calpastatin and Arthritis--Rheumatoid

Topics: Acute-Phase Proteins; Antibodies, Antinuclear; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Calcium-Binding Proteins; Disease Progression; Filaggrin Proteins; Humans; Interleukin-1; Intermediate Filament Proteins; Keratins; Matrix Metalloproteinase 3; Peptides, Cyclic; Predictive Value of Tests; Prognosis

Topics: Animals; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Calcium-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Humans

Topics: Antigens, CD; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Calcium-Binding Proteins; Cytokine Receptor gp130; Enzyme-Linked Immunosorbent Assay; Follistatin-Related Proteins; Humans; Immunoblotting; Membrane Glycoproteins

Recently identified autoantibodies in rheumatoid arthritis are targeting autoantigen molecules, such as calpastatin, a novel form of soluble gp130, and follistatin-related protein, that appear to play a role in protecting effects in joint inflammation. These autoantibodies inhibit the function of target molecules and may be involved in the pathogenic mechanisms by upregulating inflammation and joint destruction. Another autoantibody targets filaggrin, a citullurinated protein distributed in keratinated epithelia. Although the pathogenic effect of anti-filaggrin antibody is not elucidated, this autoantibody has a potentiality of a new diagnostic marker of RA because of its high sensitivity and specificity.

Topics: Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Biomarkers; Calcium-Binding Proteins; Filaggrin Proteins; Follistatin; Humans; Intermediate Filament Proteins

Topics: Animals; Arthritis, Rheumatoid; Calcium-Binding Proteins; Calpain; Humans

We have previously described that novel autoantibodies to calpastatin (endogenous inhibitor for calcium-dependent neutral protease, calpain) were detected in patients with rheumatoid arthritis (RA) and other disorders. Since calpain is thought to mediate inflammatory process and cartilage destruction, autoantibodies to its inhibitor protein, calpastatin, may be involved in the pathogenic mechanism of rheumatoid arthritis. In the present study, we analyzed antigenic epitopes reactive with autoantibodies to calpastatin and their clinical correlation. cDNA encoding the C-terminal 178 amino acids of human calpastatin (RA-6) was digested by restriction enzymes and ligated in to pEX expression vectors. These recombinant plasmids were tranfected into E. coli POP2136 and screened by colony blots using RA sera containing anticalpastatin antibodies and a mouse monoclonal antibody. RA patient sera recognized the C-terminus of domain IV (epitope C1 ; aa. 647-673) and C-terminus of domain III (epitope C2 ; aa. 496-571), whereas the mouse monoclonal antibody recognized an entirely different region containing the calpain-binding site (epitope B2 ; aa. 572-625). To evaluate epitope reactivity of patient autoantibodies, 15 RA sera containing anti-calpastatin were reacted with epitope fusion proteins. In immunoblotting, most RA sera recognized either C1 or C2 epitopes (67% and 40%, respectively), and only one patient recognized both epitopes. B2 epitope a more progressed and sever state of arthritis than those not reacting with C1. These results suggests that anti-calpastatin antibodies may play a role in the pathogenic mechanisms of RA and their epitope reactivity may be important for disease progression.

Topics: Adult; Animals; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Calcium-Binding Proteins; Epitopes; Female; Humans; Immunoblotting; Male; Mice; Middle Aged

Topics: Animals; Apoptosis; Arthritis, Rheumatoid; Astrocytes; Calcium-Binding Proteins; Choline; Fatty Acids, Nonesterified; Humans; Immune System; Mycoplasma fermentans; Phosphorylcholine; Rats

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

Topics: Arthritis, Rheumatoid; Calcium-Binding Proteins; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Humans; Inflammation; Interleukin-17; Interleukin-6; NF-kappa B; Protein Conformation; Signal Transduction; STAT5 Transcription Factor; Th17 Cells

Topics: Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Blotting, Western; Calcium-Binding Proteins; Cytokine Receptor gp130; Enzyme-Linked Immunosorbent Assay; Follistatin-Related Proteins; Humans

