Abstract
BackgroundCytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro.MethodsMigration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining.ResultsIL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane.ConclusionWe demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.
Highlights
Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β2-integrin adhesion in granulocytes
Previous studies have shown that upstream activation of PI3K, Extracellular Signal-Regulated Kinase (ERK-1/2), or p38 Mitogen-Activated Protein Kinase (MAPK) [17] pathways caused by IL-8/CXCL8 regulates the induction of transendothelial polymophonuclear leukocytes (PMNs) migration
Transmigration caused by 100 ng/ml IL-8/ CXCL8 was substantially greater after 90 min incubation time compared to 60 min incubation time
Summary
Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β2-integrin adhesion in granulocytes. Previous studies have shown that upstream activation of PI3K, ERK-1/2, or p38 MAPK [17] pathways caused by IL-8/CXCL8 regulates the induction of transendothelial PMN migration. PLA2s are divided into five different groups; a) secretory PLA2 [12,13], b) cytosolic gIVPLA2 (gIVPLA2) [14], c) Ca2+independent PLA2, [15,16] d) platelet-activating factor hydrolyses, [17,18] and e) lysosomal PLA2 [19] Among these groups, gIVaPLA2 is thought to be a ratelimiting enzyme in eicosanoid biosynthesis [20] and to be essential in maintenance of β2-integrin adhesion in granulocytes [21,22]. We have shown that phosphorylation to activate gIVaPLA2 results from upstream phosphorylation of these kinase and that maintenance of this phosphorylated state regulates the process of β2-integrin adhesion [24,25]
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