INTEGRIN LIGAND IN GUT LAMINA PROPRIA

肠道固有层中的整合素配体

• 批准号: 2887869
• 负责人: CHRISTINA M PARKER
• 金额: $ 8.48万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887869
• 关键词: T lymphocyte; cell adhesion; expression cloning; human subject; integrins; intestinal mucosa; laboratory mouse; ligands; molecular cloning; monoclonal antibody; recombinant proteins; tissue /cell culture; transfection

Mucosal T cells subsets have distinct localization patterns, as CD8+ comprise over 90 percent of the intestinal intraepithelial T lymphocytes while CD4+ T cells predominate within the lamina propria. Little is known about the mechanisms which account for this subset specific localization. The integrin alphaEbeta7 expressed on greater than 90 percent of CD8+ lymphocytes and on 40-50 percent of CD4+ cells diffusely distributed with the intestinal mucosa but on less than 3 percent of peripheral blood lymphocytes, and binds to E-cadherin expressed on epithelial cells. Thus, it is a candidate to function in T cell/epithelial cell interactions which may modify T cell localization. To evaluate the in vivo function(s) of alphaEbeta7, integrin alphaE deficient mice were generated. Study of these animals revealed reduced numbers of T cells within intestinal epithelium and also within the lamina propria where E-cadherin is not expressed. Furthermore, freshly isolated intestinal intraepithelial lymphocytes (iIEL), which express alphaEbeta7, adhered more efficiently to lamina propria tissue sections than iIEL isolated from alphaE deficient mice. These observations led us to hypothesize that alphaEbeta7 mediates an adhesive interaction to a ligand expressed within the lamina propria. In this application, studies are proposed to demonstrate that an additional alphaEbeta7 ligand is expressed within the lamina propria and to define this ligand at the molecular level. In aim 1, alphaEbeta7 transfectants will be tested to confirm that alphaEbeta7 expression confers adhesion of transfected cells to the lamina propria and to determine if human alphaEbeta7 mediates a similar adhesive function. In addition, studies will be performed to identify a cultured cells line which expresses the putative additional alphaEbeta7 ligand, using adhesion assays to identify cell lines which support alphaEbeta7 dependent adhesion. In aim 2, anti-ligand monoclonal antibodies or a soluble recombinant form of alphaEbeta7 which binds to this ligand will be generated and used to determine the biochemical structure of the alphaEbeta7 ligand. In aim 3, the gene encoding this lamina propria ligand will be cloned using either expression cloning or ligand purification followed by amino acid sequence determination. These studies will enable the identification and characterization of a second alphaEbeta7 counter-receptor and will provide insight into the mechanisms of mucosal lymphocytes localization. As an anti-alphaE monoclonal antibody dramatically reduced intestinal inflammation in a murine inflammatory bowel disease model, these studies may have important implications in the treatment of inflammatory bowel disease.

粘膜 T 细胞亚群具有不同的定位模式，如 CD8+ 超过 90% 的肠上皮内 T 淋巴细胞 而 CD4+ T 细胞在固有层中占主导地位。 小的是 了解解释该子集特定的机制 本土化。 整合素 alphaEbeta7 表达量大于 90 CD8+ 淋巴细胞的百分比和 40-50% 的 CD4+ 细胞的弥漫性 分布于肠粘膜，但仅占不到3% 外周血淋巴细胞，并与表达的 E-钙粘蛋白结合 上皮细胞。 因此，它是在 T 中发挥作用的候选者 细胞/上皮细胞相互作用可能会改变 T 细胞的定位。 评估 alphaEbeta7、整合素 alphaE 的体内功能 产生了有缺陷的小鼠。 对这些动物的研究表明减少 肠上皮内和肠内的 T 细胞数量 固有层，其中不表达 E-钙粘蛋白。此外，新鲜 分离的肠上皮内淋巴细胞 (iIEL)，表达 alphaEbeta7，更有效地粘附到固有层组织切片 与从 alphaE 缺陷小鼠中分离的 iIEL 相比。 这些观察导致 我们假设 alphaEbeta7 介导粘附相互作用 固有层内表达的配体。 在这个应用程序中， 拟议的研究表明，额外的 alphaEbeta7 配体在固有层内表达并定义该配体 在分子水平上。 在目标 1 中，alphaEbeta7 转染子将是 测试证实 alphaEbeta7 表达赋予粘附 将细胞转染至固有层并确定是否人类 alphaEbeta7 介导类似的粘附功能。 此外，研究 将进行以确定表达的培养细胞系 假定的额外 alphaEbeta7 配体，使用粘附测定来 鉴定支持 alphaEbeta7 依赖性粘附的细胞系。瞄准目标 2、抗配体单克隆抗体或可溶性重组形式 与该配体结合的 alphaEbeta7 将被生成并用于 确定 alphaEbeta7 配体的生化结构。 瞄准目标 3、编码该固有层配体的基因将使用克隆 表达克隆或配体纯化，然后氨基酸 顺序确定。 这些研究将有助于识别 以及第二个 alphaEbeta7 反受体的表征，并将 提供对粘膜淋巴细胞定位机制的深入了解。 作为抗 alphaE 单克隆抗体，可显着减少肠道 小鼠炎症性肠病模型中的炎症，这些研究 可能对炎症性肠病的治疗具有重要意义 疾病。