| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
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.
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