Figure 4.

Co-stimulation of macrophage TNF production by citrullinated-fibrinogen-containing immune complexes is dependent on TLR4 and FcγR. A, Peritoneal macrophages from wild-type (WT), TLR4-deficient, or MyD88-deficient mice were stimulated with citrullinated fibrinogen (cFb) alone (50 μg/ml) or cFb-containing immune complexes (cFb-IC; 50 μg/ml of cFb incubated with 75 μg/ml of polyclonal anti-Fb antibody). The TLR4-agonist lipopolysaccharide (LPS, 100 ng/ml) was used as a positive control B, RAW 267.4 macrophages were pretreated for 30 minutes with the TLR4-inhibitor CLI-095 (0.5μg/ml) or media control and then stimulated with cFb alone (10μg/ml) or cFb-IC (10 μg/ml of cFb incubated with 50 μg/ml of polyclonal anti-Fb antibody). C, RAW 267.4 macrophages were pretreated for 30 minutes with FcγR-blocking antibody (10μg/ml), and then both pretreated and untreated, control wells were washed twice with serum-free media and stimulated with cFb alone (10μg/ml) or cFb-IC (10μg/ml of cFb incubated with 50 μg/ml of polyclonal anti-Fb antibody). TNF levels in culture supernatants following 16–18 hours of stimulation were measured by ELISA. Results are representative of experiments performed at least twice. Values are the mean and SEM of triplicate cultures.