Adaptive immunity in rheumatoid arthritis: anticitrulline and other antibodies in the pathogenesis of rheumatoid arthritis

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1 REVIEW C URRENT OPINION Adaptive immunity in rheumatoid arthritis: anticitrulline and other antibodies in the pathogenesis of rheumatoid arthritis Lars Klareskog, Khaled Amara, and Vivianne Malmström Purpose of review To describe recent progress concerning rheumatoid arthritis (RA)-associated autoantibodies, in particular antibodies to citrullinated proteins antigens. Recent findings An increasingly diverse and RA-associated repertoire of antibodies has been defined over the last few years. These antibodies are preferentially, but not exclusively, reactive with posttranslationally modified antigens. Citrullinated antigens are the most common targets, but also other modifications including homocitrullination (carbamylation) are recognized. These antibodies display varying degrees of crossreactivity, and both broadly cross-reactive and monoreactive antibodies are present. Progress, described in this review, has been made both concerning mechanisms behind the generation of these antibodies and concerning their effector functions. Summary Several different triggering mechanisms are involved in forming an antibody repertoire that evolves before the onset of clinical disease, and where antibodies with different specificities may interact directly or indirectly with target organs in causing different arthritis-associated symptoms. The increasing understanding of the role of adaptive and specific immunity in RA creates opportunities for a new generation of interventions. Keywords anticitrulline protein antibodies, cartilage-derived autoantigens, immunotherapy, posttranslational modifications INTRODUCTION Experience from animal experiments has taught us that there are many different routes to arthritis, where some involve adaptive immunity and others depend on aberrant regulation of innate immunity [1]. Aberrant immunity contributing to arthritis may have many different targets, involving ubiquitous antigens such as glucose phosphate isomerase (GPI) [2], as well as organ-specific antigens including a series of cartilage-derived proteins [3,4]. Notably, though, man-made genetic manipulations or immunization protocols are most often involved in the generation of these models, demonstrating that these animal experiments visualize potential pathways that may be involved in disease, but do not provide any information on whether these pathways are ever activated in humans. Important lessons from these models are, however, that specific immunity associated with arthritis may both induce disease and protect against disease, and that different specific immune reactions may cause joint inflammation as well as other arthritisassociated symptoms such as pain [5 ] or osteopenia [6 ]. A key message for this review is thus that there may be many routes to arthritis also in humans, and that we have to consider immunities directed toward several different antigens, organ-specific as well as ubiquitous. Furthermore, we have to acknowledge that observed arthritis-associated immunities in humans may be disease inducing, Rheumatology Unit, Department of Medicine, KarolinskaInstitutet/Karolinska University Hospital, Stockholm, Sweden Correspondence to Lars Klareskog, Rheumatology Unit, Department of Medicine, KarolinskaInstitutet/Karolinska University Hospital, Stockholm , Sweden. Tel: / ; lars.klare skog@ki.se Curr Opin Rheumatol 2014, 26:72 79 DOI: /BOR Volume 26 Number 1 January 2014 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

2 Adaptive immunity in rheumatoid arthritis Klareskog et al. KEY POINTS ACPAs and other autoantibodies are associated with different subsets of RA. Antibodies with different specificities are associated with different genetic variants and with different environmental factors. ACPAs and other autoantibodies present in RA patients have different fine specificities and different degrees of cross-reactivity with various citrullinated and/or otherwise posttranslationally modified peptides/ proteins. Different ACPAs and other RA-associated antibodies have different capacities to cause and/or contribute to different arthritis-associated symptoms, and specific mechanisms associated with a second signal may be needed before ACPAs contribute to disease symptoms. ACPAs and other RA-associated autoantibodies may cause disease symptoms, be neutral or protect against disease. Improved understanding of these aspects of adaptive immunity in RA provides new opportunities for prevention and therapy. protective or not involved at all in the disease process. The focus in this section is on immunity toward antigens that are posttranslationally modified by citrullination, but also a series of other adaptive immunities will be discussed more briefly as they may provide alternative ways of triggering or protecting against arthritis. ANTICITRULLINE AND OTHER SPECIFIC IMMUNITIES