Autoantigen In Rheumatoid Arthritis Research Articles

Protein microarray characterization of autoantibody responses in rheumatoid arthritis and systemic lupus erythematosus

In rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and other autoimmune rheumatic diseases, our understanding of the specificity of the autoantibody response and the role of autoantibodies in pathogenesis are limited. We developed antigen microarray technology to perform multiplex characterization of autoantibody responses. Antigen microarrays are produced by attaching thousands of proteins and peptides to addressable locations on the surface of solid supports using a robotic arrayer. Arrays are probed with serum from disease and control patients, followed by anti-human secondary antibodies covalently-conjugated to spectrally-resolvable fluorochromes. We initially developed a 'connective tissue disease' array containing structurally diverse autoantigens including nucleic acids, histones, hnRNPs, snRNPs, collagens, Ro, La, SCL-70, CENP-B, Jo-1, pyruvate dehydrogenase, and post-translationally-modified antigens. Array analysis of serum derived from patients with SLE, rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue diseases, scleroderma, myositidies, and primary biliary cirrhosis identified autoantibody response patterns characteristic of these diseases (Fig. ​(Fig.1).1). We are now developing 'synovial proteome' arrays to study the autoantibody responses in RA, and have produced first-generation arrays containing 450 distinct protein and peptide candidate antigens derived from synovial joints. These include native and citrulline-modified filaggrin peptides, native and deiminated fibrinogen and vimentin, glucose-6-phosphate isomerase, collagen types I, II, III, IV and V, hnRNP-A2/RA33, hnRNP-D, immunoglobulins, HCgp39, BiP, and HSPs 60, 65, 70, and 90. We probe our arrays with sera and synovial fluid from RA and control patients. We are using our arrays to examine serial serum samples from patients with early RA to: (1) identify antigens targeted early in disease, and (2) examine for evidence of B-cell epitope spreading. 'Synovial proteome' arrays represent a powerful tool to study the breadth and specificity of autoreactive B-cell responses, and to identify candidate and define relevant autoantigens in RA. Figure 1

The stress protein BiP is a major autoantigen in rheumatoid arthritis

BiP (heavy chain binding protein) is the major chaperone of the endoplasmic reticulum that interacts transiently with most of the proteins of the secretory pathway and assists in their folding. BiP's function under stress conditions is essential for cell viability and constitutively increasing or decreasing the BiP levels is detrimental to cell growth or to survival. Here, we present the RA autoantigen formerly designated p68 as identical to BiP and that autoreactivity against BiP is a specific feature of RA. p68 was isolated and proven to be identical to BiP by two different approaches (Edman sequencing and MALDI-TOF mass spectrometry). Using tissue sections, BiP has been shown to be overexpressed in the RA as compared to the OA joint. Applying immunoblots, BiP-reactive autoantibodies were present in 63% of 400 RA patients, in 7% of 200 patients with other rheumatic diseases and in 1 of 150 healthy individuals. In patients with early arthritis approximately 50% are positive. An ELISA was established to quantify anti-BiP antibodies and the data of 400 RA and 400 control patients will be presented. The majority of RA sera was found reactive with a posttranlationally modified form of BiP and the major epitope was O-linked N-acetylglucosamine. Furthermore, we present evidence that BiP-specific T cell reactivity is pathogenically altered in RA. Overt BiP-specific T cell reactivity as determined by T cell proliferation assays could be observed in two thirds of patients with RA, but neither in healthy individuals nor in patients with other rheumatic diseases. Blocking anti-HLA-DR antibodies expectedly decreased T cell proliferation indicating the presence of HLA-DR restricted effector T cells. A subset of RA patients exhibited a BiP-specifically suppressed T cell reactivity. Blocking anti-HLA-DR antibodies in these assays overcame the suppressive effect and allowed an increased proliferation. This argues strongly for the presence of BiP-specific regulatory T cells restricted for HLA-DR and BiP-specific effector T cells restricted for HLA-DP and -DQ in this subset of RA patients. These effects could not be mimicked by blocking anti-IL-10 or anti-TGF-b antibodies, implicating that other factors or also direct cell-cell-contact are required. Apparently, the healthy immune system views BiP as a component to which autoreactivity is either avoided or tightly regulated. In RA this general principle appears to have lost control. BiP-reactive may serve as a new diagnostic marker in RA, while regulatory T cells may provide means for a specific therapy.

Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthritis.

