Evidence for caspase-dependent and -independent Siglec-9-mediated neutrophil deaths. (A) The caspase inhibitors z-VAD-fmk and z-IETD-fmk completely blocked Siglec-9- and Fas receptor-mediated neutrophil deaths. However, the enhanced Siglec-9-triggered death following GM-CSF priming was only partially blocked by the 2 caspase inhibitors. Results of 20-hour cultures are shown (n = 3). Data are expressed as means ± SEM. (B) Spontaneous neutrophil death was associated with the occurrence of the 17-kDa caspase-3 cleavage product and of the 18-kDa caspase-8 fragment (both detectable in 9-hour cultures). Cleavage of both caspases was accelerated in anti-Fas mAb-treated cultures (the active fragments of caspase-3 and caspase-8 are both detectable in 3-hour cultures). In addition, active 15-kDa Bid was seen in 3-hour anti-Fas mAb-treated neutrophil cultures. In anti-Siglec-9-treated neutrophils, the 2 active caspase products were detectable in 6-hour cultures. Strikingly, although GM-CSF enhanced Siglec-9-mediated neutrophil death, the active caspase cleavage products were not seen in neutrophils cultured less than 9 hours. Immunoblots are representative of 3 independent experiments.