Redundancy of a Functional Melanocortin 1 Receptor in the Anti-inflammatory Actions of Melanocortin Peptides: Studies in the Recessive Yellow (e/e) Mouse Suggest an Important Role for Melanocortin 3 Receptor¹

Melanocortin peptides, (e.g., α-melanocyte stimulating hormone (α-MSH)³) are derived from a larger precursor called the pro-opiomelanocortin gene product and are characterized by a common amino acid motif (HFRW). These endogenous peptides have long been reported to possess anti-inflammatory effects in many experimental models of acute and chronic inflammation, including experimental bowel disease, allergy, and chronic (mycobacterium-induced arthritis) and systemic inflammation (endotoxemia) (1, 2). Melanocortins act at a subgroup of G-protein coupled receptor, termed melanocortin receptors (MC-R), of which five members have been identified so far. MC-Rs have a wide and varied distribution throughout the body (3). All MC-Rs are positively coupled to adenylate cyclase, and agonism at these receptors leads to increases in intracellular cAMP (3). Some specific actions have been attributed to specific members of the receptor family; examples being MC1-R mediated skin pigmentation, MC4-R control of obesity and MC2-R stimulation of adrenal steroidogenesis (3). It remains still unclear if the anti-inflammatory actions of melanocortin peptides are mediated by a single MC-R.

The MC1-R has long been regarded as the receptor responsible for the anti-inflammatory effects of α-MSH and related peptides (3). MC1-R mRNA, but not protein, expression has been found on monocytes, B lymphocytes, NK cells, a subset of cytoxic T cells (4), dendritic cells (5), and more recently mast cells (6). MC1-R-mediated anti-inflammatory effects appear to occur via inhibition of NF-κB activation (7, 8) and protection of IκBα degradation (9). These intracellular events would produce a reduction in the expression of proinflammatory cytokines (10) and adhesion molecules (8), thereby affecting the humoral and cellular phases of inflammation (11, 12).

Recently, our own studies have identified a putative role for MC3-R in modulating experimental inflammation (13). Selective agonists at the MC3-R (the natural γ₂-MSH) (14) and the synthetic peptide MTII (15)) displayed inhibitory activity in a murine model of monosodium urate (MSU) crystal-induced peritonitis (16). The in vivo data have been supported by the detection of both MC3-R mRNA and protein on mouse and rat peritoneal macrophages (Mφ) as well as on rat knee joint Mφ (13, 16, 17). In addition, MC3-R activation on Mφ caused cAMP accumulation and inhibition of cytokine release (13, 16, 17).

This study was planned to address the apparent dichotomy between MC1-R and MC3-R in mediating the anti-inflammatory actions of melanocortins. We have taken advantage of the recent characterization of more selective MC-R agonists, and from the availability of the recessive yellow (e/e) mouse strain. A frameshift mutation in the MC1-R gene in these animals results in a single deletion of a nucleotide at position 549; the outcome is a receptor protein with a premature termination in the fourth trans-membrane domain, thus unable to couple to adenylate cyclase and activate cAMP synthesis (18, 19). Interestingly, these mice have altered pigmentation (yellow mice) without clear defects in the immune system (18, 19), thus resembling the lack of reported phenotype associated with red hair as studied in humans (20). The results produced here with an experimental model of peritonitis show that a functional MC1-R is not necessary to elicit the anti-inflammatory actions of melanocortin peptides.

Male C57BL.6 mice (20–22 g body weight) were purchased from Tuck (Battlesbridge, Essex, U.K.) and recessive yellow (e/e) mice (18) were a kind gift from Dr. N. Levin (Trega Bioscience, San Diego, CA). Mice were maintained on a standard chow pellet diet with tap water ad libitum using a 12 h light/dark cycle. Animal experimental work was performed according to Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).

