molecule. J. Immunol. 171: 538–541. 5. Borza, D. B., E. G. Neilson, and B. G. Hudson. 2003. Pathogenesis of Goodpasture citrullinated proteins by ACPAs leads to formation of immune syndrome: a molecular perspective. Semin. Nephrol. 23: 522–531. complexes and activation of complement, with subsequent re- 6. Hughes, B. W., M. L. Moro De Casillas, and H. J. Kaminski. 2004. Pathophysiology of . Semin. Neurol. 24: 21–30. lease of C5a and further attraction and triggering of granulo- 7. Ji, H., K. Ohmura, U. Mahmood, D. M. Lee, F. M. Hofhuis, S. A. Boackle, cytes, monocytes, macrophages, and mast cells through both K. Takahashi, V. M. Holers, M. Walport, C. Gerard, et al. 2002. Arthritis critically complement receptor- and Fc␥R-dependent pathways (Fig. 3; dependent on players. Immunity 16: 157–168. 8. Monach, P. A., C. Benoist, and D. Mathis. 2004. The role of in mouse no. 6 immune complex formation). models of rheumatoid arthritis, and relevance to human disease. Adv. Immunol. 82: Now the immune response has entered a vicious circle, in 217–248. 9. Dorner, T., K. Egerer, E. Feist, and G. R. Burmester. 2004. Rheumatoid factor revis- which inflammation causes even more Ag to be made, with pos- ited. Curr. Opin. Rheumatol. 16: 246–253. sible perpetuation of the response as the result. 10. Nienhuis, R. L., and E. Mandema. 1964. A new serum factor in patients with rheu- matoid arthritis; the antiperinuclear factor. Ann. Rheum. Dis. 23: 302–305. 11. Young, B. J., R. K. Mallya, R. D. Leslie, C. J. Clark, and T. J. Hamblin. 1979. Anti- Concluding remarks keratin antibodies in rheumatoid arthritis. Br. Med. J. 2: 97–99. 12. Miossec, P., P. Youinou, P. Le Goff, and M. P. Moineau. 1982. Clinical relevance of ACPA are unique and predictive for RA and citrullinated pro- antikeratin antibodies in rheumatoid arthritis. Clin. Rheumatol. 1: 185–189. teins are found in the inflamed joint. This response fulfills thus 13. Sondag-Tschroots, I. R., C. Aaij, J. W. Smit, and T. E. Feltkamp. 1979. The antipe- rinuclear factor. 1. The diagnostic significance of the antiperinuclear factor for rheu- the first two criteria of Witebsky postulates of autoimmunity matoid arthritis. Ann. Rheum. Dis. 38: 248–251. and points toward a role for the response against citrullinated 14. Aho, K., R. von Essen, P. Kurki, T. Palosuo, and M. Heliovaara. 1993. Antikeratin Ags in the pathogenesis of RA. To fulfill all of the Witebsky and antiperinuclear factor as markers for subclinical rheumatoid disease pro- cess. J. Rheumatol. 20: 1278–1281. postulates, induction of disease upon generation of an immune 15. Vincent, C., G. Serre, F. Lapeyre, B. Fournie, C. Ayrolles, A. Fournie, and response against citrullinated Ags in experimental animal mod- J. P. Soleilhavoup. 1989. High diagnostic value in rheumatoid arthritis of antibodies to the stratum corneum of rat oesophagus epithelium, so-called “antikeratin antibod- els is required. Although this would represent an important step ies.” Ann. Rheum. Dis. 48: 712–722. forward, it will mean that we are still only beginning to under- 16. Simon, M., E. Girbal, M. Sebbag, V. Gomes-Daudrix, C. Vincent, G. Salama, and G. Serre. 1993. The cytokeratin filament-aggregating protein filaggrin is the target of stand RA and the role of the citrullinated Ag-specific immunity the so-called “antikeratin antibodies,” autoantibodies specific for rheumatoid arthritis. (Table I). J. Clin. Invest. 92: 1387–1393. 5580 BRIEF REVIEW: THE EMERGING ROLE OF ANTICITRULLINE AUTOIMMUNITY IN RA

