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* The William Harvey Research Institute, London, UK;
Merck Research Laboratories, Rahway, New Jersey, USA; and
Department of Pharmaceutical Chemistry and Toxicology, Universitá di Napoli, Naples, Italy
1Correspondence: The William Harvey Research Institute, Queen Mary School of Medicine and Dentistry, Charterhouse Sq., London EC1M 6BQ, UK. E-mail: m.perretti{at}qmul.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, [D-Trp8]--MSH activated MC3-R (as tested by accumulation of cyclic AMP) and inhibited (
50%) the release of interleukin (IL)-1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3-R null mice. In vivo, administration of 3–30 µg [D-Trp8]--MSH significantly inhibited leukocyte influx and cytokine production in a model of crystal-induced peritonitis, and these effects were absent in MC3-R null mice or blocked by coadministration of an MC3-R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (70%) by systemic or local administration of [D-Trp8]--MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3-R agonists as novel anti-inflammatory therapeutics.—Getting, S. J., Lam, C. W., Chen, A. S., Grieco, P., Perretti, M. Melanocortin 3 receptors control crystal-induced inflammation.
Key Words: neutrophil trafficking • macrophage activation • anti-inflammation • drug discovery
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. By acting on a specific family of seven transmembrane G-protein-coupled receptors, these hormones control several homeostatic functions including adrenal corticosteroid generation, skin pigmentation, and exocrine gland secretion (2)
. There are five distinct melanocortin receptors (MC-R) with a varied distribution throughout the body, being found in the central nervous system (CNS), periphery, as well as immune cells (3)
. Pharmacological characterization of each receptor has identified specific roles such that MC1-R controls skin pigmentation (4)
, MC2-R promotes adrenal steroidogenesis (5)
, and MC4-R is involved in the control of obesity (6)
and erectile dysfunction (7)
. Less clear are the physiological functions of MC3-R, though we have reported negative control of macrophage (MØ) activity (8)
, and of MC5-R, seemly involved in the control of exocrine secretion (2)
.
The anti-inflammatory activity of natural and synthetic melanocortins in experimental models of arthritis (9
, 10)
, inflammatory bowel disease (11)
, and asthma (12)
has been well established. These inhibitory/protective actions are receptor-mediated and involve, at the signaling level, cAMP accumulation, which is then associated, varying with the cell type, to reduced NF-
B activation (13
, 14)
and delayed up-regulation of heme oxygenase (HO)-1 (15)
and of the anti-inflammatory cytokine IL-10 (16)
. Altogether, these molecular events would provide a mechanistic explanation for the pan-inhibitory effect of melanocortins on proinflammatory cytokine and adhesion molecule expression.
Historically, MC1-R activation has been considered central to the anti-inflammatory actions of melanocortins, especially in the skin compartment (17)
; however, subsequent studies have pointed to MC3-R as a major determinant in bringing about anti-inflammatory actions (8
, 18
, 19)
. Indeed, the study of the actions of melanocortins in the recessive yellow e/e mouse, which bears an inactive MC1-R mutant, has corroborated the notion that MC3-R activation leads to major anti-inflammatory effects (20)
.
We recently described an analog of -MSH, [D-Trp8]--MSH that displays a high degree of specificity toward MC3-R (over the MC4-R and MC5-R) by about two orders of magnitude in the binding assay (21)
, yielding an 250-fold selectivity in the cAMP assay, as seen using transfected L cells. Further, the binding affinity and cAMP accumulation produced by [D-Trp8]--MSH at human MC3R is significantly higher with respect to the native peptide, -MSH (21)
. As human and mouse MC3-R are >95% homologous (22)
, the present study was undertaken to address for the first time the biological actions of [D-Trp8]--MSH in models of inflammation and tissue injury, integrating the pharmacological profile observed with analyses in mice nullified for MC3-R (23)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, were kindly donated by Dr. H. Chen (Merck Research Laboratories, Rahway, NJ, USA). Sprague-Dawley rats (200–250 g body wt) were purchased from Charles River (Kent, UK). Mice and rats were maintained on a standard chow pellet diet with tap water ad libitum using a 12 h light/dark cycle. WT animals were used 7 days after arrival according to guidelines laid down by the Ethical Committee for the Use of Animals, Bart’s, and The Royal London School of Medicine and Dentistry. Animal work was performed according to Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986).