An enriched population of peritoneal Mφ (>95% pure) was prepared by 2 h adherence at 37°C in 5% CO₂/95% O₂ atmosphere in RPMI 1640 supplemented with 10% FCS, by culturing 5 × 10⁶ Mφ in 24-well plates. Nonadherent cells were washed off using warm medium, and adherent cells (>95% Mφ) were then incubated with γ₂-MSH (95 μM) alone or in combination with SHU9119 (9 μM) for 15 min in RPMI 1640 medium. Cells were then stimulated with 1 mg/ml MSU crystals (a concentration chosen from previous experiments (13)), and cell-free supernatants were collected 2 h later. KC and IL-1β levels were measured by ELISA as described below.

Mφs (1 × 10⁵) were allowed to adhere in 24-well plates as above, and incubated with serum-free RPMI 1640 medium containing 1 mM isobutylmethylxanthine with γ₂-MSH (30 μg/ml equivalent to 95 μM), MTII (10 μg/ml equivalent to 9.3 μM), MS05 (30 μg/ml equivalent to 22 μM), or the direct adenylate cyclase activator forskolin (3 μM). In some experiments the effect of these peptides in the presence of 9 μM of the MC3/4-R antagonist SHU9119 or the selective MC4-R antagonist HS024 were investigated. After 30 min at 37°C, supernatants were removed and cells were washed and lysed. cAMP levels in cell lysates were determined with a commercially available enzyme immunoassay (Amersham Life Sciences, Little Chalfont, U.K.) using a standard curve constructed with 0–3,200 fmol/ml cAMP.

Peritoneal Mφ (5 × 10⁶) enriched by 2 h adherence at 37°C in 24-well plates and lysed in 1 ml of Trizol reagent (lysis buffer for RNA preparation) from Life Technologies (Paisley, U.K.) and RNA was isolated according to manufacturer's protocol. RNA was extracted with chloroform and isopropanol, precipitated with ethanol, and the pellet was resuspended in diethyl pyrocarbonate-treated water. The yield and purity of the RNA was then estimated spectrophotometrically at 260 and 280 nm. Total RNA (3 μg) was used for the generation of cDNA. PCR amplification reactions were then performed on aliquots of the cDNA. All PCR were performed using PCR beads (Pharmacia Biosystems Europe, St Albans, U.K.) in a final volume of 25 μl using a Hybaid OmniGene thermal cycler (Middlesex, U.K.). The murine MC3-R primer sequences were used as previously described (13): MC3-R, 5′-GCC TGT CTT CTG TTT CTC CG-3′ and 5′-GCC GTG TAG CAG ATG CAG TA-3′ (forward and reverse) which amplified a fragment of 820 bp in length. The cycling parameters were as follows: after an initial denaturation for 3 min at 94°C, 30 cycles of annealing at 60°C (30 s), extension at 72°C (1 min), and denaturation at 94°C (45 s), and a final further extension of 72°C for 10 min. Amplification products were visualized by ethidium bromide fluorescence in agarose gels. Images were inverted using the Graphic Converter software (version 2.1) running on a Macintosh Performa 6200 (Reine, Germany).

