Production Of Anti-citrullinated Protein Antibodies Research Articles

Synovial fluid mononuclear cells provide an environment for long-term survival of antibody-secreting cells and promote the spontaneous production of anti-citrullinated protein antibodies

ObjectivesIn rheumatoid arthritis (RA), observations point to a crucial role for (autoreactive) B cells in disease pathogenesis. Here, we studied whether cells from the synovial environment impact on the longevity...

Smoking and Air Pollution as Pro-Inflammatory Triggers for the Development of Rheumatoid Arthritis.

Smoking is now well recognized not only as a risk factor for rheumatoid arthritis (RA), but also as a determinant of disease activity, severity, response to therapy, and possibly mortality. Studies, mostly recent, which have provided significant insights into the molecular and cellular mechanisms which underpin the pathogenesis of smoking-related RA, as well as the possible involvement of other types of outdoor and indoor pollution form the basis of this review. Smoking initiates chronic inflammatory events in the lungs. These, in turn, promote the release of the enzymes, peptidylarginine deiminases 2 and 4 from smoke-activated, resident and infiltrating pulmonary phagocytes. Peptidylarginine deiminases mediate conversion of various endogenous proteins to putative citrullinated autoantigens. In genetically susceptible individuals, these autoantigens trigger the production of anti-citrullinated peptide, pathogenic autoantibodies, an event which precedes the development of RA. An increasing body of evidence has linked chronic inflammatory events in the lungs of smokers, to the production of anti-citrullinated peptide autoantibodies and development of RA. Creation of awareness of the associated risks, assessment of smoking status and implementation of compelling antismoking strategies must be included in the routine clinical management of patients presenting with suspected RA. Chronic inflammatory mechanisms operative in the lungs of smokers lead to the production of anti-citrullinated protein antibodies which, in turn, drive the development of RA. These mechanistic insights not only reinforce the association between smoking and risk for RA, but also the necessity to increase the level of awareness in those at highest risk.

Autoantibodies From Single Circulating Plasmablasts React With Citrullinated Antigens and Porphyromonas gingivalis in Rheumatoid Arthritis.

Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whether Porphyromonas gingivalis facilitates the generation of ACPAs. Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti-cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity with P gingivalis was tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase from P gingivalis. Approximately 19.5% of plasmablast-derived antibodies from anti-CCP-positive RA patients, but none from 1 anti-CCP-negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase from P gingivalis. The reactivity of ACPAs against citrullinated proteins from P gingivalis was confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity with P gingivalis antigens but completely lost their reactivity with citrullinated human antigens. These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti-P gingivalis immune responses.

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

ABSTRACTRodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregs and the role of IL-17 in connection with Treg depletion. Given the relevance of Tregs in RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregs in inflammatory arthritis. Selective depletion of Tregs was achieved using a Foxp3-DTR-eGFP mouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of the Foxp3 gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregs increased in the paw and its draining lymph node in DTHA, and depletion of Tregs resulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Treg depletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregs are important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using the Foxp3-DTR-eGFP mouse on a C57BL/6 background for Treg depletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregs and IL-17 in arthritis.

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

An Association Between Amino Acid Position 74 of HLA–DRB1 and Anti–Citrullinated Protein Antibody Levels in Japanese Patients With Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

Anti-citrullinated protein antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA), and strong associations between HLA-DRB1 alleles and ACPA levels have been detected in RA patients. We undertook this study to elucidate the associations between particular amino acid positions in HLA-DRB1 and ACPA levels in patients with RA. We analyzed ACPA data on a total of 4,371 Japanese ACPA-positive RA patients in whom HLA-DRB1 allele genotyping had been performed. Generalized linear regression analysis and omnibus testing were carried out to determine associations of HLA-DRB1 alleles, amino acid residues, or amino acid positions with levels of ACPA. HLA-DRB1*09:01 and HLA-DR15 were confirmed to be associated with ACPA levels. HLA-DRB1*08:03 and DRB1*14:06 were associated with reduced and increased ACPA levels, respectively. We detected a strong association between ACPA levels and amino acid position 74 (P = 1.9 × 10(-51) ). The association was mainly conferred by alanine residue (P = 4.5 × 10(-51) ). After adjustment for position 74, amino acid positions 60 and 57 were found to be associated with ACPA levels. Amino acid positions 74 and 57 had previously been reported to be associated with susceptibility to ACPA-positive RA in Asians. Combinations of the amino acid residues at position 74 and position 60 or 57 could induce improvement in Akaike's information criterion comparable to that induced by the 5 significant HLA-DRB1 alleles (HLA-DRB1*08:03, DRB1*09:01, DRB1*14:06, DRB1*15:01, and DRB1*15:02). Amino acid position 74 in HLA-DRB1 is strongly associated with ACPA levels in ACPA-positive RA, as well as with RA susceptibility. The mechanisms of ACPA production and susceptibility to ACPA-positive RA seem to partly overlap.