In an attempt to isolate disease-associated autoantigens in rheumatoid arthritis (RA), we cloned a new autoantigen named gp130-RAPS, which is a novel soluble form of the IL-6 signal-transducing molecule gp130. gp130-RAPS is a 50-kDa protein translated from alternatively spliced mRNA and has a truncated form of gp130 with a unique sequence, Asn-Ile-Ala-Ser-Phe (NIASF), in its COOH-terminus. We observed serum antibodies to this NIASF sequence frequently in patients with RA, but not in those with other systemic rheumatic diseases or in healthy subjects. In RA, detection of those antibodies was significantly associated with disease activity indices such as serum C-reactive protein (CRP) levels, erythrocyte sedimentation rate, blood platelet counts, and serum IL-6 concentration. In vitro experiments revealed that gp130-RAPS inhibited IL-6 activity, and this inhibition was neutralized by antibodies to the COOH-terminus of gp130-RAPS derived from patients with RA. Thus, autoantibody to gp130-RAPS may play an important role in the progression of RA by promoting IL-6 activity. Inspection of autoantibodies to gp130-RAPS may become a practical clinical test for RA. gp130-RAPS and its autoantibody provide a new clue to the complicated pathogenesis of RA.

Synthetic peptides that inhibit binding of the collagen type II 261–273 epitope to rheumatoid arthritis-associated HLA-DR1 and -DR4 molecules and collagen-specific T-cell responses

Copolymer 1 [Cop 1, poly (Y, E, A, K)] is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS), a disease that is linked to HLA-DR2 (DRB1∗1501). Another copolymer [poly (Y, A, K)] was also identified that binds to rheumatoid arthritis (RA)-associated HLA-DR1 (DRB1∗0101) or HLA-DR4 (DRB1∗0401) molecules and inhibits the response of HLA-DR1- and -DR4-restricted T cell clones to an immunodominant epitope of collagen type II (CII) 261–273 (a candidate autoantigen in RA). In the present study various peptides have been synthesized based on binding “motifs” of Cop 1 for HLA-DR1 and -DR4 molecules. Those peptides with K at P-1 or K at P8 were particularly effective as inhibitors of binding of CII 261–273, of Cop 1 and of the influenza virus hemagglutinin peptide 306–318 to these class II proteins. Moreover, several of them were also potent inhibitors of the CII 261–273-reactive T cell clones. These findings suggest that small peptides or their more stable derivatives may be able to substitute for copolymers in the treatment of RA, and by implication of MS.

Human cartilage gp-39+,CD16+ monocytes in peripheral blood and synovium: correlation with joint destruction in rheumatoid arthritis.

To investigate the expression of human cartilage (HC) gp-39, a possible autoantigen in rheumatoid arthritis (RA), in peripheral blood and synovium, to characterize its cellular source, and to analyze correlations with clinical features. The expression of HC gp-39 in synovium and peripheral blood mononuclear cells (PBMC) was assessed by immunohistochemistry and flow cytometry. Synthesis and secretion were investigated by both reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. PBMC expressing HC gp-39 were increased in RA patients compared with spondylarthropathy patients (P = 0.0029) and with healthy control subjects (P = 0.0013). HC gp-39+ cells were also slightly overrepresented in RA synovium (P = 0.01). In both blood and synovium, HC gp-39+ cells were identified as CD14dim,CD16+ monocytes, a phenotype which can differentiate from classic CD14++ monocytes by maturation in vitro. HC gp-39 messenger RNA was detected in RA synovium and PBMC, and PBMC secreted HC gp-39 in vitro. The number of HC gp-39+ PBMC correlated with serum levels of C-reactive protein (r = 0.39, P = 0.003) and HC gp-39 (r = 0.52, P = 0.014). HC gp-39 expression in RA synovial lining correlated with joint destruction (r = 0.77, P < 0.001). CD16+ monocytes, a cellular source of HC gp-39 in vivo, are overrepresented in both RA peripheral blood and synovial tissue. The presence of HC gp-39+ cells in RA synovium is correlated with the degree of joint destruction. These data support a role of these cells in the local autoimmune response that leads to chronic inflammation and joint destruction.

Tissue-associated autoantigens in rheumatoid arthritis. Tissue-antigens detected by autoantibodies in synovial fluid and sera of RA patients.

Tissue-associated autoantigens in rheumatoid arthritis. Tissue-antigens detected by autoantibodies in synovial fluid and sera of RA patients.

