Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.     

Rheumatoid factor,not antibodies against citrullinated proteins,is associated with baseline disease activity in rheumatoid arthritis clinical trials

IntroductionAlthough the prognostic value of rheumatoid factor (RF) and autoantibodies against citrullinated proteins (ACPAs) in patients with rheumatoid arthritis (RA) is well established, their association with RA disease activity remains unclear. Here, we investigate this association in a large study using data from clinical trials.MethodsWe used baseline data from four recent randomized controlled clinical trials of RA. We investigated individual and composite measures of disease activity. The relationship of RF and ACPAs with these measures was investigated by using stratified analysis (comparing four groups of patients according to the presence or absence of RF and ACPAs) and matched analysis (disease activity levels compared between patients negative and patients highly positive for one autoantibody who were matched for levels of the other autoantibody as well as for age, gender, and duration of RA).ResultsA total of 2118 patients were analysed in the different cohorts. In the stratified analysis, RF⁺ patients, regardless of ACPA status, had the highest levels of disease activity, whereas ACPA⁺ patients had disease activity that was similar to or lower than that of ACPA⁻ patients, both in the presence and in the absence of RF. When matched for ACPA levels, patients with highly positive RF had significantly higher disease activity for all composite indices compared with patients who were RF⁻ (P = 0.0067), whereas ACPA-highly-positive and ACPA-negative patients matched for RF levels had similar disease activity, again even with the tendency toward lower disease activity for ACPA⁺ patients (P = 0.054).ConclusionThe data presented suggest that the presence of RF has a clear association with higher levels of disease activity but that the presence of ACPAs has not and even appears to be associated with lower disease activity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0736-9) contains supplementary material, which is available to authorized users.     

Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint

Introduction

A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).

Methods

Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.

Results

Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.

Conclusions

We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.     

Use of total lymphoid irradiation (TLI) in studies of the T cell-dependence of autoantibody production in rheumatoid arthritis

The effect of total lymphoid irradiation (TLI) on T cell-dependent and -independent humoral immune responses was studied in patients with intractable rheumatoid arthritis (RA). The serum levels of several autoantibodies and of antibodies to diphtheria (DT) and tetanus (TT) toxoids and to pneumococcal polysaccharide (PPS; 12 antigenic types) were studied before and after TLI. In addition, the patients were given a booster injection of DT and TT and a single injection of pneumococcal vaccine after radiotherapy. Antibody levels to DT and TT decreased about twofold after TLI and did not rise significantly (p greater than 0.05) after a booster injection. However, there was no reduction in antibody levels to PPS after TLI, and a significant rise in titers was observed after a single vaccination (p less than 0.01). The serum levels of rheumatoid factor (RF), anti-nuclear antibody (ANA), and granulocyte associated IgG rose slightly after TLI. Thus, the autoantibodies and antibodies to polysaccharides appear to be relatively independent of helper T cell function, which is markedly reduced after TLI. On the other hand, antibodies to protein antigens such as DT and TT appear to be more closely dependent upon T helper function in man, as has been reported in rodents. The findings suggest that T cell-independent autoantibody responses alone do not maintain the joint disease activity in RA, because improvement in joint disease after TLI has been reported.     

The <Emphasis Type="Italic">PTPN22</Emphasis>1858C/T polymorphism is associated with anti-cyclic citrullinated peptide antibody-positive early rheumatoid arthritis in northern Sweden

