TLR4/C5aR cosignaling on macrophages enhances their capacity to differentiate Th17 cells. (A) Thioglycollate-elicited peritoneal macrophages from WT mice were stimulated with C5a, LPS, LPS + C5a, or vehicle control (medium) for 20 hours, and IL-6 levels in the supernatant were measured by ELISA. (B) Macrophages were stimulated for 20 hours as in panel A, and naive CD4⁺ T cells from WT mice were then added to the culture and activated by anti-CD3 for 3 days in the presence of recombinant TGF-β1. Some wells were treated with a C5aR antagonist at the time of macrophage stimulation by LPS and C5a (Ant-0 hour) or at the time of T-cell addition and activation (Ant-20 hour). Th17-cell frequencies were determined by FACS after intracellular staining. (C) Macrophage and CD4⁺ T-cell cocultures were set up and analyzed as in panel B except that CD4⁺ T cells were from C5aR^−/− mice. Data are representative of 2 independent experiments. Wells were set up in triplicates, and values shown are mean ± SEM. *P < .01 by Student t test.