Marijuana is the most widely used illicit drug in the United States. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been shown to be immunosuppressive and to alter the functional activities of macrophages and other immune cells. The goal of this project is to define the effect of THC on the expression of cytokines and nitric oxide by astrocytes and microglial cells. The rationale for examining the effect of THC on these cytokines and NO is that these factors have been implicated in playing a major role in the causation of a variety of nervous system disorders, including AIDS dementia. The hypothesis to be tested is that THC alters the expression of cytokines and/or nitric oxide by astrocytes and microglial cells and that it does so by a cannabinoid receptor-mediated process. Astrocytes and microglial cells from Balb/c mice, and human U373MG astrocytoma cells will serve as in vitro models for assessing the effect of THC on the production of interleukin-1 (IL1), interleukin-6 (IL6), tumor necrosis factor (TNF), and nitric oxide (NO) elicited by interferon-gamma plus bacterial lipopolysaccharide. First, we will define the effect of THC on the expression of IL1, IL6, TNF, and NO. Immunosorbent assays will be employed for assessment of human and murine cytokines. An assay which employs the Griess reagent will be used to assess NO-2 production. Second, we will identify the step at which THC alters cytokine and/or nitric oxide production. Radiolabel biosynthetic incorporation pulse and pulse-chase experiments will be performed to establish whether expression of cytokines and NO-related events is affected at the transcriptional, translational, or post-translational levels. Third, we will define the role of a cannabinoid receptor in altering nitric oxide and/or cytokine expression by astrocytes and microglial cells. Functional implication of a role of a cannabinoid receptor in mediating cytokine expression will be achieved using pertussis toxin uncoupling and betat(2)cAMP reconstitution experiments. Implication of a functional role for a cannabinoid receptor will be followed by studies to establish functional linkage between receptor expression and alterations in NO and/or cytokine production. Experiments will include CB1 receptor selective antagonism with SR141716A, structure activity relationships for demonstrating stereoselectivity with enantiomeric pairs of THC analogs, """"""""blocking"""""""" of receptors with specific antibodies, and antisense inhibition of cannabinoid message functionality. Finally, we will perform experiments to relate functional linkage to receptor expression. Mutational reverse transcriptase-PCR (MRT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA) will be performed to discriminate between CB1 and CB2 cannabinoid receptor expression. In this fashion, the kinetics of receptor expression as they relate to macrophage activation state and to NO and/or cytokine production will be defined. Furthermore, co-expression of CB1 and CB2 receptors can be recognized which would facilitate interpretation of outcomes resulting from functional linkage studies.
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