Effects of TNFα on neutrophil apoptosis. (A) Neutrophils were incubated for 6 hours (□) or 20 hours (▩) in the absence and presence of GM-CSF (50 U/mL), TNFα (10 ng/mL), or CHX (10 μg/mL), prior to measurement of apoptosis by assessment of morphology. There was a significant difference (*P < .05) between apoptosis of controls and TNFα-treated neutrophils showing initial increase in neutrophil apoptosis, whereas at 20 hours it acted as a survival factor. Values shown are means (± SD, n = 5). (B) Neutrophils were incubated for either 2 hours or 18 hours as indicated, and apoptosis was determined by measuring the binding of FITC–annexin V and flow cytometry. Values shown are means (± SD, n = 4) and * shows a P value of less than .015 between 2-hour control and 2-hour plus TNFα. (C) Neutrophils were stained with MitoTracker red after culture for 2 hours in the absence (control, □) or presence (10 ng/mL, ■) of TNFα. Graph shows percentage of cells that had either lost (negative) or had decreased (dim) or enhanced (bright) fluorescence, compared with that measured in the cell population at time zero. Insert shows representative confocal images. Values shown are means (± SD, n = 5). (D) Mean fluorescence of 6 individual cells whose fluorescence either decreased or was enhanced following TNFα (10 ng/mL) treatment, together with representative confocal images of MitoTracker red fluorescence.