Endothelial Myosin Light Chain Kinase Regulates Neutrophil Migration Across Human Umbilical Vein Endothelial Cell Monolayer This information is current as Hajime Saito, Yoshihiro Minamiya, Michihiko Kitamura, Satoshi of October 2, 2021. Saito, Katsuhiko Enomoto, Kunihiko Terada and Jun-ichi Ogawa J Immunol 1998; 161:1533-1540; ; http://www.jimmunol.org/content/161/3/1533 Downloaded from References This article cites 45 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/161/3/1533.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Endothelial Myosin Light Chain Kinase Regulates Neutrophil Migration Across Human Umbilical Vein Endothelial Cell Monolayer1 Hajime Saito,* Yoshihiro Minamiya,2* Michihiko Kitamura,* Satoshi Saito,* Katsuhiko Enomoto,† Kunihiko Terada,‡ and Jun-ichi Ogawa* 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 Downloaded from (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/ http://www.jimmunol.org/ calmodulin-dependent MLCK in endothelial cells regulates neutrophil transendothelial migration. The Journal of Immunology, 1998, 161: 1533–1540. critical step in acute inflammation is migration of the discontinuities at the tricellular corner where the majority of neu- circulating neutrophil from the vascular compartment trophils migrate across (14). A through a gap between adjacent endothelial cells into Phosphorylation of myosin II light chain (MLC) by a calcium/ surrounding tissue (1–3). Substantial evidence has accumulated calmodulin-dependent kinase is considered to be an essential step demonstrating that adhesion molecules, such as b2 integrin (4–7), in contraction of smooth muscle cells (15–18) and other cells (19– ICAM-1 (6–8), and platelet endothelial cell adhesion molecule-1 21) through interaction of actin and myosin (22, 23) and formation by guest on October 2, 2021 (PECAM-1)3 (9–11), are required for initiation of neutrophil of myosin II filaments (24–27). There have also been reports that transendothelial migration. Mechanisms involved after neutrophil the actin filament binds directly to the adherence junction-associ- adhesion have received less attention. In particular, signaling ated protein, a-catenin (28, 29), and that a-spectrin, which is mechanisms triggered within endothelial cells during neutrophil cross-linked to the actin filament, binds to the tight junction asso- transmigration are not well understood. Huang et al. have provided ciated-protein, ZO-1 (30). Histamine and thrombin-induced phos- data indicating that cytosolic calcium-dependent endothelial sig- phorylation of MLC have been shown to initiate endothelial cell naling results in creation of gaps at junctions between adjacent retraction (31–35) and to result in disassembly of the adherence endothelial cells through which neutrophils are able to pass (12). junction complex (36). Taken together, these findings support a There are two kinds of endothelial cell junctions (ECJ) described hypothesis that phosphorylation of MLC induces opening gaps be- as tight junctions and adherence junctions (13, 14), both displaying tween endothelial cells associated with tricellular corners and with ECJ. In the present study we examine the hypothesis that MLCK in endothelial cells plays an active role in transendothelial migration † ‡ *Second Department of Surgery, First Department of Pathology, and First Depart- of neutrophils. To assess MLC phosphorylation during neutrophil ment of Biochemistry, Akita University School of Medicine, Hondo Akita City, Japan transmigration, we used an in vitro model consisting of a mono- Received for publication September 29, 1997. Accepted for publication April 8, 1998. layer of HUVEC cultured on amniotic membrane (37). We also The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance investigated the effect of inhibition of MLCK on migration of neu- with 18 U.S.C. Section 1734 solely to indicate this fact. trophils across a HUVEC monolayer. Our results are in accord 1 This work was supported by Grant-in-Aid for Scientific Research (C) from the with a calcium/calmodulin-dependent MLCK acting to regulate Ministry of Education, Science, Sports, and Culture of Japan 08671507 (Y.M.). transendothelial neutrophil migration. 2 Address correspondence and reprint requests to Dr. Yoshihiro Minamiya, Assistant Professor of Pulmonary Surgery, Second Department of Surgery, Akita University School of Medicine, 1-1-1 Hondo Akita City 010-8543, Japan. E-mail address: [email protected]. Materials and Methods 3 Abbreviations used in this paper: PECAM-1, platelet endothelial cell adhesion mol- Abs and reagents ecule-1; ECJ, interendothelial junction; MLC, myosin II light chain; MLCK, myosin light chain kinase; anti-M II pAb, anti-human platelet myosin II rabbit polyclonal Ab; Anti-human platelet myosin II rabbit polyclonal Ab (32) (anti-M II pAb) 21 was kindly provided by Dr. Robert Wysolmerski (St. Louis University, St. LTB4, leukotriene B4; [Ca ]i, intracellular calcium ion; BAPTA/AM, bis-(O-amino- 9 9 phenoxyl)ethane-N, N, N , N -tetraacetic acid acetoxymethyl ester; IC50, 50% inhib- Louis, MO). The anti-chicken myosin light chain mouse mAb MY-21, itory concentration. which cross-reacts with human MLC, was purchased from Sigma (St. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1534 ENDOTHELIAL CELLS REGULATE NEUTROPHIL MIGRATION Louis, MO). Leukotriene B4 (LTB4) was kindly provided by Ono Phar- at room temperature with anti-M II pAb diluted with PBS containing 0.1% maceutical (Osaka, Japan). Rhodamine-phalloidin and bis-(O-aminophe- BSA (final Ab concentration: 0.75 mg/ml). After rinsing with PBS con- noxyl) ethane-N, N, N9, N9-tetraacetic acid acetoxymethyl ester (BAPTA/ taining 0.1% BSA, the membrane was incubated at room temperature for AM) were purchased from Molecular Probes (Eugene, OR). 1 h with secondary Ab, anti-rabbit IgG pAb conjugated with rhodamine. Trifluoperazine and ML-9 were purchased from Calbiochem (La Jolla, CA) After washing with PBS, several drops of 90% glycerol/10% PBS con- and Seikagaku Kogyo (Tokyo, Japan), respectively. taining 0.1 M N-propylgallate were added and the membrane was covered with a coverslip. Finally, the membrane was examined using a confocal Assay of transendothelial migration of neutrophils laser scanning microscope system (LSM 410, Zeiss, Oberkochen, Ger- many) coupled to a Axioverd 135 fluorescence microscope (Zeiss). HUVEC culture. HUVEC were harvested by perfusion of umbilical vein with 0.25% trypsin (Life Technologies, Grand Island, NY) according to the Myosin light chain phosphorylation modified method of Jaffe et al. (38). Cell preparations were transferred to 60-mm plastic tissue culture dishes coated with type I collagen (Sigma), To analyze myosin phosphorylation, myosin was immunoprecipitated as and HUVEC were grown in M199 medium (Life Technologies) supple- described by Wysolmerski and Lagunoff (35, 41) with minor modifications. mented with 20% FCS, 100 U/ml penicillin G, and 100 mg/ml streptomycin A confluent monolayer of HUVEC on an amniotic culture ring (diameter 32 while being maintained at 37°C in a humidified 5% CO2, 95% air atmo- 90 mm) was labeled with [ P]orthophosphoric acid (DuPont-NEN, Bos- sphere. HUVEC were then seeded onto acellular human amniotic tissue ton, MA) as follows. HUVEC were washed twice with