rhC5a enhances FcγRIII-dependent IC activation of AMs in vitro. (a) MH-S AM cells were cultured in medium containing 1% FCS, stimulated (black bars, +rhC5a) or not (white bars, –rhC5a) with 50 ng/ml recombinant human C5a for 2 hours, and assayed for rhC5a-dependent changes in FcγRII/III mRNA normalized to β-tubulin by TaqMan RT-PCR. (b) FCS-cultured MH-S cells (medium control, open circles) were incubated for the indicated time points with rhC5a (filled circles), heat-aggregated IgG (IC, open squares), or the combination of both stimuli (filled squares) and analyzed for production of MIP-2/TNF-α mRNA by TaqMan RT-PCR (upper panels) and MIP-2/TNF-α protein by ELISA (lower panels). Results are expressed as means ± SEM from three independent experiments performed in duplicate. Significant differences were determined by Student’s t test (*P < 0.05; **P < 0.001). Note the more rapid induction of MIP-2 and TNF-α mRNA correlating with significantly increased MIP-2/TNF-α protein concentrations in culture supernatants of ICs + rhC5a as compared with IC treatment groups.