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. ? ? ?
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