Date Published: January 14, 2010
Publisher: Public Library of Science
Author(s): Maria Teresa Cencioni, Valerio Chiurchiù, Giuseppina Catanzaro, Giovanna Borsellino, Giorgio Bernardi, Luca Battistini, Mauro Maccarrone, Olivier Jacques Manzoni. http://doi.org/10.1371/journal.pone.0008688
Abstract: Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB1R and CB2R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB1 and CB2 receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.
Partial Text: Anandamide (N-arachidonoylethanolamine, AEA) is an endogenous arachidonic acid derivative which functionally belongs to the family of endocannabinoids (ECs), an evergrowing class of lipid mediators isolated from brain and peripheral tissues , . AEA exerts its action through binding and activation of cannabinoid receptors, CB1R and CB2R . These receptors are differentially distributed among cells, with CB1R being preferentially expressed in the brain and in other neural tissues, whilst CB2R is found mainly in cells of the immune system , . The presence of endogenous ligands for the cannabinoid receptors has increased awareness of the potential importance of endocannabinoids in regulating and fine-tuning several processes, including neuroprotection  and immune responses . When stimulated, immune cells up-regulate particular receptors or express new ones. This scenario has been demonstrated also for CBRs, since several stimuli have been reported to differently modulate both cannabinoid receptor subtypes in immune cells, like mouse splenocytes, Jurkat T-cells, mouse macrophages and microglia, as well as human tonsillar B cells and dendritic cells . In addition, it has been shown that chronic marijuana smoking increases CB1R and CB2R mRNA in peripheral blood mononuclear cells . Modulation of CBRs gives support to a regulatory role of endocannabinoids in immune responses, however the molecular details of this activity have yet to be fully elucidated. So far, AEA has been detected in different immune cells, including dendritic cells, macrophages, microglia, and lymphocytes . Moreover, activation of lymphocytes and other immune cells causes a rapid and robust increase in AEA levels, further supporting a role for AEA in immune regulation . For instance, AEA suppresses LPS-induced nitric oxide production by mouse peritoneal macrophages , and LPS-induced cytokine mRNA expression in rat microglial cells . In addition, AEA decreases mitogen-induced T and B cell proliferation , suppresses CD8 T lymphocyte migration , and induces apoptosis in macrophages  and dendritic cells . In the present study we sought to better define the role of AEA in modulating immune functions of primary human T cells, with a special focus on the key regulators of autoimmune inflammation: Th17 lymphocytes .
Since its identification from porcine brain, AEA has been shown to exert cannabimimetic activities such as the induction of hypothermia, analgesia, and motor effects. Although first described in the nervous system, it is becoming increasingly clear that AEA plays an important role in modulating the immune system. The present study clearly shows that AEA is immunosuppressive when added to activated T-lymphocytes, and that it acts mainly through CB2R. In the attempt to shed some light on such immunomodulatory activity of AEA, we first collected evidence for the alteration of the expression of CB1R and CB2R, as a result of cell activation. In fact, it is well-documented that immune cells, when activated, can modulate specific receptors in order to trigger and sustain an efficient immune response. Our data not only demonstrate by means of several techniques that CB2R is by far more abundant than CB1R in primary human T-cells, but also that both receptors are recruited on the cell surface upon activation. To date there is evidence showing that classical antigenic stimuli, such as LSP or anti-CD3, can modulate both CB1R and CB2R expression, however there are discrepancies in the literature especially because of the different experimental models used, spanning from Jurkat cells to mouse splenocytes . Thus, our analysis of CB1R and CB2R seems to represent the first evidence of a redistribution of CBRs with an increased localization on the cell surface, particularly in the case of CB2R. In addition, [35S]GTPγS binding data seem to support a receptor “sensitization” in a highly purified population of primary human immune cells, that is CD3+-T lymphocytes. This seems of interest, since it allows a better understanding of the role of CB1versus CB2 receptors in endocannabinoid-mediated immunomodulation of lymphocytes. Increasing evidence supports indeed that both receptors are involved in central nervous system autoimmunity, having particular beneficial effects in the