To test whether HLA-DR alleles influence the production of particular autoantibodies in rheumatoid arthritis (RA) patients, we screened synovial proteins with sera of RA patients homozygous for different HLA-DR alleles by using 2D blots. We found that sera of RA patients homozygous for HLA-DRB1*0404 recognised a 100-kDa synovial protein identified as calpastatin. We studied B and T cell epitopes on calpastatin and their association with HLA-DRB1*0404.. The frequency of positive sera in patients expressing different RA-associated HLA-DR allele combinations was calculated by inhouse ELISA using purified synovial calpastatin or calpastatin peptides encompassing the entire calpastatin protein as immunosorbent. Interaction between calpastatin peptides and HLA-DR alleles was tested by a direct binding assay. T cell responses to calpastatin were measured in RA patients and controls.. We found that RA-associated HLA-DR alleles are associated with presence of autoantibodies to synovial calpastatin in RA patients' sera. HLA-DRB1*0404 is strongly associated with antisynovial calpastatin in RA sera. One linear B cell epitope is preferentially associated with HLA-DRB1*0404. Multiple peptides from calpastatin bind every tested HLA-DR allele associated or not with RA. Peptides from domain 1 and 4 of calpastatin are the best HLA-DR allele binders. The T cell response to calpastatin is frequent in RA patients and independent of the HLA-DR background.. HLA-DRB1*0404 is strongly associated with anticalpastatin antibodies in rheumatoid arthritis.

Topics: Alleles; Arthritis, Rheumatoid; Autoantibodies; B-Lymphocytes; Calcium-Binding Proteins; Case-Control Studies; Cell Proliferation; Cells, Cultured; Chi-Square Distribution; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Epitopes; Genetic Predisposition to Disease; HLA-DR Antigens; HLA-DRB1 Chains; Humans; Protein Binding; Protein Structure, Tertiary; Synovial Membrane; T-Lymphocytes

Rheumatoid factor (IgM-RF) has been widely used to diagnose rheumatoid arthritis (RA) in clinical practice. We investigated the RA diagnostic performances of anti-cyclic citrullinated peptide antibody (anti-CCP), matrix metalloproteinase-3 (MMP-3), anti-agalactosyl IgG antibody (CA*RF), and anti-calpastatin antibody (ACA) in comparison with IgM-RF. Among 68 RA patients, IgM-RF was positive in 31 (45.6%) and negative in 37 (54.4%). In the IgM-RF-positive group, positivity in anti-CCP, CA*RF, and ACA was 97%, 100%, and 97%, respectively, although that in MMP-3 (74%) was inferior to the others. On the other hand, in the IgM-RF-negative group, positivity in anti-CCP, MMP-3, and ACA was 73%, 81%, and 86%, respectively, although that in CA*RF was only 59%. We conclude that the combination of IgM-RF and anti-CCP/ACA will provide an accurate diagnosis of RA in clinical practice.

Topics: Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Calcium-Binding Proteins; Humans; Immunoglobulin M; Peptides, Cyclic; Rheumatoid Factor

To develop a quantitative method of measuring autoantibodies against human calpastatin in rheumatoid arthritis (RA) and to determine their diagnostic value compared with other autoimmune and articular diseases.. We performed a highly sensitive ELISA for IgG and IgM anticalpastatin autoantibodies in human sera using human erythrocyte calpastatin as an antigen. Samples were diluted 1:2000 for the measurement of IgG and 1:400 for IgM.. IgG anticalpastatin antibodies were found in the sera of 48 of 58 patients (82.8%) with RA. In contrast, IgG anticalpastatin antibodies were found in the sera of only 2 of 11 (8.3%) patients with osteoarthritis (OA). Compared to sera from patients with other autoimmune diseases, anticalpastatin antibody sensitivity for RA was better than that of systemic lupus erythematosus (5.6%), systemic sclerosis (0%), mixed connective tissue disease (0%), and Sjögren's syndrome (20%). IgG anticalpastatin antibodies also showed high specificity (96.1%) for RA. Almost 90% of patients with RA were positive for IgG or IgM anticalpastatin antibodies.. We have developed a simple, sensitive, specific, and quantitative ELISA for anticalpastatin antibodies that may have a high diagnostic value for RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Blotting, Western; Calcium-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Female; Humans; Immunoglobulin G; Immunoglobulin M; Male; Middle Aged; Osteoarthritis; Sensitivity and Specificity

