ENDOTHELIAL MYOSIN LIGHT-CHAIN KINASE REGULATES NEUTROPHIL MIGRATION ACROSS HUMAN UMBILICAL VEIN ENDOTHELIAL-CELL MONOLAYER

Although extravasation of neutrophils is a critical step in acute infl ammation, the role of the endothelial cytoskeleton in neutrophil trans migration has not been fully investigated. We used an in vitro model o f neutrophil transmigration across a monolayer of HUVEC cultured on am niotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayer 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 rhoda mine-phalloidin and anti-myosin Ab staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipita tion of P-32-labeled HUVEC with anti-myosin polyclonal Ab, Normally, n eutrophil migration induced F-actin formation, myosin filament formati on, and MLC phosphorylation in HUVEC, When HUVEC was pretreated with t he myosin light chain kinase (MLCK) inhibitor, ML-9, neutrophil migrat ion was diminished and F-actin formation, myosin filament formation, a nd 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 calmodul in antagonist, trifluoperazine, had similar effects, These results ind icate that a calcium/calmodulin-dependent MLCK in endothelial cells re gulates neutrophil transendothelial migration.