Background/Purpose: The endocannabinoid system (ECS) is comprised of two evolutionary conserved cannabinoid receptors 1 and 2 (CB1 and CB2) which participate in pain management through G_αi/o mediated signaling. Recent studies show that the expression of CB2, not CB1, is upregulated at both the mRNA and protein levels in rheumatoid arthritis synovial fibroblasts (RASFs) as compared to normal synovial fibroblasts, however, the role the ECS receptors in pro-inflammatory cytokine signaling in RASFs remains poorly understood. In the present study, we used a selective agonist of CB2 (JWH-133) to characterize the role of CB2 activation in interleukin-1β (IL-1β)-induced inflammation in human RASFs.

Methods: Human RASFs were obtained from patients diagnosed with RA according to the ACR guidelines (8 female, 2 male, average age 47.7 ± 5.7 years). Human RASFs were pre-treated with JWH-133 (10-20 µM) for 10 minutes prior to the addition of IL-1β (10 ng/mL) stimulation for 30 minutes for signaling studies or for 24 hours to evaluate the inflammatory mediators. The role of CB2 in IL-1β signaling was examined by employing a small interfering RNA (siRNA) method or using overexpression plasmid specific for CB2. Conditioned media was used for the quantification of IL-6 and IL-8 by ELISA and cell lysates were prepared for the analysis of IL-1β signaling proteins and nuclear translocation of transcription factors NF-κBp65 or AP-1 using Western immunoblotting. To study protein-protein interactions, RASFs treated with JWH-133 or IL-1β were incubated with TAK-TAB_V5 protein. Associated partners were pulled down by immunoprecipitation of V5 beads and analyzed using a Western blotting method.

Results: Pretreatment of JWH-133 exacerbated IL-1β-induced IL-6 and IL-8 production by 21% and 12%, respectively, in human RASFs (p<0.05; n=3). Furthermore, we observed that JWH-133 selectively increased the expression of IL-1β-induced Cox-2 by >2-fold (p<0.05; n=3), suggesting a proinflammatory role of CB2 in IL-1β signaling in human RASFs. Knockdown of CB2 using siRNA inhibited IL-1β-induced IL-6 and IL-8 production by more than 50% and completely abrogated Cox-2 expression in RASFs (p<0.05; n=3). In contrast, the overexpression of CB2 further increased IL-1β-induced IL-6 and IL-8 by approximately 3-fold and 4-fold, respectively (p<0.05; n=3), confirming its proinflammatory role in RASF mediated pain and inflammation. Analysis of immunoprecipitation studies revealed that CB2 associates with active TAK1 upon JWH-133 stimulation to coordinate with IL-1β signaling pathways as observed with an increased nuclear translocation of NF-κBp65 and AP-1.

Conclusion: Our study provides novel evidence that CB2 elicits proinflammatory role in IL-1β signaling in RASFs and suggests CB2 as a potential therapeutic target in the management of pain and inflammation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-the-role-of-endocannabinoid-system-using-jwh-133-a-selective-cannabinoid-cb2-receptor-agonist-in-il-1%ce%b2-induced-inflammation-in-human-rheumatoid-arthritis-synovial-fibroblast/