Abstract | Introduction: Infectious arthritis occurs when E.coli or other microorganisms lead to joint infection and increases in pro-inflammatory cytokines. Investigating how the body naturally produced anti-inflammatory proteins inhibit these pathways, may lead to the development of new therapies for these pathologies (1). The melanocortin agonists α-MSH and D[Trp8]-γ-MSH (2) display an important role in inhibiting inflammatory mediators and inducing pro-resolving anti-inflammatory pathways, in models of acute and chronic inflammation (1). The anti-inflammatory effects of these peptides are via a family of G-protein coupled receptors, of which five melanocortin receptors (MC) have been identified, with both MC1 and MC3 being promising candidates for modulation of the host inflammatory response in arthritic pathologies (3). This study aims to determine whether the MC3/4 antagonist SHU9119 modulates the anti-inflammatory, pro-resolving effects of α-MSH and D[Trp8]-γ-MSH in lipopolysaccharide (LPS) stimulated chondrocytes. Methods: Human C20/A4 cell-line chondrocytes were plated at 1x106 cells/well in 24-well plates and were pre-treated with the pan-melanocortin agonist α-MSH (3µg/ml) (Sigma–Aldrich Inc. Poole, UK), and the MC3 agonist D[Trp8]-γ-MSH (3 µg/ml) (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30mins prior to 0.1µg/ml of LPS (E.coli;111.60) (Sigma–Aldrich Inc. Poole, UK) stimulation for 6h. In separate experiments the cells were pre-treated with the MC3/4 antagonist SHU9119 (10µg/ml) (Bachem, Bubendorf, Switzerland) 1h prior to LPS stimulation (Time 0h). Cells were harvested to determine the anti-inflammatory protein heme-oxygenase 1 (HO-1) expression by western blot, whilst cell-free supernatants were collected and analysed for IL-6 and IL-8 release by ELISA. Data are expressed as Mean ± SD of n=4 determination in quadruplicate. *P<0.05 vs. control. Results: Cytokine analysis: LPS 0.1ug/ml caused a maximal release of IL-6 and IL-8 with 112.3±6.1 and 314.7±1.9 pg/ml respectively (P<0.05 vs. control). Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused a significant reduction in IL-6 (61% and 70% respectively) and IL-8 (71% and 59% respectively) following LPS stimulation. The antagonist SHU9119 completely attenuated the effect of α-MSH on IL-6 and IL-8 release, whilst significantly reducing the effect of D[Trp8]-γ-MSH for both cytokines. HO-1 expression: LPS caused a 30% (0.70 fold) reduction in HO-1 protein expression compared to control, whilst pre-treatment of cells with α-MSH, and D[Trp8]-γ-MSH caused a significant increase in HO-1 expression with a 1.25 and 1.57 fold increase respectively. Pre-treatment with the antagonist SHU9119 inhibited both α-MSH and D[Trp8]-γ-MSH induced HO-1 expression. Conclusion: In conclusion α-MSH and D[Trp8]-γ-MSH significantly inhibit proinflammatory cytokine release whilst inducing the anti-inflammatory protein HO-1. These effects are attenuated in the presence of the MC3/4 antagonist SHU9119, thus suggesting a potential role for the MC3 receptor in mediating their anti-inflammatory effects in this model. |
---|