Is there a link between carbamylation and citrullination in periodontal disease and rheumatoid arthritis?

The remarkable similarity in inflammatory response and pathology of periodontal disease and rheumatoid arthritis has been recognized for several decades. However, how these two disease may be interrelated has been less clear. During the pathogenesis of rheumatoid arthritis there is a preclinical immunological phase which precedes the clinical manifestation of rheumatoid arthritis. During this phase serum autoantibodies appear many years before the clinical signs and symptoms of rheumatoid arthritis become apparent. To date, the two best studied autoantibodies have been rheumatoid factor and anti-citrullinated protein antibodies (ACPA). Of these the production of ACPA has been considered very important due to their high predictive value in future manifestation of rheumatoid arthritis. Citrullination is a common post-translational modification of proteins based on the enzymatic conversion of arginine into citrulline. Extra-articular citrullination and production of ACPA, as a priming immunological experience, is well documented in many tissues including the inflamed gingival tissues associated with periodontal disease. More recently, carbamylation of proteins has also been implicated in the pathogenesis of rheumatoid arthritis in a manner similar to citrullination. Carbamylation is a post translational modification of proteins by an enzyme-independent modification of lysine residues against which autoantibodies are subsequently induced. In this article we hypothesise that, like citrullination, carbamylation of proteins and associated antibody production during the gingival inflammation associated with gingivitis and periodontitis may play a role in the pathogenesis of rheumatoid arthritis.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation

ObjectivesImmunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs...

Identification of secreted phosphoprotein 1 gene as a new rheumatoid arthritis susceptibility gene

To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.

Structural Changes and Antibody Enrichment in the Lungs Are Early Features of Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

It has been suggested that immunologic events in the lungs may be involved in triggering immunity, in particular production of anti-citrullinated protein antibodies (ACPAs) during early phases of rheumatoid arthritis (RA). The aim of this study was to investigate the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status. High-resolution computed tomography (HRCT) was used to examine the lungs of 105 patients with early, untreated RA (70 with ACPA-positive RA and 35 with ACPA-negative RA) and 43 healthy individuals. Bronchoscopy with collection of bronchoalveolar lavage (BAL) fluid and mucosal bronchial biopsy specimens was performed in 23 RA patients. The presence of citrullinated proteins in the bronchial tissue was detected by immunohistochemical staining. ACPAs (detected with an anti-cyclic citrullinated peptide 2 test) and total Ig levels were determined in the sera and BAL fluid of RA patients. HRCT imaging revealed that 63% of ACPA-positive RA patients had parenchymal lung abnormalities, compared with only 37% of ACPA-negative RA patients and 30% of healthy controls (each P < 0.05). These significant differences remained after adjustment for smoking status. Airway changes detected by HRCT were more frequent in RA patients than in healthy controls (66% versus 42%; P < 0.05), but there was no difference between ACPA-positive and ACPA-negative RA patients. Immunohistochemical studies of the bronchial tissue showed increased staining for citrullinated proteins in ACPA-positive RA patients compared with ACPA-negative RA patients (P < 0.05). ACPA levels were relatively higher in the BAL fluid as compared with the sera of ACPA-positive RA patients, suggesting that there is local production of ACPAs in the lungs of these patients. The presence of ACPAs is associated with parenchymal lung abnormalities, site-specific citrullination, and antibody enrichment in the lungs early in the development of ACPA-positive RA.

THU0008 Meta-analysis of the association of smoking and PTPN22 R620W genotype on autoantibody status and radiological erosions in rheumatoid arthritis

BackgroundSmoking has been linked to production of anti-citrullinated protein antibodies (ACPA) in HLA-DRB1 shared epitope positive patients which has also been associated with a worse severity of rheumatoid arthritis (RA)...

