It has been shown that GPR receptors coupled to G

It has been shown that GPR55 receptors coupled to G12/13 proteins down-regulate reactive oxygen species (ROS) production and degranulation in human neutrophils [9]. MCs degranulation stimulated by IgE/Ag also requires the generation of ROS [64]; therefore, the possibility to explore the inhibitory actions of AEA on ROS production in MCs is an attractive idea. On the other hand, there is evidence that activation of another G12/13-coupled receptor (LPA5) by lysophosphatidic mitotic inhibitor inhibits B cell antigen receptor (BCR) by diminishing calcium mobilization. Our data support the hypothesis that GPR55 and other receptors coupled via PTX insensitive proteins (particularly G12/13) can modulate selected effector functions in immune cells. Interestingly, we observed that not only GPR55 but also CB2 receptor antagonism blocked the inhibitory actions of AEA, HU308 and LPI on FcεRI-induced degranulation. These results strongly suggest that a CB2-GPR55 heteromer mediates this inhibition. Our findings are in line with the observation that a CB2 receptor antagonist prevents the inhibitory effects of 2-AG on histamine release in MCs [59] and also blocks the effects of the CB2 receptor agonist S-777469 on skin MCs [65]. However, the participation of GPR55 was not addressed in those studies. As to the mechanism behind the inhibitory actions of AEA, we found that it does not block early FcεRI-induced phosphorylation events, but prevents calcium-dependent cytokine production. FcεRI signal transduction system is composed by multiple branches connecting the IgE/Ag-dependent crosslinking of the receptor with degranulation, lipid mediator release and cytokine production [26,66,67]. One of the most important initial events is the tyrosine phosphorylation of protein kinase Lyn that continues with the activation of protein kinase Syk. Then, tyrosine phosphorylation of distinct adapters allows the recruitment of effector enzymes, such as phospholipase C (PLC)γ, which controls protein kinase C activation and calcium release from intracellular stores. Our data show that AEA does not affect those early events on FcεRI signaling, but importantly blocks cytokine mRNA production. It is well known that a number of transcription factors, implicated in the accumulation of IL-2, IL-4, TNF and other cytokines, are regulated by calcium mobilization in MCs. Present results indicate that the point(s) of the inhibitory actions of anandamide is (are) located downstream the early kinase activation and before the activation of transcription factors. These results are in line with data generated in T cells, where Δ-9-tetrahydrocannabinol suppressed the DNA-binding activity of NFAT and NFκB transcription factors on the CD40L gene promoter and inhibited T-cell receptor (TCR)-induced calcium rise without significant impairment of ZAP70, PLCγ1/2, Akt and GSK3β phosphorylation [68]. Our data add to the description of inhibitory actions of endocannabinoids on the activation of transcription factors involved in cytokine synthesis in immune cells. They also suggest that common negative regulatory pathways operate in innate and adaptive immune cells. The formation of active dimers between distinct GPCRs has been identified as a biochemical mechanism that modifies the intensity and quality of signals generated by a given ligand [69]. It is known that CB2 and GPR55 receptors are co-expressed in several cell types, such as neutrophils and cancer cells [70,71]. These receptors activate different pathways via ligand- and concentration-specific crosstalk [72]. The formation of CB2 and GPR55 receptor heterodimers is capable of down-regulating MC activation via non-classical mechanisms in response to cannabinoids, opening the possibility of using CB2 receptor agonists to trigger GPR55-mediated inhibitory effects to limit MC-dependent inflammatory reactions, where the levels of LPI could not reach the threshold to trigger GPR55 activation.