The PTPN22 1858C/T polymorphism has been associated with several autoimmune diseases including rheumatoid arthritis (RA). We have shown that carriage of the T variant (CT or TT) of PTPN22 in combination with anti-cyclic citrullinated peptide (anti-CCP) antibodies highly increases the odds ratio for developing RA. In the present study we analysed the association between the PTPN22 1858C/T polymorphism and early RA in patients from northern Sweden, related the polymorphism to autoantibodies and the HLA-DR shared epitope, and analysed their association with markers for disease activity and progression. The inception cohort includes individuals who also donated samples before disease onset. A case–control study was performed in patients (n = 505; 342 females and 163 males) with early RA (mean duration of symptoms = 6.3 months) and in population-based matched controls (n = 970) from northern Sweden. Genotyping of the PTPN22 1858C/T polymorphism was performed using a TaqMan instrument. HLA-shared epitope alleles were identified using PCR sequence-specific primers. Anti-CCP2 antibodies were determined using enzyme-linked immunoassays. Disease activity (that is, the number of swollen and tender joints, the global visual analogue scale, and the erythrocyte sedimentation rate) was followed on a regular basis (that is, at baseline and after 6, 12, 18 and 24 months). Both the 1858T allele and the carriage of T were associated with RA (χ² = 23.84, P = 0.000001, odds ratio = 1.69, 95% confidence interval = 1.36–2.11; and χ² = 22.68, P = 0.000002, odds ratio = 1.79, 95% confidence interval = 1.40–2.29, respectively). Association of the 1858T variant with RA was confined to seropositive disease. Carriage of 1858T and the presence of anti-CCP antibodies was independently associated with disease onset at an earlier age (P < 0.05 and P < 0.01, respectively), while the combination of both resulted in an even earlier age at onset. Smoking was identified as a risk factor independent of the 1858T variant and anti-CCP antibodies.     

Contribution of Peptide Backbone to Anti-Citrullinated Peptide Antibody Reactivity

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting approximately 1–2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs), which have been found in up to 70% of RA patients’ sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g. why some Cit-Gly-containing sequences are not targeted by ACPAs.     

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.     

S-Nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.     

Model-based inference of neutralizing antibody avidities against influenza virus

To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n = 45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities (ρ = 0.54, 95% CI = [0.31, 0.70], P < 10⁻⁴). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.     

How citrullination invaded rheumatoid arthritis research

Citrullination and the immune response to citrullinated proteins have been fundamental for the early recognition of rheumatoid arthritis by serological tests and a better understanding of its pathophysiology. In the first years after the initial publications, the focus was on the antibodies directed to citrullinated proteins. It is now realized that citrullinating enzymes and citrullinated proteins may have important roles in the maintenance of the inflammatory processes in the joints. There is also accumulating evidence for a direct role of citrullination in tissue destruction in the rheumatoid synovium. Here we will discuss the development and importance of anti-citrullinated protein antibodies in rheumatoid arthritis as well as recent findings implicating citrullination in the pathophysiology of rheumatoid arthritis.The first indication that patients with rheumatoid arthritis (RA) produce antibodies to a specific autoantigen was published in 1964 by two Dutch scientists, Nienhuis and Mandema. The exact nature of this antigen, the so-called perinuclear factor, remained unclear for decades. In 1978, the target of seemingly unrelated RA-specific autoantibodies (that is, keratin) was identified. Almost 15 years later, Guy Serre’s group convincingly showed that both antigens were identical to the cytokeratin filament-aggregating protein filaggrin (reviewed in [1]). Our own previously published results had shown that the newly made precursor of filaggrin in cultured buccal mucosa cells (that is, profilaggrin) did not react with RA antibodies [2]. This prompted us to consider the possibility that a post-translational modification of filaggrin, absent on newly made profilaggrin, was required for the formation of the antigenic target of these antibodies. Since 1994, we have tested several likely modifications using synthetic peptides. Indeed, citrullination, the enzymatic conversion of peptidylarginine into peptidylcitrulline, turned out to be essential to make peptides reactive with RA autoantibodies. We subsequently developed an enzyme-linked immunosorbent assay with citrullinated peptides and confirmed that the anti-peptidylcitrulline activity was specific for RA [3]. Our further work was directed to the development of the CCP2 test, using cyclic citrullinated peptides (CCPs) selected from random peptide libraries [4].The discovery of CCP/protein as the most prominent RA-specific antigen had great impact on RA diagnostics and our understanding of RA pathophysiology. The following milestones can be noted (see [5] also).1. After decades of intensive research by many groups, a specific diagnostic test for RA had finally been developed. The CCP2 test has a specificity of more than 95%, is very sensitive (~75%), and is still considered the gold standard in RA autoantibody testing. Since 2010, anti-citrullinated protein antibodies (ACPAs) have been included in the new American College of Rheumatology/European League Against Rheumatism classification criteria for RA.2. Recently, an international reference preparation for ACPAs was evaluated by the International Committee for the Standardization of Autoantibodies in Rheumatic and Related Diseases [6]. It is available for the scientific community via the Centers for Disease Control and Prevention (Atlanta, GA, USA).3. A positive CCP2 test predicts the development of RA, often years before clinical confirmation (reviewed in [5]). It appears that time to RA diagnosis is shorter in patients with high anti-CCP2 titers at enrollment as compared with those with low titers [7].4. ACPA-positive RA is characterized by a more severe disease course. Early treatment of ACPA-positive individuals appears to be very effective.5. ACPA-negative patients (about 25% of the total RA population) generally display a much milder course of disease. About 35% of such ACPA-negative patients produce anti-carbamylated protein antibodies. Interestingly, the chemical product of carbamylation (that is, lysine converted to homocitrulline) is structurally very similar to citrulline [8].6. Specific human leukocyte antigen (HLA) genes (DRB1 shared epitope (SE) alleles) not only are the most important genetic risk factor for RA but also are strongly associated with the production of ACPAs.7. The best known environmental risk factor for RA, cigarette smoking, is a risk factor only for ACPA-positive and not for ACPA-negative RA [9]. There is increasing evidence that smoking acts as a trigger for anti-citrulline immunity and does so mainly in the context of certain HLA genes and certain other genetic risk factors.8. ACPAs and citrullinated antigens form immune complexes which stimulate the inflammatory process. Continuous production of such immune complexes ultimately results in the chronic inflammation, characteristic for RA (Figure 1).Open in a separate windowFigure 1Citrullination-related immunity and pathophysiology in rheumatoid arthritis. In genetically susceptible individuals, an environmental factor may initiate a primary inflammation, which can occur in various tissues, and trigger the immune response to citrullinated proteins (left). The resulting anti-citrullinated protein/peptide antibodies (ACPAs) are distributed through the circulation and may form immune complexes with citrullinated proteins produced in an inflamed synovium, thereby boosting the inflammatory process. This will be associated with the infiltration and activation of neutrophils, macrophages, and lymphocytes; cell death; extracellular DNA trap formation; the activation and release of peptidylarginine deiminases (PADs); de novo citrullination; and diversification of the ACPA response. Besides the common inflammation-associated mediators of tissue destruction (not shown), ACPAs and PADs can be directly involved in these processes. HLA, human leukocyte antigen.     

