Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonyl-glycerol is mediated through down-regulation of the nuclear factor of activated T cells

2-Arachidonyl-glycerol (2-Ara-G1) recently was identified as a putative endogenous ligand for cannabinoid receptor types CB1 and CB2 by competitive binding. More recent immune function assays demonstrated that 2-Ara-G1 possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-G1 was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-G1 to mouse splenocyte cultures suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 secretion and steady state mRNA expression in a dose-dependent manner. 2-Ara-G1 also produced a marked inhibition of IL-2 promotor activity as determined by transient transfection of EL4.IL-2 cells with a pIL-2-CAT construct. 2-Ara-G1 at 5, 10, 20, and 50 mu M suppressed phorbol-12-myristate-13-acetate plus ionomycin-induced IL-2 promotor activity by 18%, 28%, 39%, and 54%, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2 by 2-Ara-G1, the DNA-binding activity of transcription factors, nuclear factor of activated T cells (NF-AT), nuclear factor for immunoglobulin kappa chain in B cells (NF-kappa B/Rel), activator protein-1 (AP-1), octamer, and cAMP-response element binding protein was evaluated by electrophoretic mobility shift assay in mouse splenocytes. In addition, a reporter gene expression system for p(NF-kappa B)(3)-CAT, p(NF-AT)(3)-CAT, and p(AP-1)(3)-CAT was used in transiently transfected EL4.IL-2 cells to determine the effect of 2-Ara-G1 on promoter activity for each of the specific transcription factors. 2-Ara-G1 reduced both the NF-AT-binding and promoter activity in a dose-dependent manner and, to a lesser degree, NF-kappa B/Rel-binding and promoter activity. No significant effect was observed on octamer-and cAMP-response element-binding activity. AP-1 DNA-binding activity was not inhibited by 2-Ara-G1, but a modest inhibition of promoter activity was observed.