Presence of antibodies against cyclic citrullinated peptides in patients with 'rhupus': a cross-sectional study

'Rhupus' is a rare condition sharing features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). If rhupus is a distinctive entity, an overlap between RA and SLE or a subset of SLE is currently debated. This study was performed to explore the prevalence of antibodies against cyclic citrullinated peptides (anti-CCP antibodies) in rhupus. Patients meeting American College of Rheumatology criteria for RA, SLE, or both were included. Clinical and radiographic features were recorded and sera were searched for anti-CCP antibodies, rheumatoid factor, antinuclear antibodies, anti-extractable nuclear antigens, and antibodies against double-stranded DNA (anti-dsDNA antibodies). Seven patients for each group were included. Clinical and serological features for RA or SLE were similar between rhupus and RA patients, and between rhupus and SLE patients, respectively. Values for anti-CCP antibodies obtained were significantly (p < 0.05) higher in RA (6/7) and rhupus (4/7) than in SLE patients (0/7) and healthy subjects (0/7). Our data support the possibility that rhupus is an overlap between RA and SLE, because highly specific autoantibodies for RA (anti-CCP) and for SLE (anti-dsDNA and anti-Sm) are detected in coexistence.     

Elevated levels of serum antibodies against alpha-1, 6-glucan in patients with systemic lupus erythematosus or rheumatoid arthritis

This study was undertaken to investigate whether levels of anti-alpha-1, 6-glucan antibodies in human sera correlate with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Serum samples were collected from patients with SLE (n = 30), RA (n = 30) and healthy adult volunteers. IgG, IgA and IgM levels against alpha-1, 6-glucan were measured using enzyme linked immunosorbent assays. Anti-alpha-1, 6-glucan IgG prevalence was raised in patients with active SLE (73.3%) and RA (60%) compared with healthy controls (13.3%). Strong correlation between anti-alpha-1,6-glucan-IgG levels and anti-perinuclear factor (r = 0.642; p<0.05) in RA patients or anti-nuclear antibodies (r = 0.675; p<0.05) in SLE patients was observed. No significant differences in anti-alpha-1,6-glucan-IgA or-IgM levels were noted between different groups. We conclude that anti-alpha-1,6-glucan-IgG levels were significantly elevated in patients with SLE or RA and positively correlated with disease activity.     

Increased concentrations of antibody against heat shock protein in patients with myeloperoxidase anti-neutrophil cytoplasmic autoantibody positive microscopic polyangiitis

To determine serum antibody against human and bacterial heat shock protein (HSP) 60/70 in myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibody (ANCA) positive microscopic polyangiitis (MPA), 58 patients with MPO-ANCA positive MPA, 48 with RA (rheumatoid arthritis) and 40 with SLE (systemic lupus erythematosus) were studied. Serum antibodies against HSP (human HSP 70, human HSP 60, Mycobacterium HSP 70, and Escherichia coli HSP 60) were measured by sandwich ELISA. The frequency of anti-human HSP 60/70 antibody positive patients was significantly greater in MPO-ANCA positive MPA than SLE and healthy controls. Anti-human HSP 60/70 antibody titers in patients with MPO-ANCA positive MPA were significantly higher than those of healthy controls; anti-bacterial HSP 60/70 antibody titers were also higher. There was a significant correlation between titers of anti-human HSP 70 antibody and anti-Mycobacterium HSP 70 antibody. A correlation was also found between titers of anti-human HSP 70 antibody and anti-human HSP 60 antibody. Anti-human and bacterial HSP 60/70 antibody titers changed in parallel with disease activity in patients with antibody positive MPA. The anti-HSP antibody titer was also increased in patients with RA and SLE. These results suggest that an immunological background via anti-HSP 60/70 antibodies might be associated with pathogenesis in MPO-ANCA positive MPA.     

Determining oxidant and antioxidant status in patients with genital warts

Abstract

Objectives

Warts are abnormal skin growths caused by human papilloma virus (HPV) infections within the skin of patients. Genital warts usually appear in the perianal and perigenital regions. Asymptomatic warts may be activated after years and may damage natural immunity. The inflammation that occurs during this process may lead to an imbalance between the prooxidant and the antioxidant systems. The aim of this study was to investigate erythrocyte glutathione peroxidase (GSH-Px) activity, serum paraoxonase enzyme levels, and oxidative stress levels in patients with genital warts.

Patients and Methods

In total, 32 patients with genital warts and 35 healthy subjects were included in this study. Erythrocyte GSH-Px activity, serum catalase activity, and paraoxonase enzyme, and malondialdehyde (MDA) levels were determined.

Results

Erythrocyte GSH-Px activity, serum MDA levels, and catalase activity were significantly higher in patients with genital warts than in controls (P < 0.01, P < 0.05, and P < 0.05, respectively). However, serum paraoxonase enzyme levels were not significantly different between groups (P > 0.05). Serum triglyceride levels were significantly lower in patients with genital warts than in controls (P < 0.01). However, there were no statistically signi?cant differences between groups with respect to total cholesterol, high-density lipoprotein cholesterol, or low-density lipoprotein cholesterol levels (all P > 0.05).

Conclusions

Our data suggest that oxidative stress is increased in genital warts. Increased oxidative stress levels may contribute to the pathogenesis of genital warts, and prolonged HPV infection due to chronic inflammation could also affect oxidative stress.     

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Reactive oxygen species, lipid peroxides and essential fatty acids in patients with rheumatoid arthritis and systemic lupus erythematosus

We studied free radical generation, lipid peroxidation and the levels of essential fatty acids and of their metabolites in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Both superoxide and hydrogen peroxide generation by peripheral leukocytes but not malondialdehyde levels, as measured by thiobarbituric acid assay, were found to be significantly enhanced both in RA and SLE. Fatty acid analysis of the plasma PL fraction revealed that both LA and ALA metabolites are significantly decreased in RA and SLE compared to controls. These results suggest that essential fatty acid metabolism is altered in RA and SLE.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of ³⁵S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.     

No evidence for a putative involvement of platelet-activating factor in systemic lupus erythematosus without active nephritis

BACKGROUND: Platelet-activating factor (PAF) seems to be implicated in systemic lupus erythematosus (SLE) patients with associated renal diseases. AIMS: In this study, we ensured the role of PAF in SLE patients without renal complications. METHODS: Blood PAF and acetylhydrolase activity, plasma soluble phospholipase A(2), and the presence of antibodies against PAF were investigated in 17 SLE patients without active nephritis and in 17 healthy controls. RESULTS: Blood PAF levels were not different (p=0.45) between SLE patients (6.7+/-2.8 pg/ml) and healthy subjects (9.6+/-3.1 pg/ml). Plasma acetylhydrolase activity (the PAF-degrading enzyme) was significantly (p=0.03) elevated in SLE patients (57.8+/-6.4 nmol/min/ml) as compared with controls (37.9+/-2.6 nmol/min/ml). Plasma soluble phospholipase A(2) (the key enzyme for PAF formation) was not different (p=0.6) between SLE patients (59.1+/-5.1 U/ml) and controls (54.7+/-2.4 U/ml). Antibodies against PAF were detected only in 3/17 SLE patients. Flow cytometry analysis did not highlight PAF receptors on circulating leukocytes of SLE patients. CONCLUSION: This clinical study highlights no evidence for a putative important role of PAF in SLE patients without active nephritis.     

Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a recently identified proinflammatory cytokine of the TNF superfamily. Studies have indicated that TWEAK plays an important role in renal, vascular injury and immune disease. The aim of this study was to explore the expression of the TWEAK in peripheral blood mononuclear cells (PBMCs) and analyze the correlation between TWEAK and disease activity and renal damage of SLE. The expression of TWEAK in PBMCs was determined by RT-PCR and western blot. SLE disease activity was evaluated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) 2000 score. Next were analyzed the correlations of TWEAK mRNA and protein to serum IL-10, MCP-1 and some laboratory parameters of SLE disease activity. Subjects comprised 48 patients with SLE including 25 patients with renal damage and 23 without, 20 patients with rheumatoid arthrithis (RA) and 15 healthy controls. The results showed that TWEAK expressions in PBMCs from SLE patients were significantly higher than that in RA patients or healthy controls, especially higher in those patients with renal disease. Elevated production of TWEAK is correlated positively and significantly with SLEDAI, proteinuria, serum anti-dsDNA, IL-10 and MCP-1, but inversely associated with serum complements. Our results suggested that TWEAK in PBMCs is positively related to SLE disease activity and might be involved in the pathogenesis of SLE.     

