TNF-α induces neutrophil apoptosis independently of Bid. (A) TNF-α stimulation results in neutrophil cell death. Primary human neutrophils were stimulated with TNF-α (50 ng/mL) or anti-FAS antibody (1 μg/mL). Twenty-four hours after neutrophil isolation, viability was assessed by ethidium bromide staining and flow cytometry (n > 10) (left panel). Eight hours after neutrophil isolation, neutrophils were harvested on glass plates, fixed, and stained. Neutrophils with apoptotic features are marked with arrows. A representative section in 1 of 4 independent experiments is shown (right panel). (B) TNF-α–induced murine neutrophil death is independent of Bid. After isolation of neutrophils from wild-type or bid^−/− mice, cells were left untreated or stimulated with TNF-α (50 ng/mL) or FAS ligand (100 ng/mL). After 24 hours of culture, neutrophil apoptosis was assessed by annexin V–propidium iodide staining and flow cytometry (n = 5). (C) Cytochrome c is not released in TNF-α–stimulated neutrophils. Primary human neutrophils were stimulated with TNF-α or anti-FAS antibody for 4 hours. Cytochrome (Cyt) c release from mitochondria was analyzed by flow cytometry. Shown are a quantification of cytochrome c release (n = 3) (left panel) and representative flow cytometry diagrams (right panel).