Calpastatin is the natural inhibitor of calpains, a protease that is overexpressed in rheumatoid synovial tissue and plays a key role in cartilage destruction. Autoantibodies to calpastatin (ACAST) were recently detected in rheumatoid arthritis (RA). Our aim was to determine their prevalence and their clinical significance.. ACAST were detected in a solid-phase enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the 27 C-terminal amino acids of calpastatin (CAST-C27) as the antigen. All sera reacting with this peptide also bound to purified erythrocyte calpastatin in an ELISA and/or an immunoblot assay. The frequencies and clinical significance of ACAST-C27 were assessed in sera from a well-documented population of 102 community-recruited patients (76 females; mean age 50 yr) with RA that had been evolving for <5 yr (median 2 yr) (group 1), 109 healthy blood donors, 289 patients with non-RA rheumatic disease and 88 community cases of very early (median 4 months) arthritis, i.e. 58 RA and 30 non-RA patients (group 2).. The sensitivity of ACAST-C27 for RA was 19.5% (20/102) in group 1 and 10.3% (6/58) in group 2. These antibodies were also found in patients with anti-double-stranded DNA-positive systemic lupus erythematosus (SLE) (15.5%) and patients with anti-Ro-positive Sjögren's syndrome (18.5%). However, they were not detected in cases of rheumatism resembling early RA, i.e. peripheral spondylarthropathies. ACAST-C27 were not detected in the 30 non-RA patients of group 2. They were predominantly of immunoglobulin isotype G3 and exclusively expressed lambda chains. Among ACAST-C27-positive sera, eight out of 20 (group 1) and four out of six (group 2) were negative for rheumatoid factor and anti-keratin antibodies/antiperinuclear factor. No relationship was found between ACAST-C27 and clinical, biological or radiological findings.. ACAST-C27 are detected only in a restricted set of connective tissue diseases and therefore appear to be specific for RA when antibodies that are usually associated with SLE or primary Sjögren's syndrome are negative. Because of their presence in community cases of very early RA, particularly in some seronegative forms, ACAST-C27 may be useful in discriminating recent-onset RA from the more common non-RA rheumatic diseases, such as spondylarthropathies.

Topics: Adult; Aged; Amino Acid Sequence; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Calcium-Binding Proteins; Connective Tissue Diseases; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Immunoglobulin Light Chains; Longitudinal Studies; Male; Middle Aged; Molecular Sequence Data; Prospective Studies; Protein Structure, Tertiary; Radiography; Sensitivity and Specificity; Seroepidemiologic Studies

STATEMENT OF FINDINGS: An inception cohort of 238 patients having peripheral joint synovitis of less than 12 months duration was evaluated clinically and followed prospectively for 1 year to determine the clinical significance of a number of rheumatoid arthritis (RA) associated autoantibodies. Serum samples collected at the time of the initial evaluation were tested for rheumatoid factor (RF) and antibodies to Sa (anti-Sa), RA-33, (pro)filaggrin [antifilaggrin antibody (AFA)], cyclic citrullinated peptide (anti-CCP), calpastatin, and keratin [antikeratin antibody (AKA)]. RF had a sensitivity of 66% and a specificity of 87% for RA. Anti-Sa, AFA, and anti-CCP all had a specificity of more than 90%, but a sensitivity of less than 50% for this diagnosis. Overall, there was a high degree of correlation between AFA, AKA, anti-Sa or anti-CCP, this being highest between anti-Sa and anti-CCP (odds ratio, 13.3; P < 0.001). Of the 101 patients who were positive for at least one of these four autoantibodies, 57% were positive for only one. Finally, anti-SA identified a subset of predominantly male RA patients with severe, erosive disease. Anti-SA, AFA and anti-CCP are all specific for early RA but, overall, have little additional diagnostic value over RF alone. Although these antibodies may preferentially recognize citrullinated antigens, the modest degree of concordance between them in individual patient sera suggests that it is unlikely a single antigen is involved in generating these responses.

