MODULATION OF CIITA BY CANNABINOIDS IN HUMAN MICROGLIA

描述（由申请人提供）：大麻素被确定为具有免疫调节作用，然而，大麻素受体参与免疫功能的特定方面已在少数研究中得到证实。大麻素的许多作用是由两种G蛋白偶联受体介导的，称为CB 1和CB 2。大多数关于大麻素免疫调节作用的现代研究都集中在CB 2受体上，因为它主要在免疫细胞上表达。我们已经表明，CB 1受体在小胶质细胞中表达，也表达MHC II类抗原。II类MHC基因的调节主要发生在转录水平，并且非DNA结合蛋白，II类反式激活因子（CIITA），已被证明是II类转录的主激活因子。我们最近的数据表明，大麻素通过在转录水平上对CIITA的作用影响MHC II类分子的表达。已知CIITA基因由多个启动子控制。CIITA的干扰素（IFN）-γ诱导型表达主要由启动子IV调节，启动子IV含有三个结合位点，分别结合转录因子STAT-1 α、USF-1和IRF-1和IRF-2的IFN-γ激活序列、E盒和IFN调节因子位点。STAT-1、USF-1和IRF-1和IRF-2对启动子的协同激活导致CIITA的表达，然后CIITA激活II类MHC基因的转录。本提案将阐明大麻素介导CIITA在人类小胶质细胞中表达的机制。为了实现我们的目标，我们将从人脑中分离和研究小胶质细胞的原代培养物。将用IFN-g活化的细胞暴露于大麻素激动剂和拮抗剂，以确定大麻素受体活化对CIITA mRNA和蛋白质水平的刺激的影响。该提案的具体目的是1）确定大麻素对CIITA的作用是否是受体介导的，2）确定特定转录因子与CIITA启动子的结合是否受大麻素的影响。从我们拟议的试点研究中获得的数据将为我们的长期计划提供有价值的信息，以确定大麻素受体在小胶质细胞中的功能意义。小胶质细胞是中枢神经系统感染的第一道防线，与包括艾滋病在内的各种免疫性疾病有关。

DESCRIPTION (provided by applicant): Cannabinoids are established as being immunomodulatory, however, the involvement of the cannabinoid receptors in specific aspects of immune function has been demonstrated in few studies. Many effects of cannabinoids are mediated by two G-protein coupled receptors, designated CB1 & CB2. Most modern studies on immunomodulatory effects of cannabinoids have been focused on CB2 receptor, since it is primarily expressed on immune cells. We have shown that CB1 receptor is expressed in microglia that also express MHC class II antigen. The regulation of class II MHC genes occur primarily at the transcriptional level, and a non-DNA-binding protein, class II transactivator (CIITA), has been shown to be the master activator for class II transcription. Our recent data indicate that cannabinoids affect MHC class II expression through actions on CIITA at the transcriptional level. The CIITA gene is known to be controlled by multiple promoters. Interferon (IFN)- g -inducible expression of CIITA is primarily regulated by promoter IV which contains three binding sites, an IFN-g activation sequence, an E Box and an IFN regulatory factor site that bind the transcription factors STAT-la, USF-1 and IRF-1 & IRF-2 respectively. Synergistic activation of the promoter by STAT-1, USF-1 and IRF-1 and IRF-2 leads to expression of CIITA, which then activates transcription of class II MHC genes. The present proposal will elucidate the mechanism by which the cannabinoids mediate the expression of CIITA in human microglia. To address our goal, we will isolate and study primary cultures of microglial cells from human brain. Cells activated with IFN-g will be exposed to cannabinoid agonists and antagonists to determine the effect of cannabinoid receptor activation on stimulations of CIITA mRNA and protein levels. The specific aims of the proposal are 1) Determine whether cannabinoid effects on CIITA are receptor mediated and 2) Determine whether the binding of specific transcription factors to CIITA promoter is affected by cannabinoids. Data obtained from our proposed pilot study will provide valuable information for our long-range plan to define the functional significance of cannabinoid receptors in microglia. Microglia form the first line of defense during infection in the CNS and are involved in various immune diseases including AIDS.