The anti-inflammatory effects of melanocortin peptides in lipopolysaccharide activated chondrocytes

Vedia Can1, Magdalena Kaneva2, Mark Kerrigan3, Ian Locke1, Stephen Getting1. 1University of Westminster, School of Life Sciences, W1W 6UW, London, UK, 2William Harvey Research Institute, Department of Biochemical Pharmacology, EC1M 6BQ, London, UK, 3University of Greenwich, School of Science, ME4 4TB, Chatham, UK

Introduction

Infectious (septic) arthritis occurs when bacteria such as E.coli or other microorganisms infect the joint leading to inflammation and release of pro-inflammatory cytokines. Harnessing the body’s natural anti-inflammatory proteins to target this underlying inflammatory component may provide an effective treatment1.

Melanocortin peptides display potent anti-inflammatory effects in models of experimental inflammation1, with effects being mediated via activation of a family of G-protein coupled melanocortin receptors (MC). To date five have been identified, with MC1 and MC3 being the most promising candidates for modulation of the host inflammatory response. This study aims to determine whether melanocortin peptides inhibit pro-inflammatory cytokine release and induce anti-inflammatory pro-resolving proteins in a model of lipopolysaccharide (LPS) stimulated chondrocytes.

Methods

Human C20/A4 cell-line chondrocytes2 were plated at 1x106 cells/well in 24-well plates and stimulated with 0.1-3μg/ml of LPS (E.coli;111.60) for 6h, to determine the release of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. In separate experiments, chondrocytes were pre-treated with the pan-melanocortin agonist α-MSH (3μg/ml), the MC3 agonist D[Trp8]-γ-MSH3 (3 μg/ml) and c-terminal peptide of α-MSH KPV (4μg/ml) (all dissolved in PBS) for 30mins prior or 2h after LPS (0.1μg/ml) stimulation for 6 h. Following stimulation, cells were harvested to determine heme-oxygenase 1 (HO-1) expression by western blot. Cell-free supernatants were analysed for IL-6 and IL-8 release by ELISA. Data are expressed as Mean ± SD of n=4 determination in triplicate. *P<0.05 vs. appropriate control.

Results

LPS (0.1μg/ml) caused a maximal release of IL-6 and IL-8 with 93.6 ± 6.1 and 316.1 ± 2.1pg/ml respectively (P<0.05 vs. control). Higher concentrations of LPS caused a reduction in release of these cytokines at this time-point. Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused a significant reduction in IL-6 and IL-8 release following LPS stimulation (0.1μg/ml) with α-MSH causing a 30% and 49% reduction in IL-6 and IL-8 with 65.6 ± 6.9 and 160.3 ± 19.2pg/ml respectively (P<0.05). Whilst the selective MC3 agonist D[Trp8]-γ-MSH causd a 60% and 29% reduction in IL-6 and IL-8 with 37.8 ± 3.5 and 226.4 ± 8.4pg/ml respectively (P<0.05 vs. control), the peptide KPV failed to inhibit either IL-6 or IL-8.

Pre-treatment of C-20/A4 chondrocytes with melanocortin peptides inhibited LPS induced cytokine release. Next, we investigated the effect of therapeutic peptide treatment on IL-8 release with the melanocortin peptides being administered 2h after LPS stimulation. α-MSH and D[Trp8]-γ-MSH causing a 23% and 30% reduction in IL-8 release down to 245.0 ± 16.8 and 220.9 ±13.8pg/ml; P≤0.05, respectively.

LPS caused a 21% (0.79 fold) reduction in HO-1 protein expression compared to control, whilst pre-treatment of cells with α-MSH, D[Trp8]-γ-MSH and KPV caused a significant increase in HO-1 expression with a 1.22,1.59 and 1.2 fold increase respectively.

Conclusion

These results suggest a role for melanocortin peptides at inhibiting pro-inflammatory cytokine release whilst inducing pro-resolving anti-inflammatory proteins following LPS stimulation of human C-20/A4 chondrocytes. Overall suggesting a potential therapeutic application of these peptides in arthritic pathologies.

[1] Getting SJ, Pharmacol Ther 111:1-15,2006

[2] Kaneva M, et al.,Br J. Pharmacol. 167(1):67-79,2012

[3]Getting SJ, et al.,FASEB J 20:2234-41,2006