Topics: Acute Disease; Adult; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Calcium-Binding Proteins; Citrulline; Coenzyme A Ligases; Cohort Studies; Epitopes; Female; Filaggrin Proteins; Histocompatibility Testing; Humans; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Peptides, Cyclic; Predictive Value of Tests; Proteins; Rheumatoid Factor; Seroepidemiologic Studies; Synovitis

Our previous reports revealed that calpain has proteoglycanase activity and exists in synovial fluid in osteoarthritis and rheumatoid arthritis. We examined the effects of cytokines on expression of the calpain-calpastatin system in fibroblastic synoviocytes (FLS). Primary cultures of human FLS from osteoarthritis (OA) and rheumatoid arthritis (RA) patients were stimulated with inflammatory cytokines and the amounts of m-calpain and calpastatin mRNAs expressed were determined by Northern blotting. Northern blots were subjected to computerized densitometer and band intensities were determined. Interleukin-1 (IL-1) down-regulated m-calpain and tissue-type calpastatin mRNA expression in OA and RA FLS. In RA FLS, although IL-6 did not alter m-calpain mRNA expression, IL-1 + tumor necrosis factor (TNF) and IL-1 + transforming growth factor (TGF) down-regulated m-calpain mRNA expression. These results provide new information about the effects of inflammatory cytokines on calpain and calpastatin system in OA and RA pathology.

Topics: Arthritis, Rheumatoid; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cytokines; Down-Regulation; Gene Expression Regulation, Enzymologic; Humans; Interleukin-1; Kinetics; Osteoarthritis; RNA, Messenger; Synovial Fluid; Transforming Growth Factors; Tumor Necrosis Factor-alpha

To detect immunoglobulin isotype-specific autoantibodies to native human calpastatin in patients with rheumatic diseases, we performed immunoblot analysis using the heated HeLa cell extracts to enrich heat-resistant calpastatin. The calpastatin molecule that was apparently migrated to 110 kD by SDS-PAGE was confirmed to react with monoclonal anti-human calpastatin antibody in immunoblotting. IgG antibodies to calpastatin were detected in 22 of 48 sera (46%) from patients with RA, whereas only 20% (5/25), 11% (2/19) and 13% (2/15) of sera from SLE, SSc and PM/DM had IgG anti-calpastatin antibodies, respectively. IgM antibodies were also found in 40% (19/48) of RA and 12% (3/25) of SLE patients but not detected in sera from patients with other rheumatic diseases. IgA antibodies were found in only one RA and one SLE serum. In RA, 7 of 48 sera (15%) had IgM antibodies alone, but all SLE sera with IgM antibodies had IgG antibodies. Thus, anti-calpastatin autoantibodies were detected by using the native human calpastatin. Although these autoantibodies were found in patients with various rheumatic diseases, they were present in RA patients at the highest frequency. In particular, the presence of IgM antibodies appeared to be more specific in RA patients.

Topics: Arthritis, Rheumatoid; Autoantibodies; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; HeLa Cells; Hot Temperature; Humans; Immunoblotting; Immunoglobulin Isotypes; Tissue Extracts