In vitro MØ activation
An enriched population of peritoneal macrophages (MØ) (>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 x 106 MØ in 24-well plates. Nonadherent cells were washed off using warm media, and adherent cells (>95% MØ) were then incubated with [D-Trp8]--MSH (0.3 to 30 µg/ml) or 
-MSH (10 µg/ml). Cells were then stimulated with 1 mg/ml monosodium urate crystals (MSU, a concentration chosen from previous studies; ref. 20
) and cell-free supernatants collected 2 h later. Levels of the chemokine KC (CXCL1) and the pluripotent cytokine IL-1ß were measured by ELISA as described below.
Models of inflammation
Crystal peritonitis was induced by injection of 3 mg MSU crystals in 0.5 ml PBS as reported (20)
. At the 6 h time point, animals were killed by CO2 exposure, and peritoneal cavities were washed with 3 ml of PBS containing 3 mM ethylenediaminetetraacetate (EDTA) and 25 U/ml heparin. In some cases, [D-Trp8]--MSH (3–30 µg/mouse) was given at time 0. Aliquots of lavage fluid were then stained with Turk’s solution and differential cell counts were performed using a Neubauer hemocytometer and a light microscope (Olympus B061): leukocytes were identified as >95% neutrophils by light microscopy determination. Lavage fluids were then centrifuged at 400 g x 10 min and supernatants were stored at –20°C prior to biochemical determinations.
Rat knee joint
Rats were anesthetized with halothane and MSU crystals (20 mg/ml) were injected into the synovial space of the right knee in a volume of 50 µl sterile PBS (equivalent to 1.0 mg per joint). The left knee was injected with sterile PBS alone. At 16 h the animals were killed and the knee joint was exposed to measure the arthritic score according to ref. 24
and the size of the joint using a caliper, as described (9)
. Both knee joints were excised and lavaged with 1 ml PBS containing EDTA (3 mM) and heparin (25 U/ml). Lavage fluids were then centrifuged at 400 g x 10 min and the resulting pellet was stained with Turk’s solution for neutrophil number quantification. For each rat, data are reported as 105 PMN per joint, being corrected for the PBS value as measured in the left knee joint. Similarly, joint size is reported as an increase in the MSU-injected joint above the value measured in the PBS-treated joint, as measured using a caliper.
Biochemical analyses
MC3-R mRNA and protein expression
The MØ pattern of expression for MC3-R message and protein was determined as recently described (15)
. Primers were purchased from Applied Biosystems (Cheshire, UK). Classic 18s rRNA primers and random primers were purchased from Promega (Southampton, UK). The total volume of each reaction was made up to 50 µl with RNA-free distilled water. The cycle parameter were as follows; initial denaturation for 3 min at 94°C, followed by 30 cycles of denaturation (94°C for 45 s, annealing (60°C for 30 s), extension (72°C for 1 min), and a final extension of 72°C for 10 min. Amplified products were visualized by ethidium bromide fluorescence in 1% agarose gels. For Western blot, a recently described and validated anti-MC3-R rabbit serum was used (16)
. Briefly, proteins were resolved on a 10% polyacrylamide gel, and membranes were blocked overnight and incubated with an anti-MC3-R (1:500) rabbit serum; nonspecific antibody (Ab) binding was washed prior to the addition of goat anti-rabbit Ab (1:2000). Membranes were further washed and specific Ab binding was detected by an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Buckinghamshire, UK). After detection, bound antibodies were removed by incubating the membranes in acidic glycine solution (100 mM glycine, pH 2.5). The membranes were subsequently reprobed for detection of -tubulin (1:5000; Sigma-Aldrich Co. Ltd., Gillingham, UK).
Intracellular cAMP accumulation
MØ (1x105 per well) were incubated in serum-free RPMI 1640 media containing 1 mM isobutylmethylxantine (IBMX) and 1–30 µg/ml [D-Trp8]--MSH or the direct adenylate activator forskolin (3 µM). In a separate experiment, a selected concentration of 10 µg/ml [D-Trp8]--MSH was incubated in the presence of an anti-MC3-R polyclonal antibody (pAb) (1:50 to 1:200) (15)
or equal dilutions of nonimmune rabbit serum (Sigma-Aldrich). In all cases cells were washed after 30 min at 37°C and lysed, and cAMP levels in cell lysates were determined with a commercially available enzyme immunoassay (EIA) (Amersham Ltd,. Little Chalfont, Buckinghamshire, UK) using a standard curve constructed with 0–3200 fmol/ml cAMP.
