zVAD-sensitized, TNF-α–stimulated neutrophils undergo atypical cell death characterized by the emergence of early apoptoticlike cells that heralds the appearance of necroticlike cells.

(A) Neutrophils were preincubated with or without zVAD (100 μM) for 1 hour. Subsequently, at time 0, neutrophils were stimulated with TNF-α (10 ng/mL) or equivalent volume of buffered saline. Cell death was analyzed by dual-parameter flow cytometry (see “Materials and methods”) at indicated times. In each panel, the percentages of cells in the left lower (live), the right lower (early apoptotic), and the right upper (primary necrotic and late apoptotic/secondary necrotic) quadrants are indicated. Results shown are representative of 3 experiments. Note that, with typical neutrophil apoptosis such as constitutive or induced by TNF-α alone, there was progression from early apoptosis (annexin V–positive, PI-negative) to secondary necrosis (annexin V–positive, PI-positive) during the 6-hour time course examined. Note also that, with zVAD-preincubation, TNF-α–stimulated, dying (annexin V–FITC–positive) neutrophils were predominantly early apoptotic (PI-negative) at 2 and 3 hours but by 6 hours had progressed, leading to the accumulation of PI-positive cells (similar to that noted in Figure 2A-B). (B) Diff-Quick–stained cytocentrifuge preparations of neutrophils preincubated with or without zVAD and harvested at 4 hours after the addition of TNF-α or buffered saline were examined by light microscopy (magnification, × 1000). Note that TNF-α alone induced classic apoptotic nuclear changes. Similar to some of the features observed by TEM (shown in panel C), zVAD-sensitized, TNF-α–stimulated neutrophils frequently displayed apoptoticlike cell shrinkage and nuclear fragmentation and condensation (black arrow). In addition, necroticlike changes including cell and nuclear swelling that, at times, occurred in cells with fragmented nuclei (white arrow) were observed. (C) Neutrophils preincubated with zVAD were fixed at 2 (i,ii), 3 (iii,iv), and 6 (viii) hours after TNF-α stimulation and examined by TEM (magnification, × 10 000). Some of the notable cellular changes are shown. Also shown are neutrophils harvested after a 6-hour incubation with zVAD (v), cell culture medium (vi), or TNF-α (vii) alone. While some zVAD-preincubated neutrophils appeared to have been activated with significant cytoplasmic vacuolizations as early as 1 hour after TNF-α stimulation (not shown), many assumed atypical cell shapes and appeared to be undergoing extensive cytoplasmic fragmentation (i) and degranulation (ii) at 2 hours. Subtle but definite smoothing out of the outer nuclear envelopes was also noted (i,ii). Additionally, loss of the cell surface microvilli was frequently observed (i,iii). At 3 hours, nuclear lobes in many cells appeared to be fragmented, and nuclear chromatin condensed (iii). Furthermore, confirming the gross visual and light microscopic observations that significant cellular debris accumulated in neutrophil samples incubated with zVAD and TNF-α, many cellular fragments that appeared to remain membrane-bound were noted by TEM (iv). In addition, some necrotic cells were noted (not shown). In contrast, neutrophils incubated with zVAD alone appeared relatively normal, with preserved cell surface microvilli and nuclear morphology (v). Classically apoptotic neutrophils were observed when aged in culture medium alone (vi). Neutrophils stimulated with TNF-α alone also showed typical apoptotic nuclear changes (vii). Interestingly, similar nuclear changes could be seen at this time point in TNF-α–stimulated neutrophils in the presence of zVAD (viii).