Autoantibodies against calpastatin have recently been described to be highly prevalent in sera of patients with rheumatoid arthritis (RA). When the sera of 45 patients with RA were analysed for autoantibodies against calpastatin by a newly developed enzyme-linked immunosorbent assay (ELISA), only four sera (8.9%) tested positive, which is not significantly different from the frequency observed in healthy controls. Since the ELISA is based on a synthetic peptide containing the C-terminal 27 amino acids of calpastatin bound to the solid phase, this negative result might be the consequence of the small antigen used. Therefore, a systematic analysis of the epitopes for autoantibodies in calpastatin was performed using sera from RA patients and healthy individuals. Recombinant fusion proteins containing fragments of calpastatin or the complete protein were produced and sera analysed by Western blots. In the N-terminal portion (amino acids 1-369), at least two major epitopes exist, against which 65% of normal sera as well as 76% of RA sera show reactivity in Western blot assays. These epitopes are not useful for clinical diagnostics. Only five out of 45 (11.1%) RA sera reacted against the C-terminal portion (amino acids 363-708) of calpastatin, while four out of 52 (7.7%) control sera showed reactivity. Three of the five RA sera and two out of four control sera had autoantibodies against the C-terminal 27 amino acids of calpastatin. These three patient sera had already been tested positive in the ELISA. The fourth patient positive in the ELISA was Western blot negative. The differences between the group of RA patients and controls are not statistically significant. When the clinical characteristics of the four patients with autoantibodies against the carboxyl end of calpastatin were analysed, it became apparent that all four had significantly elevated C-reactive protein (>50 mg/l). This observation might indicate that calpastatin autoantibodies are found in RA patients with more active disease. Thus, while the majority of RA patients do not have an increased prevalence of calpastatin autoantibodies, it cannot be ruled out definitively that a small subgroup may be characterized by autoantibodies to the C-terminus of calpastatin.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Arthritis, Rheumatoid; Autoantibodies; C-Reactive Protein; Calcium-Binding Proteins; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Severity of Illness Index

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Arthritis, Rheumatoid; Autoantibodies; Base Sequence; Binding, Competitive; Calcium-Binding Proteins; Calpain; DNA, Complementary; HeLa Cells; Humans; Immunoglobulin G; Molecular Sequence Data; Muscles; Recombinant Fusion Proteins; Rheumatic Diseases; Species Specificity; Swine

RA is the most frequent and most destructive inflammatory arthropathy. Rheumatoid factors, in spite of their lack of disease specificity, are important serological markers for RA and appear important in its immunopathogenesis as well. In search of more disease-specific autoimmune systems, we have screened a human placenta lambda gt11 cDNA expression library using selected sera from patients with classical erosive RA. We have identified one clone (RA-1) that is recognized by three of five screening sera. The 950-bp insert shows a complete nucleotide sequence homology to the cDNA encoding the two COOH-terminal domains of calpastatin. The deduced open reading frame of the RA-1 cDNA predicts a 284-amino acid protein, with a calculated mol wt of 35.9 kD. Calpastatin is the natural inhibitor of calpains, which are members of the cysteine proteinases recently implicated in joint destruction in rheumatic diseases. The two domains encoded by the RA-1 clone each contain the structural features associated with the inhibitory activity of human calpastatin. By Western blotting, 45.5% or 21/44 RA sera specifically recognized both the fusion and the cleaved recombinant protein. This is in contrast to 4.7% (2/43) in nonrheumatoid sera and 0/10 in normal sera. Anticalpastatin autoantibodies could represent a disease-associated marker in chronic erosive arthritis of the rheumatoid type and could hypothetically play a dual pathogenic role, directly via an immune interference and indirectly through an immune complex mechanism.

Topics: Amino Acid Sequence; Arthritis, Rheumatoid; Autoantigens; Base Sequence; Calcium-Binding Proteins; DNA, Complementary; Gene Library; Humans; Molecular Sequence Data; Open Reading Frames; Recombinant Fusion Proteins; Serine Proteinase Inhibitors

Extracellular location of calpain and calpastatin was demonstrated in the cell-free synovial fluid obtained from the knee joint of healthy adult humans and several patients with rheumatoid arthritis. Calpains I and II and a few molecular species of calpastatin were identified by chromatographies on DEAE-cellulose and on Ultrogel AcA 34 columns as well as by immunoelectrophoretic blot analysis. Calpains I and II in the synovial fluid of the patients increased 6.7 times and 3.5 times, respectively, compared with those of the control subjects. With the patients, shortening of the heavy subunits of calpains was noted. Calpastatin also increased in the patients, but it showed rather extensive fragmentation.

Topics: Adult; Arthritis, Rheumatoid; Blotting, Western; Calcium-Binding Proteins; Calpain; Chromatography, DEAE-Cellulose; Extracellular Space; Female; Humans; Knee Joint; Molecular Weight; Synovial Fluid