The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated in 1995 from rat brain and canine gut as an endogenous ligand for the cannabinoid receptors. 2-AG is rapidly formed from arachidonic acid-containing phospholipids through increased phospholipid metabolism, such as enhanced inositol phospholipid turnover, in various tissues and cells upon stimulation. 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Notably, anandamide, another endogenous ligand for the cannabinoid receptors, often acts as a partial agonist at these cannabinoid receptors, whereas 2-AG acts as a full agonist in most cases. The results of structure-activity relationship studies suggested that 2-AG rather than anandamide is the true natural ligand for both the CB1 and the CB2 receptors. Evidence is gradually accumulating which shows that 2-AG plays physiologically essential roles in diverse biological systems. For example, several lines of evidence indicate that 2-AG plays an important role as a retrograde messenger molecule in the regulation of synaptic transmission. 2-AG has also been shown to be involved in the regulation of various types of inflammatory reactions and immune responses. In this review, we focused on 2-AG, and summarized information concerning its biosynthesis, metabolism, bioactions and physiological significance, including our latest experimental results.     

Activation by 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, of p42/44 mitogen-activated protein kinase in HL-60 cells

2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.     

The 2-arachidonoylglycerol effect on myosin light chain phosphorylation in human platelets

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B₂ formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.     

2-Arachidonoyl-sn-glycero-3-phosphate,an arachidonic acid-containing lysophosphatidic acid: occurrence and rapid enzymatic conversion to 2-arachidonoyl-sn-glycerol,a cannabinoid receptor ligand,in rat brain

A substantial amount of lysophosphatidic acid (LPA) (15.66 nmol/g tissue) was found to occur in the brain isolated from rats killed in liquid nitrogen. We found that a significant portion of brain LPA was accounted for by the arachidonic acid-containing species (5.4%). We obtained evidence that both 2-arachidonoyl species and 1-arachidonoyl species of LPA are present. The occurrence of 2-arachidonoyl LPA in the brain (0.53 nmol/g tissue) is a notable observation, because of its structural resemblance to 2-arachidonoyl-sn-glycerol (2-AG), an endogenous cannabinoid receptor ligand. We then examined the biological activity of 2-arachidonoyl LPA and compared it with that of 2-AG using neuroblastoma x glioma hybrid NG108-15 cells which express both the LPA receptor and cannabinoid CB1 receptor. We found that 2-arachidonoyl LPA interacts with the LPA receptor(s) to elicit the elevation of intracellular free Ca(2+) concentrations, whereas 2-AG interacts exclusively with the cannabinoid CB1 receptor. Next, we examined the possible metabolic relationship between 2-arachidonoyl LPA and 2-AG and obtained clear evidence that rapid enzymatic conversion of 2-arachidonoyl LPA to 2-AG took place in the brain homogenate. It is noteworthy that two types of endogenous ligands, that interact with different types of receptors, are closely related metabolically and rapidly interconvert.     

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

Alpha-methylated derivatives of 2-arachidonoyl glycerol: synthesis, CB1 receptor activity, and enzymatic stability

Alpha-methylated analogues of the endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), were synthesized aiming to the improved enzymatic stability of 2-AG. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The CB1 receptor activity was determined by the [35S]GTPgammaS binding assay, and the enzymatic stability of alpha-methylated analogues was determined in rat cerebellar membranes. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are clearly more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.     

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.     

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.     

Cannabimimetic fatty acid derivatives in cancer and inflammation

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or 2-AG can be induced by stimulation with either Ca(2+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-2H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-2H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(2) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and 2-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.     

Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Selective CB2 receptor activation ameliorates EAE by reducing Th17 differentiation and immune cell accumulation in the CNS

CB2, the cannabinoid receptor expressed primarily on hematopoietic cells and activated microglia, mediates the immunoregulatory functions of cannabinoids. The involvement of CB2 in EAE has been demonstrated by using both endogenous and exogenous ligands. We showed previously that CB2 selective agonists inhibit leukocyte rolling and adhesion to CNS microvasculature and ameliorate clinical symptom in both chronic and remitting-relapsing EAE models. Here we showed that Gp1a, a highly selective CB2 agonist, with a four log higher affinity for CB2 than CB1, reduced clinical scores and facilitated recovery in EAE in conjunction with long term reduction in demyelination and axonal loss. We also established that Gp1a affected EAE through at least two different mechanisms, i.e. an early effect on Th1/Th17 differentiation in peripheral immune organs, and a later effect on the accumulation of pathogenic immune cells in the CNS, associated with reductions in the expression of CNS and T cell chemokine receptors, chemokines and adhesion molecules. This is the first report on the in vivo CB2-mediated Gp1a inhibition of Th17/Th1 differentiation. We also confirmed the Gp1a-induced inhibition of Th17/Th1 differentiation in vitro, both in non-polarizing and polarizing conditions. The CB2-induced inhibition of Th17 differentiation is highly relevant in view of recent studies emphasizing the importance of pathogenic self-reactive Th17 cells in EAE/MS. In addition, the combined effect on Th17 differentiation and immune cell accumulation into the CNS, emphasize the relevance of CB2 selective ligands as potential therapeutic agents in neuroinflammation.