We used a protocol recently validated for rat and mouse peritoneal Mφ (16). Protein was isolated from samples of wild-type and recessive yellow (e/e) mice peritoneal Mφ and spleens in PBS containing EDTA (3 mM), leupeptin (0.39 mg/ml) and PMSF (10 mM). Protein levels were then determined (Bio-Rad protein assay; Bio-Rad, Hercules, CA) and 50 μg of protein was mixed with 0.125 M Tris-HCl (pH 6.8), 2 mM EDTA, 4% sodium dodecyl sulfate (SDS), 10% mercaptoethanol, 20% glycerol and boiled for 10 min before loading and running on an 10% polyacrylamide gel (Protogel; National Diagnostics, Ashby De La Zouche, Leicestershire, U.K.) for 60 min at 100 V. Protein was transferred onto polyvinylidine difluoride membranes (Amersham Life Sciences) by semidry blotting (Bio-Rad) for 60 min using a Tris/glycine buffer containing 20% methanol. Membranes were then blocked overnight at 4°C by immersion in a 5% nonfat dried milk solution made up in PBS containing 0.1% Tween 20. Membranes were then incubated for 2 h at 4°C in a 5% nonfat dried milk solution with an affinity-purified goat polyclonal Ab (1/200 final dilution; Autogen Bioclear, Mile Elm Calne, U.K.) raised against a peptide mapping human MC3-R. This goat polyclonal MC3-R Ab showed cross-reactivity with mouse and rat, but did not cross-react with MC1-R, MC2-R, MC4-R, and MC5-R of any species (data supplied by the manufacturer). Following one 15 min and three 5 min washes in PBS and Tween 20 (0.1%), the membrane was incubated for 1 h with an HRP-conjugated donkey anti-goat IgG secondary Ab (1/5000 in 0.1% BSA in PBS and 0.1% Tween). After another 15 min and three 5 min washes in PBS/Tween), blots were incubated with ECL solution (Amersham Life Sciences) for 1 min and then exposed to autoradiographic film for detection of chemiluminescence. Cruz m.w. markers were also used (Autogen Bioclear).

We used a protocol recently validated for rat knee joint Mφ (17). Peritoneal Mφ from wild-type and recessive yellow mice (e/e) were lavaged as described above, and cells were fixed with a mixture of freshly prepared 3% (w/v) paraformaldehyde and 0.05% (v/v) glutaraldehyde in PBS, pH 7.2, for 4 h at 4°C, washed briefly in PBS, and transferred to a solution of 2.3 M sucrose (in PBS) at 4°C overnight. The cryoprotected cells were slam-frozen (Reichert MM80E; Leica, Milton Keynes, U.K.), freeze-substituted at −80°C in methanol for 48 h, and embedded at −20°C in LRGold acrylic resin (London Resin Company, Reading, U.K.) in a Reichert freeze-substitution system. Ultrathin sections (50–80 nm) were prepared by use of a Reichert Ultracut-S ultratome and incubated at room temperature for 2 h with a polyclonal goat anti-MC3-R Ab (dilution 1/200; Autogen Bioclear) or goat anti-MC3-R preabsorbed with MC3-R blocking peptide (20 μg/ml; Autogen Bioclear) followed by protein A linked to 15 nm gold (British Biocell, Cardiff, U.K.) for 1 h. As additional negative control sections were incubated with nonimmuno goat serum (1/200, Sigma-Aldrich, Poole, Dorset, U.K.) in place of the primary Ab. The serum and antiserum were diluted in 0.1 M phosphate buffer containing 0.1% egg albumin. After immuno-labeling sections were lightly counterstained with uranyl acetate and lead citrate and examined with a JEOL 1010 transmission electron microscope (JEOL, Peabody, MA).

PMN recruitment into the peritoneal cavity was elicited by MSU crystals as recently reported (21). Mice were treated i.p. with 3 mg of MSU crystals in 0.5 ml PBS, and peritoneal cavities lavaged at 2–96 h post challenge with 3 ml PBS containing EDTA (3 mM) and heparin (25 U/ml). Aliquots of the lavage fluids were then stained with Turk's solution (0.01% crystal violet in 3% acetic acid), and differential cell counts were performed by light microscopy using a Neubauer hematocytometer. Data are reported as 10⁶ PMN per mouse. Lavage fluids were then centrifuged at 400 × g for 10 min and supernatants were stored at −20°C before biochemical determinations (see below).

