z-VAD-fmk augmentation of TNF alpha-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 microM) enhances TNF alpha-induced neutrophil apoptosis, lower concentrations (1-30 microM) 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-fmk inhibited 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.
Cowburn
Andrew S
AS
Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom. asc32@cam.ac.uk
White
Jessica F
JF
Deighton
John
J
Walmsley
Sarah R
SR
Chilvers
Edwin R
ER
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000276:immunology; D017382:Reactive Oxygen Species / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone