Cannabinoid receptors CB1 and CB2: a characterization of expression and adenylate cyclase modulation within the immune system.

Cannabinoid receptor (CB) expression was characterized in immunological cell and tissue preparations. Northern analysis revealed approximately 6-kb transcripts for CB1 (brain-type) in mouse spleen and brain and in rat cerebellum. CB1 was not detected in mouse thymus or rat spleen RNA by Northern analysis. CB2 (peripheral) was detected as a approximately 4-kb transcript in mouse spleen and thymus and as approximately 2.4-kb transcripts in rat spleen. Quantitation of CB2 transcripts in mouse spleen and thymus revealed approximately 4 x 10(3) and approximately 4 x 10(2) molecules/100 ng RNA, respectively, with no quantifiable CB2 in mouse brain. Conversely, CB1 was expressed in mouse brain (approximately 2 x 10(5) molecules/100 ng RNA) with lower expression in mouse spleen (approximately 2 x 10(2) molecules/100 ng RNA) and was not quantifiable in mouse thymus. Competition binding in intact mouse splenocytes demonstrated that nonradiolabeled cannabinoids CP-55940, Win-55212-2, CP-56667, delta 9-THC, and cannabinol all competed for receptor binding with 3H-CP-55940, a high-affinity nondiscriminating CB1 and CB2 receptor ligand. Based on previous findings which demonstrated a marked inhibition of T-cell-dependent immune responses by cannabinoids, primary T cells and several T-cell lines were characterized. Radioligand binding analysis identified 100-300 cannabinoid receptor binding sites/cell with an approximate Kd of 200-700 pM in purified splenic T cells which also exhibited cannabinoid-induced inhibition of adenylate cyclase. Northern analysis of human T-cell lines revealed approximately 2.4-kb CB2 mRNA transcripts but no CB1 in HPB-ALL cells, a cell line which also exhibited inhibition of adenylate cyclase by delta 9-THC. Conversely, Jurkat E6-1 cells expressed an unusual mRNA banding pattern for CB2 expressing three distinct transcript sizes, none of which were 2.4 kb, the size for human CB2. Jurkat also did not express CB1 mRNA and did not exhibit inhibition of adenylate cyclase when treated with delta 9-THC. Collectively, these results provide further evidence that CB2 is the predominant cannabinoid receptor within the immune system and that this form of the receptor is expressed on T cells.

Attenuation of inducible nitric oxide synthase gene expression by delta 9-tetrahydrocannabinol is mediated through the inhibition of nuclear factor- kappa B/Rel activation.

delta 9-Tetrahydrocannabinol (delta 9-THC) a prototypic compound belonging to the family of agents known as cannabinoids, produces a wide variety of biological effects, including inhibition of immune function. The putative mechanism for cannabinoid biological action involves binding to cannabinoid receptor types 1 and 2 (CB1 and CB2) to negatively regulate adenylate cyclase and inhibit intracellular signaling via the cAMP cascade. In the current study, we show that delta 9-THC produces a marked inhibition of inducible nitric oxide synthase (iNOS) transcription and nitric oxide production by the macrophage line RAW 264.7 in response to lipopolysaccharide (LPS). Analysis of RAW 264.7 cell RNA demonstrated transcripts for CB2 but not CB1. Treatment of RAW 264.7 with delta 9-THC inhibited forskolin-stimulated cAMP production in a dose-related manner, verifying the expression of functional cannabinoid receptors by this cell line. iNOS transcription, which is regulated in part by the nuclear factor-kappa B/Rel (NF-kappa B/Rel) family of transcription factors, has been shown to be under the control of the cAMP signaling cascade. We demonstrate that delta 9-THC inhibits the activation and binding of NF-kappa B/Rel proteins to their cognate DNA site, kappa B, in response to LPS stimulation. LPS treatment of RAW 264.7 cells also induced the activation of the cAMP cascade, as indicated by an increase in binding of nuclear factors to the cAMP response element. Activation of CRE binding proteins was inhibited by delta 9-THC. Forskolin treatment of RAW 264.7 cells induced both kappa B and cAMP response element binding activity and was likewise inhibited by delta 9-THC. Collectively, this series of experiments indicates that NF-kappa B/Rel is positively regulated by the cAMP cascade to help initiate iNOS gene expression in response to LPS stimulation of macrophages. This activation of iNOS is attenuated by delta 9-THC through the inhibition of cAMP signaling.

Molecular analysis of the glycoprotein C-negative phenotype of attenuated Marek's disease virus.

Serial passage of oncogenic serotype 1 Marek's Disease virus (MDV) in cultured avian cells results in attenuation of viral oncogenicity and pathogenicity. Coincident with attenuation, expression of MDV glycoprotein C (gC) is significantly reduced. Regulation of MDV gC may have important implications for virus-host interactions and have important consequences in the process of viral attenuation. To investigate the mechanism by which MDV gC expression is reduced during attenuation of the very virulent serotype 1 MDV strain Md11, protein levels, gene structure, steady-state RNA levels, and transcription rates of MDV gC from oncogenic and attenuated isolates of Md11 were determined. Comparison of these data with similar studies on MDV proteins whose expression is not altered during attenuation indicates that reduced expression of MDV gC is directly related to reduction in transcription rate of the MDV gC gene in attenuated Md11. Reduced transcription rates and lack of any gross structural alterations in the attenuated MDV gC gene suggest that MDV regulatory protein(s) which interact with the MDV gC promoter are altered during attenuation of MDV strain Md11. This conclusion is supported by DNA sequence data which indicate that the promoters of oncogenic and attenuated Md11 gC genes are identical.

Marek's disease virus-mediated enhancement of avian leukosis virus gene expression and virus production.

Direct interaction between two viruses in coinfected cells may promote replication and pathogenesis of one or both virus types. Synergism between herpesviruses and retroviruses is an important factor in diagnosis, treatment, and prevention of animal and human diseases. In birds, Marek's disease virus (MDV) may be an important cofactor in avian leukosis virus induced disease. Infection of susceptible cells with non-oncogenic serotype 2 MDV, an avian herpesvirus, and Rous-associated virus type 2 (RAV-2 ALV), a leukemogenic avian retrovirus, results in enhanced (greater than 3-fold) transcription of retroviral genes, relative to infection with ALV alone. A direct relationship between concentrations of retroviral gene expression and amount of input MDV suggests that MDV-encoded or -induced factors are responsible for enhanced ALV gene expression, ultimately leading to increased accumulation of ALV-specific RNA (greater than 5-fold) and protein (greater than 10-fold). At lower doses of input MDV, ALV virus production increased over 3-fold, relative to cells infected with ALV alone. Interactive laser cytometry was used to detect accumulation of both MDV and ALV antigens within single cells from coinfected cultures. These results suggest a direct role for MDV-encoded or -induced factors in enhancement of ALV gene expression and demonstrate the importance of herpesviruses as cofactors in retrovirus replication and pathogenesis in coinfected cells.