Anti-type II collagen antibodies are associated with early radiographic destruction in rheumatoid arthritis

Introduction

We have previously reported that high levels of antibodies specific for native human type II collagen (anti-CII) at the time of RA diagnosis were associated with concurrent but not later signs of inflammation. This was associated with CII/anti-CII immune complex (IC)-induced production of pro-inflammatory cytokines in vitro. In contrast, anti-cyclic citrullinated peptide antibodies (anti-CCP) were associated both with late inflammation and late radiological destruction in the same RA cohort. We therefore hypothesized that anti-CII are also associated with early erosions.

Methods

Two-hundred-and-fifty-six patients from an early RA cohort were included. Baseline levels of anti-CII, anti-CCP and anti-mutated citrullinated vimentin were analyzed with ELISA, and rheumatoid factor levels were determined by nephelometry. Radiographs of hands and feet at baseline, after one and after two years were quantified using the 32-joints Larsen erosion score.

Results

Levels of anti-CII were bimodally distributed in the RA cohort, with a small (3.1%, 8/256) group of very high outliers with a median level 87 times higher than the median for the healthy control group. Using a cut-off discriminating the outlier group that was associated with anti-CII IC-induced production of proinflammatory cytokines in vitro, baseline anti-CII antibodies were significantly (p = 0.0486) associated with increased radiographic damage at the time of diagnosis. Anti-CII-positive patient had also significantly increased HAQ score (p = 0.0303), CRP (p = 0.0026) and ESR (p = 0.0396) at the time of diagnosis but not during follow-up. The median age among anti-CII-positive subjects was 12 years higher than among the anti-CII-negative patients.