Immune response to nucleic acid antigens and native DNA by human peripheral blood lymphocytes in vitro

The immunogenicity of DNA fragments (either oligonucleotide (oligo) or total DNA digest) covalently linked to keyhole limpet hemocyanin (oligo-KLH or DNA-KLH) was tested with peripheral blood lymphocytes (PBL) from 63 systemic lupus erythematosus patients (SLE) in vitro. PBL from 10 normal individuals and 11 rheumatoid arthritis (RA) patients served as controls. Antibodies to three nucleic acid antigens (oligo, denatured DNA (d-DNA), and native DNA (n-DNA] were assayed in supernatants of cultured lymphoid cells by a sensitive solid-phase radioimmunoassay. More than 50% of SLE and RA patient lymphoid cells formed spontaneous antibodies to one or several nucleic acid antigens. In contrast, only two normals did. After in vitro challenge with oligo-KLH or DNA-KLH, cultured lymphocytes of more than 50% of SLE patients formed antibodies to one or several nucleic acid antigens. Similar results were obtained in PBL from RA patients. In SLE patients, the response to both antigens was either monospecific or polyspecific, but DNA-KLH appeared to raise a greater proportion of antibody to n-DNA than oligo-KLH. A greater proportion of patients with active disease responded in vitro compared with those with inactive disease. A mixture of oligo together with KLH was not immunogenic in vitro. Oligo-KLH or DNA-KLH did not raise antibody to an irrelevant antigen, ovalbumin. Of particular interest, PBL from seven of 10 normal subjects formed antibody to n-DNA after challenge in vitro with oligo-KLH. The data support the view that DNA fragments could be an important immunogen in SLE. Furthermore, this study provides an in vitro model to test the tolerogenicity of similar fragments of DNA linked to self carrier molecules such as gamma-globulin.     

Detection of antiendothelial cell antibodies in patients with connective tissue diseases by flow cytometry and their relation to endothelial cell activation

Antiendothelial cell antibodies (AECA) have been detected by flow cytometry analysis in 23 out of 80 patients with connective tissue diseases. Ten out of 19 serum samples from patients with systemic lupus erythematosus (SLE) were positive. These antibodies were not detectable in healthy donors. We examined the capacity of serum samples to induce endothelial cell activation by modulating cell adhesion molecule expression on human umbilical vein endothelial cells. We found that sera from both AECA-positive and AECA-negative patient groups induced a significantly higher expression of E-selectin compared to healthy controls (P<0.05). There were no differences in the ICAM-1 on VCAM-1 expression. Our data suggest that increased E-selectin expression in activated endothelium in patients with various connective tissue disorders is not related to the production of AECA.     

Anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis: relation with clinical features, cytokines and HLA-DRB1

The specificity and sensitivity of anti-cyclic citrullinated peptide antibodies (anti-CCP) was examined in Latin-American patients with rheumatoid arthritis (RA). The variables considered included: 1) relation with the activity of disease, 2) extra-articular manifestations (EAM), 3) synthesis of cytokines (IL-4, IL-10, IL-12, TNF-alpha, and IFN-gamma) and IgM and IgA rheumatoid factor (RF), and 4) the association with HLA-DRB1 polymorphism. Seventy-nine RA patients were assessed (69 with established RA, and 10 with recent-onset RA not receiving any treatment), 56 with ankylosing spondylitis (AS), 25 with systemic lupus erythematosus (SLE), 50 with primary Sj?gren's syndrome (pSS), and 10 healthy individuals. Of the 69 patients with established RA, 36 were reexamined 2 years later. The activity of the RA was measured by criteria adopted by the American College of Rheumatology. Anti-CCP2, RF and cytokines levels were determined by ELISA. HLA genotypes were established by first, PCR sequence amplification using sequence-specific primers and then, complete sequencing of the product. Anti-CCP antibodies were observed in 96% of patients with RA during the first evaluation and in 86% at the second evaluation (p = 0.12). No significant change in antibody titre was observed between the two evaluations (131 +/- 58.7 and 130.6 +/- 67.1 IU, respectively). The overall sensitivity and specificity was 94% and 92%, respectively; however, at titres > 60 IU, the values were 84% and 95%, respectively. The anti-CCP likelihood ratio positive test was 12 and the likelihood ratio negative test was 0.06. The positive predictive value was 87%, and the negative predictive value was 96%. Anti-CCP antibodies were observed in 12% of SLE and pSS patients, in 2% of AS patients, and in 10% of healthy controls. In RA patients, these antibodies were not associated with the activity of disease, EAM or HLA-DRB1 alleles; no significant correlation was observed between antibody titre and cytokines level. Although anti-CCP antibodies have potential as a diagnostic tool for RA, they are not useful for monitoring clinical activity or predicting the clinical course of disease. Antibody synthesis is HLA-DRB1 independent and not correlated with Th1/Th2 cytokines.     

