Loss of the early proapoptotic effect of TNF-α in human neutrophils after delayed addition of TNF-α or pretreatment with PAF. (A) To assess the effect of delayed addition of TNF-α, human neutrophils were incubated at 5 × 106/mL in the absence (▧) or presence of 12.5 ng/mL TNF-α added either at the beginning of the incubation period (▧) or after a delay of 6 hours (▪). The extent of apoptosis was then assessed morphologically at the time points indicated. All data values represent the mean ± SEM of n = 3 separate experiments, each performed in triplicate (*P < .05 compared with time-matched controls). Where not shown, SEM values are less than 2% of means. (B) Human neutrophils (5 × 106/mL) were incubated in MDM containing 10% autologous serum in the presence or absence of PAF (1 μmol/L) for 5 minutes before being placed in 96-well flexiwell plates for a 6-hour incubation with 12.5 ng/mL TNF-α or buffer. Apoptosis was then determined on cytocentrifuge preparations using standard morphological criteria. Values represent mean ± SEM of three separate experiments, each performed in triplicate (*P < .05 compared with controls).