ASSOCIATED WITH RHEUMATOID ARTHRITIS Since the original description of antibodies to citrullinated antigens in a subpopulation of RA patients [7,8], it has become clear that citrullinated epitopes of a large number of autoantigens as well as antigens derived from microorganisms can be recognized by antibodies that are reasonably specific for RA. Thus, a fast-growing literature has demonstrated antibodies in RA patients against citrullinated extracellular and/or cell surface bound ubiquitous molecules such as fibrinogen, vimentin and alpha-enolase (see review [9]), against cartilagespecific molecules such as collagen II [10], against stress proteins such as BiP [11] against enzymes such as peptidylarginine deiminases (PADs) that mediate citrullination [12] and intracellular molecules such as histones [13 ] and also to GPI [14]. Also reactivities against bacterial-derived molecules, such as porphyromonasgingivalis-derived enolase and PADs, have been observed [15,16 ]. More recently, antibodies also against other posttranslationally modified antigens, such as carbamylated (homocitrullination of lysine) proteins have been described [16 ]. Antibodies to the different citrullinated and carbamylated antigens show a certain degree of cross-reactivity, and with more extensive studies [17,18 ] a complex pattern is now emerging with some antibodies being broadly cross-reactive between different citrullinated and some carbamylated antigens, others being much more private. These observations thus add to previous knowledge of reactivities in RA against nonmodified (auto) antigens with rheumatoid factors described as early as 1939 as prime example, but also including collagen type II and other cartilage-derived molecules and GPI. In most of these cases, however, specificity for RA is less than in the case of anticitrulline immunity. Notably, additional antibody reactivities were recently described against other modifications of collagen II [19]. Taken together, these data demonstrate the tendency of the immune system to recognize posttranslationally modified self-antigens. Also, a large number of different antigens are recognized by the antibody repertoire in RA and, most likely, additional immunities will be discovered. A major current task for research into autoimmunity and RA is subsequently to understand why and how the described autoimmunities are triggered, how they relate to the pathogenesis of different subsets of RA, and how to design strategies for the detection of additional adaptive immune reactions, which may be involved in the development or protection against RA. Below follow some comments on recent progress in these areas. INDUCTION AND REGULATION OF RA- RELATED ADAPTIVE IMMUNITY A key observation concerning the relationship between adaptive immunity and RA is that most of the antibody reactivities described above, including Anti-citrullinated protein antibodies (ACPAs) as well as antibodies to carbamylated antigens, rheumatoid factors and other nonmodified antigens occur before the onset of disease [20,21 ], and in most individuals the autoantibody repertoire is developed already at the onset of disease, with very few individuals becoming autoantibody positive later during the disease course [22]. Recent studies [23,24,25 ] using different multiplex technologies have shown that these autoantibodies emerge gradually, with a clear spreading of reactivities from one initially recognized epitope toward reactivities to many different epitopes closer to disease onset. So far, no consistent pattern of timing of the appearance of antibodies has been seen, and we therefore have few clues as to any common original initiating ß 2013 Wolters Kluwer Health Lippincott Williams Wilkins 73 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

3 Medical physiology and rheumatic diseases immunity. Instead, certain patterns of antibodies have been seen closer to disease onset suggesting that certain combinations of autoimmunities may be particularly arthritogenic [25 ]. Of particular interest has been the observation that a combination of high titers of ACPAs and the presence of antibodies to several citrullinated epitopes together with certain clinical symptoms, in particular arthralgia, provide a high likelihood for the development of clinical arthritis in the near future [26]. At the same time, it is has become clear that only a minority of those individuals who develop anticitrulline immunity will develop arthritis, and that a large proportion also of those ACPApositive individuals with arthralgia will never develop RA [26,27 ]. Thus, we need to understand what determines the emergence of the autoimmunity to citrullinated and other autoantigens; which factors determine who of the autoantibody-positive individuals will develop arthritis; and who may eventually actively be protected against arthritis. A very interesting recent observation is that potentially modifiable factors such as obesity and smoking are highly predictive for which ACPA-positive