17. Schellekens, G. A., B. A. de Jong, F. H. van den Hoogen, L. B. van de Putte, and with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum. 50: W. J. van Venrooij. 1998. Citrulline is an essential constituent of antigenic determi- 709–715. nants recognized by rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 101: 40. Suzuki, A., R. Yamada, X. Chang, S. Tokuhiro, T. Sawada, M. Suzuki, M. Nagasaki, 273–281. M. Nakayama-Hamada, R. Kawaida, M. Ono, et al. 2003. Functional haplotypes of 18. Girbal-Neuhauser, E., J. J. Durieux, M. Arnaud, P. Dalbon, M. Sebbag, C. Vincent, PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated M. Simon, T. Senshu, C. Masson-Bessiere, C. Jolivet-Reynaud, et al. 1999. The with rheumatoid arthritis. Nat. Genet. 34: 395–402. targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies 41. Barton, A., J. Bowes, S. Eyre, K. Spreckley, A. Hinks, S. John, and J. Worthington. are posttranslationally generated on various sites of (pro)filaggrin by deimination of 2004. A functional haplotype of the PADI4 gene associated with rheumatoid arthritis arginine residues. J. Immunol. 162: 585–594. in a Japanese population is not associated in a United Kingdom population. Arthritis 19. Sebbag, M., M. Simon, C. Vincent, C. Masson-Bessiere, E. Girbal, J. J. Durieux, and Rheum. 50: 1117–1121. G. Serre. 1995. The antiperinuclear factor and the so-called antikeratin antibodies are 42. Caponi, L., E. Petit-Teixeira, M. Sebbag, F. Bongiorni, S. Moscato, F. Pratesi, the same rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 95: 2672–2679. C. Pierlot, J. Osorio, S. Chapuy-Regaud, M. Guerrin, et al. 2005. A family based study 20. Vossenaar, E. R., N. Despres, E. Lapointe, A. van der Heijden, M. Lora, T. Senshu, shows no association between rheumatoid arthritis and the PADI4 gene in a white W. J. van Venrooij, and H. A. Menard. 2004. Rheumatoid arthritis specific anti-Sa French population. Ann. Rheum. Dis. 64: 587–593. antibodies target citrullinated . Arthritis Res. Ther. 6: R142–R150. 43. Hoppe, B., G. A. Heymann, F. Tolou, H. Kiesewetter, T. Doerner, and A. Salama. 21. Union, A., L. Meheus, R. L. Humbel, K. Conrad, G. Steiner, H. Moereels, H. Pottel, 2004. High variability of peptidylarginine deiminase 4 (PADI4) in a healthy white G. Serre, and F. De Keyser. 2002. Identification of citrullinated rheumatoid arthritis- population: characterization of six new variants of PADI4 exons 2–4 by a novel hap- specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection lotype-specific sequencing-based approach. J. Mol. Med. 82: 762–767. by line immunoassay. Arthritis Rheum. 46: 1185–1195. 44. van Gaalen, F. A., J. van Aken, T. W. Huizinga, G. M. Schreuder, F. C. Breedveld, 22. Asaga, H., M. Yamada, and T. Senshu. 1998. Selective deimination of vimentin in E. Zanelli, W. J. van Venrooij, C. L. Verweij, R. E. Toes, and R. R. de Vries. 2004. calcium -induced apoptosis of mouse peritoneal macrophages. Biochem. Association between HLA class II genes and autoantibodies to cyclic citrullinated pep- Biophys. Res. Commun. 243: 641–646. tides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum. 50: 23. Tarcsa, E., L. N. Marekov, G. Mei, G. Melino, S. C. Lee, and P. M. Steinert. 1996. 2113–2121. Protein unfolding by peptidylarginine deiminase: substrate specificity and structural 45. Zanelli, E., F. C. Breedveld, and R. R. de Vries. 2000. HLA class II association with relationships of the natural substrates trichohyalin and filaggrin. J. Biol. Chem. 271: rheumatoid arthritis: facts and interpretations. Hum. Immunol. 61: 1254–1261. 30709–30716. 46. Vossenaar, E. R., and W. J. van Venrooij. 2004. Citrullinated proteins: sparks that Downloaded from 24. Wang, Y., J. Wysocka, J. Sayegh, Y. H. Lee, J. R. Perlin, L. Leonelli, L. S. Sonbuchner, may ignite the fire in rheumatoid arthritis. Arthritis Res. Ther. 6: 107–111. C. H. McDonald, R. G. Cook, Y. Dou, et al. 2004. Human PAD4 regulates histone 47. Tsuji, Y., M. Akiyama, K. Arita, T. Senshu, and H. Shimizu. 2003. Changing pattern arginine methylation levels via demethylimination. Science 306: 279–283. of deiminated proteins in developing human epidermis. J. Invest. Dermatol. 120: 25. Cuthbert, G. L., S. Daujat, A. W. Snowden, H. Erdjument-Bromage, T. Hagiwara, 817–822. M. Yamada, R. Schneider, P. D. Gregory, P. Tempst, A. J. Bannister, and 48. Wood, D. D., and M. A. Moscarello. 1989. The isolation, characterization, and lipid- T. Kouzarides. 2004. Histone deimination antagonizes arginine methylation. Cell aggregating properties of a citrulline containing basic protein. J. Biol. Chem. 118: 545–553. 264: 5121–5127.

