Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

Thomas W. Klein, Ph.D.

Committee Member

Raymond Widen, Ph.D.

Committee Member

Shyam Mohapatra, Ph.D.

Committee Member

Jun Tan, Ph.D.

Keywords

CP55940, CB2, IL-4, IgE, TLR4, CSR

Abstract

Cannabinoid treatment increases Th2 activity and previous reports showed B cells express the highest level of CB2 mRNA relative to other immune cells suggesting that cannabinoids play a critical role in B cell activation and maturation. To examine the direct effect of cannabinoids on B cell antibody class switching, mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB1 selective agonist, methanandamide, or the CB2 selective agonist, JW015. The cultures were then analyzed at different times by flow cytometry for expression of B cell surface markers, such as CD19, CD138, CD40, MHCII, CD23, CD80, CD45R, immunoglobulins produced such as IgM, IgE, IgD, and IgG1, and Toll-like receptors such as TLR 2 and 4. Cells treated with CP55940 showed an increase in surface expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants analyzed by ELISA. In addition, CB2 receptors were increased on B cells following stimulation with IL-4 and anti-CD40 and the class switching effect of CP55940 was attenuated by the CB2 antagonist, SR144528.

We also observed that cannabinoid treatment of B cells modulates cell functions other than antibody class switching such as surface marker and TLR expression. CP55940 caused a significant increase in surface expression of TLR 4, but had no effect on other markers. Additional experiments with cannabinoid receptor selective agonists and antagonists suggested both CB1 and CB2 receptors were involved in the TLR effect. Receptor involvement and Gi coupling was supported by our findings that cannabinoids inhibit intracellular cAMP levels in forskolin stimulated B cells, and increasing intracellular cAMP with forskolin suppressed IgE antibody class switching in activated B cell cultures. These results suggest cannabinoids negatively regulate cAMP in B cells resulting in increased IgE. In conclusion, cannabinoids can directly affect the function of B cells by inducing antibody class switching to IgE and TLR4 expression through mechanisms involving CB1 and CB2 receptors suggesting the endocannabinoid system may be an important regulator of humoral immunity and the allergic response.

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