The natural hormone α-MSH (10 μg per mouse equivalent to 6 nmol), the relatively selective MC3-R agonist γ₂-MSH (YVMGHFRWDRFG) (30 μg per mouse equivalent to 95 nmol) (14), the mixed MC3/4-R agonist MTII (Ac-Nle-cyclo[-Asp-His-d-Phe-Arg-Trp-Lys-NH₂) (10 μg per mouse equivalent to 9.3 nmol) (15), the pan-MC-R agonist HP228 (Ac-Nle-Gln-His-d-Phe-Arg-d-Trp-Gly-NH₂; 15 μg per mouse equivalent to 15.2 nmol) (22), and the selective MC1-R agonist MS05 (H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂; 1–100 μg per mouse equivalent to 0.66–66 nmol) (23) were administered s.c. 30 min before MSU crystals. In some experiments, agonist effect was tested in the presence of the MC3/4-R antagonist SHU9119 (Ac-Nle-cyclo[-Asp-His-d-2-Nal-Arg-Trp-Lys-NH₂) or selective MC4-R antagonist HS024 (Ac-Cys-Nle-Arg-d-2-Nal-Arg-Trp-Lys-Cys-NH₂) (16) of which 9 nmol were given i.p. 30 min before MSU crystals. MTII, α-MSH, γ₂-MSH, HP228, SHU9119, and HS024 were purchased from Bachem (Saffron Walden, Essex, U.K.), stored at −20°C before use, and dissolved in sterile PBS (pH 7.4). MS05 was kindly provided by Melacure Therapeutics (Uppsala, Sweden).

Murine KC and IL-1β levels in peritoneal lavage fluids were determined using commercially available ELISA purchased from R&D Systems (Abingdon, U.K.). In brief, lavage fluids (50 μl) were assayed for each cytokine and compared with a standard curve constructed with 0–1 ng/ml of the standard cytokine. The ELISAs showed negligible (<1%) cross-reactivity with several murine cytokines and chemokines (data as furnished by manufacturer).

Data are reported as mean ± SE of n distinct observations. Statistical differences were calculated on original data by ANOVA followed by Bonferroni test for intergroup comparisons (24), or by unpaired Student's t test (two-tailed) when only two groups were compared. A threshold value of p < 0.05 was taken as significant.

RT-PCR, Western blotting, and electron microscopy analyses were used to monitor MC-R expression in resident peritoneal Mφ and as a positive control spleen (Western blotting only) from both wild-type and recessive yellow (e/e) mice. RNA extracted from murine peritoneal Mφ showed the presence of MC3-R by RT-PCR in either mouse strain (Fig. 1,a). To determine whether this message was translated to protein we used Western blotting and electron microscopy. Expression of MC3-R in wild-type and recessive yellow (e/e) mice peritoneal Mφ and spleen was monitored by Western blotting analysis. Western blotting confirmed the presence of the MC3-R in protein extracts prepared from mouse peritoneal Mφ and spleen, with a band of the right m.w. (43 kDa) being obtained (Fig. 1,b). Electron microscopy was then used to highlight MC3-R gold immunolabeling which was predominantly located on the plasma membrane with a particular higher density in correspondence of membrane protrusions in wild-type (Fig. 1,c) and recessive yellow (e/e) mice (Fig. 1,d). This immunostaining was specific for MC3-R insofar as it was absent when the primary Ab was preabsorbed with the blocking peptide in wild-type (Fig. 1,e) or recessive yellow (e/e) mice (Fig. 1,f). As a final control, cells stained with nonimmune goat IgG did not display immuno-gold labeling on their plasma membrane in wild-type (Fig. 1,g) or recessive yellow (e/e) mice (Fig. 1 h).

Receptor functionality was determined quantifying cAMP accumulation in peritoneal Mφ. In C57 wild-type mice the natural and synthetic MC3-R agonists, γ₂-MSH and MTII, caused significant increases in cAMP accumulation with a 450% and 420% increase above basal levels (73 ± 12 fmol/well). A similar observation was noted with respect to the recessive yellow (e/e) mice in which a 436% and 400% increase above basal levels (80 ± 11 fmol/well) was measured for γ₂-MSH and MTII, respectively (Fig. 2,a). These increases in both wild-type and recessive yellow (e/e) mice were blocked in the presence of the MC3/4-R antagonist SHU9119 (Fig. 2,b) but not the selective MC4-R antagonist HS024 (Fig. 2,c). The effect of forskolin was retained in the presence of either antagonist. The selective MC1-R agonist MS05 failed to elicit any increase in cAMP in either type of Mφ (Fig. 2, a–c).