ELISA measurements
Levels of the CXC chemokine KC (CXCL1) or IL-1ß levels in the lavage fluids were quantified with QuantikineTM ELISA purchased from R&D Systems (Oxfordshire, UK). The ELISAs showed negligible (<1%) cross-reactivity with several murine cytokines and chemokines (data as furnished by the manufacturer).
Drug treatment
[D-Trp8]--MSH (3–100 µg), the mixed MC3/4-R agonist MTII (Ac-Nle-c[-Asp-His-D-Phe-Arg-Trp-Lys-NH2 at a dose of 10 µg per mouse equivalent to 9.3 nmol) (25)
, or PBS (100 µl) were administered subcutaneously (s.c.) or intra-articularly (i.a.). In some experiments agonist effect was tested in the presence of the MC3/4-R antagonist SHU9119 (Ac-Nle-c[-Asp-His-D-2-Nal-Arg-Trp-Lys-NH2) (26)
or the selective MC4-R antagonist HS024 (Ac-Cys-Nle-Arg-D-2-Nal-Arg-Trp-Lys-Cys-NH2) (27)
given intraperitoneally (i.p.) intraperitoneal at the dose of 9 nmol 30 min prior to MSU crystals. Doses were selected from our earlier studies and from preliminary dose-response curves (9
, 20)
. MTII, -MSH, SHU9119, and HS024 were purchased from Bachem Ltd. (Saffron Walden, Essex, UK), stored at –20°C prior to use, and dissolved in sterile PBS (pH 7.4). [D-Trp8]--MSH was synthesized in our laboratory by solid-phase peptide synthesis as previously reported (21)
.
Statistics
Data are reported as mean ± SE of n distinct observations. Statistical differences were calculated on original data by ANOVA, followed by a Bonferroni test for intergroup comparisons or by an unpaired Student’s t test (2-tailed) when only two groups were compared. A threshold value of P < 0.05 was taken as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MSH on macrophage from WT and MC3-R null mice-MSH provoked rapid intracellular accumulation of cAMP with optimal concentrations of 10–30 µg/ml (equivalent to 4.7 to 14 µM; Fig. 1A). At 10 µg/ml, [D-Trp8]--MSH provoked a response not dissimilar from the one produced by forskolin, a direct activator of adenylate cyclase, or by -MSH (10 µg/ml; data not shown). The effect of [D-Trp8]--MSH was absent when MØ collected from MC3-R null mice was used: these cells expressed MC1-R but not MC3-R (Fig. 1B
). Addition of a specific anti-MC3-R pAb, but not of nonimmune rabbit serum, in the culture abrogated the effect of [D-Trp8]--MSH, (Fig. 1A
). The difference between specific and nonspecific serum was evident at the 1:100 dilution; addition of a less diluted preparation of the inactive serum provoked minimal, yet not significant, variation in intracellular accumulation of cAMP (Fig. 1A
).
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[D-Trp8]--MSH signaling in the MØ was associated with an inhibitory effect on proinflammatory cytokine release. Figure 2
reports these data, with a concentration-dependent reduction in the release of KC (CXCL1) and IL-1ß; -MSH, used as positive control, was equally effective. The latter melanocortin retained significant efficacy in MC3-R null MØ, whereas [D-Trp8]--MSH failed to affect KC (Fig. 2A
) and IL-1ß (Fig. 2B
) release in this genotype.
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This set of data was also supported by pharmacological analyses. Co-addition of the mixed MC3/4-R antagonist SHU9119 (9 µM), but not of the selective MC4-R antagonist HS024, abrogated the inhibitory effects of [D-Trp8]--MSH on both KC (from 420±40 to 220±15 pg/ml in the presence of 10 µg/ml [D-Trp8]--MSH [P<0.05] to 405±18 pg/ml when SHU9119 was added together with [D-Trp8]--MSH) and IL-1ß (220±25 pg/ml down to 96±14 pg/ml with 10 µg/ml [D-Trp8]--MSH, and back to 244±44 for [D-Trp8]--MSH+ SHU9119) release (data are mean ±SE of 3 experiments).
Effects of [D-Trp8]--MSH on acute inflammation in WT and MC3-R null mouse
In line with previous studies (28)
, i.p. injection of urate crystals provoked intense accumulation of blood-borne PMN, reliably assessed at the 6 h time point (Fig. 3A). Pretreatment with [D-Trp8]--MSH attenuated cell influx at all doses tested; this inhibition was associated with lower exudate levels of KC and IL-1ß (Fig. 3B, C
). The synthetic melanocortin MTII, partially selective for MC3-R, was also tested here, producing inhibitory effects similar to those attained by [D-Trp8]--MSH.
