Mass-spectrometric identification of carbamylated proteins present in the joints of rheumatoid arthritis patients and controls.

OBJECTIVES: Antibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in the sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint. METHODS: We obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA. RESULTS: Immunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins. CONCLUSIONS: We conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA.

Anti-carbamylated protein (anti-CarP) antibodies precede the onset of rheumatoid arthritis.

OBJECTIVE: The presence of anti-citrullinated protein antibodies (ACPA) and IgM-rheumatoid factor (IgM-RF) years before the clinical diagnosis of rheumatoid arthritis (RA) suggests they are possibly involved in the pathogenic process underlying RA. In this study, we analysed whether anti-carbamylated protein (anti-CarP) antibodies, a novel autoantibody system against carbamylated proteins, can also be detected in healthy individuals before they developed RA. METHODS: Multiple sera from asymptomatic blood donors prior to the onset of their RA symptoms and sera from age-matched and sex-matched controls were tested for the presence of antibodies directed against carbamylated-fetal calf serum (Ca-FCS), carbamylated-fibrinogen (Ca-Fib), cyclic citrullinated-peptide 2 and IgM-RF. RESULTS: Anti-Ca-FCS and anti-Ca-Fib antibodies were each present in 27% and 38% of the last serum samples of blood donors prior to the diagnosis of RA. Both anti-Ca-FCS and anti-Ca-Fib antibodies could be detected many years before the onset of RA. Anti-CarP antibodies as well as ACPA are, on average, detected earlier than IgM-RF. CONCLUSIONS: In addition to ACPA and IgM-RF, also the newly identified anti-CarP antibodies appear many years before the diagnosis of RA.

Low-avidity anticitrullinated protein antibodies (ACPA) are associated with a higher rate of joint destruction in rheumatoid arthritis.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Previously we have shown that ACPA display a considerably lower avidity as compared with antibodies against recall antigens. Nonetheless, ACPA-avidity did vary between patients. As antibody mediated effects are influenced by antibody-avidity, we now investigated ACPA-avidity in relation to biological activity and clinical outcome. METHODS: We determined the avidity of ACPA and related this with severity of joint damage in two Dutch early-RA cohorts containing 199 and 132 patients respectively. Differences in effector functions of low- and high-avidity ACPA were studied. RESULTS: Extensive variation in ACPA-avidity between patients was observed. This allowed the analysis of the relationship between avidity and severity. The presence of low-avidity ACPA is associated with a higher rate of joint destruction. This finding was replicated in an independent cohort. Analysis of the properties of low-versus high-avidity ACPA revealed that low-avidity ACPA are less hampered in their ability to bind 'new' citrullinated antigens. Although no differences could be observed regarding cellular activation via Fc-γ receptors, low-avidity ACPA were more potent in activating the complement system. CONCLUSIONS: Patients with low-avidity ACPA display a higher rate of joint destruction. Low-avidity ACPA display a higher potency to interact with more citrullinated antigens in time and show that low-avidity ACPA are more potent in complement activation. These data indicate that (low) avidity impacts on the biological activity of ACPA and associates with a worse radiological outcome.

Anti-carbamylated protein antibodies are present in arthralgia patients and predict the development of rheumatoid arthritis.

OBJECTIVE: Recently, we discovered a new autoantibody system in rheumatoid arthritis (RA): anti-carbamylated protein (anti-CarP) antibodies. These antibodies have value in predicting joint destruction; however, it is not clear whether they are present before the diagnosis of RA and whether they have value as predictors of RA development. Therefore, we studied whether anti-CarP antibodies are present in patients with arthralgia and whether their presence is associated with the development of RA. METHODS: Sera from 340 arthralgia patients who did not have clinical signs of arthritis but who were positive for IgM rheumatoid factor (IgM-RF) and/or anti-cyclic citrullinated peptide 2 (anti-CCP-2) and 32 healthy controls were tested for anti-CarP IgG antibodies. Of the patients with arthralgia, 111 were IgM-RF positive/anti-CCP-2 antibody negative and 229 were anti-CCP-2 antibody positive. Patients were observed for the development of RA (based on the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria) during a median followup period of 36 months. Cox proportional hazards regression analysis was performed to compare the risk of developing RA between arthralgia patients who were positive for anti-CarP antibodies and those who were negative for anti-CarP antibodies during followup. RESULTS: Anti-CarP antibodies were present in the sera of 39% of the patients. One hundred twenty patients developed RA, after a median of 12 months (interquartile range [IQR] 6-24). The presence of anti-CarP antibodies was associated with the development of RA in the entire arthralgia cohort after correction for RF and anti-CCP-2 antibody status (hazard ratio 1.56 [95% confidence interval 1.06-2.29], P=0.023), as well as in the anti-CCP-2 antibody-positive subgroup (odds ratio 2.231 [95% confidence interval 1.31-3.79], P=0.003). CONCLUSION: Anti-CarP antibodies are present in patients with arthralgia, and their presence predicts the development of RA independent of anti-CCP-2 antibodies.

