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 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 monolayer of HUVEC cultured on amniotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayer in response to a gradient leukotriene B4 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 Ab staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of 32P-labeled HUVEC with anti-myosin polyclonal Ab. 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.
Second Department of Surgery, Akita University School of Medicine, Hondo Akita City, Japan.
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H
Saito
Y
Y
Minamiya
M
M
Kitamura
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S
Saito
K
K
Enomoto
K
K
Terada
J
J
Ogawa
D000199:Actins / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007975:Leukotriene B4 / Q000494:pharmacology; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology*; D010766:Phosphorylation; D014471:Umbilical Veins
D000199:Actins; D018994:Myosin Light Chains; D007975:Leukotriene B4; D009219:Myosin-Light-Chain Kinase; D009218:Myosins