Inhibition of interleukin-2 secretion by 2-arachidonyl glycerol and anandamide occurs through peroxisome proliferator activated receptor gamma independently of the cannabinoid receptors

2-Arachidonyl glycerol (2-AG) and anandamide (AEA) are endogenous arachidonic acid derivatives which activate the cannabinoid receptors, CB1 and CB2, hence termed endocannabinoids. 2-AG and AEA modulate a variety of immunological responses, including induction of transient calcium influx in HL-60 cells, modulation of lymphocyte proliferation, and suppression of cytokine production. As is observed with the plant-derived cannabinoids, 2-AG and AEA suppress interleukin (IL)-2 production. The overall goal of these studies was to determine the mechanism of the inhibitory effects of AEA and 2-AG upon IL-2 secretion. The suppression of IL-2 by 2-AG and AEA in splenocytes derived from CB1/CB2 null mice coupled with the failure of the CB 1/CB2 antagonists to block the decrease in IL-2 by AEA and 2-AG, demonstrates that the cannabinoid receptors are not involved. Interestingly, arachidonic acid causes a concentration-dependent suppression of IL-2 secretion, which was similar to that of structurally-related AEA and 2-AG. The decrease in IL-2 by AEA and 2-AG was partially reversed by pretreatment with the nonspecific cyclooxygenase (COX) inhibitor, flurbiprofen, as well as the COX-2 specific inhibitor, NS398, suggesting that COX-2 metabolites of 2-AG and AEA are responsible for the inhibitory effects upon IL-2, rather than the parent molecules. Because peroxisome proliferator activated receptor gamma (PPARgamma) activation has been correlated with IL-2 suppression in T cells and a number of COX metabolites are known PPARgamma agonists, the ability of 2-AG to activate PPARgamma was investigated. Both 2-AG and 2-AG ether, a non-hydrolyzable analogue of 2-AG, activate PPAR, as evidenced by differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARgamma-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARgamma in IL-2 suppression by 2-AG and 2-AG ether was examined. IL-2 suppression by 2-AG and 2-AG ether in activated T cells was blocked by T0070907, a potent PPARgamma-specific antagonist. Similarly, T0070907, also blocked AEA-mediated IL-2 suppression. Additionally, 2-AG was also found to inhibit the transcriptional activity of nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NFkappaB), transcription factors that are critical for IL-2 transcription. In addition to its effects upon IL-2, 2-AG suppresses the transcription of IL-4 and IFNgamma, cytokines that are also regulated by NFAT. Moreover, the inhibition of NFAT and (NFkappaB), transcriptional activity by 2-AG was abrogated in the presence of T0070907. Collectively, the aforementioned studies provide evidence that suppression of IL-2 by COX-2 metabolites of 2-AG and AEA is mediated through activation of PPARgamma independently of CB1/CB2. In addition, evidence is provided that PPARgamma activation by 2-AG or its metabolite inhibits the transcriptional activity of NFAT and (NFkappaB), which ultimately results in suppression of IL-2 by activated T cells.