Background/Purpose: Endocannabinoids are immunomodulatory lipid compounds that act on cannabinoid receptors type 1 and 2 but also on transient receptor potential (TRP) channels. Their action is terminated by FAAH, one major enzyme responsible for endocannabinoid degradation. COX-2, however, also degrades endocannabinoids, and products from this reaction are pro-inflammatory. This study studied a potential mode of action for the anti-inflammatory effects of endocannabinoids in synovial fibroblasts from RA and OA donors. Furthermore it is investigated how pro-inflammatory cytokines alter the responsiveness of synovial fibroblasts to (endo)cannabinoid ligands.

Methods: MMP-3 and cytokines were detected by ELISA. ERK 1/2, p38, CREB and cjun phosphorylation was assessed by proteome profiler analysis and cell-based ELISA. Cannabinoid receptors 1 and 2, TRPA1, TRPV1, and COX-2 were detected by western blotting and cell-based ELISA. The XCELLigence system was used to determine EC50 values for CB₁/CB₂/TRPV1/TRPA1 with/without cytokine stimulation. 

Results: The endocannabinoid arachidonylethanolamide (anandamide, AEA) and related eicosanoids palmitoylethanolamide (PEA), oleoylethanolamide (OEA) and N-arachidonylglycine (NAGly) reduced TNF induced production of IL-6, IL-8 and MMP-3. The effects of AEA, PEA and OEA were significantly enhanced by addition of the COX-2 inhibitor nimesulide but not by FAAH inhibiton. The effects of all compounds tested were not inhibited by CB₁ or CB₂ antagonism but were blocked by TRPV1 and TRPA1 antagonists in RASFs and OASFs. In the case of AEA, COX-2 inhibition reversed the effects of TRPA1 antagonism. Quantification of CB₁, CB₂, COX-2, TRPV1 and TRPA1 revealed a significant stimulatory influence of pro-inflammatory cytokines and hypoxia on observed protein levels. Furthermore, high AEA concentrations (>1µM) induced cell death only when combined with the intracellular calcium chelating agent BAPTA-AM. Analysis of intracellular signaling pathways revealed an inhibitory effect of AEA on p38 and ERK1/2 phosphorylation after TNF stimulation.

Conclusion: Under hypoxic conditions, Endocannabinoids promote an anti-inflammatory phenotype in RASFs and OASFs by activating/desensitizing TRPV1 and TRPA1. As a consequence, MAP kinase signaling is reduced as demonstrated after AEA treatment. Furthermore, COX-2 and FAAH inhibition are necessary to fully exploit the therapeutic potential of endocannabinoids. This might be important in RA where low oxygen and abundant cytokine expression up-regulate COX-2 in the joint. In addition, pro-inflammatory cytokines increase the efficacy of endocannabinoids due to upregulation of target receptors.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anandamide-and-related-eicosanoids-decrease-the-production-of-pro-inflammatory-cytokines-in-synovial-fibroblasts-by-a-cox-2-dependent-mechanism-involvement-of-calcium-and-trp-channels/