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首页> 外文期刊>Journal of inflammation. >Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro
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Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro

机译：胞质IVa磷脂酶A 2 介导IL-8 / CXCL8诱导的人多形核白细胞体外迁移

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Background Cytosolic 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. Methods Migration 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. Results IL-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. Conclusion We 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.
机译：背景胞质gIVaPLA2是花生四烯酸代谢产物生成和诱导粒细胞中β2-整合素粘附的关键酶。我们假设gIVaPLA2激活也是PMN在体外粘附后迁移的重要下游步骤。方法使用transwell迁移室评估由IL-8 / CXCL8引起的PMN迁移。在暴露于IL-8 / CXCL8之前，先用两种与结构无关的gIVaPLA2抑制剂花生四烯基三氟甲基酮（TFMK）或吡咯酮对PMN进行预处理。测量存在于下部腔室中的迁移的PMN的分数，作为总髓过氧化物酶含量。使用[14C-PAPC]作为特异性底物F-肌动蛋白聚合分析了GIVaPLA2酶的活性，若丹明-鬼笔环肽染色后检查了细胞结构。结果IL-8 / CXCL8诱导的PMNs迁移以浓度和时间依赖性方式引起。对于IL-8 / CXCL8刺激的PMN，其浓度足以引起MAPKs（ERK-1 / 2和p38）和Akt / PKB的上游磷酸化，观察到时间相关的磷酸化和胞质gIVaPLA2转运到细胞核。 gIVaPLA2的抑制与PMN迁移的阻滞程度相对应。 IL-8 / CXCL8激活后，均未引起AA和LTB4分泌。在不受刺激的PMN中，F-肌动蛋白弥漫地分布在细胞质中。然而，用IL-8 / CXCL8激活后，在细胞边缘周围观察到清晰的极化形态，富含F-肌动蛋白的褶皱。 gIVaPLA2的抑制作用可阻止由IL-8 / CXCL8引起的细胞形状变化和迁移，但不会引起F-肌动蛋白聚合或胞质F-肌动蛋白向PMN膜内部小叶移位。结论我们证明IL-8 / CXCL8引起a）细胞质gIVaPLA2的磷酸化和易位至细胞核，b）细胞形状改变，c）F-肌动蛋白聚合，以及d）体外PMN的化学吸引/迁移。抑制gIVaPLA2可阻止IL-8 / CXCL8引起的PMN的变形和随后的迁移。我们的数据表明，激活gIVaPLA2是体外PMN迁移的重要步骤。

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