Immune complexes containing citrullinated fibrinogen costimulate macrophages via Toll-like receptor 4 and Fcγ receptor.

OBJECTIVE Rheumatoid arthritis (RA) is associated with the presence of anti-citrullinated protein antibodies (ACPAs). Nearly two-thirds of patients with ACPA-positive RA have immune complexes that contain citrullinated fibrinogen, and these citrullinated fibrinogen-containing immune complexes (cFb-IC) can exacerbate disease in murine models of RA; however, the exact role of such ACPA ICs in RA pathogenesis has remained elusive. We undertook the present study to investigate a novel mechanism by which ACPAs specifically targeting citrullinated fibrinogen may directly stimulate macrophage tumor necrosis factor (TNF) production. METHODS Murine or human macrophages were stimulated with native fibrinogen (nFb), cFb, or in vitro-generated nFb-IC or cFb-IC, and TNF production was measured by enzyme-linked immunosorbent assay. ICs were generated with either polyclonal anti-Fb antibodies or pooled IgG from patients with ACPA-positive RA. To evaluate the role of the Toll-like receptor 4 (TLR-4)/myeloid differentiation protein (MyD88) pathway and the Fcγ receptor (FcγR) pathway in the induction of TNF by Fb and Fb-IC, parallel experiments were performed using 1) TLR-4-deficient or MyD88-deficient macrophages, and 2) inhibitors of TLR-4 or FcγR. RESULTS Citrullinated Fb stimulated macrophage TNF production more potently than did native Fb. Incorporation of cFb into ICs augmented its ability to stimulate TNF production by macrophages. Stimulation of TNF by cFb was dependent on TLR-4 and MyD88, while stimulation by cFb-IC was dependent on both TLR-4/MyD88 and FcγR. CONCLUSION We demonstrated that cFb-IC can costimulate macrophages via dual engagement of TLR-4 and FcγR, resulting in the synergistic induction of TNF production. Our findings suggest a potential role of citrullination in increasing the potency of an endogenous innate immune ligand and provide insight into the mechanism by which anticitrulline autoimmunity may contribute to the onset and propagation of inflammation in RA.