26. Vossenaar, E. R., A. J. Zendman, W. J. van Venrooij, and G. J. Pruijn. 2003. PAD, a 49. Vossenaar, E. R., S. Nijenhuis, M. M. Helsen, H. A. Van Der, T. Senshu, http://www.jimmunol.org/ growing family of citrullinating enzymes: genes, features and involvement in disease. W. B. van den Berg, W. J. van Venrooij, and L. A. Joosten. 2003. Citrullination of Bioessays 25: 1106–1118. synovial proteins in murine models of rheumatoid arthritis. Arthritis Rheum. 48: 27. Vossenaar, E. R., T. R. Radstake, A. van der Heijden, M. A. van Mansum, 2489–2500. C. Dieteren, D. J. de Rooij, P. Barrera, A. J. Zendman, and W. J. van Venrooij. 2004. 50. Linn-Rasker, S. P., A. H. van der Helm-van Mil, F. A. van Gaalen, M. Kloppenburg, Expression and activity of citrullinating peptidylarginine deiminase enzymes in mono- R. de Vries, S. le Cessie, F. C. Breedveld, R. E. Toes, and T. W. Huizinga. 2005. cytes and macrophages. Ann. Rheum. Dis. 63: 373–381. Smoking is a risk factor for anti-CCP antibodies only in RA patients that carry HLA- 28. Vossenaar, E. R., T. J. Smeets, M. C. Kraan, J. M. Raats, W. J. van Venrooij, and DRB1 shared epitope alleles. Ann. Rheum. Dis. In press. P. P. Tak. 2004. The presence of citrullinated proteins is not specific for rheumatoid 51. Gregersen, P. K. 2005. Pathways to gene identification in rheumatoid arthritis: synovial tissue. Arthritis Rheum. 50: 3485–3494. PTPN22 and beyond. Immunol. Rev. 204: 74–86. 29. Chapuy-Regaud, S., M. Sebbag, D. Baeten, C. Clavel, C. Foulquier, F. De Keyser, 52. Goedvolk, C. A., I. A. von Rosenstiel, and A. P. Bos. 2003. Immune complex associ- and G. Serre. 2005. Fibrin deimination in synovial tissue is not specific for rheumatoid ated complications in the subacute phase of meningococcal disease: incidence and lit-

arthritis but commonly occurs during synovitides. J. Immunol. 174: 5057–5064. erature review. Arch. Dis. Child. 88: 927–930. by guest on September 23, 2021 30. Masson-Bessiere, C., M. Sebbag, E. Girbal-Neuhauser, L. Nogueira, C. Vincent, 53. Lawley, T. J., L. Bielory, P. Gascon, K. B. Yancey, N. S. Young, and M. M. Frank. T. Senshu, and G. Serre. 2001. The major synovial targets of the rheumatoid arthritis- 1984. A prospective clinical and immunologic analysis of patients with serum sickness. specific antifilaggrin autoantibodies are deiminated forms of the ␣- and ␤-chains of N. Engl. J. Med. 311: 1407–1413. fibrin. J. Immunol. 166: 4177–4184. 54. Nandakumar, K. S., L. Svensson, and R. Holmdahl. 2003. Collagen type II-specific 31. Reparon-Schuijt, C. C., W. J. van Esch, C. van Kooten, G. A. Schellekens, -induced arthritis in mice: description of the disease and the in- B. A. de Jong, W. J. van Venrooij, F. C. Breedveld, and C. L. Verweij. 2001. Secretion fluence of age, sex, and genes. Am. J. Pathol. 163: 1827–1837. of anti-citrulline-containing peptide antibody by B in rheumatoid arthri- 55. Kouskoff, V., A. S. Korganow, V. Duchatelle, C. Degott, C. Benoist, and D. Mathis. tis. Arthritis Rheum. 44: 41–47. 1996. Organ-specific disease provoked by systemic autoimmunity. Cell 87: 811–822. 32. Masson-Bessiere, C., M. Sebbag, J. J. Durieux, L. Nogueira, C. Vincent, 56. Wipke, B. T., Z. Wang, J. Kim, T. J. McCarthy, and P. M. Allen. 2002. Dynamic E. Girbal-Neuhauser, R. Durroux, A. Cantagrel, and G. Serre. 2000. In the rheuma- visualization of a joint-specific autoimmune response through positron emission to- toid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and con- mography. Nat. Immunol. 