treatment of multiple sclerosis (MS). Using animal models of MS like experimental autoimmune encephalomylelitis (EAE), CB1R and CB2R have been shown to be specific markers of MS plaque cell subtypes ; furthermore, CB1R has been proven to mediate suppression of CNS autoimmune inflammation on neurons, whilst CB2R had the same anti-inflammatory role on autoreactive T cells . The present results provide evidence that AEA is able to suppress anti-CD3/anti-CD28-induced T-cell proliferation mainly via CB2 receptors. In fact, this activity of AEA was minimized by the CB1R and CB2R antagonists SR1 and SR2, with a major effect exerted by SR2, and was also mirrored by the CB2R agonist JWH-015, but not by the CB1R agonist ACEA. At any rate, the anti-proliferative action of AEA reported herein is in accordance with a previous report by Schwarz and colleagues , where the suppression of proliferation by µM concentrations of AEA was not associated to the induction of apoptosis. Indeed, they demonstrated that AEA is capable of inducing apoptosis only when used at high doses and even that was at least in part responsible for the complete inhibition of cell proliferation observed at high concentrations. The ability of AEA to suppress T-cell proliferation was also substantiated by its ability to markedly inhibit IL-2 release from activated T-lymphocytes, again in a CB2R-mediated manner. This result is consistent with a previous report, where inhibition of IL-2 release from phytohemagglutinin-stimulated PBMCs was found to be mediated by CB2R . Incidentally, here we could demonstrate that TRPV1 was not engaged in this activity of AEA. In additional experiments, the immunosuppressive effect of AEA was corroborated by a detailed analysis of the production of the major cytokines involved in the regulation of T-lymphocyte responses. Indeed, AEA-induced inhibition of TNF-α and IFN-γ release was predominantly mediated by CB2R, since cytokine suppression was not significantly reversed by the CB1R antagonist SR1. However, based on the partial effect of the latter compound, and on the fact that specific CB1 and CB2 ligands like ACEA and JWH-015 can only provide indirect evidence of the involvement of one receptor subtype over the other, a possible contribution of CB1R to the activity of AEA cannot be ruled out. In particular, JWH-015 is also a partial agonist of CB1R, although with lower affinity than for CB2R . In addition, there are reports that even AEA might not act as a physiological agonist of CB2R , although this endocannabinoid has been shown to exert manifold CB2R-dependent activities in experimental paradigms, and more recently its localization within biological membranes has led to a reconsideration of its role as a true CB2R agonist in vivo, . At any rate, it should be stressed that there are several studies documenting alterations in cytokines release induced by endocannabinoids on immune cells , , but none of them has ever been performed on primary human T-lymphocytes. The capacity of anandamide to suppress the proinflammatory response of T-cells is of pivotal importance, because not only it implies a role in inhibiting IFN-γ-mediated T-helper 1 (Th-1) responses, but it could also suggest a potential down-stream effect of this endocannabinoid also in modulating the cross-talk between T-lymphocytes and several other immune cells, including B-cells, macrophages and neutrophils. Activation of these cells is crucial in several immune-mediated diseases, and in most recent years a minor subset of T cells has gained centre stage in the study of the pathogenesis of immune disorders. These cells produce IL-17, a potent cytokine which contributes to host defense against extracellular pathogens and which has been clearly shown to be involved in the development of autoimmune diseases . Current research is investigating the possibility to interfere with the function of these cells, and the finding that a natural endogenous compound such as anandamide exerts a suppressive – but not cytotoxic – effect also on cells with a central role in the induction of autoimmunity, represents a promising beginning for a new avenue of research. It should be underlined that most immunosuppressive therapies involve the use of compounds which are cytotoxic for T-cells, thus exposing the patients to increased risk of infections. The finding that AEA preserves cell viability whilst containing the proinflammatory response represents an innovative approach in the effort to avoid autoimmune reactivity without affecting protective immune responses. On this basis, the present evidence for an immunosuppressive effect of AEA also on IL-17 production seems very timely, and is suggestive of new therapeutic approaches that could potentially target autoimmune diseases.