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
Abstract
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 μm 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 κ chain in B cells (NF-κB/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-κB)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-κB/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.
Footnotes
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Send reprint requests to: Dr. Norbert E. Kaminski, Department of Pharmacology and Toxicology, B330 Life Sciences Building, Michigan State University, East Lansing, MI 48824.
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This work was supported in part by National Institutes of Health Grant 5P01-DA09789–02.
- Abbreviations:
- CB1
- cannabinoid receptor type 1
- 2-Ara-Gl
- 2-arachidonyl-glycerol
- CB2
- cannabinoid receptor type 2
- IL-2
- interleukin-2
- Δ9-THC
- Δ9-tetrahydrocannabinol
- CBN
- cannabinol
- NF-AT
- nuclear factor of activated T cells
- NF-κB
- nuclear factor for immunoglobulin κ chain in B cells
- IκB
- inhibitors of nuclear factor for immunoglobulin κ chain in B cells
- AP-1
- activator protein-1
- AP-1c
- activator protein-1 consensus site
- AP-1p
- activator protein-1 proximal site from the IL-2 promoter
- Oct
- octamer
- CRE
- cAMP-response element
- CREB
- cAMP-response element-binding protein
- CAT
- chloramphenicol acetyltransferase
- p(NF-κB)3-CAT
- CAT plasmid with three copies of nuclear factor for immunoglobulin κ chain in B cells consensus site
- p(NF-AT)3-CAT, CAT plasmid with three copies of NF-AT consensus site
- p(AP-1)3-CAT, CAT plasmid with three copies of activator protein-1 consensus site
- PMA
- phorbol-12-myristate-13-acetate
- Io
- ionomycin
- CsA
- cyclosporin A
- NA
- naive
- VH
- vehicle
- ELISA
- enzyme-linked immunosorbent assay
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- EMSA
- electrophoretic mobility shift assay
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- Received August 25, 1997.
- Accepted January 8, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
