CD200-CD200R1 interaction contributes to neuroprotective effects of anandamide on experimentally induced inflammation.

The endocannabinoid anandamide (AEA) is released by macrophages and microglia on pathological neuroinflammatory conditions such as multiple sclerosis (MS). CD200 is a membrane glycoprotein expressed in neurons that suppresses immune activity via its receptor (CD200R) mainly located in macrophages/microglia. CD200-CD200R interactions contribute to the brain immune privileged status. In this study, we show that AEA protects neurons from microglia-induced neurotoxicity via CD200-CD200R interaction. AEA increases the expression of CD200R1 in LPS/IFN-γ activated microglia through the activation of CB(2) receptors. The neuroprotective effect of AEA disappears when microglial cells derive from CD200R1(-/-) mice. We also show that engagement of CD200R1 by CD200Fc decreased the production of the proinflammatory cytokines IL-1ß and IL-6, but increased IL-10 in activated microglia. In the chronic phases of Theiler's virus-induced demyelinating disease (TMEV-IDD) the expression of CD200 and CD200R1 was reduced in the spinal cord. AEA-treated animals up-regulated the expression of CD200 and CD200R1, restoring levels found in sham animals together with increased expression of IL-10 and reduced expression of IL-1ß and IL-6. Treated animals also improved their motor behavior. Because AEA up-regulated the expression of CD200R1 in microglia, but failed to enhance CD200 in neurons we suggest that AEA-induced up-regulation of CD200 in TMEV-IDD is likely due to IL-10 as this cytokine increases CD200 in neurons. Our findings provide a new mechanism of action of AEA to limit immune response in the inflamed brain.

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.

Understanding microglia-neuron cross talk: relevance of the microglia-neuron cocultures.

Microglia-neuron interaction is a complex process involving a plethora of ligands and receptors. The outcome of this intricate process will depend on the prevailing signals (i.e., whether the microglial cells will produce pro-inflammatory cytokines and/or phagocyte a dying neuron or whether it will produce neurotrophic factors and support neuronal growth, among other possible scenarios). In order to study this complex process, several tools have been developed, ranging from in vivo models (knockout and knock-in mice, conditional transgenic mice, imaging techniques) to in vitro models (microglia-neuron cocultures, transwell cell cultures). Here we describe a protocol for primary microglia-neuron coculture. this coculture allows to combine neurons and microglial cells coming from wild-type and KO mice, making this coculture a useful method to study in vitro the interaction of different sets of ligand-receptor.

The endocannabinoid anandamide from immunomodulation to neuroprotection. Implications for multiple sclerosis.

Over the last decade, the endocannabinoid system (ECS) has emerged as a potential target for multiple sclerosis (MS) management. A growing amount of evidence suggests that cannabinoids may be neuroprotective during CNS inflammation. Advances in the understanding of the physiology and pharmacology of the ECS have potentiated the interest of several components of this system as useful biological targets for disease management. Alterations of the ECS have been recently implicated in a number of neuroinflammatory and neurodegenerative conditions, so that the pharmacological modulation of cannabinoid (CB) receptors and/or of the enzymes controlling synthesis, transport, and degradation of these lipid mediators is considered an option to treat several neurological diseases. This chapter focuses on our current understanding of the function of anandamide (AEA), its biological and therapeutic implications, as well as a description of its effects on neuroimmune modulation.