Priming of human polymorphonuclear leukocytes with granulocyte‐macrophage colony‐stimulating factor involves protein kinase C rather than enhanced calcium mobilisation

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Abstract
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 Ca2+‐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 Ca2+‐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 γg/ml) and cycloheximide (50 γg/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 simultanous pretreat‐ment 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.

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