nonarthritis patients with arthralgia will subsequently develop clinical arthritis [27 ]. Autoimmunity against citrullinated autoantigens may develop as a result of gene environment interactions, in which smoke and other airway irritants cause an increased expression of the citrullinated autoantigens in lungs, in parallel with the activation of antigen-presenting cells in the same compartment (see recent reviews [28,29]). This leads in some individuals to a local activation of immunity against citrullinated antigens in the lungs, with formation of local germinal centers in bronchi and to local production of ACPAs in lungs [30]. Whereas this mechanism generates immunity toward certain epitopes of citrullinated autoantigens, immunity against other epitopes is not related to smoke exposure [18,31 ]. This issue is not yet fully resolved for carbamylated antigens. Thus, there are clearly different mechanisms behind the development of different specific RA-associated immune reactions. A particularly interesting and recently described putative mechanism for a spread of immunity to certain citrullinated antigens, was the recognition that antibodies that cross-react with PAD3 and PAD4, greatly enhance the enzymatic activity of PAD4, thereby creating a positive feedback system for increased citrullination at sites where the anti- PAD4 antibodies are produced [32 ]. Still other mechanisms of initiation of immunity to citrullinated antigens have been proposed, in particular a classical molecular mimicry - based mechanism in which periodontitis-associated Porphyromonas gingivalis bacteria have been shown to produce citrullinated enolase in which antibodies generated against this bacterial product may crossreact with related endogenous citrullinated alphaenolase peptides [15]. Mechanisms that may be responsible for other autoimmunities such as anticartilage immunity are so far less extensively investigated and little is known about the mechanisms that may trigger these immunities. So, in conclusion, immunity to citrullinated antigens represent a prototypic mechanisms for triggering of immunity to posttranslatonally modified proteins. Mechanisms that are involved in controlling such immunity at the level of T cells, that is thymic tolerance and peripheral tolerance are so far mostly unknown. Thus, it is not known to what extent citrullination and negative selection of T cells reactive with citrullinated self-peptides occur in thymus. T cells with reactivity to citrullinated peptides have indeed been demonstrated to a larger extent in RA patients than in genotype matched healthy controls [33], but whether such cells are subject to similar types of regulation as T cells to conventional (non-posttranslationally modified) antigens is not yet known. An interesting line of investigation into potential mechanisms behind T-cell activation to citrullinated peptides has been proposed by the group of Ireland and Unanue [34 ] who found that citrullinated peptides, potentially presented to T cells are generated during autophagy. Whereas there is still much work to be done to investigate mechanisms behind breakdown of tolerance and activation of T cells to posttranslationally modified proteins/peptides, an interesting parallel exists to a better characterized situation in the setting of coeliac disease. Here, the main T-cell reactivity is against exogenous gluten antigens, which have been enzymatically modified by tissue transglutaminase. Here, pathogenic T cells mainly recognize such modified exogenous antigen toward which no negative selection occurs in the thymus. In the case of coeliac disease, it is those T cells that provide help to B cells producing autoantibodies against tissue transglutaminase [35]. There is thus a need to characterize the T-cell repertoire in the initiating organs, such as the lung in RA, as well as in target organs, such as the joint, while keeping an open mind to the possibility that T-cell regulation and T-cell targets may differ from classical autoantigens. Thus, it is feasible that the regulation of immunity to posttranslationally modified self-antigens may involve T cells that are not subject to the same restrictions as T-cells reactive with nonmodified self-antigens. As illustrated by the so far described reactivities to P. gingivalis-derived molecules [15,16 ], we also have to consider microbes and 74 Volume 26 Number 1 January 2014 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