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Next, MC3-R null mice were tested observing no differences in markers of inflammation compared with WT mice (Fig. 3)
. However, the anti-inflammatory activities of both [D-Trp8]--MSH and MTII were no longer detected in MC3-R null mice. These experiments began by using the canonical dose of 10 µg per mouse [D-Trp8]--MSH; we then increased doses to 30 µg per mouse (corresponding to 14 nmol), finding no efficacy when MC3-R gene was nullified (Fig. 3)
. Therefore, this receptor is a major arm of the anti-inflammatory circuit operative in the mouse peritoneal cavity after melanocortin administration.
In a separate set of experiments, [D-Trp8]--MSH was given, producing 
50% reduction in PMN migration and KC or IL-1ß levels (Fig. 4
). As demonstrated in in vitro settings, SHU9119 antagonized the anti-inflammatory actions of [D-Trp8]--MSH on PMN (Fig. 4A
), KC (Fig. 4B
), and IL-1ß (Fig. 4C
) levels, whereas HS024 did not (data not shown).
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Effects of [D-Trp8]--MSH on experimental arthritis in the rat
We concluded the study by determining the efficacy of [D-Trp8]--MSH in a relevant model of pathology, and the choice fell on gouty arthritis (9)
. Joint deformation was observed 16 h postinjection of urate crystals (Fig. 5A), and this morphological changes were associated with marked PMN influx (Fig. 5B
) and edema/erythema formation (joint size; Fig. 5C
). [D-Trp8]--MSH significantly inhibited this response when given systemically or directly into the joint (Fig. 5)
. Of interest, a dose as low as 3 µg (1.4 nmol) was highly effective when given i.a. Similar to experimental peritonitis, SHU9119 (10 µg or 9 nmol) abrogated the joint protection afforded by [D-Trp8]--MSH with, for instance, an inhibition from 45 ± 10% to 5 ± 3% for PMN influx, and from 65 ± 12% to –5 ± 15% for joint size ([D-Trp8]--MSH alone or with SHU9119, respectively; P<0.05; n=6 in both cases).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 29)
, displaying minimal alterations under specific experimental conditions, but have never been tested for anti-inflammation. These new data add strength to the hypothesis that an inhibitory signal, activated by MC3-R agonists, could be exploited for the development of new therapeutics.
Since the seminal work of Lipton, Catania, and colleagues, the anti-inflammatory actions of melanocortin peptides have been partially unraveled (30)
. Of interest, ACTH was originally shown to possess anti-arthritic properties in patients suffering of gouty arthritis (31)
; the shorter form -MSH, which corresponds to the first 13 aa of ACTH, possesses anti-inflammatory activities in several experimental models, as summarized above. The fact that specific cellular targets could bring about these protective effects has raised hopes for innovative drug discovery. However, of the five melanocortin receptors identified so far, MC2-R is the only one that shows selectivity toward ACTH (2
, 22)
. In the field of experimental inflammation, MC1-R has always been put forward as the main molecular target for melanocortins (3)
. Expressed by melanocytes, hence responsible for -MSH actions on skin color, MC1-R expression has also been detected in PMN (32)
, MØ (33)
, mast cells (34)
, and endothelial cells (35)
. However, the lack of selective melanocortin peptides has opened the possibility that some of the observed inhibitory effects could be due to other members of the melanocortin receptor family.
In our own studies we could demonstrate MC1-R and MC3-R expression by rodent MØ (9
, 15)
, with the latter receptor being detected on MØ membrane protrusions, as shown by immuno-gold labeling by electron microscopy (EM) (20)
. These ultrastructural studies paved the way to the testing of several natural and synthetic melanocortins in models of acute inflammation, finding inhibitory effects (36
, 37)
. Treatment of mice with these peptides reduced inflammatory responses by 50%, in line with the hypothesis that endogenous inhibitory pathways act as modulators, rather than tout-court strong inhibitors, of the host response. Analyses of melanocortins’ effects in mice bearing a mutated and inactive MC1-R highlighted a major functional role for MC3-R in the inhibitory control exerted on MØ and the ensuing proinflammatory host response (20)
. Here we have challenged this hypothesis further by testing a new melanocortin receptor ligand, [D-Trp8]--MSH, which shows high selectivity at human MC3-R (21)
. This pharmacological approach has been integrated with a colony of MC3-R null mice (23)
. Since cells from these animals expressed MC1-R mRNA to an apparent similar degree to WT cells, and were clearly deficient in MC3-R mRNA and protein, their use would provide functional relevance to MC3-R biology in the context of inflammation.
