Regulation of Phosphatidylinositol 3-Kinase Activity and Phosphatidylinositol 3,4,5-Trisphosphate Accumulation by Neutrophil Priming Agents

Neutrophil priming by agents such as TNF-α and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-α or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10–60 s), which paralleled the augmented superoxide anion (O2−) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2− release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2− generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-α alone caused a 3-fold increase in PI3-kinase activity in p110γ PI3-kinase immunoprecipitates. p21ras activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2− generation, that PI3-kinase-γ is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21ras underlie this response.