Proteinase Modulation During T-Cell-Endothelial Adhesion

描述(申请人摘要)：我们的假设是粘附性 T细胞和内皮细胞的分子依赖性调节不仅调节 黏附功能，还能启动特定的蛋白酶诱导，表面 组装和激活，以促进轮回和变化 在T细胞的粘附性中，它影响细胞在 发炎的部位。T细胞在血管内皮细胞中的迁移 层和迁移到底层和周围的细胞外基质是 T细胞与内皮细胞的黏附启动，由特定配体介导 位于T细胞[VLA-4(A4B1)]和内皮细胞表面 细胞(VCAM-1)。我们已经证明了这种受体/配体的结合 配对引起基质金属蛋白酶-2表达和激活的变化，与 粘附性T细胞群体中侵袭性表型的表现 内皮细胞的“激活”表型。由此产生的蛋白质分解 基底膜和间质基质成分被认为促进T 细胞从受影响的血管渗出并向血管部位渗出 炎症和血管生成。在本提案中，我们将：1)确定、比较 并对比T细胞中MT1-MMP2/TIMP-2/MMP2三元复合体的特征。 刺激后的淋巴细胞和内皮细胞。2)继续我们的 MT1-基质金属蛋白酶和基质金属蛋白酶-2启动子及其各自的特性 相关转录因子。3)确定、描述、比较和 比较MT1-基质金属蛋白酶和基质金属蛋白酶-2的信号转导途径 T淋巴细胞的诱导、复合体形成、激活和聚集 内皮细胞。这些目标将通过以下几个方面结合起来实现 方法学包括利用抗原特异性的体外培养模型 小鼠T细胞克隆和品系；体内过继转移小鼠模型 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、酶谱分析、反向 酶谱，免疫沉淀，Western印迹，Northern印迹， 选定基因产物的转染和稳定表达，组织学， 免疫组织化学、MALDI-TOF、DNA阵列分析和选择的应用 转基因和基因敲除小鼠。这些实验将导致更好的 了解T细胞在局部的迁移和相互作用 细胞外基质与肿瘤治疗新方法的发展 调节选定的蛋白酶/酶抑制剂级联系统中的 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.