Caspase activation during neutrophil apoptosis. (A) Neutrophils were isolated from human peripheral blood and then cultured at 37° C for up to 20 hours. Apoptosis was determined by examination of nuclear morphology in differentially stained cells (• with dotted line) or detection of active caspase 3 in cells by immunostaining and FACS analysis (▪ with solid line), or by measurement of annexin V–PE binding by FACS (○ with dashed line). (B) Caspase activity was determined in an enzymatic assay using lysates from freshly isolated neutrophils (0 hours) and neutrophils cultured for 9 hours or 20 hours. Caspase activity was measured by detecting release of fluorochrome from tetrapeptide substrates preferentially cleaved by caspase 3 (▪), caspase 8 (□), or caspase 9 (▨). Data for panels A and B are mean ± SD of 3 separate experiments. (C) Lysates were prepared from freshly isolated 0-, 3-, 6-, 9-, 12-, or 18-hour cultured apoptotic neutrophils and analyzed by Western blotting for the presence of caspase 8 and caspase 3. The full-length 32-kDa and 56-kDa (pro.) and cleaved 17-kDa and 41/42-kDa (act.) forms of caspase 3 and 8, respectively, are indicated. Representative of 3 experiments. (D) Neutrophils were cultured for 20 hours in the absence or presence of the caspase 8 inhibitor IETD-fmk (20 μM) or the caspase 3 inhibitor DEVD-fmk (50 μM), and apoptosis was measured by analysis of cell morphology. (E) Neutrophils were cultured in the absence or presence of antagonistic antibodies to CD95 (clone ZB4, 100 ng/mL), TNFα (100 ng/mL), or membrane-bound TRAIL (1 μg/mL) for 20 hours, and apoptosis was determined by analysis of cell morphology. Data for panels D and E are mean ± SD of 3 separate experiments. *P < .01. (F) Freshly isolated neutrophils were lysed and incubated with an anti-CD95 antibody (Apo-1). CD95 immunoprecipitates were then analyzed by Western blotting for the presence of FADD and pro-caspase 8. A representative of 3 experiments is shown.