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Neutrophil Transmigration under Shear Flow Conditions In Vitro Is Junctional Adhesion Molecule-C Independent¹

Monica Sircar^*,, Paul F. Bradfield, Michel Aurrand-Lions, Richard J. Fish, Pilar Alcaide^*, Lin Yang^*, Gail Newton^*, Deanna Lamont^*, Seema Sehrawat^*, Tanya Mayadas^*, Tony W. Liang^¶, Charles A. Parkos^||, Beat A. Imhof and Francis W. Luscinskas^2,*

^* Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; Health, Science and Technology Program, Harvard Medical School, Boston, MA 02115; Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland; Service of Angiology and Haemostasis, Department of Internal Medicine, Geneva University Hospital, Geneva, Switzerland; ^¶ Raven Biotechnologies, South San Francisco, CA 94080; and ^|| Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-, IL-1, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN ₂ integrins during transendothelial migration.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants DK72564, HL72124, and DK61379 (to C.A.P.), HL36028 (to T.M.), and HL36028, HL53993, and HL56985 (to F.W.L.); Swiss National Science Foundation Grants 310000-112551/I (to M.A.-L.) and 3100A0-100697 (to B.A.I.); and grants from Oncosuisse (to B.A.I.) and Thorn Foundation (to M.A.-L.).

² Address correspondence and reprint requests to Dr. Francis W. Luscinskas, Brigham and Women’s Hospital, Harvard University, Vascular Research Division, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: fluscinskas{at}rics.bwh.harvard.edu

³ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; JAM, junctional adhesion molecule; TEM, transendothelial migration; VE, vascular endothelial; DPBS, Dulbecco’s PBS; vWF, von Willebrand factor; RT, room temperature; IP, immunoprecipitation; EGFP, enhanced GFP; DIC, differential interference contrast; TER, transendothelial electrical resistance; WPB, Weibel-Palade bodies.

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