Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Dr. Guy A. Cabral

Second Advisor

Dr. Francine Marciano-Cabral

Third Advisor

Dr. Richard T. Marconi

Fourth Advisor

Dr. Stephen T. Sawyer

Fifth Advisor

Dr. Kathleen L. McCoy

Sixth Advisor

Dr. Darrell L. Peterson

Abstract

Cannabinoids have been shown to modulate the immune system in vitro and in animal models. A major area of interest is how cannabinoids impact the brain. A whole variety of neuropathies or brain disorders, such as AIDS dementia, Parkinson’s disease, Multiple Sclerosis and Alzheimer’s disease, are associated with a hyperinflammatory response within the brain. Microglia, the resident macrophages of the brain, are the major cell type responsible for the persistent elicitation of pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, TNFa) and other mediators. In vitro experiments have demonstrated that the partial exogenous cannabinoid agonist delta-9-tetrahydrocannabinol (D9-THC) and the potent synthetic exogenous cannabinoid agonist CP55940 down-regulate the robust production of pro-inflammatory cytokines elicited in response to bacterial lipopolysaccharide (LPS) at the mRNA level. These observations suggest that cannabinoids, devoid of psychotropic properties, have the potential to betherapeutic agents. These highly lipophilic compounds can pass through the blood brain barrier and act through specific cannabinoid receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 and CB2 are expressed in the brain and the periphery, respectively, and may serve as molecular targets for ablating chronic brain inflammation. Electrophoretic mobility shift assays (EMSA) were used to assess the effects of D9-THC and CP55940 on the LPS-induced binding interactions of the universal transcription factor NFkB to its cognate promoter binding site in BV-2 microglial-like cells. EMSA analyses demonstrated that the D9-THC and CP55940 down-regulated LPS-induced NFkB binding in BV-2 cells in a biphasic manner. Furthermore, reporter activity assays determined that D9-THC and CP55940 attenuated LPS-induced, NFkB transcriptional activity in the same biphasic manner. We then determined the specificity in which cannabinoids inhibit NFkB function. Real-Time RT-PCR analysis demonstrated that BV-2 cells did not express CB1 mRNA, but they do express CB2 mRNA when untreated and stimulated with IFN-g or LPS. We performed specificity studies using CB1 and CB2 selective agonists and antagonists with our reporter activity assays. The CB1-selective agonist ACEA did not affect NFkB transcriptional activity but the CB2-selective agonist O-2137 exerted a significant decrease in activity. Furthermore, the CB1 antagonist SR141716A could not reverse the inhibitory effects of CP55490 but those effects were blocked by the CB2 antagonist SR144528. Lastly, we determined the site of action in which cannabinoids inhibit NFkB function by assessing the effects of D9-THC and CP55940 on NFkB’s inhibitor protein IkBa. IkBa retains NFkB in the cytoplasm until stimulus-induced cell activation. Neither cannabinoid compound was able to inhibit the phosphorylation of IkBa, which initiates its degradation. However both cannabinoids inhibited the complete degradation of IkBa. Western immunoblot analysis also demonstrated that comparable levels of endogenous and phosphorylated p65, the transactivation subunit of the NFkB protein (p65/p50), were detected in the nucleus of LPS-stimulated BV-2 cells pre-treated with or without D9-THC. These results suggest that, in addition to inhibiting the proteolytic degradation of IkBa, there is also a mechanism of action in the nucleus that prevents the proper binding and subsequent transcriptional activity of NFkB. Collectively, these results suggest that cannabinoids suppress pro-inflammatory cytokine gene expression at the transcriptional level, but it is likely that there is more than one signal transduction pathway involved in the cannabinoid-mediated inhibition of NFkB function.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2009

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