Intraperitoneal injection of MSU crystals produced a time dependent accumulation of PMN with a similar profile in wild-type and recessive yellow (e/e) mice. MSU crystal-induced PMN accumulation was maximal at 6 h postinjection (with an approximate influx rate of 1.1 × 10⁶ PMN per hour in the 2- to 6-h time interval) (Fig. 3,a). PMN recruitment plateaued between 6 and 24 h, remaining at the same level at the later time point in wild-type mice (>6 × 10⁶ PMN per mouse). There appears to be no difference in the cell emigration at 6 and 24 h in recessive yellow (e/e) mice compared with wild type following injection of urate crystals into the peritoneal cavity. Very few resident lymphocytes or Mφ could be detected in the lavage fluids following challenge (data not shown). The maximal PMN accumulation occurred at the 6-h time point and was preceded by a marked release of the proinflammatory CXC chemokine KC, maximal at 2 h (Fig. 3 b). Again no difference in the release of KC was measured between wild-type and recessive yellow (e/e) mice.

Nonselective MC-R agonists, the synthetic peptide HP228, and the naturally occurring hormone, α-MSH, modulated the inflammatory response in wild-type mice, with inhibitions in PMN migration of 35% and 47%, for HP228 and α-MSH, respectively (Fig. 4,a). The peptides were then administered to recessive yellow (e/e) mice and similar inhibitory responses (∼50%) were observed (Fig. 4,a). HP228 and α-MSH caused a reduction in IL-1β and KC levels measured in peritoneal lavages collected from both wild-type and recessive yellow (e/e) mice (Fig. 4, b and c). In the absence of MSU crystal injection, the number of PMN and levels of IL-1β and KC were below the detection limits in either mouse strain (data not shown).

Next we evaluated the effect of more selective melanocortin peptides in this model of peritonitis. The natural (γ₂-MSH, 30 μg/mouse) and synthetic (MTII, 10 μg/mouse) MC3-R agonists inhibited both the cellular and humoral response produced by urate crystals. Fig. 5,a shows the data for C57 wild-type mice, in which MTII and γ₂-MSH significantly (p < 0.05) inhibited MSU crystal-induced PMN recruitment by 54% and 45%, respectively. Similar degrees of inhibition were observed in the recessive yellow (e/e) mice with MTII and γ₂-MSH causing a 63% and 42% reduction in PMN migration (p < 0.05). The selective synthetic MC1-R agonist MS05 (1–100 μg) did not modify the inflammatory response in either wild-type or recessive yellow (e/e) mice (Fig. 5 a).

In view of our previous studies with intact mice (16), exudate IL-1β levels were measured in recessive yellow (e/e) mice. Treatment of these mice with MTII or γ₂-MSH caused a significant reduction in IL-1β release with 62% and 40% of inhibition, respectively (Fig. 5 b).

The attenuation of MSU crystal-induced inflammation by γ₂-MSH was prevented by the MC3/4-R antagonist SHU9119 but not by the selective MC4-R antagonist HS024, both antagonist being essentially inactive when administered on their own (Fig. 6,a). In this set of experiments, γ₂-MSH inhibited MSU crystal-induced PMN recruitment by 33% and 31% in wild-type and recessive yellow (e/e) mice, respectively; this effect was abrogated by SHU9119 but not HS024 (Fig. 6,a). γ₂-MSH inhibition of PMN migration was associated with lower IL-1β levels in both wild-type and recessive yellow (e/e) mice and this action of the peptide was again blocked in the presence of the antagonist SHU9119 (Fig. 6 b).