4 Adaptive immunity in rheumatoid arthritis Klareskog et al. possibly other external agents (cf. gluten) in our search for triggers of arthritis-related immunity including anticitrulline antibodies. Furthermore, it is feasible that other nonconventional regulatory mechanisms, such as the antibody-mediated enhancement of enzymatic activities previously mentioned [32 ], may additionally contribute to the formation of pathogenic autoimmunity, and that we may consider citrulline autoimmunity as one example of an immunological accident. In such a scenario, it is no surprise that the accident involves the generation of immunity to several different citrullinated proteins. A key issue in such a situation is to understand which of these immunities may be pathogenic, neutral or protective in relation to different disease symptoms. EFFECTOR AND PROTECTOR FUNCTIONS OF AUTOIMMUNITY, IN PARTICULAR ANTICITRULLINE ANTIBODIES, IN THE CONTEXT OF ARTHRITIS-RELATED SYMPTOMS As described above, there are now a large number of autoantibodies with different specificities described in patients with RA, and thus a number of different possibilities whereby such antibodies as single antibodies or in combinations, may be involved in causing different symptoms associated with RA. Comments, here, will be restricted to effects of antibodies, acknowledging that the less well known T-cell reactivities may be crucial not only for help to antibody production but also in directly contributing to disease symptoms. Furthermore, we will not discuss the important potential of immunoglobulin-expressing B cells to function as antigen-presenting cells for T cells. There is now increasing evidence that immune reactions against citrullinated and other antigens is generated locally in joints during arthritis, for example within but probably not restricted to local germinal center-like formations that are common in antibody-positive RA cases (see review [36]). Local antibody production has been demonstrated for a series of citrullinated antigens as well as for collagen II and rheumatoid factors (see reviews in [28,29]). A notable recent addition to this concept has emerged from studies on specificity and structure of monoclonal IgGs generated from single synovial fluidderived B cells. Here, between 16 and 33% of all CD19 þ IgGþ B cells from synovial fluid were shown to produce antibodies specific for different citrullinated antigens [37 ]. These antibodies had different origins (different coding genes), they had undergone multiple mutations, and the B cells producing these antibodies were thus most probably driven toward differentiation by local T cells. They displayed varying fine specificities for different citrullinated autoantigens and were totally nonreactive with the arginine variants of tested self-peptides [37 ]. This study, together with much previous evidence demonstrates that immunity that begins elsewhere, for example in the lungs, may over years progress into a situation in which these same immune reactions occur also in joints, where structures that permit local and T cell-dependent immunity are established. In the case of collagen II autoimmunity, including autoimmunty to citrullinated collagen II, obvious explanations exist as the targets are relatively joint-specific. This is not the case for the immunity to the majority of citrullinated antigens in RA patients, in which we still have no obvious explanation as to why and how immunity to citrullinated, and possibly also carmabylated, antigens sometimes focuses to the joints. Obviously, however, most individuals with anticitrulline immunity never experience this transformation to a state of arthritis, and in most individuals who develop RA, it takes years with existing systemic anticitrulline immunity before the transformation takes place. Thus, it will still be essential to investigate in detail the ways in which different RA-associated autoimmune reactions, in particular the different anticitrulline reactions, may contribute to various disease symptoms in seropositive patients, as well as to better understand the remarkable focusing of anticitrulline immunity to joints. As emphasized above, the best-described effects are for antibodies against collagens including citrullinated collagen type II. Thus, immunization with citrullinated collagen II is more arthritogenic (in rats) than noncitrullinated collagen II [38]. Furthermore, monoclonal mouse antibodies specifically reactive with an immunodominant epitope on citrullinated collagen II cause arthritis upon infusion in mice [4]; interestingly, antibodies against this epitope are quite common in RA patients [10]. Even administration of citrullinated collagen II without adjuvant was recently described to cause arthritis [39]. It is also notable that different anticollagen II antibodies are very different concerning their disease-inducing capacity. A cocktail of several antibodies is more efficient than single antibodies, and at least antibodies against one particular epitope are protective against arthritis [40]. A few studies have indicated that also immunization with citrullinated endogenous fibrinogen [41] or bacterialderived enolase [42] may induce arthritis; these results are of great interest and are awaiting further confirmation and extension ß 2013 Wolters Kluwer Health Lippincott Williams Wilkins 75 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