Deoxyribonuclease-Inhibitory antibodies in systemic lupus erythematosus

Deoxyribonucleases (DNases) are key enzymes for digesting DNA. Abnormalities in the function of these enzymes may contribute to the development of anti-DNA antibodies in systemic lupus erythematosus (SLE). In this study, we used bovine DNase 1-coated ELISA plates to screen anti-DNase antibodies in SLE patients. About 62% of the sera of SLE patients (63/101) were positive for anti-DNase antibodies compared to only 8% of normal controls (8/98). A positive correlation was also found between the concentrations of anti-DNase and anti-DNA antibodies in sera of SLE patients. Affinity-purified anti-DNase immunoglobulin G (IgG) from pooled sera of SLE patients bound to bovine DNase as well as DNA. A synthetic peptide, corresponding to the catalytic site of DNase, was able to completely inhibit the binding of anti-DNase IgG to DNase. In addition to bovine DNase, the anti-DNase IgG also bound to and inhibited the enzymatic activities of DNase present in streptococcal supernatants and human urine. Immunization of lupus-prone NZB/NZW mice with bovine DNase enhanced the production of anti-DNase and DNA antibodies, and accelerated the occurrence of proteinuria. Taken together, these results suggest that DNase-inhibitory antibodies which recognize a conserved epitope near the catalytic site of DNase may act in the pathogenesis of SLE.     

Low Incidence of Rheumatoid Factor and Autoantibodies in Nigerian Patients with Rheumatoid Arthritis

Sera from 53 Nigerian patients satisfying the American Rheumatism Association criteria for a diagnosis of definite or probable rheumatoid arthritis and sera from sick and healthy Nigerian controls were tested for rheumatoid factor, autoantibodies, and immunoglobulin levels. Rheumatoid factor and autoantibodies were found no more frequently in patients with rheumatoid arthritis than in controls. These findings confirm the clinical impression that Nigerian patients with polyarthritis satisfying the criteria for a diagnosis of rheumatoid arthritis differ from Caucasian patients with the disease in a number of important respects. They suggest that either these patients do not have rheumatoid arthritis but a distinct clinical syndrome or that in Nigeria the course of rheumatoid arthritis is modified by genetic or environmental factors.     

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. I. Differences in reactivity with Poly (U) and Poly-(A) Poly (U).

In a previous study, all 40 sera from patients with scleroderma, 20 of 40 sera from SLE patients, but none of 40 sera from normal controls, were found to have antibodies to ssRNA. All scleroderma sera were also found to react with HSA-coupled uridine and UMP and their reaction with HSA-coupled uridine and UMP and their reaction with ssRNA could be inhibited by uracil, uridine, and UMP. To characterize further these uracil-specific anti-RNA antibodies found in scleroderma and compare them with the anti-RNA antibodies found in SLE, we tested their reactivity with Poly (U) and with Poly (A)-Poly (U) and all but one failed to react with Poly (A)-Poly (U). This same serum was the only one in which the reaction with Poly (U) could not be inhibited with uracil. Reactivity of SLE sera was strikingly different from that found in scleroderma sera. Seventeen of 34 SLE sera studied reacted with ssRNA but only four of these reacted with Poly (U). Conversely, two SLE sera that reacted with Poly (U) did not react with ssRNA. Fifteen reacted with Poly (A)-Poly (U) and only two of these failed to react with ssRNA. Five SLE sera which were reactive with ssRNA did not precipitate with Poly (A)-Poly (U). All SLE sera which reacted with Poly (U) could be inhibited with uracil, although less effectively than in scleroderma. Reactivity with Poly (A)-Poly )U) was not inhibited with uracil nor with adenosine. These findings confirm that antibodies to RNA that are found in scleroderma are directed to uracil and thus specific to ssRNA, whereas RNA antibodies found in SLE sera are heterogeneous and directed to either the base, to the site of union of the base and sugar moiety to the ribose backbone, or to the helical structure of double stranded RNA. These differences and the respective antigenic specificities of these anti-RNA antibodies found in scleroderma and SLE may be theoretically important.     

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.