Anti-carbamylated protein antibodies in systemic sclerosis.

BACKGROUND: To investigate the presence of different isotypes of anti-carbamylated protein (CarP) antibodies in systemic sclerosis (SSc) patients and its association with skin involvement. METHODS: Sera of 194 SSc patients from the Leiden CCISS cohort, fulfilling ACR/EULAR 2013 criteria and a clinical diagnosis of SSc, 83 patients with other connective tissue diseases/Raynaud's Phenomenon, 24 rheumatoid arthritis patients and 98 age and sex-matched healthy controls were tested for the presence of anti-CarP IgG, IgA and IgM, determined by ELISA. Clinical characteristics, that were evaluated in SSc patients, included age, anti-topoisomerase antibodies (ATA), anti-centromere antibodies (ACA) and modified Rodnan Skin Score (mRSS). RESULTS: The SSc patients were 55 (SD:13) years and 155 (80%) were female. Forty-four (23%) patients tested positive for ATA, and 80 (42%) ACA. The median mRSS was 2 (range: 0; 47). Prevalence of anti-CarP IgG was higher in SSc patients than in healthy controls (8% vs 3%, p = 0.007. Prevalence of anti-CarP IgA and IgM and levels of anti-CarP isotypes were comparable between SSc patients and healthy controls. Fifteen (8%) SSc patients tested positive for anti-CarP IgG, 16 (8%) for anti-CarP IgA, and 36 (19%) for anti-CarP IgM. There were no significant correlations between age and levels of anti-CarP isotypes. No correlation between anti-CarP IgG levels and mRSS was found (r = 0.141, p = 0.049), nor for anti-CarP IgM and IgA levels. Anti-CarP IgA levels were higher in ATA compared to ACA positive SSc patients (ATA: 616 aU/ml [359; 1103]; ACA: 424 aU/ml [300; 673], p = 0.015). CONCLUSION: SSc patients can test positive for Anti-CarP IgG, IgA and IgM. We do not observe a relevant clinical association between anti-CarP antibody response and skin involvement in SSc.

The ACPA recognition profile and subgrouping of ACPA-positive RA patients.

OBJECTIVE: Anticitrullinated protein antibodies (ACPA) are the most predictive factor for the development of rheumatoid arthritis (RA). Epitope spreading towards more citrullinated epitopes occurs before the onset of RA. Here, the authors investigated whether specific epitope recognition allows the identification of specific RA subgroups and whether it is associated with clinical features of RA. METHODS: The reactivity of 661 patients with RA from the Leiden Early Arthritis Clinic against several citrullinated antigens was determined by ELISA. Cluster analyses were performed to identify subgroups of patients on the basis of their ACPA recognition profile. The association of the specific reactivities with clinical characteristics was studied. RESULTS: ACPA-positive patients displayed a heterogeneous ACPA recognition profile. After performing cluster analyses, no apparent clustering of patients was found, and on the basis of the reactivities analysed, 64 different subgroups could already be identified. The extent of epitope recognition was associated with anticyclic citrullinated peptide-2 levels. The recognition of specific citrullinated epitopes was not associated with baseline characteristics. Likewise, patients with an extended fine specificity repertoire did not display differences in baseline characteristics or joint damage after 7 years of follow-up using cyclic citrullinated peptide-2 levels as a proxy, compared to ACPA-positive patients recognising fewer peptides. CONCLUSION: These data show that the ACPA response is highly diverse with respect to recognition of specific citrullinated epitopes. Furthermore, the authors' data indicate that clinical correlates in established ACPA-positive RA are independent from the specific (group of) citrullinated peptides recognised.

The interaction between HLA shared epitope alleles and smoking and its contribution to autoimmunity against several citrullinated antigens.

