FIG. 4.

The physiological form of properdin (P₃), when bound to C. pneumoniae, induces complement activation using normal human serum. The P₃ form of properdin was isolated from purified properdin by ion-exchange chromatography, followed by size exclusion chromatography, as described in Materials and Methods. (A and B) C. pneumoniae bacteria (50 × 10⁶ IFU) were incubated with (Cpn + P₃) or without (Cpn) 3 μg P₃ in 100 μl HBSS-0.1% BSA for 30 min at 4°C. C. pneumoniae bacteria were then washed, and 5% normal human serum was added in the presence of HBSS-0.1% BSA-Mg-EGTA or HBSS-0.1% BSA-EDTA. These mixtures were incubated for the indicated times at 37°C. The particles were then washed with cold HBSS plus EDTA, and C3b deposition was assessed by flow cytometry using an FITC-labeled anti-C3 antibody. Red, 0 min; green, 5 min; dark blue, 10 min; brown, 15 min; purple, 30 min; light blue, 60 min. (C) C3b deposition was graphed as mean fluorescence intensity, obtained from panels A and B, versus time.