5 Medical physiology and rheumatic diseases A recently described effect of ACPAs representing a potentially important mechanism in arthritis (Kleyer and Schett, pp of this issue) is the demonstration that antibodies against certain citrullinated antigens, including citrullinated vimentin may cause osteoclast activation [6 ]. If this direct method of activation of a single-cell type can be showntobeuniquefortheosteoclast,thesefindings have obvious implications for the understanding of an early sequence of events in seropositive RA, with the potential that initial inflammatory and osteoclast-dependent events may take place in the bone marrow and in the cartilage bone pannus junction. These events may hypothetically initiate a further spread of inflammation in the synovium in which other cells as well as other immune reactions may contribute to the perpetuation of inflammation. A most obvious question is, however, whether and how the most commonly found anticitrulline antibodies in RA patients, which are directed against noncartilage self-molecules, can activate synovial inflammation in a more direct way, in addition to the effects on osteoclasts. So far, experiments aimed at directly transferring arthritis to mice with the help of anticitrulline antibodies from serum of RA patients have generally failed to directly induce arthritis (our own unpublished observations). Similarly, mouse monoclonal antibodies against citrullinated fibrinogen failed to induce arthritis upon direct transfer experiments in mice, but were able to enhance the development of arthritis when a mild synovitis had been induced with anticollagen II antibodies [43]. These observations are compatible with the fact that ACPAs may indeed exist over many years without causing disease, and that the majority of individuals with ACPAs actually do not develop arthritis. Thus, something in addition to the mere presence of these antibodies appears to be needed to eventually make these ACPAs contribute to the development of synovitis. Here, recently published studies have suggested the need for such an additional mechanism (compatible with a previously proposed second signal [29]). Thus, ACPAs from RA patients were shown to activate the production of neutrophil extracellular traps (NETs) from neutrophils [44 ]. Such NETs contain molecules needed for innate antimicrobial defense including LL37 and some of the molecules ion the NETs are indeed citrullinated [45]. In a report from Khandpur et al. [44 ], these NETs were shown to have potent macrophage activating properties. It is thus feasible that the formation of NETs, possibly as a consequence of an antimicrobial defense in joints might function as an intermediate step between ACPAs and effector functions in the joint. As neutrophil activation and thus release of NETs is a well known feature of many joint inflammatory reactions, this may also be an additional mechanism able to focus the effector functions of anticitrulline immunity and ACPAs to joints. Additional potential effector mechanisms along the same lines are proposed from the observation that citrullinated fibrinogen may bind to receptors, in particular toll-like receptors on fibroblasts, including synovial fibroblasts, and that antibodies to epitopes on the citrullinated fibrinogen may enhance the activation of these cells [46]. This proposed mechanism is indeed similar to the mechanism that has been proposed and widely verified in systemic lupus erythematosus (SLE), where dsdna has been shown to bind to receptors on plasmacytoid dendritic cells and where binding of anti-dsdna antibodies to the cell-bound dsdna, and subsequent engagement of Fc receptors, mediate the production of alpha-interferon from these target cells for the anti-dsdna antibodies [47]. Several additional effector mechanisms remain possible and are currently subject to active investigation in many laboratories. It is feasible that these studies will show that different antibodies may contribute to different symptoms in RA patients, and possibly explain why different patients experience different clinical problems, sometimes dominated by inflammation in joints, in other cases more associated with extra-articular manifestations (including inflammation in lungs), in other cases with very active bone destruction and in still others with pain as a major component. CONCLUSION The interesting scenario now emerging is that immune reactions, including antibodies with different fine specificities, may contribute to different RA-related symptoms, and also that the presence of antibodies with certain specificities or other unique features may in fact protect against these symptoms. If the findings summarized above are replicated, we may be able both to predict disease development and symptomatology with the help of antibody analysis, and possibly also influence disease course by the administration of antibodies with certain features. Such protective effects may depend both on specificity as suggested both from previous experiments with anticollagen antibodies [40] and from a recent study [48] on effects of monoclonal antibodies reactive with different citrullinated peptides. Protective effects may thus depend on specificity but may also depend on other features, in particular glycosylation. A particularly interesting 76 Volume 26 Number 1 January 2014 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

