GRANULOMA REGULATION THROUGH SOMATOSTATIN RECEPTOR SSTR2

通过生长抑素受体 SSTR2 调节肉芽肿

• 批准号: 2904947
• 负责人: DAVID E ELLIOTT
• 金额: $ 12.37万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2904947
• 关键词: Crohn's disease; T lymphocyte; antisense nucleic acid; blocking antibody; flow cytometry; genetically modified animals; granuloma; hamsters; hormone receptor; immunomodulators; immunoprecipitation; immunoregulation; interferon gamma; laboratory mouse; leukocyte activation /transformation; peptide hormone analog; phosphorylation; polymerase chain reaction; protein biosynthesis; protein isoforms; receptor binding; receptor expression; schistosomiasis; somatostatin; western blottings

Granulomatous inflammation is characteristic of many diseases like Crohn's disease, sarcoidosis and primary biliary cirrhosis. Granulomas are focal collections of inflammatory cells, tightly regulated by T cells. In turn, other inflammatory cells types regulate granuloma T cells. Diseases like IBD develop when there is disruption of this cross regulatory circuitry. Much of this regulatory circuitry proceeds through release of neurokines and other lymphokines. Somatostatin (SOM) is one such neurokine that alters lymphocyte responses. Granuloma macrophages secrete SOM. Murine granuloma T cells display SOM receptors as do cells in human granulomas. Five distinct SOM receptors (SSTR) are cloned, sequenced, and characterized. Our current evidence indicates that SSTR2 is the only SOM receptor expressed on granuloma T cells. In addition, granuloma cells express both isoforms of SSTR2. SOM inhibits interferon- gamma (IFN-gamma) release from granuloma T cells. SOM may prove to be an important regulator of chronic inflammation, since IFN-gamma is a pivotal cytokine that regulates many inflammatory cell types. Therefore, to further elucidate the importance of SSTR2 in inflammation, this project will pursue these four hypotheses: 1.) SOM regulates IFN-gamma release from granuloma T cells through interaction with SSTR2, 2.) SSTR2 expression is regulated by T cell activation, 3) The two isoforms of SSTR2 differ in ability to inhibit IFN-gamma release, 4) SOM controls granulomatous responses in vivo by interaction with SSTR2. Murine schistosomiasis provides a well defined natural model system to study granulomas in the intestines and liver. Granulomas in murine schistosomiasis express SOM, SSTR2 and other aspects of the SOM circuit. Thus, we will use cells isolated from these granulomas to achieve the following aims: Aim 1: Determine if SOM interacts directly with T cell SSTR2 to regulate IFN-gamma release without the interposed participation of another cell type using receptor blocking antibody, specific agonists, flow cytometry and cloned T cells with abrogated SSTR2 display. Aim 2: Determine if T cell activation affects SSTR2 mRNA and protein synthesis, and SSTR2 phosphorylation, and using quantitative RT-PCR, nuclear run-off analysis, flow cytometry, western blotting and immunoprecipitation. Aim 3: Determine if the two isoforms of SSTR2 are differentially regulated and have different capacity to inhibit T cell IFN-gamma release using isoform specific quantitative RT-PCR and immunoprecipitation, and selective transfection of the isoforms. Aim 4: Determine if intralesional SOM interacts with SSTR2 to regulate granulomas in vivo using SSTR2 blocking antibodies and transgenic mice with selectively impaired T cell SSTR2 display. Our hope is that these discoveries will lead to new therapies for IBD.

肉芽肿性炎症是许多疾病的特征，例如 克罗恩病、结节病和原发性胆汁性肝硬化。肉芽肿 是炎症细胞的集中集合，受到 T 细胞的严格调控 细胞。反过来，其他炎症细胞类型调节肉芽肿 T 细胞。当这种交叉受到破坏时，就会出现炎症性肠病等疾病 调节电路。大部分调节电路通过 神经因子和其他淋巴因子的释放。 生长抑素（SOM）是其中之一 这种改变淋巴细胞反应的神经因子。肉芽肿巨噬细胞 分泌SOM。鼠肉芽肿 T 细胞与细胞一样显示 SOM 受体 在人类肉芽肿中。克隆了五种不同的 SOM 受体 (SSTR)， 测序并表征。我们目前的证据表明 SSTR2 是肉芽肿 T 细胞上表达的唯一 SOM 受体。此外， 肉芽肿细胞表达 SSTR2 的两种亚型。 SOM 抑制干扰素 肉芽肿 T 细胞释放γ (IFN-γ)。 SOM 可能被证明是 慢性炎症的重要调节因子，因为 IFN-gamma 是关键的 调节多种炎症细胞类型的细胞因子。因此，要 进一步阐明SSTR2在炎症中的重要性，该项目 将追求这四个假设：1.) SOM 调节 IFN-gamma 释放 通过与 SSTR2 相互作用，从肉芽肿 T 细胞中产生，2.) SSTR2 表达受 T 细胞激活调节，3) 的两种亚型 SSTR2 抑制 IFN-γ 释放的能力不同，4) SOM 对照 通过与 SSTR2 相互作用产生体内肉芽肿反应。鼠类 血吸虫病提供了一个明确的自然模型系统来研究 肠和肝脏肉芽肿。小鼠肉芽肿 血吸虫病表达SOM、SSTR2和SOM电路等方面。 因此，我们将使用从这些肉芽肿中分离的细胞来实现 以下目标： 目标 1：确定 SOM 是否直接与 T 细胞相互作用 SSTR2 无需干预即可调节 IFN-γ 释放 使用受体阻断抗体、特异性激动剂来检测另一种细胞类型， 流式细胞术和具有废除的 SSTR2 显示的克隆 T 细胞。目标 2： 确定 T 细胞激活是否影响 SSTR2 mRNA 和蛋白质合成， 和 SSTR2 磷酸化，并使用定量 RT-PCR、核流失 分析、流式细胞术、蛋白质印迹和免疫沉淀。目的 3：确定 SSTR2 的两种亚型是否受到差异调节 并具有不同的抑制 T 细胞 IFN-γ 释放的能力 亚型特异性定量 RT-PCR 和免疫沉淀，以及 异构体的选择性转染。目标 4：确定病灶内是否存在 SOM 与 SSTR2 相互作用，利用 SSTR2 调节体内肉芽肿 阻断抗体和 T 细胞选择性受损的转基因小鼠 SSTR2 显示。我们希望这些发现能够带来新的成果 IBD 的治疗方法。