Ethanol Effects on Human Astroglial Chemokine Expression

描述(由申请人提供)：白细胞和小胶质细胞的募集对中枢神经系统(CMS)的免疫活性很重要。星形胶质细胞衍生的趋化因子主要负责这些免疫细胞的趋化作用。星形胶质细胞趋化因子在许多生理事件中也是必不可少的，包括中枢神经系统的发育。长期酗酒会导致大脑病理，并可能损害中枢神经系统的免疫功能。这些酒精效应可能部分源于星形胶质细胞趋化因子表达的变化，因为星形胶质细胞是趋化因子的主要来源，在组织动态平衡、损伤反应和免疫中起着重要作用。然而，酒精对人脑星形胶质细胞中趋化因子表达的影响知之甚少。在体外细胞迁移实验中，我们证实了脂多糖+白介素LP刺激的人A172星形胶质细胞对人U937白细胞的趋化作用。星形胶质细胞诱导的白细胞趋化作用部分是对星形胶质细胞分泌的趋化因子干扰素诱导蛋白-10(IP-10)的反应。然而，当星形胶质细胞在刺激前长期暴露在50 mM乙醇中时，IP-10的产生和白细胞的趋化作用都会减少。我们假设酒精暴露抑制了人脑星形胶质细胞中趋化因子的表达。导致星形胶质细胞对白细胞和小胶质细胞的趋化作用减弱。星形胶质细胞趋化因子表达减少也可能导致乙醇诱导的中枢神经系统病理，包括神经元发育改变。具体目的1：确定慢性酒精暴露和酒精戒断对促炎诱导的星形胶质细胞介导的白细胞和小胶质细胞趋化的影响。人类星形胶质细胞和A172细胞将在体外被脂多糖、肽聚糖、细胞因子和/或HIV-1蛋白Tat和gp120刺激，以诱导趋化因子的产生。人类U937白细胞和CHME-5小胶质细胞对星形胶质细胞暴露介质的趋化作用将被用来识别对乙醇敏感的刺激。特定目的2：确定慢性酒精暴露和酒精戒断对促炎诱导的人脑星形胶质细胞趋化因子表达的影响。趋化因子的表达将通过检测RNA(核糖核酸酶保护法)和分泌蛋白(ELISA)水平来评估。星形胶质细胞中乙醇敏感趋化因子(S)的鉴定将为深入研究乙醇调节趋化因子表达的机制提供起点。这些信息将有助于深入了解乙醇对神经病理和中枢神经系统免疫功能的影响。预计这些信息最终将有助于开发新的脑部病理和中枢神经系统免疫反应的药物操作，特别是与酒精滥用有关的药物。

DESCRIPTION (provided by applicant): Recruitment of leukocytes and microglia is important to immunocompetence in the central nervous system (CMS). Astrocyte-derived chemokines are largely responsible for chemotaxis of these immune cells. Astroglial chemokines are also essential to many physiological events, including CNS development. Chronic, abusive alcohol consumption causes brain pathologies and may compromise CNS immunocompetence. These alcohol effects may stem in part from changes in astroglial chemokine expression, as astroglia are a major source of chemokines and are instrumental in tissue homeostasis, response to injury and immunity. However, little is known about ethanol effects on chemokine expression in human astroglia. We have demonstrated in an in vitro cell migration assay, that media from lipopolysaccharide (LPS) + interleukin (IL)-lp-stimulated human A172 astroglia induces chemotaxis of human U937 leukocytic cells. Astrogial-induced leukocyte chemotaxis is partly in response to the chemokine, interferon-y inducible protein-10 (IP-10) secreted by the astroglia. Yet, when astroglia were chronically exposed to 50 mM ethanol prior to stimulation, IP-10 production and leukocyte chemotaxis were reduced. We hypothesize that ethanol exposure inhibits chemokine expression in human astroglia. resulting in reduced astroglial-mediated chemotaxis of leukocytes and microglia. Reduced astroqlial chemokine expression may also contribute to ethanol-induced CNS pathologies, including altered neuronal development. SPECIFIC AIM 1: Identify the effect of chronic ethanol exposure, and ethanol withdrawal, on proinflammatory-induced astroglial-mediated chemotaxis of leukocytes and microglia. Human astrocytes and A172 cells will be stimulated in vitro with LPS, peptidoglycan, cytokines and/or the HIV-1 proteins, Tat and gp120, to induce chemokine production. Chemotaxis of human U937 leukocytes and CHME-5 microglia in response to astroglialexposed media will be used to identify the stimuli that are ethanol-sensitive. SPECIFIC AIM 2: Identify the effect of chronic ethanol exposure, and ethanol withdrawal, on proinflammatory-induced chemokine expression in human astroglia. Chemokine expression will be assessed by measuring mRNA (RNase protection assay) and secreted protein (ELISA) levels. Identification of the ethanol-sensitive chemokine(s) in astroglia will provide a starting point for an indepth investigation into the mechanism by which ethanol modulates chemokine expression. This information will lend significant insight into the consequences of ethanol on neuropathology and CNS immunocompetence. It is anticipated that this information will ultimately be instrumental in the development of novel pharmacological manipulations of brain pathology and immune responsiveness in the CNS, especially as related to alcohol abuse.