A role for melanocortin peptides in modulating oxidative stress induced inflammation in A549 cells

Stephen Getting, Magdalena Kaneva, Mark Kerrigan

University of Westminster, London, UK

Melanocortin peptides exert their anti-inflammatory effects via activation of a subgroup of G-protein coupled receptors1. To date five melanocortin receptors (MCR) have been identified with the MC1 and MC3R being proposed to bring about the anti-inflammatory effects of melanocortins2. Here we have used an in vitro model of oxidative stress to determine whether melanocortin peptides can switch off inflammatory cytokine release from activated epithelial cells.

RT-PCR and western blotting were used to determine MC1 and 3R expression in A549 cells. Cells were plated at 1 x106/well in 24 well plates and pretreated with 1-30 μg/ml of the pan melanocortin agonist α-MSH and the selective MC3R agonist dTrp8-γ-MSH1 for 30 mins prior to determination of cAMP accumulation by EIA. In separate experiments cells were pretreated with 1-30 μg/ml of α-MSH and dTrp8-γ-MSH for 30 mins prior to stimulation with either PBS or H2O2 (20-200μM) for 4h. Cell free supernatants were collected and nitric oxide (NO) levels assessed by nitrite quantification by the Greiss method and the cytokines IL-8 and MCP-1 levels determined in cell-free supernatants by commercially available ELISA. Data is expressed as Mean ± SD of n=4 determination in triplicate. *P<0.05 vs. appropriate control.

RT-PCR and western blotting showed the presence of mRNA and protein for MC1 and MC3R in A549 cells. Functionality of the receptors was determined by stimulating the cells with α-MSH and dTrp8-γ-MSH, resulting in a concentration dependent increase in cAMP accumulation, with a maximal accumulation of 330 ± 20 and 180 ± 10 fmol/well at 10 μg/ml for α-MSH and dTrp8-γ-MSH respectively (n=4, *P<0.05). We next determined whether α-MSH and dTrp8-γ-MSH could modulate H2O2 stimulated release of nitrite, IL-8 and MCP-1. Stimulation of cells with H2O2 resulted in a maximal release of IL-8 (610 ± 50 pg/ml, *P<0.05 vs. control, n=4) and MCP-1 (1435 ± 53 pg/ml, * P<0.05 vs. control, n=4) at 200 μM. Pre-treatment of cells with 10 μg/ml α-MSH and dTrp8-γ-MSH prior to H2O2 stimulation resulted in significant reductions in all parameters measured with an 85% and 82% reduction in IL-8 and MCP-1 by α-MSH, whilst the selective MC3R agonist dTrp8-γ-MSH caused a significant 88% and 71% (*P<0.05 vs. control n=4) reduction in both IL-8 and MCP-1 respectively.

Collectively these data have identified functionally active MC1 and MC3R on the human epithelial cell line A549. Hydrogen peroxide induced nitrite; IL-8 and MCP-1 were abrogated in the presence of α-MSH and dTrp8-γ-MSH. Therefore, therapeutic targeting of MCR may be beneficial in the treatment of inflammatory lung disease, such as in asthma and COPD.

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

Getting SJ, et al., Pharmacol Ther 111: 1-15, 2006.