3: 366–372. stitute a higher proportion of IgG than in synovial fluid and serum. Clin. Exp. Immu- 57. Wipke, B. T., Z. Wang, W. Nagengast, D. E. Reichert, and P. M. Allen. 2004. Staging nol. 119: 544–552. the initiation of autoantibody-induced arthritis: a critical role for immune complexes. 33. Miossec, P., P. Youinou, P. Le Goff, and M. P. Moineau. 1982. Clinical relevance of J. Immunol. 172: 7694–7702. antikeratin antibodies in rheumatoid arthritis. Clin. Rheumatol. 1: 185–189. 58. Dahlback, B. 2005. Blood coagulation and its regulation by anticoagulant pathways: 34. Schellekens, G. A., H. Visser, B. A. de Jong, F. H. van den Hoogen, J. M. Hazes, genetic pathogenesis of and thrombotic diseases. J. Intern. Med. 257: F. C. Breedveld, and W. J. van Venrooij. 2000. The diagnostic properties of rheuma- 209–223. toid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum. 43: 59. Shi, Y., J. E. Evans, and K. L. Rock. 2003. Molecular identification of a danger signal 155–163. that alerts the immune system to dying cells. Nature 425: 516–521. 35. Wener, M. H., K. Hutchinson, C. Morishima, and D. R. Gretch. 2004. Absence of 60. Scaffidi, P., T. Misteli, and M. E. Bianchi. 2002. Release of chromatin protein antibodies to cyclic citrullinated peptide in sera of patients with virus in- HMGB1 by necrotic cells triggers inflammation. Nature 418: 191–195. fection and . Arthritis Rheum. 50: 2305–2308. 61. Savill, J. S., A. H. Wyllie, J. E. Henson, M. J. Walport, P. M. Henson, and C. Haslett. 36. Palosuo, T., R. Tilvis, T. Strandberg, and K. Aho. 2003. Filaggrin related antibodies 1989. phagocytosis of aging neutrophils in inflammation: programmed among the aged. Ann. Rheum. Dis. 62: 261–263. cell death in the leads to its recognition by macrophages. J. Clin. Invest. 83: 37. Rantapaa-Dahlqvist, S., B. A. de Jong, E. Berglin, G. Hallmans, G. Wadell, 865–875. H. Stenlund, U. Sundin, and W. J. van Venrooij. 2003. Antibodies against cyclic 62. Nakayama-Hamada, M., A. Suzuki, K. Kubota, T. Takazawa, M. Ohsaka, citrullinated peptide and IgA rheumatoid factor predict the development of rheuma- R. Kawaida, M. Ono, A. Kasuya, H. Furukawa, R. Yamada, and K. Yamamoto. 2005. toid arthritis. Arthritis Rheum. 48: 2741–2749. Comparison of enzymatic properties between hPADI2 and hPADI4. Biochem. Bio- 38. Nielen, M. M., D. van Schaardenburg, H. W. Reesink, R. J. van de Stadt, phys. Res. Commun. 327: 192–200. I. E. van der Horst-Bruinsma, M. H. de Koning, M. R. Habibuw, 63. Vincent, C., L. Nogueira, M. Sebbag, S. Chapuy-Regaud, M. Arnaud, O. Letourneur, J. P. Vandenbroucke, and B. A. Dijkmans. 2004. Specific autoantibodies precede the D. Rolland, B. Fournie, A. Cantagrel, M. Jolivet, and G. Serre. 2002. Detection of symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. antibodies to deiminated recombinant rat filaggrin by enzyme-linked immunosorbent Arthritis Rheum. 50: 380–386. assay: a highly effective test for the diagnosis of rheumatoid arthritis. Arthritis Rheum. 39. van Gaalen, F. A., S. P. Linn-Rasker, W. J. van Venrooij, B. A. de Jong, 46: 2051–2058. F. C. Breedveld, C. L. Verweij, R. E. Toes, and T. W. Huizinga. 2004. Autoantibodies 64. Lee, D. M., and P. H. Schur. 2003. Clinical utility of the anti-CCP assay in patients to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with rheumatic diseases. Ann. Rheum. Dis. 62: 870–874.