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-Gl) 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-Gl possessed immunomodulatory activity. Because several plant-derived cannabinoids inhibit interleukin-2 (IL-2) expression, 2-Ara-Gl was investigated for its ability to modulate this cytokine. The direct addition of 2-Ara-Gl 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-Gl 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-Gl at 5, 10, 20, and 50 microM 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-Gl, 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-Gl on promoter activity for each of the specific transcription factors. 2-Ara-Gl 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-Gl, but a modest inhibition of promoter activity was observed.
Ouyang
Y
Y
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824, USA.
Hwang
S G
SG
Han
S H
SH
Kaminski
N E
NE
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D001095:Arachidonic Acids; D002186:Cannabinoids / Q000378:metabolism; D002478:Cells, Cultured; D016384:Consensus Sequence; D003433:Crosses, Genetic; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects*; D063388:Endocannabinoids; D005260:Female; D005989:Glycerides / Q000378:metabolism / Q000494:pharmacology*; D051863:Host Cell Factor C1; D007376:Interleukin-2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000276:Adjuvants, Immunologic; D001095:Arachidonic Acids; D002186:Cannabinoids; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D063388:Endocannabinoids; D005989:Glycerides; C497693:Hcfc1 protein, mouse; D051863:Host Cell Factor C1; D007376:Interleukin-2; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C094503:glyceryl 2-arachidonate