Role of C5-derived fragments in complement-dependent neutrophil activation. (A) PMNs (2 × 106/mL in HBSS2+) were preincubated with DMSO 1/104 or 10μM C5aR-antagonist W-54011 for 5 minutes, then preactivated with different conditions as in Figure 2, and finally incubated with one-third NHS. The control was buffer-treated PMNs incubated in NHS-EDTA (white bars). CD11b (left) and cell-bound C3d (right) were measured by flow cytometry and expressed as mean plus or minus SD of MFI (n = 3 or 4) and statistical analysis comparison performed between samples with C5aRA and the corresponding DMSO control. *P < .05. ***P < .001. The EDTA control is not shown in the C3d panel because it is close to zero. (B) Similar analysis, where concentrated PMNs were pretreated with anti-CD88 or the IgG1 control, for 15 minutes at room temperature, then diluted to 2 × 106/mL in HBSS2+ and preactivated as in Figure 2, then incubated with one-third of the volume of NHS with EGTA/Mg. (C-D) PMNs were preactivated as in Figure 2, then incubated with one-third vol/vol NHS, C5-depleted serum, or with C5-depleted serum supplemented with 100 μg/mL final concentration of purified C5 (D). Results are mean plus or minus SD MFI (n = 3 or 4) and statistical analysis comparison performed between samples with C5− serum and the corresponding NHS control. *P < .05. ***P < .001.