6 Adaptive immunity in rheumatoid arthritis Klareskog et al. example of effects of different glycosylation patterns have been provided from studies, on antibodies that have been modified by enzymes from bacteria, in which these enzymes appear to have evolved during evolution to modify IgG antibodies so that they do not bind complement or Fc receptors [49]. Thus, antibodies with glycosylation modified by one of these enzymes, EndoS, are not able to exert any effector functions in arthritis, and may instead be able to protect against disease [49]. A very recent study [50 ] indicates, that such deglycosylated IgG molecules may indeed inhibit immune complex dependent inflammation even when the deglycosylated antibodies are not directed to the targets of the pathogenic antibodies. But complexity does not stop here. It appears that also ACPAs other than those of the IgG class may have important effects on arthritis. Thus IgE ACPAs have been suggested to be critical for the degranulation of mast cells in joints, and therefore contribute to the development of synovitis [51]. In addition, IgA ACPAs have been described and suggested to mediate protective effects [52]. The present review summarizes a number of features that make seropositive RA a very interesting prototypic disease for analysis and manipulation of antibody-dependent inflammation, and some of these features associated with potential effector functions are summarized in the list below. Tentative effector functions of ACPA are: (1) Osteoclast activation [6 ]: ACPA production in response to certain citrullinated proteins, mainly vimentin may induce bone loss, providing a link between the adaptive immune system and bone. (2) Activation of NETs production from neutrophils [44 ]: As neutrophil activation and thus release of NETs is a feature of many inflammatory reactions in joints, this may be an additional mechanism able to focus the effector functions of anticitrulline immunity and ACPAs to joints. It has been suggested that externalization of citrullinated epitopes and immunostimulatory molecules contributes to the NETosis, which may promote aberrant adaptive and innate immune responses in the joint, and therefore perpetuate pathogenic mechanisms in this disease. (3) Macrophages activation [46]: Antibodies against citrullinated fibrinogen may enhance the activation of macrophages by binding to their receptors, in particular toll-like receptors. (4) ACPA may have protective effects depending on the specificity and/or other features, mainly glycosylation of their Fc parts. Thus, antibodies with certain glycosylation patterns are not able to exert any effector functions in arthritis, and may instead be able to protect against disease [49]. (5) Mast cells degranulation: it has been suggested by Schuerwegh et al. [51] that IgE ACPAs are critical for the degranulation of mast cells in joints and therefore contribute to the development of synovitis, and also IgA ACPAs have been described and suggested to mediate protective effects. These features include a gradual emergence of several different disease-specific antibodies, the presence of such antibodies long before joint symptoms, production of at least some of these antibodies in initiator organs such as the lungs as well as in effector organs such as the joint and access to experimental systems where effector as well as protective effects of these antibodies can be analysed. The situation is thus very attractive for testing of interventions in the various defined phases of RA development (see [53]). It is our hope, therefore, that the rapid advancement of our understanding of adaptive and specific immunity in cause and pathogenesis will soon evolve into a new phase in which a series of interventions can be tested for their abilities to prevent or treat major subpopulations of RA. Acknowledgements We are indepted to several members of our laboratory for discussions where concepts of the present review have evolved. Conflicts of interest The work from our own laboratory that is cited in this review was supported by funds from the Swedish Research Council (VR), the European Research Council (ERC), the SSF-funded program PRIMI, the Swedish VRsupported Linneaus consortium Ceric and the Swedish Rheumatism Association (patient organization). Financial support as well as important intellectual stimulus was provided from the IMI-supported program BTCure There are no conflicts of interest. REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Kollias G, Papadaki P, Apparailly F, et al. Animal models for arthritis: innovative tools for prevention and treatment. Ann Rheum Dis 2011; 70: ß 2013 Wolters Kluwer Health Lippincott Williams Wilkins 77 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

