GRANULOMA REGULATION THROUGH SOMATOSTATIN RECEPTOR SSTR2

肉芽肿性炎症是许多疾病的特征，如 克罗恩病、结节病和原发性胆汁性肝硬变。肉芽肿 是炎性细胞的焦点集合，由T细胞严密调控 细胞。反过来，其他类型的炎性细胞调节T肉芽肿 细胞。像IBD这样的疾病是在这种交叉中断时发生的 调节电路。这种调节电路的大部分都是通过 释放神经因子和其他淋巴因子。生长抑素(SOM)是一种 这种改变淋巴细胞反应的神经因子。肉芽肿巨噬细胞 秘而不宣。小鼠肉芽肿T细胞与DO细胞一样表达SOM受体 在人类肉芽肿中。克隆了5个不同的SOM受体(SSTR)， 进行了排序，并进行了表征。我们目前的证据表明，SSTR2 是肉芽肿T细胞上唯一表达的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-干扰素。SOM可能会被证明是一个 慢性炎症的重要调节因子，因为干扰素-γ是一个关键的 调节多种炎性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 将追寻以下四个假设：1)SOM调节干扰素-γ的释放 通过与SSTR2的相互作用从肉芽肿T细胞中分离出来。SSTR2 表达受T细胞激活的调节，3)两种亚型 SSTR2抑制干扰素-γ释放的能力不同，4)SOM对照 与SSTR2相互作用引起的体内肉芽肿反应。小鼠 血吸虫病为研究提供了一个定义良好的自然模型系统 肠道和肝脏的肉芽肿。小鼠肉芽肿 血吸虫病表达SOM、SSTR2等方面的SOM回路。 因此，我们将使用从这些肉芽肿中分离出来的细胞来实现 以下目标：目标1：确定SOM是否与T细胞直接作用 SSTR2在没有干预参与的情况下调节干扰素-γ的释放 另一种细胞类型使用受体阻断抗体，特定的激动剂， 流式细胞术和SSTR2显示缺失的克隆T细胞。目标2： 确定T细胞激活是否会影响SSTR2基因和蛋白质的合成， 和SSTR2的磷酸化，并使用定量RT-PCR，核径流 分析、流式细胞术、免疫印迹和免疫沉淀。目标 3：确定SSTR2的两种异构体是否存在差异调节 并具有不同的抑制T细胞干扰素-γ释放的能力 异构体特异性定量RT-PCR和免疫沉淀 选择性地转染异构体。目标4：确定病灶内是否 SOM与SSTR2相互作用通过SSTR2调节体内肉芽肿 封闭抗体与选择性受损T细胞转基因小鼠 显示SSTR2。我们希望这些发现将带来新的 IBD的治疗方法。

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.