Cytokine and chemokine release from adherent Mφ in vitro was evaluated as marker cell activation. C57 wild-type Mφ incubation with γ₂-MSH significantly reduced MSU crystal-elicited KC and IL-1β release (Table I). A similar profile was observed in Mφ taken from recessive yellow (e/e) mice. In line with the in vivo data, γ₂-MSH inhibition of KC and IL-1β release by Mφ collected from either wild-type and recessive yellow (e/e) mice was abolished by coincubation with 9 μM SHU9119 (Table I).

In this study we have used an integrated approach with ultrastructural in vitro and in vivo analyses to demonstrate that a functional MC1-R is not necessary for the anti-inflammatory efficacy of melanocortin peptides. This does not appear to be the case for MC3-R, thus possibly explaining why MC1-R deficiency or lack of function does not cause clear immunological defects in rodents or humans.

This study was prompted by the unclear role that distinct MC-Rs might play in mediating the anti-inflammatory properties of melanocortin peptides. We have previously shown that MC3-R is present on rodent resident peritoneal Mφ by Western blotting and RT-PCR, and proposed that its activation down-regulated the experimental inflammatory response (13, 16). However, it has been suggested by other studies (3, 25) that MC1-R could be responsible for the anti-inflammatory effects displayed by α-MSH and other melanocortin peptides. Here we sought to address this discrepancy using a panel of more selective melanocortin agonists and antagonists and, more importantly, mice with a defected MC1-R. The recessive yellow (e/e) mouse has a frameshift mutation that leads to the expression of a nonfunctional MC1-R (18).

Initially, we monitored the basal conditions in the recessive yellow (e/e) mouse. We have previously shown that MC3-R is expressed on mouse resident peritoneal Mφ by RT-PCR and Western blotting (13, 16) and on rat knee joint Mφ by electron microscopy (17). Thus, we confirmed first the presence of MC3-R mRNA in Mφ taken from these mice using RT-PCR. Western blotting analysis confirmed that message was translated to protein in both wild-type and recessive yellow (e/e) mice peritoneal Mφ and, as a positive control, spleens. We then used electron microscopy to visualize a punctuate distribution of the receptor on the Mφ plasma membrane and its microvilli, in cells taken from wild type as well as from the mice bearing a nonfunctional MC1-R. Finally, MC3-R was functionally intact on these cells, since melanocortin agonists elicited equal levels of intracellular cAMP. Together these data indicate that MC3-R is not only expressed on peritoneal Mφ from wild-type and recessive yellow (e/e) mice but is fully functional, such that cAMP formation occurs after agonist activation. In addition, the recessive yellow (e/e) mouse did not display any signs of on-going inflammatory response; analysis of mesenteries of wild-type and recessive yellow (e/e) mice by intravital microscopy did not show an augmented interaction between circulating white blood cells and the postcapillary endothelium, nor higher numbers of leukocytes in the interstitium (data not shown). A lack of difference between the two mouse strains was also observed in terms of MSU crystal-induced peritonitis (assessed as degree of PMN migration and release of KC). The crystals produced an intense and long-lasting accumulation of blood-borne PMN into the peritoneal cavity, previously characterized (21), with essentially no difference between wild-type and recessive yellow (e/e) mice, not only with regards to the maximal responses, but also for the time-course profiles. Together these data indicate that an alteration in MC1-R functionality does not lead to subtle changes in animal homeostasis as well as in the host response to crystal injury.

Satisfied with the background conditions, we then tested the effect of several melanocortin peptides in these two mouse strains. Systemic administration of the nonselective melanocortin agonists α-MSH (7, 8, 9, 10) and HP228 (22) inhibited MSU crystal-induced PMN migration and this was associated with a reduction in IL-1β and KC in the inflammatory exudates. These peptides were equally active in wild-type and recessive yellow (e/e) mice. Another study has reported the anti-inflammatory effect of α-MSH in a model of LPS induced brain inflammation in recessive yellow (e/e) mice (9), strongly indicating that involvement of another MC-R also in this experimental condition. Whereas α-MSH anti-inflammatory actions have been reported in several studies (13, 25, 26, 27, 28) this is the first study that the nonselective peptide HP228 has been shown to inhibit PMN migration, adding HP288 to the growing list of melanocortins able to down-regulate experimental inflammation. Similarly to α-MSH, HP228 fully retained its anti-inflammatory efficacy in recessive yellow (e/e) mice.

The lack of involvement of MC1-R in the experimental peritonitis was further substantiated with the use of more selective melanocortin peptides, and we chose γ₂-MSH (putative endogenous agonist at MC3-R (14)), MTII (long lasting MC3-R activator (15)) and MS05 (selective MC1-R activator (23)). Systemic administration of MTII and γ₂-MSH before MSU crystal injection attenuated PMN migration equally in wild-type and recessive yellow (e/e) mice and this was associated with a reduction in IL-1β exudates levels. This data is in agreement with previous studies in this model (16). As mentioned in the introduction, melanocortin inhibition of IL-1β is not surprising, and it likely is due to cAMP-mediated inhibition of transcription factor functions (3, 29). Aside from our previous study (16), γ₂-MSH inhibition of LPS-induced IL-1β gene expression has also been documented (30). Also, our own in vitro data and those published previously all agree for an exquisite inhibitory action of melanocortins on cytokine/chemokine synthesis and release (3, 13, 16, 26, 27, 28, 31).

The proposition that MC3-R is the predominant anti-inflammatory receptor for melanocortins (13, 16, 17) is supported by the fact that the selective MC1-R agonist MS05 (23) was inactive in both wild-type and recessive yellow (e/e) mice. This selective agonist has been reported to down-regulate TNF-α-induced E-selectin, VCAM and ICAM mRNA, and protein expression in human dermal vascular endothelial cells (32). These in vitro data have been extrapolated to explain a potential anti-inflammatory role for MC1-R. In our experimental conditions, though, MC1-R does not appear to be active. A lack of involvement of MC1-R has also been observed when assessing the protective effects of melanocortins in a model of myocardial ischemia/reperfusion-induced arrhythmias (33). The central role of MC3-R was also supported by the experiments with the MC-R antagonists. The MC3/4-R antagonist SHU9119, but not the selective MC4-R antagonist HS024, abrogated cAMP accumulation produced by the different agonists on Mφ in vitro and PMN accumulation in vivo. These results were equally obtained in wild-type and recessive yellow (e/e) mice.

It is worth noting a potential extrapolation of the data here presented to the human system. Several human MC1-R single mutation have been reported within the Northern European population with 75% of individuals showing some allelic variants (3) and similar single nucleotide polymorpisms as well as frame shift mutations have been identified in other mammals (3). The importance of these mutations lies in the fact that these receptors are nonfunctional and in turn lead to a red or blond hair coloration, lighter skin types and less ability to tan (34, 35). To date, no clear affection of the immune system has been reported in these subjects. Similarly, no correlation between this phenotype and a higher risk of inflammatory disorders has been made (3).

In conclusion, we have demonstrated here that MC3-R activation modulates the host inflammatory response in this experimental model of peritonitis, and that this role is not solely played in mice with a nonfunctional MC1-R (recessive yellow (e/e) but also in intact wild-type mice. The apparent lack of involvement of MC1-R in this specific model was reinforced by the experiment with the selective MC1-R agonist (MS05). These findings highlight the involvement of MC3-R in modulating the inflammatory response. With the development of more selective compounds and the use of knockout mice for the MC3-R (36, 37) this scientific challenge can be addressed clearly in a conclusive manner.

We thank Drs. R de Médicis and A Lussier (University of Sherbrooke, Sherbrooke, Canada) for the supply of MSU crystals. Recessive yellow (e/e) mice were a kind gift from Dr. Nancy Levin (Trega Bioscience).