Joint-specific and systemic autoreactivity in the development of inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a prevalence of approximately 1%. RA primarily affects peripheral joints, where an inflammatory synovitis may result in cartilage destruction, bone erosion, and ultimately joint deformity and loss of joint function. The aetiology of RA is unknown and the pathogenesis is only poorly understood [1]. What is known is that certain major histocompatibility complex (MHC) class II genes, in particular those encoding the β-chain of the DR1 and DR4 molecules, confer inherited susceptibility to RA [2]. The MHC association strongly suggest that RA is an autoimmune T-cell dependent disorder, in which the disease-associated MHC class II molecules present autoantigenic peptides to autoreactive T cells, that in turn mediate the inflammatory process. The identity of the MHC class II bound autoantigen(s) in RA is unknown, but collagen type II (CII) has been a candidate primarily because it is the major protein component of articular cartilage and because it can induce arthritis in nonhumanized and humanized animal models of RA [3]. The involvement of CII in RA has, however, had a rather chequered history. Although there is now general consensus that humoral immunity to CII is associated with RA, it has been much more difficult to establish a direct role for CII-reactive T cells in the pathogenesis of RA (for example, see [4,5]). Moreover, treatment of RA patients with oral CII has not been as successful as expected on the basis of results obtained from similar trials in collageninduced arthritis in animals [6].

Presence of rheumatoid arthritis (RA) synovial autoantigen recognized by T cells in RA joints

The disease-specific antigen in rheumatoid arthritis (RA) is still unknown. Analyses of the T cell receptor (TCR) repertoire suggested that certain antigen-driven T cells infiltrate into the legion. Efforts to establish T cell lines or clones which recognize RA-specific antigen showed that multiple antigen-reactive T cells were detected in inflamed joints. These antigens fall in two categories, one is foreign antigens which induce molecular mimicry and the other is autoantigens. To date, few etiological studies have shown convincing relationships between RA and infectious diseases. Further, lymphocytes reactive to candidate RA autoantigens were often observed in other autoimmune patients. To determine the primary and RA-specific autoantigen, we focused on T cells specific for RA synovial nurse cells. In the present review, we summarize previous studies on tissue-infiltrating T cells in RA and our recent studies on RA synovial nurse cell-specific autoantigen recognized by T cell clones established from RA joints.

Serum YKL-40 concentrations in patients with rheumatoid arthritis: relation to disease activity.

YKL-40, also called human cartilage glycoprotein-39, is secreted by chondrocytes, synovial cells, macrophages and neutrophils. Studies have shown that YKL-40 is an autoantigen in rheumatoid arthritis (RA). We evaluated whether serum YKL-40 was related to disease activity in patients with RA. Serum YKL-40 was determined by radioimmunoassay in 156 patients with RA during a 1 yr longitudinal study. Serum YKL-40 was increased in 54% of the patients with clinically active disease. Patients with clinically active disease initially who became inactive after 12 months had a significant decrease in serum YKL-40 (-30%, P < 0.002) and patients who changed from inactive to active disease had an increase in serum YKL-40. Patients who remained active had unchanged serum YKL-40 during the study. Serum YKL-40 decreased rapidly (-24% after 7 days, P < 0.01) during prednisolone therapy, and more slowly in patients treated with methotrexate only (-15% after 60 days, P < 0.01). Patients with early RA (disease duration < 3 yr, n = 50) and a persistently elevated serum YKL-40 were at risk of radiological disease progression as determined by Larsen score. Serum YKL-40 varies according to disease activity in RA, but provides in some respect information different from conventional markers. Our previous studies are consistent with a local release of YKL-40 in the arthritic joint followed by a secondary increase in serum YKL-40. YKL-40 may prove to be a new tool for the study of disease activity and pathophysiology of RA.

T-cell anergy induced by clonotype-specific antibodies: modulation of an autoreactive human T-cell clone in vitro.

Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T-cell clone recognizing an epitope of human cartilage gp-39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin-2 (IL-2) gene transcription. In addition, a diminished interferon-gamma (IFN-gamma) production was found. Our data indicate that anergy was not a result of T-cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL-2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb-induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide-induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases.

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones.

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of L-tyrosine, L-glutamic acid, L-alanine, and L-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261-273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261-273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice.

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261-273 (CII 261-273). We have defined MHC and T cell receptor contacts in CII 261-273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abbeta0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.

Matrix Metalloproteinase MMP-19 (RASI-1) is Expressed on the Surface of Activated Peripheral Blood Mononuclear Cells and is Detected as an Autoantigen in Rheumatoid Arthritis

In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patient was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, T H1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.

Promoter analysis of the matrix metalloproteinase RASI-1, a novel autoantigen in rheumatoid arthritis

Promoter analysis of the matrix metalloproteinase RASI-1, a novel autoantigen in rheumatoid arthritis

Promoter analysis of the matrix metalloproteinase RASI-1, a novel autoantigen in rheumatoid arthritis

Promoter analysis of the matrix metalloproteinase RASI-1, a novel autoantigen in rheumatoid arthritis