GM-CSF enhances superoxide anion production and PtdIns(3,4,5)P3 accumulation in peripheral blood neutrophils by a PI3-kinase–dependent mechanism.
(A) Freshly isolated neutrophils (1 × 106 cells) were incubated in the presence or absence of 100 ng/mL GM-CSF or 200 U/mL TNF-α for 30 minutes at 37°C before stimulation with 100 nM fMLP or buffer for 10 minutes, as indicated. Superoxide anion release was determined as detailed in the “Materials and methods” section. GM-CSF and TNF-α significantly enhanced fMLP-stimulated superoxide anion generation. The PI3-kinase inhibitors LY294002 (10 μM) and wortmannin (100 nM) abolished superoxide anion generation under all conditions studied. Data represent the mean ± SD of 2 independent experiments, each performed in triplicate. (B) GM-CSF (100 ng/mL)–stimulated PI3-kinase activity was determined by measuring PtdIns(3,4,5)P3 accumulation at 30 minutes following cytokine addition, as described in the “Materials and methods” section. GM-CSF significantly enhanced PtdIns(3,4,5)P3accumulation compared with time-matched controls. Data represent values from 7 independent experiments, each performed in duplicate, together with mean ± SEM values. **P > .005.
