Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB(1) receptors.

BACKGROUND: VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using in vitro and in vivo approaches. METHODS: i) in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) in vivo: CB(1) receptor deficient mice (Cnr1(-/-)) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. RESULTS: Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB(1) receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB(1) receptor deficient mice (Cnr1(-/-)), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1(-/-) mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB(1) receptor exacerbated neuroinflammation. CONCLUSIONS: Our results suggest that CB(1) receptor dependent VCAM-1 inhibition is a novel mechanism for AEA-reduced leukocyte transmigration and contribute to a better understanding of the mechanisms underlying the beneficial role of endocannabinoid system in the Theiler's virus model of MS.

The endocannabinoid anandamide downregulates IL-23 and IL-12 subunits in a viral model of multiple sclerosis: evidence for a cross-talk between IL-12p70/IL-23 axis and IL-10 in microglial cells.

Theiler's virus (TMEV) infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases due to its anti-inflammatory properties by regulating cytokine network. IL-12p70 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of MS. In the present study we showed that the endocannabinoid anandamide (AEA) downregulated the gene expression of IL-12p70 and IL-23 forming subunits mRNAs in the spinal cord of TMEV-infected mice and ameliorated motor disturbances. This was accompanied by significant decreases on the serological levels of IL-12p70/IL-23 and more interestingly, of IL-17A. In contrast, serum levels of IL-10 resulted elevated. In addition, we studied the signalling pathways involved in the regulation of IL-12p70/IL-23 and IL-10 expression in TMEV-infected microglia and addressed the possible interactions of AEA with these pathways. AEA acted through the ERK1/2 and JNK pathways to downregulate IL-12p70 and IL-23 while upregulating IL-10. These effects were partially mediated by CB2 receptor activation. We also described an autocrine circuit of cross-talk between IL-12p70/IL-23 and IL-10, since endogenously produced IL-10 negatively regulates IL-12p70 and IL-23 cytokines in TMEV-infected microglia. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the CNS. Accordingly, pharmacological modulation of endocannabinoids might be a useful tool for treating neuroinflammatory diseases.