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

Abstract

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocytemacrophage 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 pg/ml) and cycloheximide (50 pg/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 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 Caz+ requirement of phospholipase A l .The human cytokine granulocyte-macrophage colonystimulating factor (GM-CSF) is a 22-kDa glycoprotein produced by activated T-lymphocytes, endothelial cells, fibroblasts, monocytes and macrophages [l-31. It stimulates the proliferation and differentiation of erythroid and myeloid stem cells [4-71.GM-CSF is not found in the circulation at detectable levels, suggesting a paracrine action. Apart from its differentiating activity on the precursor cells, it is also capable of acting directly on mature effector cells like eosinophils, neutrophils and monocytes. Such immediate effects on human Correspondence fa V. Ullrich,

Lymphocytes stimulate expression of 5-lipoxygenase and its activating protein in monocytes in vitro via granulocyte macrophage colony-stimulating factor and interleukin 3.

“…Studies in conditioned HL-60 cells and in GM-CSF-conditioned human neutrophils have not demonstrated regulation of 5-lipoxygenase mRNA expression (9,19). Many previous studies have addressed the regulation of 5-lipoxygenase activity and, depending on the cell type and the duration of study, have reported changes at the level of arachidonic acid release (17,(31)(32)(33), 5-lipoxygenase (9,19,21,34), or FLAP (10,20). In our model of primary cell isolates conditioned with lectin-activated lymphocyte supernatant, we have found regulation of 5-lipoxygenase activity at the level of protein and mRNA expression of both 5-lipoxygenase and FLAP, but found no increase in arachidonic acid release.…”

Multiple Phenotypic Changes Define Neutrophil Priming

“…Direct activation of neutrophils by fMLF does not lead to the detectable release of leukotrienes, but priming with GM-CSF, LPS, or TNFα followed by fMLF stimulation significantly increases LTB 4 release (see Table 1; DiPersio et al, 1988a; Schatz-Munding and Ullrich, 1992; Palmantier et al, 1994; Seeds et al, 1998; Zarini et al, 2006). All three of these priming agents activate PLA 2 and increase AA release without increasing intracellular Ca 2+ (DiPersio et al, 1988b; Schatz-Munding and Ullrich, 1992; Zarini et al, 2006).…”

“…All three of these priming agents activate PLA 2 and increase AA release without increasing intracellular Ca 2+ (DiPersio et al, 1988b; Schatz-Munding and Ullrich, 1992; Zarini et al, 2006). The elevation in available AA substrate leads to prolonged activation of 5-LO and enhanced production of downstream lipid mediators (Surette et al, 1993, 1998; Doerfler et al, 1994).…”

TLR2 ligands augment cPLA2α activity and lead to enhanced leukotriene release in human monocytes

“…Growth factors, cytokines, phorbol esters, LPS, and EBV have been identified and characterized as priming agents [22]. GM-CSF and TNF-␣ priming has been studied widely in neutrophils [23][24][25][26][27][28], and it has been implicated in an enhanced endogenous generation of AA [23,24,26,27]. Furthermore, GM-CSF has been shown to enhance LT synthesis, also when exogenous substrate was used to circumvent cPLA 2 activity [22,29].…”