The NADPH oxidase mediates intracellular oxidant production but not cell death in zVAD-sensitized, TNF-α–stimulated neutrophils.

Neutrophils isolated from healthy donors or 2 patients with X-linked chronic granulomatous disease (X-CGD) were preincubated with or without zVAD for 30 minutes. Subsequently, TNF-α was added at time 0. At 1 and 4 hours, intracellular production of reactive oxygen species was quantified by the accumulation of DCF fluorescence with flow cytometry (see “Materials and methods”). (A) Results from a healthy donor and patient 1 with documented X91⁺ subtype of X-CGD and complete absence of NADPH oxidase activity. Dotted contours indicate resting neutrophils; bolded contours, neutrophils stimulated with TNF alone; filled-in contours, neutrophils stimulated with TNF in the presence of zVAD. (B) Results of neutrophil survival in both patients by dual-parameter flow cytometry performed at 4 hours. Cytocentrifuge preparations of the neutrophils from patient 1 were stained with Diff-Quick at 6 hours (C; magnification, × 400). Note that the apoptoticlike (closed arrow) and the necroticlike (open arrow) cell death that occurred in patient 1 was similar to that described above.