Cartilage-binding antibodies induce pain through immune complex-mediated activation of neurons.
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ABSTRACT:
SUBMITTER: Bersellini Farinotti A
PROVIDER: S-EPMC6683987 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Antibodies binding to cartilage proteins are present in the blood and synovial fluid of early rheumatoid arthritis patients. In order to develop animal models mimicking the human disease, we have characterized the arthritogenic capacity of monoclonal antibodies directed towards different joint proteins in the cartilage. METHODS:Purified antibodies specific to unmodified or citrullinated collagen type II (CII), collagen type XI (CXI), and cartilage oligomeric matrix protein (COMP) were produced as culture supernatant, affinity purified, pooled as antibody cocktails (Cab3 and Cab4), and injected intravenously into mice to induce arthritis. An adjuvant (lipopolysaccharide or mannan) was subsequently injected intraperitoneally on either day 5 or day 60 to enhance arthritis. Antibody binding and complement activation on the cartilage surface were analyzed by immunohistochemical methods. Bone erosions and joint deformations were analyzed by histological assessments, enzyme-linked immunosorbent assays, and micro-CT. Luminex was used to detect CII-triple helical epitope-specific antibody responses. RESULTS:The new cartilage antibody cocktails induced an earlier and more severe disease than anti-CII antibody cocktail. Many of the mouse strains used developed severe arthritis with 3 antibodies, binding to collagen II, collagen XI, and cartilage oligomeric matrix protein (the Cab3 cocktail). Two new models of arthritis including Cab3-induced LPS-enhanced arthritis (lpsCAIA) and Cab3-induced mannan-enhanced arthritis (mCAIA) were established, causing severe bone erosions and bone loss, as well as epitope spreading of the B cell response. Cab4, with addition of an antibody to citrullinated collagen II, induced arthritis more efficiently in moderately susceptible C57BL/6?J mice. CONCLUSIONS:The new mouse model for RA induced with cartilage antibodies allows studies of chronic development of arthritis and epitope spreading of the autoimmune response and bone erosion.
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Project description:Complex I (NQR) is a critical site of superoxide (O2-*) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYICGEETALIESIEGK(219) of 51-kDa protein and (361)VDSDTLCTEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O2-* generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated O2-*generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.
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Project description:ObjectiveAutoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.MethodsAnti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.ResultsNET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.ConclusionIgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.
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