Anandamide inhibits IL-12p40 production by acting on the promoter repressor element GA-12:: possible involvement of the COX-2 metabolite prostamide E2

The eCB [endoCB (cannabinoid)] system is being considered as a novel therapeutic target for immune disorders. Cytokines of the IL-12 (interleukin-12) family have essential functions in cell-mediated immunity. In the present study, we have addressed the mechanisms of action of the eCB AEA (anandamide) on the regulation of IL-12p40 in activated microglia/macrophages. We demonstrated that AEA can inhibit the expression of p35, p19 and p40 subunits, which form the biologically-active cytokines IL-12 and IL-23 in microglia stimulated with LPS (lipopolysaccharide)/IFN gamma (interferon gamma). Additionally, we have provided evidence that AEA reduces the transcriptional activity of the IL-12p40 gene in LPS- and IFN gamma-co-activated cells, and this is independent of CB or vanilloid receptor activation. Site-directed mutageneis of the different elements of the p40 promoter showed that AEA regulates IL-12p40 expression by acting on the repressor site GA-12 (GATA sequence in IL-12 promoter). Prostamide E-2 (prostaglandin E-2 ethanolamide), a product considered to be a putative metabolite of AEA by COX-2 (cyclo-oxygenase 2) oxygenation, was also able to inhibit the activity of the IL-12p40 promoter by acting at the repressor site. The effects of AEA and prostamide E-2 on p40 transcription were partially reversed by an antagonist of EP2 (prostanoid receptor-type 2), suggesting the possibility that prostamide E-2 may contribute to the effects of AEA on IL-12p40 gene regulation. Accordingly, the inhibition of COX-2 by NS-398 partially reversed the inhibitory effects of AEA on IL-12p40. Overall, our findings provide new mechanistic insights into the activities of AEA in immune-related disorders, which may be relevant for the clinical management of such diseases.