Activation of PI 3-kinase is involved in PF-4/TNF-induced neutrophil exocytosis. (A) Effect of PI 3-kinase inhibitor wortmannin on PF-4/TNF-induced lactoferrin release in human neutrophils. Neutrophils were preincubated for 15 minutes in the presence or absence of increasing concentrations of wortmannin followed by stimulation with PF-4 (4 μM) and TNF (9 ng/mL) alone or in combination for 30 minutes at 37°C. Cells that received stimulus alone and were left without inhibitor served as controls. Lactoferrin release was determined in cell supernatants by the use of a sandwich ELISA system. Assay backgrounds (2.5% ± 0.4%) determined in samples of unstimulated cells run in parallel were subtracted. Data represent mean ± SD of 4 independent experiments, each performed in duplicate. Asterisk (*) indicates significant differences (P < .03) between inhibitor-treated and untreated samples based on the data from 4 individual experiments. (B) Phosphorylation of AKT kinase in response to stimulation with PF-4 or TNF. Neutrophils were stimulated with either PF-4 (4 μM) or TNF (9 ng/mL) for the times indicated. Phosphorylation of AKT kinase was evaluated by Western blot analysis using an antiserum against the Ser473-phosphorylated AKT kinase. Blots were stripped and reprobed with anti-AKT kinase antiserum to confirm equal protein loading. The data from one representative experiment of 3 are given.