Cannabinoid-induced immune suppression in mouse splenocytes involves both cannabinoid receptor-dependent and -independent mechanisms

Cannabinoids are a group of structurally related compounds derived from the Cannabis sativa plant. The immunosuppressive effects of cannabinoids are putatively mediated through G protein-coupled receptors (CB1 and CB2) that exhibit inhibition of adenylate cyclase activity in lymphocytes. The CB2 receptor is primarily localized to lymphoid tissue, whereas the CB1 receptor has been characterized in the CNS with lower expression levels in lymphoid tissues. The immunosuppressive effects of cannabinoid compounds include inhibition of interleukin-2 (IL-2) production in phorbol ester plus calcium ionophore (PMA/Io)-stimulated lymphocytes. The mechanism of IL-2 inhibition, including the role of the cannabinoid receptors; however, is not yet understood. The following objectives are utilized to test this hypothesis: Immune modulation by cannabinoid compounds is mediated via cannabinoid receptors (CB1 and/or CB2), resulting in disruption of activator protein-1 (AP-1) transcription factor binding in the promoter region of immune system genes, such as IL-2. The first objective was to characterize the effects of cannabinoids on AP-1 transcription factor activity in primary mouse SPLC. Cannabinoid compounds inhibited PMA/Io-stimulated AP-1 transcription factor binding to both the consensus AP-1 and proximal AP-1 sites. Cannabinoid compounds also inhibited PMA/Io-stimulated nuclear factor of activated T cells (NF-AT) transcription factor binding activity to the distal NF-AT site, which depends on cooperative binding with AP-1 proteins. The decrease in transcription factor binding activity was due, in part, to inhibition of PMA/Io-stimulated protein expression of the AP-1 components, c-fos and c-jun. This inhibition of c-fos and c-jun protein expression was not due to inhibition of steady state mRNA expression, suggesting that cannabinoids inhibited post-translational modifications of c-fos and c-jun. Indeed, cannabinoids inhibited the activation of extracellular signal-regulated mitogen activated protein kinase (ERK MAPK). The second objective was to characterize the role of the cannabinoid receptors in cannabinoid-induced inhibition of PMA/Io-stimulated IL-2 production. Cannabinoids such as cannabinol (CBN), cannabidiol (CBD) and the WIN-55212 stereoisomers inhibited PMA/Io-stimulated IL-2 production with a rank order potency of WIN-2 ≈ CBD > WIN-3 > CBN. This inhibition; however, was not attenuated in the presence of both cannabinoid receptor antagonists. With the demonstration that cannabinoid-induced inhibition of PMA/Io-stimulated IL-2 production was independent of the cannabinoid receptors, other T cell targets were examined. Modulation of intracellular calcium seemed a likely candidate to explain the pleiotropic effects of these compounds. Thus, the third objective was to determine the effect of cannabinoids on intracellular calcium concentration. Cannabinoid compounds elevated intracellular calcium concentration in a cannabinoid receptor-dependent manner in resting SPLC. Furthermore, the mechanism of cannabinoid-induced immune modulation is both cannabinoid receptor-dependent and -independent.