A5.16 TNF-α Response of Synovial Fluid Monocyte-Macrophages to ACPA Immune Complexes

Abstract

Background Autoantibodies to citrullinated proteins (ACPA) are specifically associated to rheumatoid arthritis (RA) and produced in the inflamed synovium where citrullinated fibrin, their main antigenic target, is abundant. Using a human in vitro model we showed that macrophages generated by differentiation of blood monocytes from healthy individuals or patients with RA secrete TNF-α in response to immune complexes formed by ACPA and citrullinated fibrinogen (ACPA-IC). Moreover while in both healthy individuals and RA patients the TNF-α response of macrophages was much higher than that of their monocyte precursors, the TNF-α production of blood monocytes and monocyte-derived macrophages from RA patients did not differ from that observed with the healthy controls. Objectives To further assess the impact of ACPA-IC on joint inflammation, we evaluated the TNF-α response they prompt in monocyte-macrophages isolated from the synovial fluid (SF) of patients with RA and with other arthritides. Materials and Methods SF samples were obtained from 7 patients with RA (4 ACPA-positive, 3 ACPA-negative) and 8 ACPA-negative control patients with various arthritides. Polymorphonuclear cells were eliminated by centrifugation over Ficoll or capture with CD15-conjugated magnetic beads then monocyte-macrophages further purified using CD14-beads (median purity: 95%). The purified cells were stimulated with IC generated by capture of ACPA from IgG fractions prepared from RA sera, on immobilised citrullinated fibrinogen, as described (Clavel, Arthritis Rheum, 2008). Results With SF monocyte-macrophages from both RA and control patients, secretion of TNF-α sometimes occurred in the absence of any stimulation. However in RA patients, irrespective of their ACPA status, TNF-α secretion increased when the cells were cultured on ACPA-IC (median (range) = 16 (2–110) pg/ml). Such activation was also observed with the SF monocyte-macrophages from control patients (184 (33–638) pg/ml). In the whole series of SF samples, the increase in TNF-α secretion was found to be highly significant (p Conclusions In contrast with our previous observations on CD14-positive blood monocytes and on derived macrophages, it appears that CD14-positive monocyte-macrophages can be pre-activated in the SF and secrete TNF-α spontaneously, but that they can nevertheless be further activated by ACPA-IC. These properties are not restricted to RA patients and seem to be characteristic for the SF CD14-positive monocyte-macrophages. Since citrullinated fibrin and ACPA have been described in the SF of RA patients, it is highly probable that ACPA-IC play a direct pro-inflammatory role in the SF by inducing or enhancing TNF-α secretion by the SF monocyte-macrophages.