Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts.

BACKGROUND: In systemic sclerosis (SSc), autoantibodies provide the most accurate tool to predict the disease subset and pattern of organ involvement. Scleroderma autoantibodies target nucleic acids or DNA/RNA-binding proteins, thus SSc immune complexes (ICs) can embed nucleic acids. Our working hypothesis envisaged that ICs containing scleroderma-specific autoantibodies might elicit proinflammatory and profibrotic effects in skin fibroblasts. METHODS: Fibroblasts were isolated from skin biopsies obtained from healthy subjects and patients with diffuse cutaneous SSc (dcSSc). ICs were purified by polyethylene-glycol precipitation from sera of SSc patients bearing different autoantibodies. ICs from patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS) and from normal healthy subjects (NHS) were used as controls. After incubation with ICs, fibroblasts were evaluated for ICAM-1 expression, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-2, tumor growth factor (TGF)-ß1 and Pro-CollagenIα1 secretion, collagen (col)Iα1, mmp-1, toll-like receptor (tlr)2, tlr3, tlr4, tlr7, tlr8, tlr9, interferon (ifn)-α, ifn-ß and endothelin-1 mRNA, and NFκB, p38MAPK and SAPK-JNK activation rate. Experiments were also performed after pretreatment with DNase I/RNase and NFκB/p38MAPK inhibitors. RESULTS: The antigenic reactivity for each SSc-IC mirrored the corresponding serum autoantibody specificity, while no positivity was observed in NHS-ICs or sera. SSc-ICs but not NHS-ICs increased ICAM-1 expression, stimulated IL-6, IL-8, MMP-2, MCP-1, TGF-ß1 and Pro-CollagenIα1 secretion, upregulated et-1, ifn-α, ifn-ß, tlr2, tlr3 and tlr4, and activated NFκB, p38MAPK and SAPK-JNK. tlr9 was significantly upregulated by ARA-ICs, mmp-1 was significantly induced by ACA-ICs whereas colIα1 was not modulated by any SSc-ICs. SLE-ICs and PAPS-ICs significantly upregulated MMP-2 and activated NFκB, p38MAPK and SAPK-JNK. SLE-ICs and PAPS-ICs did not affect colIα1, mmp-1 and Pro-CollagenIα1. DNase I and RNase treatment significantly reduced the upregulation of study mediators induced by SSc-ICs. Pretreatment with NFκB/p38MAPK inhibitors suggested that response to anti-Th/To-ICs was preferentially mediated by p38MAPK whereas ATA-ICs, ACA-ICs and ARA-ICs engaged both mediators. In dcSSc fibroblasts, stimulation with SSc-ICs and NHS-ICs upregulated IL-6 and IL-8. CONCLUSIONS: These data provide the first demonstration of the proinflammatory and profibrotic effects of SSc-ICs on fibroblasts, suggesting the potential pathogenicity of SSc autoantibodies. These effects might be mediated by Toll-like receptors via the interaction with nucleic acid fragments embedded in SSc-ICs.

Comparison of Serological Biomarkers in Rheumatoid Arthritis and Their Combination to Improve Diagnostic Performance.

Introduction: The diagnosis of rheumatoid arthritis (RA) is based on a combined approach that includes serological markers such as rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA). The goal of this study was to evaluate the clinical performance of several RF and ACPA immunoassays for the diagnosis of RA, as well as the diagnostic value of a combinatory approach with these markers. Methods: The study cohort included 1,655 patients from the Swiss Clinical Quality Management registry with sera from 968 patients with RA and 687 disease controls, including patients with axial spondyloarthritis (n = 450) and psoriatic arthritis (n = 237). ACPA were determined by anti-CCP2 IgG enzyme-linked immunosorbent assay (ELISA), QUANTA Flash® CCP3 IgG [chemiluminescent immunoassay (CIA)], and QUANTA Lite® CCP3 IgG ELISA. RF was determined by ELISA (QUANTA Lite® RF IgM, RF IgA, and RF IgG) and with two research use only CIAs (QUANTA Flash® RF IgM and RF IgA). Results: All three ACPA assays showed good discrimination between RA patients and controls and good clinical performance. Overall, CCP3 performed better than CCP2. More pronounced differences were observed between the RF assays. We observed that CIA platforms for both RF IgM and RF IgA showed better performance than the ELISA platforms. Excellent and good total agreements were found between ELISA and CIA for CCP3 (total agreement 95.3%, kappa = 0.90), and between CCP2 and CCP3 ELISA (total agreement 86.6%, kappa = 0.73), respectively. RF IgM CIA and ELISA had a good qualitative agreement (86.5%, kappa = 0.73); RF IgA CIA and ELISA showed a moderate total agreement (78.5%, kappa = 0.53). When combinatory analyses were performed, the likelihood of RA increased with dual positivity and triple positivity and combining different markers resulted in higher odds ratio than the individual markers in all cases. Conclusion: ACPA and RF showed good clinical performance in this large Swiss cohort of RA patients and controls. Overall, the performance of CCP3 was superior to CCP2. The combination of these biomarkers in an interval model represents a potential tool for the diagnosis of RA patients.