ENDOTHELIAL CELL BIOLOGY IN INFLAMMATION

该提案针对白细胞中的内皮细胞（EC）生物学 运输和炎症，重点是血管粘连的作用 受体和激活因子在控制淋巴细胞归巢。 I. 粘膜血管地址素MAdCAM-1的生物学特性将在下文中详细描述。 探索通过：1）在原位视频显微镜分析的 MAdCAM-1对L-选择素介导的淋巴细胞滚入的贡献 派伊尔集合淋巴结（PP）-高内皮微静脉（HEV）; 2）测定 MAdCAM-1结合和呈递淋巴细胞活化能力 趋化因子家族的趋化因子; 3）免疫组织学评估 MAdCAM-1变体形式的生理分布; 4） MAdCAM-1敲除小鼠的形态和功能分析;和5） 这些研究扩展到人。 分子和功能 外周淋巴结地址素（PNAd）-HEV配体的生物学 L-选择素的结构分析 HEV的选择素结合硫酸化糖表位，利用新的 一组抗PNAd糖型MAb：2）鉴定显性PNAd 负责HEV糖位呈递的糖蛋白：PNAd 在体内归巢研究中具有可证实的生理相关性的gp 3）基因的鉴定 编码参与PNAd糖基合成的酶，或其它HEV 分化抗原，用抗PNAd进行哺乳动物表达克隆 糖表位单克隆抗体;并通过分离HEV特异性cDNA， 杂交选择 三. HEV或EC相关因子 负责快速G蛋白介导的淋巴细胞活化， 归巢将通过免疫组织学和体内抗体来鉴定 趋化因子对HEV的抑制研究;如果通过 产生能够抑制活化步骤的MAb， 体外人工血管模型中淋巴细胞与EC的相互作用。 四. 体外迁移和/或趋化性测定将确定是否 MAdCAM-1可作为α 4 β 7+淋巴细胞的选择性底物 爬行，与局部化学引诱物协调作用， 淋巴细胞亚群的趋化性或趋触性。 诉 血管地址素和其他EC粘附的发育调节 受体将进行免疫组织学研究;和已知的或 新的细胞因子在地址调节将探索通过在体外 培养的EC系的研究，包括一种新的转化的HEV细胞 线;以及在细胞因子敲除小鼠的分析中。 六. 的 生理作用、功能和分子生物学（如有说明） 新鉴定的参与白细胞运输的EC抗原将 这些包括MECA-32抗原（抗体， 抑制淋巴细胞归巢）; PA 67抗原（参与T细胞 粘附到活化的小鼠EC）;和92-ELAM（参与粘附到活化的小鼠EC）。 单核细胞）。 七.最后，血管选择素在淋巴细胞中的作用 将探索向发炎关节的贩运。 拟议的研究应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运输中的作用 正常和病理性免疫反应。

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.