TNF-α–induced neutrophil apoptosis is dependent on MAPK p38 and class IA PI3K activity. (A) Inhibition of p38 and PI3K before TNF-α stimulation results in neutrophil survival. Before TNF-α (50 ng/mL) or anti-FAS antibody (1 μg/mL) stimulation, primary human neutrophils were incubated for 30-60 minutes with vehicle (Medium) or with small-molecule inhibitors against PI3K (100nM wortmannin), p38 (1μM SB203580), MEK (50μM PD98059), SAPK/JNK (10μM SP600125), or JAK/STAT (25μM AG490). Viability was assessed by ethidium bromide staining and flow cytometry after 24 hours (n ≥ 3). (B) Class IA PI3Ks are death molecules after TNF-α stimulation. Neutrophils were incubated with vehicle (Control, Medium) or different PI3K inhibitors before TNF-α (50 ng/mL) stimulation. Viability was assessed by ethidium bromide staining and flow cytometry after 24 hours. The following inhibitors were used: the broad-spectrum PI3K inhibitor wortmannin (100nM), the class IA PI3K-selective inhibitor PI103 (100nM), the p110β isoform-selective inhibitor TGX221 (100nM), the p110δ isoform-selective inhibitor IC87114 (1μM), and the p110γ isoform-selective inhibitor AS604850 (1 and 10μM) (n ≥ 4). (C) Activation of p38 and PI3Ks is independent of caspase activity. Neutrophils were pretreated with or without VAD for 60 minutes and subsequently stimulated for 15 minutes with 50 ng/mL of TNF-α or 1 μg/mL of anti-FAS antibody. Cell lysates were analyzed by immunoblotting for phosphorylated Ser473 AKT (an indirect readout for PI3K activity) or phosphorylated Thr180/Tyr182 p38. p38, AKT, and GAPDH protein levels were analyzed as loading controls. A representative immunoblot is shown, and protein expression levels were quantified relative to the control condition (n = 3).