The role of 5-HT2 receptors in inflammation

GMK Ekström, C Wenglén, A Sjödin, M Siller, AC Ryde, N Palmqvist. AnaMar AB, S-223 70, Sweden

Background. Serotonin, 5-HT (5-hydroxytryptamine) is believed to play a role in inflammatory processes by e.g., modulating cytokine expression. Targeting of the involved 5-HT receptors could offer novel opportunities for the treatment of inflammatory disorders. The aim of our study was to evaluate the role of 5-HT and the 5-HT2 receptor family in inflammation models by using commercially available receptor selective agonists and antagonists. In vitro, the modulation of IL-6 production in synoviocytes was studied, whereas in vivo, effects on the inflammatory cytokine response after systemic lipopolysaccharide (LPS)-challenge were analysed.

Methods. Primary synoviocytes were isolated from pannus tissue obtained from inflamed knee joints of antigen-induced arthritic rats (females, Dark Agouti). The culture, a mixture of fibroblasts and macrophages, was used after 7 days ex vivo. The cells were incubated with LPS (50 ng/ml) alone or in combination with 5-HT or 5-HT2 receptor agonists (1 µM), and 5-HT2 receptor antagonists (0.1-10 µM) were added as appropriate. After three days exposure, supernatants were collected, and the IL-6 content measured by ELISA. The compounds used were (agonist and antagonist): 5-HT2A: TCB-2 and 4F 4PP; 5-HT2B: α-Methyl-5-HT and RS 127445; 5-HT2C: CP 809101 and RS 102221.To evaluate the contribution of 5-HT2 receptor signalling in vivo, the following receptor antagonists were used: 5-HT2A: ketanserin; 5-HT2B: RS 127445; 5-HT2C: SB 242084. Female BALB/c mice (n=10/group) were given 10mg/kg antagonist, subcutaneously, 15 minutes before intraperitoneal injection of LPS. After further 90 minutes blood was collected and the content of TNF-α and IL-6 in serum was analysed with ELISA.

Statistical calculations were made using one-way ANOVA followed by Bonferroni’s post test.

Results. 5-HT and all three 5-HT2 receptor agonists more than doubled the production of IL-6 in LPS-stimulated pannus-derived synoviocytes. 5-HT: 145% increase (p<0.001), TBC-2: 168% (p<0.001), α-Methyl-5-HT: 149% (p<0.001), CP809101:128% (p<0.001). Further, each 5-HT2 receptor antagonist dose-dependently suppressed the IL-6 production induced by 5-HT or the corresponding 5-HT2 receptor agonist. With 5-HT stimulation: 4F 4PP, 0.1-10 µM, suppression 60-83% (p<0.001 in all concentration); RS 127445, 10 µM, suppression 35% (p<0.001); RS102221, 0.1-10 µM, suppression 23-76% (p<0.001 in all concentration). With TCB-2: 4F 4PP, 0.1-10 µM, suppression 29-82% (p<0.001 in all concentration). With α-Methyl-5-HT: RS 127445, 10 µM, suppression 23% (p<0.05). With CP 809101: RS 102221, 10 µM, suppression 52% (p<0.001).

In vivo, antagonists to the 5-HT2A, 5-HT2B and 5-HT2C receptors efficiently decreased the production of IL-6 and TNF-α induced by systemic LPS exposure in mice. IL-6: Ketanserin 78% suppression (p<0.001), RS 12744 37% (p<0.05), SB 242084 64% (p<0.01). TNF-α: Ketanserin 95% suppression (p<0.001), RS12744 76% (p<0.001), SB242084 84% (p<0.001).

Conclusion. Our results confirm that 5-HT is a pro-inflammatory mediator and demonstrate the central role of the 5-HT2 receptors in inflammation. Interestingly, all three 5-HT2 receptor subtypes seem to be of importance, but further studies employing alternative approaches are warranted. The obtained results support the development of 5-HT2 receptor antagonists for clinical use in inflammatory diseases.