Cannabinoids ablate release of TNFα in rat microglial cells stimulated with lypopolysaccharide

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Abstract
Upon activation, brain microglial cells release proinflammatory mediators, such as TNFα, which may play an important role in eliciting neuroinflammatory processes causing brain damage. As cannabinoids have been reported to exert anti-inflammatory and neuroprotective actions in the brain, we here examined the effect of both synthetic and endogenous cannabinoids on TNFα release elicited by bacterial endotoxin lypopolysaccharide (LPS) in cultured microglia. Exposure of primary cultures of rat cortical microglial cells to LPS significantly stimulated TNFα mRNA expression and release. The endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG), as well as the synthetic cannabinoids (+)WIN 55,212-2, CP 55,940, and HU210, inhibited in a concentration-dependent manner (1–10 μM) the LPS-induced TNFα release. Unlike the high-affinity cannabinoid receptor agonist (+)WIN 55,212-2, the low-affinity stereoisomer (−)WIN 55,212-2 did not exert any significant inhibition on TNFα release. Given this stereoselectivity, the ability of (+)WIN 55,212-2 to inhibit LPS-induced TNFα release from microglia is most likely receptor-mediated. By RT-PCR we found that the two Gi/o protein-coupled cannabinoid receptors (type 1 and 2) are both expressed in microglial cultures. However, selective antagonists of type 1 (SR141716A and AM251) and type 2 (SR144528) cannabinoid receptors did not affect the effect of (+)WIN 55,212-2. Consistent with this finding is the observation that the ablative effect of (+)WIN 55,212-2 on LPS-evoked release of TNFα was not sensitive to the Gi/o protein inactivator pertussis toxin. In addition, the cAMP elevating agents dibutyryl cAMP and forskolin both abolished LPS-induced TNFα release, thus rendering unlikely the possibility that (+)WIN 55,212-2 could ablate TNFα release through the inhibition of adenylate cyclase via the Gi-coupled cannabinoid receptors type 1 and 2. In summary, our data indicate that both synthetic and endogenous cannabinoids inhibit LPS-induced release of TNFα from microglial cells. By showing that such effect does not appear to be mediated by either CB receptor type 1 or 2, we provide evidence suggestive of the existence of yet unidentified cannabinoid receptor(s) in brain microglia. GLIA 41:161–168, 2003.