z-VAD-fmk augmentation of TNFα-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species

In most cell types constitutive and ligand-induced apoptosis is a caspase-dependent process. In neutrophils, however, the broad-spectrum caspase inhibitor z-VAD-fmk enhances tumor necrosis factor-alpha (TNF alpha)-induced cell death, and this has been interpreted as evidence for caspase-dependent and -independent cell death pathways. Our aim was to determine the specificity of the effect of z-VAD-fmk in neutrophils and define the potential mechanism of action. While confirming that z-VAD-fmk (> 100 mu M) enhances TNF alpha-induced neutrophil apoptosis, lower concentrations (1-30 mu M) completely blocked TNF alpha-stimulated apoptosis. Boc-D-fmk, a similar broad-spectrum caspase inhibitor, and z-IETD-fmk, a selective caspase-8 inhibitor, caused a concentration-dependent inhibition of only TNF alpha-stimulated apoptosis. Moreover, the caspase-9 inhibitor, Ac-LEHD-cmk, had no effect on TNF alpha-induced apoptosis, and z-VAD-fmk and Boc-D-fmkinhibited TNF alpha-stimulated reactive oxygen species (ROS) generation. These data suggest that TNF alpha-induced apoptosis in neutrophils is fully caspase dependent and uses a mitochondrial-independent pathway and that the proapoptotic effects of z-VAD-fmk are compound specific and ROS independent.