ENDOTHELIAL CELL BIOLOGY IN INFLAMMATION

这项建议针对白细胞中的内皮细胞(EC)生物学 贩运和炎症，侧重于血管粘连的作用 淋巴细胞归巢调控中的受体和激活因子。 I.粘膜血管地址素的生物学，MAdCAM-1将是 探索通过：1)原位视频显微镜分析 MadCAM-1在L-选择素介导的淋巴细胞滚入中的作用 Peyer‘s补片(PP)-高内皮微静脉(HEV)；2)测定 MAdCAM-1结合和激活淋巴细胞的能力 趋化因子家族的趋化因子；3)免疫组织学评估 MAdCAM-1变异型的生理分布；4) MAdCAM-1基因敲除小鼠的形态和功能分析 将这些研究扩展到人类。II.分子和功能 外周淋巴结寻址蛋白的生物学--HEV配体 L--选择素--通过以下几个方面进行研究：1)L的结构分析-- HEV的选择素结合硫酸糖苷利用一种新的优势 抗Pnad糖型单抗小组：2)显性Pnad的鉴定 负责HEV：PNAD糖体提呈的糖蛋白(GP‘s) 在活体归巢研究中具有可证明的生理相关性的GP 将作为分子克隆的靶点；3)基因鉴定 编码参与Pnad糖蛋白合成的酶，或其他HEV 分化抗原，用抗PNAD的哺乳动物表达克隆 糖蛋白表位单抗；通过分离HEV特异性cDNAs 杂交选择。三、HEV或EC相关因子(S) 负责G蛋白介导的淋巴细胞的快速激活 归巢将通过免疫组织学和体内抗体来确定 趋化因子对HEV的抑制研究；如果通过 能够抑制激活步骤的单抗的生产 体外人工血管模型中淋巴细胞与内皮细胞的相互作用。 四、体外迁移和/或趋化试验将决定 MAdCAM-1可作为α4β7+淋巴细胞的选择性底物 爬行，与局部趋化物质协同作用调节 淋巴细胞亚群的趋化性或趋化性。五、关于 血管地址蛋白和其他EC黏附的发育调节 将对受体进行免疫组织学研究；已知或 通过体外实验探索寻址蛋白调节中的新细胞因子 培养EC株的研究，包括一种新的转化的HEV细胞 在对细胞因子基因敲除小鼠的分析中也是如此。六、 生理作用(S)，功能，以及分子生物学(如果有指示) 新发现的与白细胞运输有关的EC抗原将 探索：这些包括MECA-32抗原(其抗体 抑制淋巴细胞归巢)；PA 67抗原(参与T细胞 与激活的小鼠EC的黏附)；和92-ELAM(参与黏附 单核细胞)。最后，血管选择素在淋巴细胞中的作用 将调查贩运到发炎关节的情况。 拟议的研究应该扩大我们对关键作用的理解 血管内皮细胞在调节淋巴细胞转运中的作用 正常和病理免疫反应。

This proposal targets endothelial cell (EC) biology in leukocyte trafficking and inflammation, focusing on the role of vascular adhesion receptors and activating factors in the control of lymphocyte homing. I. The biology of the mucosal vascular addressin, MAdCAM-1 will be explored through: 1) In situ videomicroscopic analyses of the contribution of MAdCAM-1 to L-selectin-mediated lymphocyte rolling in Peyer's patch (PP)-high endothelial venules (HEV); 2) determination of the ability of MAdCAM-1 to bind and present lymphocyte activating chemoattractants of the chemokine family; 3) immunohistologic assessment of the physiologic distribution of MAdCAM-1 variant forms; 4) morphologic and functional analyses of MAdCAM-1 knockout mice; and 5) extension of these studies to man. II. The molecular and functional biology of the peripheral lymph node addressin (PNAd)--HEV ligands for the L-selectin--will be studied through: 1) structural analyses of L- selectin-binding sulfated glycotopes of HEV, taking advantage of a novel panel of anti-PNAd glycotype MAbs: 2) identification of dominant PNAd glycoproteins (gp's) responsible for glycotope presentation by HEV: PNAd gp's with demonstrable physiologic relevance in in vivo homing studies will be targeted for molecular cloning; 3) identification of genes encoding enzymes involved in PNAd glycotope synthesis, or other HEV differentiation antigens, by mammalian expression cloning with anti-PNAd glycotope MAbs; and through isolation of HEV-specific cDNA's by hybridization selection. III. HEV- or EC-associated factor(s) responsible for rapid G-protein-mediated activation of lymphocytes during homing will be identified through immunohistologic and in vivo antibody inhibition studies of chemokines on HEV; and if indicated through production of MAbs capable of inhibiting the activation step in lymphocyte interactions with EC in in vitro artificial vessel models. IV. In vitro migration and/or chemotaxis assays will determine whether MAdCAM-1 can serve as a selective substrate for alpha4Beta7+ lymphocyte crawling, acting coordinately with local chemoattractants in regulating the chemotaxis or haptotaxis of lymphocyte subsets. V. The developmental regulation of vascular addressins and of other EC adhesion receptors will be studied immunohistologically; and the role of known or novel cytokines in addressin regulation will be explored through in vitro studies of cultured EC lines, including a novel transformed HEV cell line; and also in analyses of cytokine knockout mice. VI. The physiologic role(s), functions, and if indicated, the molecular biology of newly identified EC antigens involved in leukocyte trafficking will be explored: These include the MECA-32 antigen (antibodies to which inhibit lymphocyte homing); the PA 67 antigen (involved in T cell adhesion to activated mouse EC); and 92-ELAM (involved in adhesion of monocytes). VII. Finally, the role of vascular selectins in lymphocyte trafficking to inflamed joints will be explored. The proposed studies should expand our understanding of the critical role of the vascular endothelium in regulating lymphocyte trafficking during normal and pathologic immune responses.