Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.
Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.
Schatz-Munding
M
M
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Ullrich
V
V
D000470:Alkaloids / Q000494:pharmacology; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D015289:Leukotrienes / Q000096:biosynthesis; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D002413:Cations, Divalent; D015289:Leukotrienes; D011994:Recombinant Proteins; D003609:Dactinomycin; D016718:Arachidonic Acid; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003513:Cycloheximide; D001094:Arachidonate 5-Lipoxygenase; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium