Anti-inflammatory effects of the MC3R agonist D-TRP8-γ-MSH in a model of experimental gouty peritonitis

1Getting SJ, 2Grieco P and 1Perretti M, 1William Harvey Research Institute, Charterhouse Square, London, UK, 2University of Naples, Naples, Italy

M elanocortin peptides exhibit potent anti-inflammatory effects via activation of members of a family of G-protein coupled receptors, termed MC-Rs. Amongst the 5 members of the family, MC1R and, from our group, MC3R expressed on the macrophage (MØ) have been proposed as functional targets. Activation of these receptors with non-selective ligands inhibit neutrophil (PMN) migration and chemokine (KC) release in C57Bl6 (WT) as well as in mice bearing a non-functional MC1R (mutant MC1R) (Getting et al., 2003). We now challenge the hypothesis centred on MC3R as a major determinant for the anti-inflammatory actions of melanocortin using the selective MC3R agonist D-TRP 8-γ-MSH (Grieco et al., 2000).

Methods. In vitro: MØ from mutant MC1R or WT mice were cultured as previously described (Getting et al., 2003) and treated with PBS or D-TRP 8-γ-MSH (0.3-30 μg/ml) alone or in the presence of an anti-MC3R neutralizing rabbit serum (1:50 to 1:200) for 30 min, cAMP accumulation was measured by EIA. MØ were also treated with D-TRP 8-γ-MSH (10 μg/ml) ± the mixed MC3/4R antagonist SHU9119 (10 μg/ml) or the selective MC4R antagonist HS024 (10 μg/ml) for 30 min prior to 1 mg/ml urate crystal addition. Supernatants were collected 2h later and analyzed for KC content by ELISA. In vivo: male WT or mutant MC1R mice (24-30g body weight, n=6 per group) were injected s.c. with D-TRP 8-γ-MSH (10 μg) or PBS, 30 min prior to urate crystal injection (3 mg in 0.5 ml sterile PBS i.p.). Some mice were pre-treated 30 min earlier with SHU9119 (10 μg) or HS024 (10 μg). In separate experiments the anti-inflammatory effect of D-TRP 8-γ-MSH was evaluated in the presence of the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX, 10µmol/kg i.p.) given 8h after urate treatment (Lam et al., 2005). Peritoneal cavities were lavaged at 6 or 24h and cell counts and KC determined as previously described (Getting et al., 2003). Values were analyzed by ANOVA and Bonferroni test (*P<0.05 taken as significant).

D-TRP 8-γ-MSH caused a concentration dependent increase in cAMP levels 10 m g/ml causing the maximal effect both in WT and mutant MC1R MØ: 74.5 ± 31% and 52.2 ± 6.9% of forskolin response, respectively. This increase was abrogated by the top concentration of anti-MC3R antibody. KC release from cultured MØ by urate crystals was attenuated by D-TRP 8-γ-MSH (42±5%, n=6; *P<0.05) in WT and 31±6%, n=6; *P<0.05 in mutant MC1R. SHU9119, but not HS024, blocked this effect in both cell genotype. In vivo, D-TRP 8-γ-MSH inhibited KC release and PMN accumulation elicited by urate crystals both in WT and mutant MC1R mice (~40-50% inhibitions; n=6; *P<0.05). Finally, treatment with the HO-1 inhibitor ZnPPIX abrogated the anti-inflammatory effects of D-TRP 8-γ-MSH: inhibition of PMN migration vanished from 50% to 0% (*P<0.05).

In conclusion, MC1R appears redundant for mediating the anti-inflammatory effects of melanocortins, whereas these new data with a selective MC3R reinforce the pivotal role played by this receptor and of its downstream effectors cAMP and HO-1.

Getting, SJ. et al., (2003) J. Immunol170, 3323-3330;
Grieco et al., (2000) J. Med. Chem.43:4998-5002;
Lam, CW. et al., (2005) J. Immunol.174, 2297-2304.

This work was supported by the Research Advisory Board , St. Bartholomew’s and the Royal London Charitable Foundation (RAB04/PJ/4)