Conclusion

In contrary to anti-CCP, anti-CII-positive patients with RA have increased joint destruction and HAQ score at baseline. Anti-CII thus characterizes an early inflammatory/destructive phenotype, in contrast to the late appearance of an inflammatory/destructive phenotype in anti-CCP positive RA patients. The anti-CII phenotype might account for part of the elderly acute onset RA phenotype with rather good prognosis.     

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides—viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein–Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4—were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10⁻⁶ M > K_D > 10⁻⁷ M) and interacted with HCP1 with higher apparent affinity (K_D = 10⁻⁸ M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.     

Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA.In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n = 60), healthy controls (n = 40), systemic lupus erythematosus (n = 20), Sjögren’s syndrome (n = 40)) were screened for antibody reactivity.Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity.Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.     

Autoantibody profile in rheumatoid arthritis during long-term infliximab treatment

The aim of the present study was to investigate the effect of long-term infliximab treatment on various autoantibodies in patients with rheumatoid arthritis. Serum samples from 30 consecutive patients, who were prospectively followed during infliximab and methotrexate therapy for refractory rheumatoid arthritis, were tested at baseline and after 30, 54 and 78 weeks. At these points, median values of the Disease Activity Score were 6.38 (interquartile range 5.30–6.75), 3.69 (2.67–4.62), 2.9 (2.39–4.65) and 3.71 (2.62–5.06), respectively. Various autoantibodies were assessed by standard indirect immunofluorescence and/or ELISA. Initially, 50% of patients were positive for antinuclear antibodies, and this figure increased to 80% after 78 weeks (P = 0.029). A less marked, similar increase was found for IgG and IgM anticardiolipin antibody titre, whereas the frequency of anti-double-stranded DNA antibodies (by ELISA) exhibited a transient rise (up to 16.7%) at 54 weeks and dropped to 0% at 78 weeks. Antibodies to proteinase-3 and myeloperoxidase were not detected. The proportion of patients who were positive for rheumatoid factor (RF) was similar at baseline and at 78 weeks (87% and 80%, respectively). However, the median RF titre exhibited a progressive reduction from 128 IU/ml (interquartile range 47–290 IU/ml) to 53 IU/ml (18–106 IU/ml). Anti-cyclic citrullinated peptide (CCP) antibodies were found in 83% of patients before therapy; anti-CCP antibody titre significantly decreased at 30 weeks but returned to baseline thereafter. In conclusion, the presence of anti-double-stranded DNA antibodies is a transient phenomenon, despite a stable increase in antinuclear and anticardiolipin antibodies. Also, the evolution of RF titres and that of anti-CCP antibody titres differed during long-term infliximab therapy.     

Alpha-actinin-binding antibodies in relation to systemic lupus erythematosus and lupus nephritis

This study investigated the overall clinical impact of anti-α-actinin antibodies in patients with pre-selected autoimmune diseases and in a random group of anti-nuclear antibody (ANA)-positive individuals. The relation of anti-α-actinin antibodies with lupus nephritis and anti-double-stranded DNA (anti-dsDNA) antibodies represented a particular focus for the study. Using a cross-sectional design, the presence of antibodies to α-actinin was studied in selected groups, classified according to the relevant American College of Rheumatology classification criteria for systemic lupus erythematosus (SLE) (n = 99), rheumatoid arthritis (RA) (n = 68), Wegener's granulomatosis (WG) (n = 85), and fibromyalgia (FM) (n = 29), and in a random group of ANA-positive individuals (n = 142). Renal disease was defined as (increased) proteinuria with haematuria or presence of cellular casts. Sera from SLE, RA, and Sj?gren's syndrome (SS) patients had significantly higher levels of anti-α-actinin antibodies than the other patient groups. Using the geometric mean (± 2 standard deviations) in FM patients as the upper cutoff, 20% of SLE patients, 12% of RA patients, 4% of SS patients, and none of the WG patients were positive for anti-α-actinin antibodies. Within the SLE cohort, anti-α-actinin antibody levels were higher in patients with renal flares (p = 0.02) and correlated independently with anti-dsDNA antibody levels by enzyme-linked immunosorbent assay (p < 0.007) but not with other disease features. In the random ANA group, 14 individuals had anti-α-actinin antibodies. Of these, 36% had SLE, while 64% suffered from other, mostly autoimmune, disorders. Antibodies binding to α-actinin were detected in 20% of SLE patients but were not specific for SLE. They correlate with anti-dsDNA antibody levels, implying in vitro cross-reactivity of anti-dsDNA antibodies, which may explain the observed association with renal disease in SLE.     

