The role of PECAM-1 on neutrophil transendothelial migration

Research Project

Project/Area Number	09671206
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	General surgery
Research Institution	Akita University
Principal Investigator	KAMATA Shuichi Akita Univ.Sch.of Med.Research Associate, 医学部, 助手 (00224650)
Co-Investigator(Kenkyū-buntansha)	KAWAI Hideki Akita Univ.Sch.of Med.Research Associate, 医学部, 助手 (20291271) MINAMIYA Yoshihiro Akita Univ.Sch.of Med.Asst.Prof., 医学部, 講師 (30239321) 北村道彦秋田大学, 医学部, 助教授 (10153131)
Project Period (FY)	1997 – 1998
Project Status	Completed (Fiscal Year 1998)
Budget Amount *help	¥3,000,000 (Direct Cost: ¥3,000,000) Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000) Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
Keywords	neutrophil / transendothelial migration / myosin light chain kinase / endothelial cell / PECAM-1 / myosin light chain kinese
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 monolayer of human umbilical endothelial cells (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 antibody staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of [32P] labeled HUVEC with anti-myosin polyclonal 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.