title: Granulocyte-macrophage colony-stimulating factor primes phospholipase D activity in human neutrophils in vitro: role of calcium, G-proteins and tyrosine kinases.
abstract: The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human peripheral blood neutrophils primes phospholipase D (PLD) to subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). The present investigation was directed at the elucidation of the pathway(s) involved in the regulation of the activity of PLD in untreated as well as in GM-CSF-primed neutrophils. Pretreatment with pertussis toxin (PT) totally inhibited fMLP-induced activation of PLD in control or GM-CSF-treated cells. PT did not affect the activation of PLD by PMA but inhibited the priming effect of GM-CSF. Activation of PLD by fMLP was dose-dependently inhibited by erbstatin, an inhibitor of tyrosine kinases. Furthermore, pre-incubation with GM-CSF accelerated the tyrosine phosphorylation response to fMLP (as analysed by protein immunoblot with antiphosphotyrosine antibodies). In PMA-stimulated neutrophils, erbstatin antagonized the priming effect of GM-CSF on PLD without affecting the direct effects of the phorbol ester. Buffering cytoplasmic calcium with the chelator BAPTA inhibited fMLP-induced activation of PLD as monitored by the formation of phosphatidylethanol. The stimulation of PLD by PMA was partially attenuated in BAPTA-loaded cells while the priming effect of GM-CSF was abolished. Thus, priming of human neutrophil PLD by GM-CSF may be mediated by G-proteins, by increases in the levels of cytosolic free calcium, and by stimulation of protein kinase C and/or tyrosine kinase(s).
authors.affiliation: Centre de Recherche en Inflammation, Immunologie et Rhumatologie, Centre Hospitalier de l'Université Laval, Ste-Foy, Québec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Bourgoin
authors.forename: P E
authors.initials: PE
authors.lastname: Poubelle
authors.forename: N W
authors.initials: NW
authors.lastname: Liao
authors.forename: K
authors.initials: K
authors.lastname: Umezawa
authors.forename: P
authors.initials: P
authors.lastname: Borgeat
authors.forename: P H
authors.initials: PH
authors.lastname: Naccache
mesh_terms: D002118:Calcium / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D006873:Hydroquinones; D010712:Phosphatidic Acids; D010566:Virulence Factors, Bordetella; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; C048212:erbstatin