FRI0005 Production of anti-citrullinated protein antibodies by B cell subsets isolated from peripheral blood of patients with rheumatoid arthritis

BackgroundAnti-citrullinated protein antibodies (ACPA) are among the most important molecular candidates that could drive the inflammatory immune response in a subset of patients with rheumatoid arthritis (RA). Although many aspects...

Longterm Effects of Rituximab on B Cell Counts and Autoantibody Production in Rheumatoid Arthritis: Use of High-sensitivity Flow Cytometry for More Sensitive Assessment of B Cell Depletion

To assess the efficacy and safety of longterm rituximab (RTX) therapy for rheumatoid arthritis (RA) and study correlations among B cell depletion, clinical response, and autoantibody production. Seventy-seven patients with moderate or high RA activity received RTX and were re-treated every 6 months regardless of clinical response. All patients received at least 5 cycles. We assessed 28-joint Disease Activity Score (DAS28), IgM rheumatoid factor (RF), and anticitrullinated protein antibody (ACPA) levels at baseline, after 15 days, and then every 6 months for 24 months. Absolute CD19+ B lymphocyte counts were determined in 50 patients using high-sensitivity flow cytometry (hsFACS) by reading 100,000 events. After 6, 12, 18, and 24 months, 51.6%, 51.9%, 73.3%, and 83.8% of patients, respectively, showed good European League Against Rheumatism responses. Significant and sustained decreases in IgM RF and ACPA levels were observed as early as 6 months and 12 months, respectively. The baseline mean absolute B cell number was 0.234 g/l. B cell numbers diminished significantly after the very first infusion by Day 15 (0.104 g/l; p = 0.007); they further decreased until 24 months (0.0013 g/l; p < 0.001). One RTX infusion resulted in incomplete depletion in 76.7% of patients. Upon RTX treatment, changes in CD19+ B cell numbers positively correlated with changes in DAS28 (r = 0.963, p = 0.008) and IgM RF (r = 0.859, p = 0.028), but not with changes in ACPA production (r = 0.726, p = 0.102). The correlations between B cell numbers and DAS28 were observed in both ACPA-seropositive (r = 0.999, p < 0.0001) and ACPA-negative patient subpopulations (r = 0.962, p = 0.009). The correlation between CD19+ cell numbers and IgM RF was observed only in the ACPA-positive population (r = 0.944, p = 0.005) but not in seronegative patients (r = 0.398, p = 0.435). No safety issues arose. In RA, clinical response to RTX is associated with the extent of B cell depletion and with autoantibody production. Changes in CD19+ B cell numbers correlate with those in disease activity and, in seropositive patients, also with IgM RF, but not with ACPA production. We found that hsFACS may be a useful method to more accurately assess incomplete B cell depletion.

New insights into the genetics of immune responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is a common autoimmune disease with a strong genetic component. Numerous aberrant immune responses have been described during the evolution of the disease. In later years, the appearance of anti-citrullinated protein antibodies (ACPAs) has become a hallmark for the diagnosis and prognosis of RA. The post-translational transformation of arginine residues of proteins and peptides into citrulline (citrullination) is a natural process in the body, but for unknown reasons autoreactivity towards citrullinated residues may develop in disposed individuals. ACPAs are often found years before clinical manifestations. ACPAs are present in about 70% of RA patients and constitute an important disease marker, distinguishing patient groups with different prognoses and different responses to various treatments. Inside the human leukocyte antigen (HLA) region, some HLA-DRB1 alleles are strongly associated with their production. Genome-wide association studies in large patient cohorts have defined a great number of single nucleotide polymorphisms (SNPs) outside of the HLA region that are associated with ACPA positive (ACPA+) RA. The SNPs are generally located close to or within genes involved in the immune response or signal transduction in immune cells. Some environmental factors such as tobacco smoking are also positively correlated with ACPA production. In this review, we will describe the genes and loci associated with ACPA+ RA or ACPA- RA and attempt to clarify their potential role in the development of the disease.

