INTEGRIN LIGAND IN GUT LAMINA PROPRIA

肠道固有层中的整合素配体

• 批准号: 6171107
• 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/6171107
• 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.

粘液T细胞亚群具有不同的定位模式，如CD8 + 包括超过90%的肠上皮内T淋巴细胞， 而CD4 + T细胞在固有层内占优势。 之甚少 已知的机制，占这个子集的具体 本地化 整合素α Ebeta7在大于90 CD8+淋巴细胞和40 - 50%的CD4+细胞弥漫性 分布在肠粘膜上，但不到3%的 外周血淋巴细胞，并结合表达的E-钙粘蛋白， 上皮细胞 因此，它是在T中起作用的候选者 细胞/上皮细胞相互作用，其可以改变T细胞定位。 评价α E β 7、整合素α E 产生缺陷小鼠。 对这些动物的研究显示， 肠上皮内和组织内的T细胞数量 固有层，其中E-钙粘蛋白不表达。此外，新鲜 分离的肠上皮内淋巴细胞（iIEL），表达 α E β 7，更有效地粘附于固有层组织切片 与从alphaE缺陷小鼠分离的iIEL相比。 这些观察导致 我们假设α E β 7介导了粘附相互作用， 在固有层内表达的配体。 在本申请中， 提出了一些研究来证明额外的α E β 7 配体在固有层内表达， 在分子水平上。 在目标1中，将α Ebeta7转染子 测试以证实α E β 7表达赋予粘附性， 将转染的细胞转移到固有层，并确定人 α E β 7介导类似的粘附功能。 另外研究 将进行鉴定，以鉴定表达 假定的额外的alphaEbeta7配体，使用粘附试验， 鉴定支持α E β 7依赖性粘附的细胞系。在aim中 2、抗配体单克隆抗体或可溶性重组形式 将产生与该配体结合的α E β 7，并用于 确定α E β 7配体的生化结构。 在aim中 3，编码该固有层配体的基因将使用 表达克隆或配体纯化，随后进行氨基酸 序列测定 这些研究将有助于确定 和第二α E β 7反受体的表征， 提供了深入了解粘膜淋巴细胞定位的机制。 作为抗α E单克隆抗体， 在小鼠炎症性肠病模型中，这些研究 可能对治疗炎症性肠病有重要意义 疾病