The role of endothelial cell on neutrophil transendothelial migration

尽管中性粒细胞外渗是急性炎症的关键步骤，但内皮细胞骨架在中性粒细胞迁移中的作用尚未被充分研究。我们使用了一种中性粒细胞在羊膜上培养的单层人脐静脉内皮细胞(HUVEC)间迁移的体外模型。在白三烯B_4的作用下，允许人中性粒细胞通过HUVEC单层迁移，然后在显微镜下计数迁移的中性粒细胞数量。我们还用罗丹明-鬼臼蛋白和抗肌球蛋白抗体染色观察内皮F-肌动蛋白和肌球蛋白细丝的形成。用抗肌球蛋白多克隆抗体免疫沉淀法标记HUVEC，检测内皮细胞肌球蛋白轻链(MLC)磷酸化。正常情况下，中性粒细胞迁移诱导HUVEC形成F-肌动蛋白、肌球蛋白细丝形成和MLC磷酸化。当肌球蛋白轻链激酶(MLCK)抑制剂ML-9预处理HUVEC时，中性粒细胞迁移减少，并抑制F-肌动蛋白形成、肌球蛋白细丝形成和MLC磷酸化。细胞内钙离子螯合剂双(O-氨基苯氧基)乙烷-N，N，N‘，N’-四乙酸乙酰氧甲酯(BAPTA/AM)和钙调蛋白拮抗剂三氟拉嗪对HUVEC的作用相似。这些结果表明，内皮细胞内钙/钙调蛋白依赖的MLCK调节中性粒细胞跨内皮细胞的迁移。

Although extravasation of neutrophils is a critical step in acute inflammation, the role of the endothelial cytoskeleton in neutrophil transmigration has not been fully investigated. We used an in vitro model of neutrophil transmigration across a monolayr of human umbilical endothelial cells (HUVEC) cultured on amniotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayr in response to a gradient leukotriene B_4 and then the number of migrated neutrophils were counted microscopically. We also followed endothelial F-actin and myosin filament formation using rhodamine-phalloidin and anti-myosin antibody staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of [^<32>P] labeled HUVEC with anti-myosin polyclnal antibody. Normally, neutrophil migration induced F-actin formation, myosin filament formation and MLC phosphorylation in HUVEC.When HUVEC was pretreated with the myosin light chain kinase (MLCK) inhibitor, ML-9, neutrophil migration was diminished and F-actin formation, myosin filament formation and MLC phosphorylation were inhibited. Pretreatments of HUVEC with the intracellular calcium ion chelator, bis-(O-aminophenoxyl) ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), and the calmodulin antagonist, trifluoperazine, had similar effects. These results indicate that a calcium/calmodulin-dependent MLCK in endothelial cells regulates neutrophil transendothelial migration.