Very high levels of anti–citrullinated protein antibodies are associated with HLA–DRB1*15 non–shared epitope allele in patients with rheumatoid arthritis

Production of anti-citrullinated protein antibodies (ACPAs) is an important biomarker for rheumatoid arthritis (RA). We undertook this study to determine whether genetic factors (HLA-DRB1 alleles) are associated with extreme ACPA levels in individuals with ACPA-positive RA, and to ascertain whether there are any phenotypic characteristics associated with these subgroups of RA. HLA-DRB1 allelic groups were genotyped in 1,073 ACPA-positive RA patients from the Swedish Epidemiological Investigation of Rheumatoid Arthritis study. We found that 283 patients (26.4%) had high ACPA levels (defined as >1,500 units/ml using the Euro-Diagnostica anti-CCP2 test), while the rest of the patients had moderate ACPA levels and served as the comparison group. A replication group consisted of 235 RA patients. No significant differences in baseline disease activity were observed between patients with high and those with moderate ACPA levels. However, the HLA-DRB1*15 allele was associated with high ACPA levels (P=0.0002). A similar trend was detected in HLA-DRB1*15-positive patients in the replication cohort, with meta-analysis of the discovery and replication cohorts demonstrating an overall effect of HLA-DRB1*15 on development of high ACPA levels in both the discovery and replication cohorts (P<0.0001 by Mantel-Haenszel test with a fixed-effects model). Our data indicate that HLA-DRB1*15 may promote the production of high ACPA levels. Due to the high value of ACPA level scores in the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA, the presence of HLA-DRB1*15 may, at least in part, contribute to fulfilling the criteria for RA. This illustrates the complex nature of the genetic regulation of ACPA levels. Additional mechanistic studies of the regulation of ACPAs and ACPA-positive RA are pending.

Specific expression of PAD4 and citrullinated proteins in lung cancer is not associated with anti-CCP antibody production

Anti-citrullinated protein antibodies (ACPAs), produced against citrullinated proteins, are diagnostic and prognostic markers of rheumatoid arthritis (RA). The underlying mechanism that explains the connection of smoking, citrullination [catalyzed by peptidyl arginine deiminases (PADs)] and ACPAs is still unclarified in RA. Thus, we searched for a non-arthritic model in which an increased cell death allows the formation of autoantibodies. Data supporting that lung cancer might be a good candidate are as follows: (i) smoking plays a role in its pathogenesis, (ii) the disease is frequently accompanied by paraneoplastic syndrome, (iii) smoking increases citrullination in the lung, (iv) various types of malignancies are associated with increased citrullination and (v) lung cancer tissue shows similarities with RA synovium. Serum PAD4, rheumatoid factor (RF) and ACPA levels were measured in 42 lung cancer patients; expression of cytokeratin 7 (CK7), PAD4 and citrullinated proteins was visualized in 113 lung cancer tissues. All parameters were analyzed in correlation with smoking history. None of the patients had polyarthritis or autoimmune disease. Significantly increased RF levels were associated with higher PAD4 levels in smoker lung cancer patients compared with non-smokers. Both PAD4 and citrullination immunostaining strongly correlated with that of CK7 in lung cancer, however, did not differ according to smoking history. Two of 30 smoker lung cancer patients had high anti-cyclic citrullinated peptide levels. In conclusion, PAD4 and citrullination may be helpful in distinguishing lung cancer from healthy tissue. Smoking, abnormal serum PAD4 and RF levels may not be sufficient for the production of ACPAs and development of autoimmunity.

Régulation génétique de la production des auto-anticorps au cours de la polyarthrite rhumatoïde

Rheumatoid arthritis (RA) results from interactions between genes, environment and autoimmunity. Anticitrullinated protein antibodies (ACPA) represent a sensitive and specific serological marker of RA. ACPA production has been associated with several genetic predisposing and protective factors. Within the HLA region, the HLA-DRB1 locus accounts for the most important part of the predisposition to ACPA production. Outside the HLA region, various genes, regions or locus were associated to ACPA production such as PADI4, PTPN22, CTLA4, FcγR, 6q23 region and TRAF1-C5 locus. On the other hand, protective genetic factors were reported within the HLA region, e.g. HLA-DR3 alleles, or outside this region, e.g. IRF5 and MEFV genes. Gene-gene interactions were shown to modulate ACPA production, in particular between HLA-DRB1 locus and PTPN22 gene. Furthermore, environmental and lifestyle-related factors, such as smoking, were reported to interact with predisposing genetic factors to enhance ACPA production. In the next future, a better knowledge and understanding of interactions between genetic, environmental and immunological factors may allow us to identify homogeneous subgroups of early RA patients and to move towards the development of personalized medicine.

Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients

High titers of specific anti-citrullinated protein antibodies (ACPAs) are frequently present in the serum of rheumatoid arthritis (RA) patients, but their presence in synovial fluid is less well characterized. The purpose of this study was to compare the levels of antibody to 4 well-defined citrullinated candidate RA autoantigens in serum and synovial fluid and to determine whether antibodies to one citrullinated antigen are dominant over another. Furthermore, we studied their relationships with mutated citrullinated vimentin (MCV), a newly identified RA-specific serum assay, and the classic cyclic citrullinated peptide (CCP) in the synovial fluid of well-defined HLA-DR groups. Paired serum and synovial fluid samples from 290 RA patients and serum samples from 100 age- and sex-matched healthy controls were analyzed for the presence of anti-MCV and anti-CCP antibodies and for reactivity to citrullinated fibrinogen, alpha-enolase, type II collagen, and vimentin. A total of 219 of the 290 patients were genotyped for the HLA-DR shared epitope alleles. Significantly higher proportions of antibodies against all RA-associated citrullinated antigens were found in synovial fluid as compared with serum. This was also true for the MCV and CCP responses but not for non-RA-associated anti-tetanus toxoid antibodies. As expected, we found a high correlation between citrullinated vimentin and MCV responses. All synovial fluid ACPAs were predominantly associated with HLA-DRB1*04 alleles and were confined to the CCP+/MCV+ subset of patients. MCV and CCP positivity represent a similar subset of RA patients, whereas ACPAs with different fine specificities fall into subgroups of anti-CCP+/anti-MCV+ patients. The levels of all specific ACPAs were elevated in synovial fluid, suggesting that there is local antibody production and/or retention of ACPAs at the site of inflammation governed by RA-predisposing genes.

Antibodies to citrullinated proteins: molecular interactions and arthritogenicity

The discovery of antibodies specific for citrullinated protein epitopes [anti-citrullinated protein antibodies (ACPAs)] is a hallmark for the diagnosis and prognosis of rheumatoid arthritis (RA) and will also be a useful tool for understanding the fundamental pathologic processes. There are several essential questions pertaining to ACPA that remain to be explored, such as understanding the early specificity of the underlying T-cell recognition, whether the production of ACPA is a primary or secondary process, and in the event of such antibodies being arthritogenic, whether they could possibly regulate the disease development. To answer these questions, animal models are needed, but unfortunately ACPA is not a prominent feature of any of the classical animal models of RA. However, we showed recently that ACPA can be isolated from animals susceptible to collagen-induced arthritis that are specific for citrullinated type II collagen (CII). The citrulline specificity could be visualized, and the specificity is determined primarily by a direct interaction with citrulline. We also demonstrated that these antibodies are specific for the citrullinated epitopes and are pathogenic in vivo. A new hypothesis to explain how inflammation in RA can be directed to cartilaginous joints and be self-perpetuating is suggested, which involves recognition of post-translational modifications (glycosylation and citrullination) on CII by T and B cells that can have both arthritogenic and regulatory consequences.

Synovial intracellular citrullinated proteins colocalizing with peptidyl arginine deiminase as pathophysiologically relevant antigenic determinants of rheumatoid arthritis–specific humoral autoimmunity

To address the ongoing debate concerning the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and to analyze their pathophysiologic relevance to the induction or perpetuation of the RA-specific anti-citrullinated protein antibodies (ACPAs). Synovium of 19 RA patients and 19 non-RA controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD-2), and for the free citrulline-producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients, the findings of anticitrulline staining in synovium were related to serum and synovial fluid ACPA levels, as measured by enzyme-linked immunosorbent assay. F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA samples versus 5% of control samples, whereas extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrullinated proteins but not free citrulline. Accordingly, iNOS was equally found in RA and control synovium and in intracellular citrullinated protein-positive and intracellular citrullinated protein-negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD-2, which was found in 59% of RA samples versus 17% of control samples. Independent of local disease activity, the presence of the RA-specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint. These data confirm the presence of RA-specific intracellular citrullinated proteins in synovium. The link with PAD-2 and local and systemic ACPA levels emphasizes their pathophysiologic relevance for RA-specific humoral autoimmunity.