zVAD and cycloheximide alter the kinetics of cell death response in TNF-α–stimulated neutrophils.
(A) Compilation from a series of time-course studies of cell death in TNF-α–stimulated neutrophils with and without zVAD preincubation, assayed by dual-parameter flow cytometry. Open circles represent the TNF-α–attributable effect on neutrophil survival (ie, ΔSurvival = TNFLive, the percentage of TNF-α–treated neutrophils that do not bind annexin V–FITC and exclude PI, minus CLive; the percentage of live cells cultured in medium alone) at a given time point in a particular experiment. Closed circles represent zVAD-attributable effect on cell survival in TNF-α–stimulated neutrophils relative to TNF-α alone (ie, ΔSurvival = (TNF + zVAD)Live minus TNFLive) at a given time point within the same experiment. Indicated also are median values of the net effects of TNF-α and zVAD on cell survival at each time point. The intra-assay variability for the assay was less than 2.2% (95% confidence interval) in absolute number. Additionally, a paired t test was used for comparing TNFLive versus CLive and (TNF + zVAD)Live versus TNFLive at each time point. Statistical significance (defined as P < .01) was reached in all comparisons except (TNF + zVAD)Live versus TNFLive at 2 hours (means ± SEM of 73.1% ± 2.7% versus 77.6% ± 2.3%, respectively, n = 19,P = 0.11). Thus, whereas the net cell killing effect exerted by TNF-α occurred early and decreased with time, it was not significantly protected by zVAD at 2 hours and, paradoxically, was significantly augmented and sustained by zVAD at 4 ((TNF + zVAD)Live versus TNFLive, 53.3% ± 3.5% versus 71.6% ± 3.2%, respectively) and 6 hours (52.3% ± 3.2% versus 66.8% ± 2.5%). (B) Neutrophils were preincubated with cycloheximide (CHX, 1 μg/mL) in the absence or presence of zVAD or zDEVD (100 μM) for 30 minutes before the addition of TNF-α at time 0. Cell survival was determined at 4 hours by dual-parameter flow cytometry. *P < .05 compared with TNF + CHX; n = 3.