OBJECTIVE: Recent data suggest that a gene-environment interaction between smoking and the HLA shared epitope alleles plays a role in shaping the autoimmune reaction to specific citrullinated antigens. This study was undertaken to determine the effects of HLA shared epitope alleles and tobacco exposure on the immune response against various citrullinated antigens. These associations were analyzed in the anti-citrullinated protein antibody (ACPA)-positive stratum to control for the possibility that the associations found are explained by the known interaction between HLA shared epitope alleles and tobacco exposure on ACPA status. METHODS: In 661 patients with rheumatoid arthritis, reactivity against several citrullinated antigens from vimentin, fibrinogen, enolase, and myelin basic protein was determined by enzyme-linked immunosorbent assay. The effects of the HLA shared epitope alleles and tobacco exposure were assessed by logistic regression analysis. Biologic interaction was analyzed by investigating whether the effects of the risk factors combined exhibited departure from additivity. RESULTS: A significant interaction between tobacco exposure and HLA shared epitope alleles was found for the presence of ACPA as reported previously. When these interaction effects were studied for several ACPA "fine specificities," significant interactions were noted for several citrullinated peptides. However, these interactions were not present after stratification for ACPA status, indicating that the interaction between tobacco exposure and HLA shared epitope alleles influences autoimmunity not to specific citrullinated antigens, but rather to ACPA development. CONCLUSION: Our data indicate that the gene-environment interaction between HLA shared epitope alleles and smoking does not appear to shape the reactivity of the ACPA response. These data suggest that smoking promotes nonspecific citrullination rather than citrullination of specific antigens.

Short article: Absence of serological rheumatoid arthritis biomarkers in inflammatory bowel disease patients with arthropathies.

OBJECTIVE: Biomarkers that are associated with future progression to rheumatoid arthritis (RA) and joint destruction have been discovered previously in patients with arthralgia. The present study examined these RA biomarkers in inflammatory bowel disease (IBD) patients with arthropathies. PATIENTS AND METHODS: Sera from 155 IBD patients with and 99 IBD patients without arthropathies were analyzed for immunoglobulin (Ig) M rheumatoid factor (RF), IgA-RF, anti-cyclic citrullinated peptide 2, anti-cyclic citrullinated peptide 3.1, and anti-carbamylated protein antibody positivity using enzyme-linked immunosorbent assays. The prevalence of the autoantibodies in the IBD patients was compared with the prevalence in RA patients. RESULTS: No differences were found in biomarker positivity between IBD patients with and without arthropathies. Significantly more biomarker positivity (P<0.001) was observed in RA patients compared with IBD patients with arthropathies. Also, smoking turned out to be significantly associated with positivity for IgM-RF or IgA-RF. CONCLUSION: Our findings suggest that there is no apparent clinical value in the detection of RA biomarkers in serum of IBD patients to help identify arthropathies.

The specificity of anti-carbamylated protein antibodies for rheumatoid arthritis in a setting of early arthritis.

INTRODUCTION: Anti-carbamylated protein (anti-CarP) antibodies have been described in rheumatoid arthritis (RA) and arthralgia patients at risk of developing RA. To what extent these autoantibodies are specific for RA is unknown. Therefore, we investigated the diagnostic performance of the presence of anti-CarP antibodies for RA in a setting of early arthritis. METHODS: Anti-CarP antibodies were detected using carbamylated fetal calf serum as substrate. Anti-CCP2 antibodies were measured using enzyme-linked immunosorbent assay and immunoglobulin M (IgM) rheumatoid factor (RF) as part of routine care. Sera were derived from patients in the Leiden Early Arthritis Clinic cohort obtained at inclusion. Test characteristics were determined using the fulfillment of the 2010 RA criteria after 1 year as outcome. RESULTS: In total 2086 early arthritis patients were studied regarding the presence of anti-CarP antibodies. We observed that the sensitivity and specificity of the presence of anti-CarP antibodies for RA were 44 % and 89 %, respectively. As a reference, sensitivity and specificity of the presence of anti-CCP2 antibodies were 54 % and 96 %, respectively, and of IgM-RF 59 % and 91 %. Patients harboring anti-CarP antibodies not classified as RA were mainly diagnosed with undifferentiated arthritis and less frequently reactive arthritis and psoriatic arthritis. CONCLUSION: Anti-CarP antibodies are predominantly present in RA but can also be detected in other forms of arthritis.

Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

INTRODUCTION: Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA). METHODS: Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity. RESULTS: Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level. CONCLUSIONS: We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity.

INTRODUCTION: B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells. METHODS: In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients. RESULTS: Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion. CONCLUSION: By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.

Value of anti-modified citrullinated vimentin and third-generation anti-cyclic citrullinated peptide compared with second-generation anti-cyclic citrullinated peptide and rheumatoid factor in predicting disease outcome in undifferentiated arthritis and rheumatoid arthritis.

OBJECTIVE: Autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPAs) determined by testing with second-generation anti-cyclic citrullinated peptide (anti-CCP-2) are frequently measured in clinical practice because of their association with disease outcome in undifferentiated arthritis (UA) and rheumatoid arthritis (RA). Recently, 2 new ACPA tests were developed: third-generation anti-CCP (anti-CCP-3) and anti-modified citrullinated vimentin (anti-MCV) autoantibody tests. To facilitate the decision on which autoantibody to test in daily practice, this study evaluated the capability of these autoantibodies and combinations of them to predict 3 outcome measures: progression from UA to RA, the rate of joint destruction in RA, and the chance of achieving sustained disease-modifying antirheumatic drug (DMARD)-free remission in RA. METHODS: Patients with UA (n=625) were studied for whether UA progressed to RA after 1 year. Patients with RA (n=687) were studied for whether sustained DMARD-free remission was achieved and for the rate of joint destruction during a median followup of 5 years. Positive predictive values (PPVs) for RA development and for associations with the disease course in RA were compared between single tests (anti-CCP-2, anti-CCP-3, anti-MCV, and RF) and between combinations of these tests. RESULTS: Among the single tests performed in patients with UA, anti-CCP-2 tended to have the highest PPV for RA development (67.1%), but the 95% confidence intervals of the other tests overlapped. Among the single tests in patients with RA, all 4 tests showed comparable associations with the rate of joint destruction and with the achievement of remission. In both ACPA-positive and ACPA-negative RA, the presence of RF was not associated with more joint destruction. For all outcome measures, performing combinations of 2 or 3 autoantibody tests did not increase the predictive accuracy compared with performing a single test. CONCLUSION: For clinical practice, a single autoantibody test is sufficient for risk estimation in UA and RA.

Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment.

OBJECTIVE: Anti-CD20-mediated B cell depletion with rituximab is a new and effective therapy for rheumatoid arthritis (RA). Although B cells in peripheral blood (PB) are consistently depleted in all patients, the clinical effects are more heterogeneous, possibly related to differences in the depleting effects of lymphoid or solid tissues. The aim of this study was to investigate B cell depletion in different compartments (PB, bone marrow, and synovium) and determine predictive variables for responsiveness to rituximab therapy. METHODS: Before and 12 weeks after rituximab treatment, samples of PB, bone marrow, and synovium were collected from 25 patients with RA refractory to disease-modifying antirheumatic drugs and tumor necrosis factor-blocking agents. CD19+ and CD20+ B cells in PB and bone marrow were measured by flow cytometric analysis, whereas CD79a+ and cytoplasmic CD20+ B cells in the synovium were stained by immunohistochemistry. The effects of rituximab on serum Ig and autoantibodies were measured by enzyme-linked immunosorbent assay. RESULTS: Rituximab effectively depleted the CD20+ subset of B cells in the PB, bone marrow, and synovium of RA patients. Rituximab significantly reduced autoantibody production (anti-citrullinated protein antibodies [ACPAs] and rheumatoid factor [RF]), in part due to a nonspecific decrease in total Ig production. Importantly, positivity for circulating ACPA IgM, in combination with a high infiltration of CD79a+ B cells in the synovium, but not of CD138+ plasma cells, was a predictor of clinical outcome after rituximab treatment. ACPA IgM titers were independently associated with synovial infiltration of CD20-,CD79a+ B cells, but not with CD138+ plasma cells. CONCLUSION: These data provide novel insights into the mechanisms of CD20-mediated B cell depletion in the lymphoid and solid tissues of RA patients and suggest a pivotal role for ACPA IgM-producing plasmablasts in RA.

Rap1 signaling is required for suppression of Ras-generated reactive oxygen species and protection against oxidative stress in T lymphocytes.

Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to TCR signaling in T lymphocytes. Conversely, chronic oxidative stress leads to decreased proliferative responses and enhanced transcription of inflammatory gene products, and is thought to underlie the altered pathogenic behavior of T lymphocytes in some human diseases, such as rheumatoid arthritis (RA). Although the signaling mechanisms regulating ROS production in T lymphocytes has not been identified, activation of the small GTPase Ras has been shown to couple agonist stimulation to ROS production in other cell types. We find that Ras signaling via Ral stimulates ROS production in human T lymphocytes, and is required for TCR and phorbol ester-induced ROS production. The related small GTPase Rap1 suppresses agonist, Ras and Ral-dependent ROS production through a PI3K-dependent pathway, identifying a novel mechanism by which Rap1 can distally antagonize Ras signaling pathways. In synovial fluid T lymphocytes from RA patients we observed a high rate of endogenous ROS production, correlating with constitutive Ras activation and inhibition of Rap1 activation. Introduction of dominant-negative Ras into synovial fluid T cells restored redox balance, providing evidence that deregulated Ras and Rap1 signaling underlies oxidative stress and consequent altered T cell function observed in RA.