The goal of this research is to define the effect of Delta-9- tetrahydrocannabinol (THC), the major psychoactive component of marijuana, on macrophage antigen presentation. We have shown that THC alters antigen presentation by exerting differential effects on macrophage processing of antigens via the endosomal pathway. The outcome of THC effects was dependent on the conformation of the antigen. In the present study, we will test the hypothesis that THC alters macrophage functional competence, at least in part, by influencing a subset of steps during antigen processing whose role is dictated by the structure of the antigen. First, we will determine the effect of THC on the processing of antigens ingested via the phagosomal pathway to determine whether THC affects this alternative pathway. The effect of THC on this antigen processing will be established using Bacillus subtilis as a degradative marker and a viability bacterial colony count recovery assay. Second, we will examine the effect of THC on three major aspects of antigen processing, including disruption of disulfide bonds in an antigen, enzymatic activity of acid proteases called cathepsins, and status of the intracellular reducing environment. Third, we will define the effect of THC on the co- localization of molecules involved in antigen processing and presentation. Immunoelectron microscopy will employ cross-linked 15-nm gold-labeled globulins as a tracer for antigen processing and 5-nm gold-labeled monoclonal MK-D6 anti-Ia antibody as a tracer for Class II molecules. Fourth, we will define the role of a cannabinoid receptor in altering antigen processing. Functional assessment of a role of a cannabinoid receptor in mediating antigen processing will be achieved using pertussis toxin uncoupling, reconstitution with betat/2cAMP, CB1 selective antagonism with SR141716A, and structural activity relationships for demonstrating stereoselectivity for the enantiomeric pairs (- )CP55940/(+)CP56667 and (-)HU210/(+)HU211. Implication of a functional cannabinoid receptor in mediating antigen processing will be followed by characterization of receptor protein expression using anti-cannabinoid receptor subtype antibodies to CB1, CB1a, and CB2 in Flow Cytometry and ELISA and detection of receptor subtype-specific mRNA by Mutagenic Reverse Transcription Polymerase Chain Reaction to relate receptor differential expression to macrophage activation state.
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