title: Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents.
abstract: Neutrophil priming by agents such as TNF-alpha 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-alpha 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-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) 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-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.
authors.affiliation: Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, United Kingdom.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Cadwallader
authors.forename: Alison M
authors.initials: AM
authors.lastname: Condliffe
authors.forename: Alex
authors.initials: A
authors.lastname: McGregor
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Walker
authors.forename: Jessica F
authors.initials: JF
authors.lastname: White
authors.forename: Len R
authors.initials: LR
authors.lastname: Stephens
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chilvers
mesh_terms: D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D003571:Cytochalasin B / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D003571:Cytochalasin B; D004791:Enzyme Inhibitors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007295:Inositol Phosphates; D007527:Isoenzymes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor; D047428:Protein Kinase Inhibitors; D016283:Proto-Oncogene Proteins p21(ras); D013481:Superoxides; D014409:Tumor Necrosis Factor-alpha; C060974:phosphatidylinositol 3,4,5-triphosphate; C081655:inositol-1,3,4,5-tetrakisphosphate; C501009:HRAS protein, human