Proteinase Modulation During T-Cell-Endothelial Adhesion

描述（申请人摘要）：我们的假设是， T细胞和内皮细胞的分子依赖性调节不仅调节 粘附功能，但也启动特定的蛋白酶诱导，表面 组装和激活，促进轮回，以及变化 T细胞的粘附特性影响细胞的驻留， 炎症的部位。T细胞通过内皮细胞的迁移 层和迁移到下面和周围的细胞外基质是 由特异性配体介导的T细胞粘附至内皮启动 存在于T细胞[VLA-4（a4 B1）]和内皮细胞的表面。 细胞（VCAM-1）。我们已经证明，这种受体/配体的参与， 对引起MMP-2表达和活化的变化，与 在粘附T细胞群中的侵袭性表型的表现和 在内皮细胞中的“活化”表型。产生的蛋白水解 基底膜和间质基质成分被认为是促进T 细胞外渗出受影响的血管，并向部位 炎症和血管生成。在本建议中，我们将：1）确定，比较 并对比T细胞中MT 1-MMP/TIMP-2/MMP-2三元复合物的特性 淋巴细胞和内皮细胞。2)继续我们 MT 1-MMP和MMP-2启动子的表征及其各自的 相关转录因子3)确定、描述、比较和 对比MT 1-MMP和MMP-2参与的信号转导途径 T淋巴细胞中的诱导、复合物形成、活化和聚集， 内皮细胞这些目标将通过以下措施实现： 方法学，包括利用抗原特异性 鼠T细胞克隆和系; 实验性变态反应性脑脊髓炎（EAE）和各种细胞和 分子生物学技术，包括细胞培养、酶谱、反向 酶谱法、免疫沉淀法、Western印迹法、北方印迹法、 所选基因产物的转染和稳定表达，组织学， 免疫组织化学、MALDI-TOF、DNA阵列分析和使用选择的 转基因和基因敲除小鼠。这些实验将带来更好的 理解T细胞迁移通过局部免疫系统并与局部免疫系统相互作用 细胞外基质和发展新的和新颖的疗法，针对 调节选择的蛋白酶/蛋白酶抑制剂级联系统， 关节炎、血管炎和组织排斥器官的炎症过程。

DESCRIPTION (Applicant's abstract): Our hypothesis is that adhesion molecule-dependent modulation of T cells and endothelia modulates not only adhesive functions, but also initiates specific protease induction, surface assembly, and activation which facilitates transmigration, as well as changes in adhesive properties of the T cells which affects residency of the cells at the site of inflammation. T cell transmigration through the endothelial cell layer and migration into the underlying and surrounding extracellular matrix is initiated by T cell adhesion to the endothelium, mediated by specific ligands resident on the surfaces of both the T cell [VLA-4 (a4B1)] and the endothelial cell (VCAM-1). We have demonstrated that engagement of this receptor/ligand pair evokes changes in MMP-2 expression and activation, consistent with the manifestation of an invasive phenotype in the adherent T cell population and an "activated" phenotype in the endothelial cells. Resultant proteolysis of basement membrane and interstitial matrix components is thought to facilitate T cell extravasation out of the affected vessel and toward the site of inflammation and angiogenesis. In this proposal we will: 1) determine, compare and contrast the MT1-MMP/TIMP-2/MMP-2 ternary complex characteristics in T lymphocytes and endothelial cells following their stimulation. 2) continue our characterizations of the MT1-MMP and MMP-2 promoters and their respective pertinent transcription factors. 3) identify, characterize, compare and contrast the signal transduction pathways involved in MT1-MMP and MMP-2 induction, complex formation, activation and clustering in T lymphocytes and endothelial cells. These aims will be accomplished with a combination of methodologies including an in vitro culture model utilizing antigen-specific murine T cell clones and lines; an in vivo adoptive transfer murine model of experimental allergic encephalomyelitis (EAE) and a variety of cellular and molecular biological techniques including cell culture, zymography, reverse zymography, immunoprecipitation, Western blotting, Northern blotting, transfection and stable expression of selected gene products, histology, immunohistochemistry, MALDI-TOF, DNA array analyses and the use of selected transgenic and knockout mice. These experiments will lead to a better understanding of T cell migration through and interaction with local extracellular matrix and the development of new and novel therapies directed at modulating selected proteinase/proteinase inhibitor cascade systems in the inflammatory processes of arthritis, vasculitis, and tissue rejection organ.