2-Arachidonoyl-glycerol suppresses interferon-gamma production in phorbol ester/ionomycin-activated mouse splenocytes independent of CB1 or CB2.

2-Arachidonoyl-glycerol (2-AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin-2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2-AG on interferon-gamma (IFN-gamma) expression and associated upstream signaling events. Pretreatment of splenocytes with 2-AG markedly suppressed phorbol 12-myristate 13-acetate plus calcium ionophore (PMA/Io)-induced IFN-gamma secretion. In addition, 2-AG suppressed IFN-gamma steady-state mRNA expression in a concentration-dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1(-/-)/CB2(-/-) knockout mice were used. No difference in the magnitude of IFN-gamma suppression by 2-AG in wild-type versus CB1/CB2 null mice was observed. Time-of-addition studies revealed that 2-AG treatment up to 12 h post-cellular activation resulted in suppression of IFN-gamma, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2-AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin-induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN-gamma production. Indeed, Io partially attenuated the 2-AG-induced suppression of PMA/Io-stimulated IFN-gamma production. Taken together, these data demonstrate that 2-AG suppresses IFN-gamma expression in murine splenocytes in a CB receptor-independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.