No abstract

Inducible costimulator (ICOS), a prototypic T cell costimulator, is induced on activated T cells. ICOS regulates T cell activation and Th cell differentiation and is principally involved in humoral immune responses. Previous work showed that T cell accessory function is modulated by the plant-derived cannabinoid, delta-9-tetrahydrocannabinol (Delta(9)-THC). In light of an emerging role by ICOS in T cell-mediated immunity, the objective of this study was to investigate the effect of Delta(9)-THC on ICOS in activated mouse T cells. Induction of ICOS mRNA levels by phorbol ester (PMA) plus ionomycin (Io) activation in mouse splenocytes was attenuated by Delta(9)-THC in a concentration-related manner. Similar results were obtained in the mouse T cell line, EL4.IL-2. Anti-CD3/CD28 induced ICOS expression on CD4(+) splenic T cells, which was suppressed by Delta(9)-THC in a time- and concentration-related manner. The PMA/Io-induced icos promoter luciferase reporter activity was also down-regulated by Delta(9)-THC, suggesting that the suppression of ICOS expression by Delta(9)-THC occurs at the transcriptional level. Moreover, transcriptional activation of the NFAT was also down-regulated by Delta(9)-THC as shown by a NFAT luciferase reporter assay, which is consistent with a putative role of NFAT in regulating ICOS expression. Collectively, Delta(9)-THC suppresses ICOS expression in activated T cells, and this suppression may be related, in part, to its modulation of NFAT signaling. The emerging role of ICOS in a wide range of immune-related diseases also suggests that it may represent a potential therapeutic target, which could be modulated by cannabinoid compounds.

The anti-inflammatory activity of cannabinoids has been widely demonstrated in experimental animal models and in humans. CD40-CD40-ligand (L) interactions are among the most crucial initiators of inflammation. This study investigated the effects of Δ9-THC on CD40L expression in mouse splenic T cells after activation with various stimuli. Time course studies demonstrated that peak surface expression of CD40L by CD4+ T cells after anti-CD3/CD28 or phorbol ester plus calcium ionophore (PMA/Io) occurred 8 h post activation. Peak CD40L mRNA levels were observed at 2 h post PMA/Io treatment and at 4 h post anti-CD3/CD28 treatment. Pretreatment with Δ9-THC significantly impaired the upregulation of CD40L induced by anti-CD3/CD28 at both the protein and mRNA level. By contrast, Δ9-THC did not affect PMA/Io-induced surface CD40L expression on CD4+ T cells. Additionally, Δ9-THC also attenuated anti-CD3/CD28-induced CD40L expression on CD4+ T cells derived from CB1−/−/CB2−/− mice. We investigated whether the mechanism by which Δ9-THC suppressed CD40L expression involved putative cannabinoid activation of the glucocorticoid receptor (GR). Although activation of GR resulted in suppression of CD40L induction by anti-CD3/CD28, no interaction between Δ9-THC and GR was observed by a glucocorticoid response element (GRE) luciferase reporter assay in HEK293T cells. Collectively, these results suggest that Δ9-THC targets proximal T cell receptor-associated signaling in a cannabinoid receptor- and glucocorticoid receptor-independent manner. These findings identify suppression of CD40L expression as a novel part of the mechanism by which Δ9-THC exerts anti-inflammatory activity.

The mechanisms by which cannabinoid receptors CB 1 and CB 2 modulate immune function are not fully elucidated. Critical tools for the determination of the role of both receptors in the immune system are CB 1 /CB 2 double null mice (CB 1 /CB 2 null), and previous studies have shown that CB 1 /CB 2 null mice exhibit exaggerated responses to various immunological stimuli. The objective of these studies was to determine the magnitude to which CB 1 /CB 2 null mice responded to the respiratory allergen ovalbumin (OVA) as compared with wild-type C57BL/6 mice. The authors determined that in the absence of adjuvant, both wild-type and CB 1 /CB 2 null mice mounted a marked response to intranasally instilled OVA as assessed by inflammatory cell infiltrate in the bronchoalveolar lavage fluid (BALF), eosinophilia, induction of mucous cell metaplasia, and IgE production. Many of the endpoints measured in response to OVA were similar in wild-type versus CB 1 /CB 2 null mice, with exceptions being modest reductions in OVA-induced IgE and attenuation of BALF neutrophilia in CB 1 /CB 2 null mice as compared with wild-type mice. These results suggest that T-cell responses are not universally exaggerated in CB 1 /CB 2 null mice.

We have previously reported that Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4+ T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ9-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ9-THC attenuated CD40L expression in human CD4+ T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ9-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ9-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ9-THC suppresses human T cell function.