Marijuana is the most common street drug of abuse in the United States. A major proportion of AIDS patients are frequent users of psychoactive drugs, including marijuana. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, accounts for the majority of its immunosuppressive properties. The drug also inhibits macromolecular synthesis, suggesting a mechanism by which it effects immunosuppression. Macrophage play a central role in anti-microbial activities, in antigen presentation, and in regulation of lymphocyte function. It is possible that use of marijuana may compromise the immune system resulting in a higher susceptibility to development of disease symptoms after AIDS viral infection. Such higher susceptibility could result due to failure of macrophages to synthesize and/or express molecules, such as la antigens, which play a critical role in immune responsiveness against infectious agents since processed viral antigens are presented to helper T cells in the context of the heterodimeric la molecules. Alternatively, drug-induced membrane perturbations could affect the synthesis of la molecules and/or their interaction with processed exogenous antigen within the cell. The goal of this research is to define the effect of THC on the synthesis, post-translational modification, and expression of the major histocompatibility la Class II molecules of murine macrophages. The (B6C3)F1 mouse will be employed for in vivo drug exposure and as a source of peritoneal macrophages. The macrophage line P388D1 will be used for in vitro experiments. Murine gamma interferon or ConA supernatants from murine splenocyte cultures will be used for induction of la expression. Radiolabel pulse and pulse-chase experiments of macrophages labeled with [35S]-methionine, [14C]-amino acids, or [3H]-glucosamine, and subjected to one-dimensional or two-dimensional isodalt polyacrylamide gel electrophoresis in concert within radioautography, will be performed to establish the effect of THC on the time and rate kinetics of la molecular synthesis. Antibodies will be employed for immunoprecipitation, Western immunoblotting, immunofluorescence, and immunoelectron microscopy to identify la molecules in gel profiles and to establish drug effects on their compartmentation and cell surface expression. Murine [125I]-IgG2 will be employed in binding studies, and in ultrastructural experiments, as a probe to define the effect of THC on receptor-mediated endocytosis. The functional consequences of THC-induced alteration in la expression will be assessed. The ability of THC-exposed P388D, macrophages to present peptide fragments 1-65 and 81-104 of pigeon cytochrome c to the murine normal T- helper cell clones, DB14 and A.E7, will be evaluated using a T-cell proliferative assay and measuring 3H-thymidine incorporation. To distinguish between impairment of antigen presentation versus co- stimulatory activity, peptide receptor occupation will be assessed by measuring the hydrolysis of myo-[2-3H] inositol to radiolabeled inositol phosphates. The effect of THC on the secretion of the co-stimulatory signal interleukin-1, requisite for replication of T-helper cells in addition to antigen receptor occupancy, will be established using C3H/HeJ mouse thymocyte proliferation.
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