Biomarkers of Good EULAR Response to the B Cell Depletion Therapy in All Seropositive Rheumatoid Arthritis Patients: Clues for the Pathogenesis

Objective

To find out whether a high number of auto-antibodies can increase the probability of a “good-EULAR response” and to identify the possible biomarkers of response in seropositive rheumatoid arthritis (RA) patients undergoing the B cell depletion therapy (BCDT).

Patients and Methods

One hundred and thirty-eight patients with long standing RA (LSRA), 75% non or poorly responsive to one or more TNFα blockers, all seropositive for at least one autoantibody (AAB) (RF-IgM, RF-IgA, RF-IgG, anti-MCV, ACPA-IgG, ACPA-IgA, ACPA-IgM) received one full course of BCDT. The major outcomes (moderate or good-EULAR response) were assessed after 6 months of therapy. The IL6 and BAFF levels were also determined.

Results

At a 6-month follow-up, 33 (23.9%) of the RA patients achieved a good EULAR response. Having up to 5-AABs positivity increased the chances for treatment response. After a logistic regression analysis, however, only 4 baseline factors arose as associated with a good-EULAR response: no steroid therapy (OR = 6.25), a lymphocyte count <1875/uL (OR = 10.74), a RF-IgG level >52.1 IU/ml (OR = 8.37) and BAFF levels <1011 pg/ml (OR = 7.38). When all the AABs, except for RF-IgM and ACPA-IgG, were left in the analysis, the two final predictors were no-steroid therapy and low lymphocyte count.

Discussion

The number of AABs increased the chances of being a “good-EULAR” responder. The only predictors, however, at the baseline of a good response in this seropositive cohort of RA patients were 2 simple variables – no steroids and lymphocyte count – and two laboratory assays – IgG-RF and BAFF.     

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles

Introduction

Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.

Methods

Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.

Results

Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit_1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit_1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).

Conclusions

Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.     

Rheumatoid arthritis–associated autoantibodies in non–rheumatoid arthritis patients with mucosal inflammation: a case–control study

IntroductionRheumatoid arthritis–associated autoantibodies (RA-AAB) can be present in serum years before clinical onset of rheumatoid arthritis (RA). It has been hypothesized that initiation of RA-AAB generation occurs at inflamed mucosal surfaces, such as in the oral cavity or lungs. The aim of this study was to assess systemic presence of RA-AAB in patients without RA who had oral or lung mucosal inflammation.MethodsThe presence of RA-AAB (immunoglobulin A [IgA] and IgG anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), IgM and IgA rheumatoid factor (RF), IgG anti-carbamylated protein antibodies and IgG and IgA anti-citrullinated peptide antibodies against fibrinogen, vimentin and enolase) were determined in sera of non-RA patients with periodontitis (PD, n = 114), bronchiectasis (BR, n = 80) or cystic fibrosis (CF, n = 41). Serum RA-AAB levels were compared with those of periodontally healthy controls (n = 36). Patients with established RA (n = 86) served as a reference group. Association of the diseases with RA-AAB seropositivity was assessed with a logistic regression model, adjusted for age, sex and smoking.ResultsLogistic regression analysis revealed that IgG anti-CCP seropositivity was associated with BR and RA, whereas the association with PD was borderline significant. IgA anti-CCP seropositivity was associated with CF and RA. IgM RF seropositivity was associated with RA, whereas the association with BR was borderline significant. IgA RF seropositivity was associated with CF and RA. Apart from an influence of smoking on IgA RF in patients with RA, there was no influence of age, sex or smoking on the association of RA-AAB seropositivity with the diseases. Anti-CarP levels were increased only in patients with RA. The same held for IgG reactivity against all investigated citrullinated peptides.ConclusionAlthough overall levels were low, RA-AAB seropositivity was associated with lung mucosal inflammation (BR and CF) and may be associated with oral mucosal inflammation (PD). To further determine whether mucosal inflammation functions as a site for induction of RA-AAB and precedes RA, longitudinal studies are necessary in which RA-AAB of specifically the IgA isotype should be assessed in inflamed mucosal tissues and/or in their inflammatory exudates.     