[D-Trp8]--MSH activated classical postreceptor signaling in WT MØ, an effect genuinely due to MC3-R since absent in cells taken from MC3-R null mice and blocked by an anti-MC3-R pAb. In the MØ, this compound could also elicit longer lasting MC3-R mediated responses, such as induction of the anti-inflammatory homeostatic enzyme HO-1 (S. J. Getting, C. W. Lam, and M. Perretti, unpublished results), similar to that recently reported for -MSH and MTII (15)
. [D-Trp8]--MSH inhibition of MØ-derived cytokines in in vitro settings, together with the data discussed above, justified the second part of the study, which was centered on analysis of the potential anti-inflammatory properties of [D-Trp8]--MSH.
[D-Trp8]--MSH produced inhibition of the host response to peritoneal injection of urate crystal, a model we used widely for this line of research (36
37
38)
because of its relationship with gouty arthritis, which is sensitive to the inhibitory actions of ACTH (39
, 40)
. The dose response to [D-Trp8]--MSH was relatively flat, with significant inhibitions at 3 µg per mouse; [D-Trp8]--MSH-induced inhibition of inflammatory markers (including the multipotent cytokine IL-1ß) was very similar to those attained by the better characterized compound MTII (or melanotan; in development for skin diseases). More important, [D-Trp8]--MSH inhibitions disappeared in MC3-R null mice, confirming, also in vivo settings, compound selectivity toward this receptor. The lack of efficacy in MC3-R null mice coupled with the observations made on the redundant role of MC1-R in mice bearing a mutated receptor isoform (20)
reinforces the functional role of MC3-R in bringing about the anti-inflammatory actions of this class of peptides.
In the final part of the study we tested [D-Trp8]--MSH in a model of experimental gouty arthritis. This disease is characterized by crystal deposition in the joint with marked influx of PMN, edema, and pain (41)
. Experimentally, we have reproduced it by injecting a known amount of urate crystals into the rat joint, and previously described the time profiles for cell accumulation, mediator release, and susceptibility to ACTH and -MSH (9)
. The data obtained indicated high efficacy of [D-Trp8]--MSH in suppressing all parameters of joint inflammation under analysis, with particular efficacy when given i.a. at the dose of 3 µg. The intra-articular route of administration is often used in arthritis clinics for glucocorticoid administration (42
, 43)
. We wish to propose that these collective findings would be of potential validity for therapeutic development of MC3-R agonists to treat gouty arthritis. Future studies will assess whether selective MC3-R ligands could be of use in other forms of arthritis, including rheumatoid arthritis and osteoarthritis; -MSH has been described to inhibit adjuvant arthritis in the rat (10)
.
In conclusion, we have used new experimental tools, a selective MC3-R ligand, and a colony of MC3-R null mice to reiterate the focus on this specific melanocortin receptor as the major effector for the anti-inflammatory actions of melanocortins. We propose that these new results provide strong proof-of-concept to justify innovative anti-inflammatory drug discovery centered on MC3-R, thereby capitalizing on the properties of the endogenous homeostatic axis effected by melanocortins and their receptors.
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ACKNOWLEDGMENTS |
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Received for publication May 5, 2006. Accepted for publication June 12, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MSH. Immunol. Today 18,140-145[CrossRef][Medline]This article has been cited by other articles:
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  G. S. Cooke, S. J. Campbell, S. Bennett, C. Lienhardt, K. P. W. J. McAdam, G. Sirugo, O. Sow, P. Gustafson, F. Mwangulu, P. van Helden, et al. Mapping of a Novel Susceptibility Locus Suggests a Role for MC3R and CTSZ in Human Tuberculosis Am. J. Respir. Crit. Care Med., July 15, 2008; 178(2): 203 - 207. [Abstract] [Full Text] [PDF]
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 K. L. J. Ellacott, J. G. Murphy, D. L. Marks, and R. D. Cone Obesity-Induced Inflammation in White Adipose Tissue Is Attenuated by Loss of Melanocortin-3 Receptor Signaling Endocrinology, December 1, 2007; 148(12): 6186 - 6194. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. cells not treated with the pAb (dose 0). B) Left: RT-polymerase chain reaction (RT-PCR) showing MC1-R and MC3-R message in samples prepared from the three genotypes: different amounts of cDNA were loaded (from 6 to 24 µg); right: Western blot analysis for MC3-R protein expression, and alpha-tubulin, in heart and MØ samples from two distinct preparations for each genotype.