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This article demonstrates a new feed-forward mechanism for the development of ACPAs by showing that certain antibodies against the citrullinating enzymes PAD3 and PAD4 greatly enhance the citrullinating capacity of PAD Snir O, Rieck M, Gebe JA, et al. Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB10401-positive humanized mice and rheumatoid arthritis patients. Arthritis Rheum 2011; 63: Ireland JM, Unanue ER. Processing of proteins in autophagy vesicles of antigen-presenting cells generates citrullinated peptides recognized by the immune system. Autophagy 2012; 8: This article shows that autophagy is associated with citrullination of certain endogenous proteins, and provides therefore a new potential mechanism for activation of anticitrulline immunity 35. Sollid LM, Jabri B. Triggers and drivers of autoimmunity: lessons from coeliac disease. Nat Rev Immunol 2013; 13: Pitzalis C, Kelly S, Humby F. New learnings on the pathophysiology of RA from synovial biopsies. Curr Opin Rheumatol 2013; 25: Amara K, Steen J, Murray F, et al. Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition. J Exp Med 2013; 210: This article describes the production of human monoclonal IgG antibodies from single B cells from RA synovial fluids. A large fraction (20 40%) of B cells from ACPA-positive patients by no B cells from ACPA-negative patients produced antibodies reactive with citrullinated autantigens. The data provide support for a T-cell driven production of ACPAs in inflamed joints of ACPApositive RA. 38. Lundberg K, Nijenhuis S, Vossenaar ER, et al. Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Arthritis Res Ther 2005; 7:R458 R Thiele GM, Duryee MJ, Dusad A, et al. Citrullinated mouse collagen administered to DBA/1J mice in the absence of adjuvant initiates arthritis. Int Immunopharmacol 2012; 13: Nandakumar KS, Bajtner E, Hill L, et al. Arthritogenic antibodies specific for a major type II collagen triple-helical epitope bind and destabilize cartilage independent of inflammation. Arthritis Rheum 2008; 58: Hill JA, Bell DA, Brintnell W, et al. Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice. J Exp Med 2008; 205: Kinloch AJ, Alzabin S, Brintnell W, et al. Immunization with Porphyromonas gingivalis enolase induces autoimmunity to mammalian a-enolase and arthritis in DR4-IE-transgenic mice. Arthritis Rheum 2011; 63: Volume 26 Number 1 January 2014 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.

8 Adaptive immunity in rheumatoid arthritis Klareskog et al. 43. Kuhn KA, Kulik L, Tomooka B, et al. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J Clin Invest 2006; 116: Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, et al. NETs are a source of citrullinatedautoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med 2013; 5. This article describes how ACPAs may stimulate formation of NETs and that such NETs may activate macrophages to a more inflammatory state. The findings provide a new potential link between presence of ACPAs and activation of proinflammatory events in the joint. 45. Li P, Li M, Lindberg MR, et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med 2010; 207: Sokolove J, Zhao X, Chandra PE, et al. Immune complexes containing citrullinated fibrinogen costimulate macrophages via toll-like receptor 4 and Fcg receptor. Arthritis Rheum 2011; 63: Eloranta ML, Alm GV, Rönnblom L. Disease mechanisms in rheumatology: tools and pathways plasmacytoid dendritic cells and their role in autoimmune rheumatic diseases. Arthritis Rheum 2013; 65: Chirivi RGS, Jenniskens GJ, Raats JMH. Anticitrullinated protein antibodies as novel therapeutic drugs in rheumatoid arthritis. J Clin Cell Immunol 2013; S6: Collin M, Shannon O, Björck L. IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions. Proc Natl Acad Sci U S A 2008; 105: Nandakumar KS, Collin M, Happonen KE, et al. Dominant suppression of inflammation by glycan-hydrolyzed IgG. Proc Natl Acad Sci U S A 2013; 110: This article provides evidence that EndoS-treated IgG can inhibit IgG immune complex driven joint inflammation via a new mechanisms that involves interference with immune complex formation. If confirmed and expanded to other conditions and models, these findings open new potentials for interference with immunecomplex-mediated diseases. 51. Schuerwegh AJ, Ioan-Facsinay A, Dorjée AL, et al. Evidence for a functional role of IgEanticitrullinated protein antibodies in rheumatoid arthritis. Proc Natl Acad Sci U S A 2010; 107: Svärd A, Kastbom A, Sommarin Y, Skogh T. Salivary IgA antibodies to cyclic citrullinated peptides (CCP) in rheumatoid arthritis. Immunobiology 2013; 218: Gerlag DM, Raza K, van Baarsen LG, et al. EULAR recommendations for terminology and research in individuals at risk of rheumatoid arthritis: report from the Study Group for Risk Factors for Rheumatoid Arthritis. Ann Rheum Dis 2012; 71: ß 2013 Wolters Kluwer Health Lippincott Williams Wilkins 79 Copyright Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.