Expression of the Inherently Autoreactive Idiotope 9G4 on Autoantibodies to Citrullinated Peptides and on Rheumatoid Factors in Patients with Early and Established Rheumatoid Arthritis

Background

The pre-symptomatic stage of Rheumatoid arthritis (RA) is associated with pro-inflammatory cytokines and autoantibodies. High levels and epitope spread by Rheumatoid factors (RhF) and autoantibodies to citrullinated proteins signify progression towards disease expression. In established RA, the persistence of high autoantibody levels reflects production by both long-lived plasma cells and short-lived plasmablasts. Neither the relative contributions to pathogenesis by autoantibodies from either source, nor the factors responsible for deciding the fate of autoantigen specific ‘parent’ B-cells, is understood. Phenotypic markers identifying subsets of autoreactive B-cells are therefore of interest in understanding the origin and perpetuation of the autoimmune response in RA. One such phenotypic marker is the rat monoclonal antibody, 9G4, which recognises an idiotope on immunoglobuins derived from the inherently autoreactive VH-gene, VH4-34. We therefore investigated whether the 9G4 idiotope was expressed on autoantibodies in patients with RA.

Methodology/Principal Findings

Sera from 19 patients with established RA and those with <1year history of untreated polyarthritis either resolving into RA (n = 42) or non-RA diagnosis (n = 31) were included. Autoantibodies to cyclic citrullinated peptides (CCP), RhF and co-expression of the 9G4 idiotope were measured by ELISA. 9G4 recognised a population of anti-CCP antibodies in the majority of sera from patients with established disease and also in samples from patients with early disaese. 9G4+RhF levels were generally lower and not associated with positivity for, or levels of 9G4+CCP.

Conclusions/Significance

The persistence of 9G4+ immunoglobulins, of any isotype, in serum is rare. We describe here the novel finding of 9G4 expression on anti-CCP antibodies in patients from the earliest symptoms of RA through to established disease. Our results suggest that 9G4 expression on anti-CCP autoantibodies was not due to polyclonal expansion of VH4-34-encoded immunoglobulins. These studies may therefore provide a new focus for investigation into the evolution of the autoimmune response in RA patients.     

PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a specificity of 100% for the disease

We analysed relationships between the PTPN22 1858 polymorphism and antibodies to cyclic citrullinated peptide (CCP), rheumatoid factors (RFs) and the shared epitope (SE) gene (HLA-DRB1*0404 or 0401) and determined their combined predictive value for rheumatoid arthritis (RA) in individuals who subsequently developed RA. This case-control study was nested within the Medical Biobank of Northern Sweden. Patients with RA (n = 92) were identified from amongst blood donors antedating onset of disease by a median of 2.4 (interquartile range 1.2 to 4.9) years. Matched controls were selected randomly from the same cohorts (n = 368). Anti-CCP antibodies and RFs were determined using enzyme-linked immunoassays. Genotyping was performed using an ABI PRISM 7900HT instrument and HLA-SE genes were identified using PCR sequence-specific primers. The 1858T allele and also carriage of T were associated with future onset of RA (odds ratio (OR) = 2.29, 95% confidence interval (CI) 1.45–3.61 and OR = 2.64, 95% CI 1.56–4.47, respectively). The combination of the 1858T variant and anti-CCP antibodies gave 100% specificity for the disease. None of the 368 controls expressed this combination. The PTPN22 1858T variant and anti-CCP antibodies were clearly associated (OR = 3.80, 95% CI 1.51–9.57). A combination of the PTPN22 1858T variant and anti-CCP antibodies gave a much higher relative risk (>132.03) for developing RA than the combination of the T variant and HLA-SE (OR = 7.85). The PTPN22 1858T variant was associated with future development of RA. There was an association between the T variant and anti-CCP antibodies and their combination, found only among pre-patients, gives a very high relative risk for development of RA. The combination gave a specificity of 100% for diagnosing RA.