Activators of p38 MAP kinase can serve as costimuli in PF-4–induced exocytosis. (A) Activation of p38 MAP kinase in response to different stimuli. Neutrophils were stimulated with 9 ng/mL TNF, 100 ng/mL LPS, 10 nM fMLP, or 1 μg/mL anisomycin for the times indicated and phosphorylation of p38 MAP kinase was evaluated by Western blot analysis as described in the legend to Figure 2. The data from one representative experiment of 4 are given. (B) Effect of p38 MAP kinase activators on neutrophil exocytosis. Neutrophils were preincubated for 15 minutes in the presence or absence of 10 μM SB203580 followed by stimulation with PF-4 (4 μM) or costimuli alone (9 ng/mL TNF, 100 ng/mL LPS, 10 nM fMLP, or 1 μg/mL anisomycin, respectively), or PF-4 in combination with either costimulus 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 (1.7% ± 0.5%) determined in samples of unstimulated cells run in parallel were subtracted. Data represent the mean from 3 independent experiments (each performed in duplicate) with a variation of SD between 0.4% and 1.9%. Asterisk (*) indicates significant differences (P < .03) between inhibitor-treated and untreated samples based on the data from 3 individual experiments.