Background and objectives Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients were they probably contribute to synovitis. We showed that monoclonal IgM paraproteins with RF activity from patients with mixed cryoglobulinemia enhance the FcγR-mediated proinflammatory response of macrophages to ACPA immune complexes (ACPA-IC). Our aim was to investigate the influence of IgM RF from RA patients on the FcR- and complement-dependent effects of ACPA-IC and to examine that of RA-associated IgA RF.
Materials and methods M-CSF-differentiated macrophages were stimulated in a serum-free medium by ACPA-IC formed with plate-bound citrullinated fibrinogen in the absence or presence of one of 5 IgM RF or 3 IgA RF fractions purified from RA serum pools by sequential affinity chromatography. Pro-inflammatory and immunoregulatory cytokines were assayed in the culture supernatants. The expression of IgM and/or IgA receptors was analysed by flow cytometry and their contribution to TNF-α secretion prompted by RF-containing ACPA-IC was evaluated. The complement activation capacity of ACPA-IC formed in the absence or presence of IgM or IgA RF was compared by ELISA measuring deposition of the terminal C5b–9 complex.
Results When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with an increase of the TNF-α:IL-10 ratio and of the IL-6 and IL-8 secretions, and a decrease of the IL-1Ra:IL 1β ratio. The participation of an IgM receptor in the IgM RF-mediated amplification of the inflammatory response of macrophages was excluded, notably as they did not express any established receptor for IgM. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC. LPS further amplified the TNF-α response of macrophages to IgM or IgA RF-containing ACPA-IC. Finally, the presence of IgM RF or of IgA RF increased the capacity of ACPA-IC to activate the complement cascade.
Conclusions Specifically using autoantibodies found in RA patients, the strong complement-dependent and FcR-mediated proinflammatory effect of macro-IC including both ACPA and IgM RF or IgA RF is established, strongly reinforcing the hypothesis of their high pathogenic potential. Simultaneous FcR triggering by these macro-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.