Functional Relevance of Microglial Cannabinoid Receptors

描述（由申请人提供）：本研究的目的是确定大麻中主要的精神活性和免疫抑制成分- δ -9-四氢大麻酚（THC）对大脑内小胶质细胞的影响。小胶质细胞是巨噬细胞的常驻种群，巨噬细胞是大脑免疫系统的主要组成部分。这些细胞对创伤和感染的反应是迁移到损伤部位，产生促炎细胞因子和细胞毒性物质，吞噬感染因子和受损组织。小胶质细胞也与神经病理过程有关，如艾滋病、痴呆和多发性硬化症等，都是慢性激活的结果。我们已经证明，小胶质细胞在体外表达CB1和CB2大麻素受体，后者存在于高水平时，这些细胞处于炎症状态。这些受体已被证明在大麻素介导的非神经元部位的免疫细胞功能调节中发挥作用，我们的初步数据表明它们在小胶质细胞中发挥类似的作用。我们建议评估CB1和CB2受体在大麻素介导的小胶质细胞功能调节中的功能联系。这些研究是非常重要的，因为认识到特定的活动在功能上与大麻素受体相关联，将为消融炎症过程提供治疗方案，这些炎症过程有助于艾滋病相关和其他形式的神经发病机制。要测试的假设是四氢大麻酚和其他大麻素通过大麻素受体介导的过程改变小胶质细胞的选择功能活动。为了检验这一假设，提出以下具体目标作为研究的指导方针。首先，我们将确定大麻素受体在大脑中通过小胶质细胞表达的模式。我们已经证明，小胶质细胞在体外表达CB1和CB2受体的差异与细胞激活有关，并将确定这些事件是否发生在体内。将进行器官型脑切片实验以及体内感染性研究。大麻素受体的表达将使用组织学、免疫细胞化学、药理学和分子方法进行评估。其次，我们将定义大麻素受体对小胶质细胞的功能相关性。我们已经证明，小胶质细胞在体外处于“反应”和“启动”炎症状态时，表达高水平的CB2受体。我们将通过该受体评估大麻素介导的与这些激活状态相关的功能，包括趋化性、趋化作用、吞噬作用和抗原处理。功能分析将使用成对的大麻素对映体，受体亚型特异性拮抗剂和表现出不同配体结合亲和力的大麻素来建立结构-活性关系。

DESCRIPTION (provided by applicant): The goal of this study is to define the effect of delta-9-tetrahydrocannabinol (THC), the major psychoactive and immunosuppressive component in marijuana, on microglia within the brain. Microglia are a resident population of macrophages, which constitute a major element of the immune system of the brain. These cells respond to trauma and infection by migrating to sites of injury, produce proinflammatory cytokines and cytotoxic substances, and phagocytize infectious agents and damaged tissue. Microglia, also, have been implicated in neuropathological processes, such as AIDS dementia and multiple sclerosis as a consequence of chronic activation. We have demonstrated that microglia in vitro express CB1 and CB2 cannabinoid receptors, the latter of which are present at high levels when these cells are in an inflammatory state. These receptors have been shown to play a role in cannabinoid-mediated modulation of immune cell functions at non-neuronal sites and our preliminary data suggest that they play a similar role in microglia. We propose to assess for functional linkages for CB1 and CB2 receptors in cannabinoid-mediated modulation of microglial functions. These studies are highly significant since recognition that specified activities are linked functionally to cannabinoid receptors would provide insight regarding therapeutic regimens for ablating inflammatory processes which contribute to AIDS-associated and other forms of neuropathogenesis. The hypothesis to be tested is that THC and other cannabinoids alter select functional activities of microglia and do so by a cannabinoid receptor-mediated process. In order to test this hypothesis, the following Specific Aims are proposed as guidelines to the research. First, we will define the pattern of cannabinoid receptor expression by microglia in the brain. We have demonstrated that microglia differentially express CB1 and CB2 receptors in vitro in relation to cell activation and will establish whether these events occur in vivo. Organotypic brain slice experiments as well as in vivo infectivity studies will be performed. Cannabinoid receptor expression will be assessed using histological, immunocytochemical, pharmacological, and molecular approaches. Second, we will define the functional relevance of cannabinoid receptors on microglia. We have demonstrated that microglia in vitro, when in "responsive" and "primed" inflammatory states, express high levels of the CB2 receptor. We will assess for cannabinoid-mediated effects through this receptor on functions associated with these activation states including chemotaxis, chemokinesis, phagocytosis, and antigen processing. Functional assays will employ paired cannabinoid enantiomers, receptor subtype-specific antagonists, and cannabinoids exhibiting differential ligand binding affinities to establish structure-activity relationships.