title: 2-Arachidonoyl-glycerol suppresses interferon-gamma production in phorbol ester/ionomycin-activated mouse splenocytes independent of CB1 or CB2.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, Michigan State University, East Lansing, 48824, USA. kamins11@msu.edu
authors.forename: Barbara L F
authors.initials: BL
authors.lastname: Kaplan
authors.forename: Yanli
authors.initials: Y
authors.lastname: Ouyang
authors.forename: Cheryl E
authors.initials: CE
authors.lastname: Rockwell
authors.forename: Gautham K
authors.initials: GK
authors.lastname: Rao
authors.forename: Norbert E
authors.initials: NE
authors.lastname: Kaminski
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators / Q000494:pharmacology*; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D063388:Endocannabinoids; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005989:Glycerides / Q000494:pharmacology*; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000502:physiology*; D043885:Receptor, Cannabinoid, CB2 / Q000235:genetics / Q000502:physiology*; D013154:Spleen / Q000166:cytology / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism
chemical_list: D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D004268:DNA-Binding Proteins; D063388:Endocannabinoids; D005989:Glycerides; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010703:Phorbol Esters; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D014157:Transcription Factors; D015759:Ionomycin; C016658:phorbolol myristate acetate; D007371:Interferon-gamma; C094503:glyceryl 2-arachidonate; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium