Proteinase Modulation During T-Cell-Endothelial Adhesion

T 细胞内皮粘附过程中的蛋白酶调节

• 批准号: 6731165
• 负责人: JOSEPH A MADRI
• 金额: $ 36.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731165
• 关键词: SDS polyacrylamide gel electrophoresis; T lymphocyte; biological signal transduction; cell adhesion; endopeptidases; experimental allergic encephalomyelitis; extracellular matrix; flow cytometry; gel mobility shift assay; genetically modified animals; human tissue; immunoprecipitation; inflammation; laboratory mouse; laboratory rabbit; laboratory rat; leukocyte activation /transformation; leukocyte adhesion molecules; mass spectrometry; matrix assisted laser desorption ionization; microarray technology; multiple sclerosis; northern blottings; tissue /cell culture; vascular endothelium; western blottings

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.

描述（申请人的摘要）：我们的假设是粘附力 T 细胞和内皮细胞的分子依赖性调节不仅调节 粘合功能，而且还启动特定的蛋白酶诱导、表面 组装和激活，促进轮回和变化 T 细胞的粘附特性影响细胞的驻留 炎症部位。 T 细胞通过内皮细胞的迁移 层并迁移到下面和周围的细胞外基质中 由特定配体介导的 T 细胞粘附到内皮细胞引发 驻留在 T 细胞 [VLA-4 (a4B1)] 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 这对引起 MMP-2 表达和激活的变化，与 贴壁 T 细胞群中侵袭性表型的表现以及 内皮细胞中的“激活”表型。所得蛋白水解 基底膜和间质基质成分被认为有助于 T 细胞外渗出受影响的血管并流向病变部位 炎症和血管生成。在这个提案中，我们将：1）确定、比较 并对比T中MT1-MMP/TIMP-2/MMP-2三元复合物特征 淋巴细胞和内皮细胞受到刺激后。 2）继续我们的 MT1-MMP 和 MMP-2 启动子的特征及其各自 相关转录因子。 3）识别、表征、比较和 对比MT1-MMP和MMP-2涉及的信号转导通路 T 淋巴细胞中的诱导、复合物形成、激活和聚集 内皮细胞。这些目标将通过以下措施的结合来实现： 方法包括利用抗原特异性的体外培养模型 鼠 T 细胞克隆和细胞系；体内过继转移小鼠模型 实验性过敏性脑脊髓炎（EAE）和多种细胞和 分子生物学技术，包括细胞培养、酶谱、反向 酶谱法、免疫沉淀、蛋白质印迹、北方印迹、 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 阵列分析和选择的使用 转基因和基因敲除小鼠。这些实验将带来更好的结果 了解 T 细胞迁移并与局部相互作用 细胞外基质和针对其的新疗法的开发 调节选定的蛋白酶/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。