REGULATION AND MEDIATION OF THE IMMUNE/INFLAMMATORY RESPONSE BY PGE2

前列腺素E_2(PGE_2)是一种重要的环氧合酶代谢产物。 肺，它调节平滑肌张力以及免疫/炎症 回应。了解哪些PGE2受体亚型介导这些 反应是不完整的，是孤立的细胞效应的整合 关于免疫性/过敏性疾病的多细胞病理生理学 更不为人所知。细胞内cAMP的增加一直是 与PGE2介导的抗炎作用有关。最近的 前列腺素E受体四种亚型EP1的克隆 EP2，EP3和EP4，结合药理学研究，将允许更多 对参与其中的信号转导通路有完整的了解 这些免疫调节作用。这个项目的主要假设是 EP受体亚型连接到不同效应器系统并显示 不同的调节特性；因此表达不同的EP 单个炎性细胞类型上的受体亚型决定了 各靶细胞对前列腺素E_2的生理反应。基于我们的 在文献中的初步数据和研究中，我们假设EP2 受体是PGE2抗炎作用的主要决定因素。 为了检验这些相关假设，本文提出了以下具体目标； 在具体目标1中，我们将确定EP的表达模式 相关人和小鼠免疫/炎症细胞的受体亚型 包括单核细胞、嗜酸性粒细胞、肺泡巨噬细胞、小鼠T辅助细胞 (TH)1和Th2淋巴细胞克隆和肥大细胞。由于EP的丰度 受体通常是相当低的，敏感的逆转录酶 采用链式反应(RT-PCR)和核糖核酸酶保护试验。 EP受体基因表达阳性的细胞将进行配体分析 结合、第二信使生成和使用的功能反应 亚型选择性配体。 在具体目标2中，我们将讨论EP2和受体的重要性 通过对呼吸道过敏性或病毒性炎症模型的研究获得亚型 嗜酸性粒细胞聚集和全身过敏反应。我们已经隔离了 编码小鼠EP2受体和小鼠EP2的基因 受体基因。在具体目标3中，如果基于具体目标中的调查结果 目标1和2，我们确定EP2受体确实定位于 靶细胞支持其在PGE2诱导的抗-DNA抗体中的作用 炎症反应，我们将通过以下方式产生EP2受体缺陷小鼠 标准的基因打靶方法学，从同源重组开始 在胚胎干细胞中。这个EP2缺陷的鼠标将被用作工具 用于EP2受体功能的研究。这些研究将阐明 EP2受体功能在炎症性肺病中的作用 确定该受体亚型作为开发的治疗靶点 用于治疗这些疾病的新型配体。

Prostaglandin E2 (PGE2) is an important cyclooxygenase metabolite in the lung where it regulates smooth muscle tone as well as immune/inflammatory responses. Understanding of which PGE2 receptor subtypes mediate these responses is incomplete, an the integration of isolated cellular effects into the multicellular pathophysiology of immunologic/allergic diseases is even less well understood. Increases in intracellular cAMP have been implicated in the PGE2 mediated anti-inflammatory effect. The recent cloning of four distinct prostaglandin E receptor subtypes designated, EP1, EP2, EP3 and EP4, combined with pharmacologic studies, will allow a more complete understanding of the signal transduction pathways participating in these immuno-regulatory actions. The main hypothesis of this project is that EP receptor subtypes couple to different effector systems and display differential regulatory properties; thus expression of different EP receptor subtypes on individual inflammatory cell types determines the physiologic response to PGE2 evoked in each target cell. Based upon our preliminary data and studies in the literature, we hypothesize that the EP2 receptor is a major determinant of the anti-inflammatory effects of PGE2. To test these related hypotheses the following specific aims are propose; In specific aim 1, we will determine the pattern of expression of EP receptor subtypes on relevant human and murine immune/inflammatory cells including monocytes, eosinophils, alveolar macrophages, murine T-helper (Th)1 and Th2 lymphocyte clones and mast cells. Since the abundance of EP receptors usually is quite low, sensitive reverse transcription-polymerase chain reaction (RT-PCR) and RNAse protection assays will be employed. Cells positive for EP receptor gene expression will be analyzed for ligand binding, second messenger generation and functional responses using subtype-selective ligands. In specific aim 2, we will address the importance of EP2 and receptor subtypes through studies of models of airway allergic or viral inflammation and eosinophil accumulation, and systemic anaphylaxis. We have isolated the gene encoding the murine EP2 receptor as well as the murine EP2 receptor cDNA. In specific aim 3, if based upon the findings in specific aims 1 and 2, we establish that the EP2 receptor is indeed localized on target cells in such a way as to support its role in PGE2 evoked anti- inflammatory response, we will generate EP2 receptor-deficient mice by standard gene targeting methodology, starting with homologous recombination in embryonic stem cells. This EP2 deficient mouse will be used as a tool for the study of EP2 receptor function. These studies will elucidate the role of EP2 receptor function in inflammatory lung disease and possibly identify this receptor subtype as a therapeutic target for the development of novel ligands for the treatment of these disorders.