Sircar Monica, Bradfield Paul F, Aurrand-Lions Michel, Fish Richard J, Alcaide Pilar, Yang Lin, Newton Gail, Lamont Deanna, Sehrawat Seema, Mayadas Tanya, Liang Tony W, Parkos Charles A, Imhof Beat A, Luscinskas Francis W
Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
J Immunol. 2007 May 1;178(9):5879-87. doi: 10.4049/jimmunol.178.9.5879.
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-alpha, IL-1beta, 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 beta(2) integrins during transendothelial migration.
内皮细胞连接黏附分子（JAM）-C被认为可调节中性粒细胞迁移。在本研究中，我们使用针对人JAM-C的功能阻断单克隆抗体，以确定其在流动条件下人白细胞黏附和跨内皮细胞迁移中的作用。人脐静脉内皮细胞（HUVEC）中JAM-C的表面表达始终较低，通过流式细胞术分析评估，用炎性细胞因子肿瘤坏死因子-α、白细胞介素-1β或脂多糖处理并未增加其表面表达。通过免疫荧光显微镜观察，JAM-C染色显示在静息或细胞因子激活的HUVEC上，其在细胞间连接处以稀疏形式定位。令人惊讶的是，对用去污剂通透处理的HUVEC进行染色发现，细胞内存在大量JAM-C，且与血管性血友病因子几乎没有共定位。在体外流动模型中进行的黏附研究表明，单独使用功能阻断JAM-C单克隆抗体对多形核白细胞（PMN）的黏附或迁移没有抑制作用，而抗细胞间黏附分子-1（ICAM-1）单克隆抗体则显著降低了迁移率。有趣的是，JAM-C阻断单克隆抗体与血小板内皮细胞黏附分子-1（PECAM-1）、ICAM-1和CD99阻断单克隆抗体联合使用时，可协同抑制PMN迁移。通过慢病毒JAM-C绿色荧光蛋白融合蛋白感染使JAM-C过表达，并未增加PMN的黏附或迁移程度，也未在跨内皮迁移中发挥JAM-C的作用。这些数据表明，在该模型中，JAM-C在PMN迁移中即使有作用也极小，并且在跨内皮迁移过程中，ICAM-1是PMNβ2整合素在内皮细胞上表达的首选配体。
