Functional detection of bioactive C5a in BALF from IC-challenged mice. Pulmonary IC inflammation was induced in C57BL/6 mice and assayed for IC-induced C5a (IC). Controls received anti-OVA Ab without OVA antigen (Ab). (a) Chemotactic activity was determined at the indicated times by Transwell migration assays of neutrophils (PMNs isolated from bone marrow of C57BL/6 and C5aR–/– mice or C57BL/6 PMNs preincubated with or without anti-C5aR mAb 20/70) elicited with 300 μl of BALF pools obtained from five mice of the IC and Ab treatment groups. (b) Assays using an optimal concentration of 50 ng/ml rhC5a instead of BALF served as positive controls for the indicated PMN preparations. Results are expressed as the percentage of PMNs loaded into the upper chamber that had migrated to the bottom well (means ± SEM for five individual experiments). Differences in PMN migration of C57BL/6 and C5aR–/– mice, or after anti-C5aR 20/70 mAb treatment, were significant (*P < 0.05, **P < 0.001).