TNF-α–induced neutrophil apoptosis is dependent on effector caspase activity. (A) Caspase inhibition before TNF-α stimulation results in neutrophil survival. Primary human neutrophils were pretreated with vehicle (Medium) or 10 or 50μM pan-caspase inhibitor VAD in control, TNF-α-treated, and anti-FAS antibody–treated neutrophils. Viability was assessed by ethidium bromide staining and flow cytometry after 24 hours of stimulation (n = 3 for 50μM VAD; n = 7 for 10μM VAD). (B) Caspase-8 is not activated after TNF-α stimulation. Caspase-8 activity was assessed by immunoblot analysis of BID processing. Neutrophils were harvested immediately (Fresh) or left untreated (Control) and stimulated with TNF-α (20 ng/mL) or anti-FAS antibody (1 μg/mL) for 4 hours. A representative immunoblot is shown and BID protein expression levels were quantified relative to the fresh sample (n = 3). (C) Effector caspases are activated after TNF-α stimulation. Total effector caspase activity was assessed by immunoblot analysis of lamin B processing. Neutrophils were treated as in panel B. A representative immunoblot is shown. Lamin B protein expression levels were quantified relative to the 67-kDa lamin B band in the fresh sample (n = 2).