Mouse Suggest an Melanocortin Peptides

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 This information is current as Important Role for Melanocortin 3 Receptor of September 27, 2021. Stephen J. Getting, Helen C. Christian, Connie W. Lam, Felicity N. E. Gavins, Roderick J. Flower, Helgi B. Schiöth and Mauro Perretti

J Immunol 2003; 170:3323-3330; ; Downloaded from doi: 10.4049/jimmunol.170.6.3323 http://www.jimmunol.org/content/170/6/3323 http://www.jimmunol.org/ References This article cites 36 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/170/6/3323.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Stephen J. Getting,2* Helen C. Christian,† Connie W. Lam,* Felicity N. E. Gavins,* Roderick J. Flower,* Helgi B. Schio¬th,‡ and Mauro Perretti*

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inﬂammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of

the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally Downloaded from defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M␾) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as M␾ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M␾ activation, determined as ␤ ␥ release of the CXC chemokine KC and IL-1 , was inhibited by the more selective MC3-R agonist 2-melanocyte stimulating

hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited http://www.jimmunol.org/ IL-1␤ release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the ␥ inﬂammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of 2- melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M␾ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice. The Journal of Immunology, 2003, 170: 3323Ð3330.

elanocortin peptides, (e.g., ␣-melanocyte stimulating MC-Rs have a wide and varied distribution throughout the body hormone (␣-MSH)3) are derived from a larger precur- (3). All MC-Rs are positively coupled to adenylate cyclase, and M sor called the pro-opiomelanocortin gene product and agonism at these receptors leads to increases in intracellular cAMP by guest on September 27, 2021 are characterized by a common amino acid motif (HFRW). These (3). Some specific actions have been attributed to specific members endogenous peptides have long been reported to possess anti- of the receptor family; examples being MC1-R mediated skin pig- inflammatory effects in many experimental models of acute and mentation, MC4-R control of obesity and MC2-R stimulation of chronic inflammation, including experimental bowel disease, al- adrenal steroidogenesis (3). It remains still unclear if the anti- lergy, and chronic (mycobacterium-induced arthritis) and systemic inflammatory actions of melanocortin peptides are mediated by a inflammation (endotoxemia) (1, 2). Melanocortins act at a sub- single MC-R. group of G-protein coupled receptor, termed melanocortin recep- The MC1-R has long been regarded as the receptor responsible tors (MC-R), of which five members have been identified so far. for the anti-inflammatory effects of ␣-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 † *The William Harvey Research Institute, London, United Kingdom; Department of ␬ ␬ ␣ Human Anatomy and Physiology, University of Oxford, Oxford, United Kingdom; of NF- B activation (7, 8) and protection of I B degradation (9). and ‡Department of Neuroscience, Uppsala University, Uppsala, Sweden These intracellular events would produce a reduction in the ex- Received for publication October 15, 2002. Accepted for publication January pression of proinflammatory cytokines (10) and adhesion mole- 16, 2003. cules (8), thereby affecting the humoral and cellular phases of in- The costs of publication of this article were defrayed in part by the payment of page flammation (11, 12). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recently, our own studies have identified a putative role for 1 This work was supported by the Arthritis Research Campaign U.K. (Grant G0571). MC3-R in modulating experimental inflammation (13). Selective ␥ M.P. is a Senior Fellow of the Arthritis Research Campaign, U.K. R.J.F. is a Principal agonists at the MC3-R (the natural 2-MSH) (14) and the synthetic Research Fellow of the Wellcome Trust U.K. H.S.S. was supported by the Swedish peptide MTII (15)) displayed inhibitory activity in a murine model Research Council (VR, medicin) and Melacure Therapeutics, A.B. (Uppsala, Sweden). of monosodium urate (MSU) crystal-induced peritonitis (16). The 2 Address correspondence and reprint requests to: Dr. Stephen J. Getting, Department in vivo data have been supported by the detection of both MC3-R of Biochemical Pharmacology, The William Harvey Research Institute, Bart’s and mRNA and protein on mouse and rat peritoneal macrophages The London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, ␾ ␾ London EC1 M 6BQ, U.K. E-mail address: [email protected] (M ) as well as on rat knee joint M (13, 16, 17). In addition, ␾ 3 Abbreviations used in this paper: MSH, melanocyte stimulating hormone; MC-R, MC3-R activation on M caused cAMP accumulation and inhi- melanocortin receptors; M␾, macrophage; MSU, monosodium urate. bition of cytokine release (13, 16, 17).

This study was planned to address the apparent dichotomy be- type and recessive yellow (e/e) mice peritoneal M␾ and spleens in PBS tween MC1-R and MC3-R in mediating the anti-inflammatory ac- containing EDTA (3 mM), leupeptin (0.39 mg/ml) and PMSF (10 mM). tions of melanocortins. We have taken advantage of the recent Protein levels were then determined (Bio-Rad protein assay; Bio-Rad, Her- cules, CA) and 50 ␮g of protein was mixed with 0.125 M Tris-HCl (pH characterization of more selective MC-R agonists, and from the 6.8), 2 mM EDTA, 4% sodium dodecyl sulfate (SDS), 10% mercaptoetha- availability of the recessive yellow (e/e) mouse strain. A frameshift nol, 20% glycerol and boiled for 10 min before loading and running on an mutation in the MC1-R gene in these animals results in a single 10% polyacrylamide gel (Protogel; National Diagnostics, Ashby De La deletion of a nucleotide at position 549; the outcome is a receptor Zouche, Leicestershire, U.K.) for 60 min at 100 V. Protein was transferred onto polyvinylidine difluoride membranes (Amersham Life Sciences) by protein with a premature termination in the fourth trans-membrane semidry blotting (Bio-Rad) for 60 min using a Tris/glycine buffer contain- domain, thus unable to couple to adenylate cyclase and activate ing 20% methanol. Membranes were then blocked overnight at 4¡Cby cAMP synthesis (18, 19). Interestingly, these mice have altered immersion in a 5% nonfat dried milk solution made up in PBS containing pigmentation (yellow mice) without clear defects in the immune 0.1% Tween 20. Membranes were then incubated for2hat4¡Cina5% system (18, 19), thus resembling the lack of reported phenotype nonfat dried milk solution with an affinity-purified goat polyclonal Ab (1/ 200 final dilution; Autogen Bioclear, Mile Elm Calne, U.K.) raised against associated with red hair as studied in humans (20). The results a peptide mapping human MC3-R. This goat polyclonal MC3-R Ab produced here with an experimental model of peritonitis show that showed cross-reactivity with mouse and rat, but did not cross-react with a functional MC1-R is not necessary to elicit the anti-inflammatory MC1-R, MC2-R, MC4-R, and MC5-R of any species (data supplied by the actions of melanocortin peptides. 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 Materials and Methods PBS and 0.1% Tween). After another 15 min and three 5 min washes in Male C57BL.6 mice (20Ð22 g body weight) were purchased from Tuck PBS/Tween), blots were incubated with ECL solution (Amersham Life Downloaded from (Battlesbridge, Essex, U.K.) and recessive yellow (e/e) mice (18) were a Sciences) for 1 min and then exposed to autoradiographic film for detection kind gift from Dr. N. Levin (Trega Bioscience, San Diego, CA). Mice were of chemiluminescence. Cruz m.w. markers were also used (Autogen maintained on a standard chow pellet diet with tap water ad libitum using Bioclear). a 12 h light/dark cycle. Animal experimental work was performed accord- Electron microscopy analysis. We used a protocol recently validated for ing to Home Office regulations (Guidance on the Operation of Animals, rat knee joint M␾ (17). Peritoneal M␾ from wild-type and recessive yellow Scientific Procedures Act, 1986). 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) http://www.jimmunol.org/ In vitro experimental section glutaraldehyde in PBS, pH 7.2, for4hat4¡C, washed briefly in PBS, and Primary culture of M␾: detection of KC and IL-1␤ release. An enriched transferred to a solution of 2.3 M sucrose (in PBS) at 4¡C overnight. The population of peritoneal M␾ (Ͼ95% pure) was prepared by 2 h adherence cryoprotected cells were slam-frozen (Reichert MM80E; Leica, Milton Ϫ at 37¡Cin5%CO2/95% O2 atmosphere in RPMI 1640 supplemented with Keynes, U.K.), freeze-substituted at 80¡C in methanol for 48 h, and 10% FCS, by culturing 5 ϫ 106 M␾ in 24-well plates. Nonadherent cells embedded at Ϫ20¡C in LRGold acrylic resin (London Resin Company, were washed off using warm medium, and adherent cells (Ͼ95% M␾) were Reading, U.K.) in a Reichert freeze-substitution system. Ultrathin sections ␥ ␮ then incubated with 2-MSH (95 M) alone or in combination with (50Ð80 nm) were prepared by use of a Reichert Ultracut-S ultratome and SHU9119 (9 ␮M) for 15 min in RPMI 1640 medium. Cells were then incubated at room temperature for 2 h with a polyclonal goat anti-MC3-R stimulated with 1 mg/ml MSU crystals (a concentration chosen from pre- Ab (dilution 1/200; Autogen Bioclear) or goat anti-MC3-R preabsorbed vious experiments (13)), and cell-free supernatants were collected 2 h later. with MC3-R blocking peptide (20 ␮g/ml; Autogen Bioclear) followed by KC and IL-1␤ levels were measured by ELISA as described below. protein A linked to 15 nm gold (British Biocell, Cardiff, U.K.) for 1 h. As by guest on September 27, 2021 cAMP formation. M␾s(1ϫ 105) were allowed to adhere in 24-well additional negative control sections were incubated with nonimmuno goat plates as above, and incubated with serum-free RPMI 1640 medium con- 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 taining 1 mM isobutylmethylxanthine with 2-MSH (30 g/ml equivalent to 95 ␮M), MTII (10 ␮g/ml equivalent to 9.3 ␮M), MS05 (30 ␮g/ml containing 0.1% egg albumin. After immuno-labeling sections were lightly equivalent to 22 ␮M), or the direct adenylate cyclase activator forskolin (3 counterstained with uranyl acetate and lead citrate and examined with a ␮M). In some experiments the effect of these peptides in the presence of 9 JEOL 1010 transmission electron microscope (JEOL, Peabody, MA). ␮M of the MC3/4-R antagonist SHU9119 or the selective MC4-R antagonist HS024 were investigated. After 30 min at 37¡C, supernatants were In vivo experimental section removed and cells were washed and lysed. cAMP levels in cell lysates were MSU crystal-induced peritonitis. PMN recruitment into the peritoneal determined with a commercially available enzyme immunoassay (Amer- cavity was elicited by MSU crystals as recently reported (21). Mice were sham Life Sciences, Little Chalfont, U.K.) using a standard curve con- treated i.p. with 3 mg of MSU crystals in 0.5 ml PBS, and peritoneal structed with 0Ð3,200 fmol/ml cAMP. cavities lavaged at 2Ð96 h post challenge with 3 ml PBS containing EDTA RT-PCR for MC3-R message. Peritoneal M␾ (5 ϫ 106) enriched by 2 h (3 mM) and heparin (25 U/ml). Aliquots of the lavage fluids were then adherence at 37¡C in 24-well plates and lysed in 1 ml of Trizol reagent stained with Turk’s solution (0.01% crystal violet in 3% acetic acid), and (lysis buffer for RNA preparation) from Life Technologies (Paisley, U.K.) differential cell counts were performed by light microscopy using a and RNA was isolated according to manufacturer’s protocol. RNA was Neubauer hematocytometer. Data are reported as 106 PMN per mouse. extracted with chloroform and isopropanol, precipitated with ethanol, and Lavage fluids were then centrifuged at 400 ϫ g for 10 min and supernatants the pellet was resuspended in diethyl pyrocarbonate-treated water. The were stored at Ϫ20¡C before biochemical determinations (see below). yield and purity of the RNA was then estimated spectrophotometrically at Drug treatment. The natural hormone ␣-MSH (10 ␮g per mouse equiv- 260 and 280 nm. Total RNA (3 ␮g) was used for the generation of cDNA. ␥ alent to 6 nmol), the relatively selective MC3-R agonist 2-MSH PCR amplification reactions were then performed on aliquots of the cDNA. (YVMGHFRWDRFG) (30 ␮g per mouse equivalent to 95 nmol) (14), the All PCR were performed using PCR beads (Pharmacia Biosystems Europe, mixed MC3/4-R agonist MTII (Ac-Nle-cyclo[-Asp-His-D-Phe-Arg-Trp- St Albans, U.K.) in a final volume of 25 ␮l using a Hybaid OmniGene ␮ Lys-NH2) (10 g per mouse equivalent to 9.3 nmol) (15), the pan-MC-R thermal cycler (Middlesex, U.K.). The murine MC3-R primer sequences ␮ agonist HP228 (Ac-Nle-Gln-His-D-Phe-Arg-D-Trp-Gly-NH2;15 g per were used as previously described (13): MC3-R, 5Ј-GCC TGT CTT CTG mouse equivalent to 15.2 nmol) (22), and the selective MC1-R agonist TTT CTC CG-3Ј and 5Ј-GCC GTG TAG CAG ATG CAG TA-3Ј (forward MS05 (H-Ser-Ser-Ile-Ile-Ser-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2; and reverse) which amplified a fragment of 820 bp in length. The cycling 1Ð100 ␮g per mouse equivalent to 0.66Ð66 nmol) (23) were administered parameters were as follows: after an initial denaturation for 3 min at 94¡C, s.c. 30 min before MSU crystals. In some experiments, agonist effect was 30 cycles of annealing at 60¡C (30 s), extension at 72¡C (1 min), and tested in the presence of the MC3/4-R antagonist SHU9119 (Ac-Nle-cy- denaturation at 94¡C (45 s), and a final further extension of 72¡C for 10 clo[-Asp-His-D-2-Nal-Arg-Trp-Lys-NH2) or selective MC4-R antagonist min. Amplification products were visualized by ethidium bromide fluores- HS024 (Ac-Cys-Nle-Arg-D-2-Nal-Arg-Trp-Lys-Cys-NH2) (16) of which 9 cence in agarose gels. Images were inverted using the Graphic Converter ␣ ␥ nmol were given i.p. 30 min before MSU crystals. MTII, -MSH, 2-MSH, software (version 2.1) running on a Macintosh Performa 6200 (Reine, HP228, SHU9119, and HS024 were purchased from Bachem (Saffron Wal- Germany). den, Essex, U.K.), stored at Ϫ20¡C before use, and dissolved in sterile PBS Western blotting analysis. We used a protocol recently validated for rat (pH 7.4). MS05 was kindly provided by Melacure Therapeutics (Uppsala, and mouse peritoneal M␾ (16). Protein was isolated from samples of wild- Sweden). The Journal of Immunology 3325

Cytokine quantification by ELISAs. Murine KC and IL-1␤ levels in peri- peritoneal M␾ showed the presence of MC3-R by RT-PCR in toneal lavage fluids were determined using commercially available ELISA either mouse strain (Fig. 1a). To determine whether this message purchased from R&D Systems (Abingdon, U.K.). In brief, lavage fluids (50 was translated to protein we used Western blotting and electron ␮l) were assayed for each cytokine and compared with a standard curve constructed with 0Ð1 ng/ml of the standard cytokine. The ELISAs showed microscopy. Expression of MC3-R in wild-type and recessive yel- negligible (Ͻ1%) cross-reactivity with several murine cytokines and che- low (e/e) mice peritoneal M␾ and spleen was monitored by West- mokines (data as furnished by manufacturer). ern blotting analysis. Western blotting confirmed the presence of Statistics. Data are reported as mean Ϯ SE of n distinct observations. the MC3-R in protein extracts prepared from mouse peritoneal M␾ Statistical differences were calculated on original data by ANOVA followed by Bonferroni test for intergroup comparisons (24), or by unpaired and spleen, with a band of the right m.w. (43 kDa) being obtained Student’s t test (two-tailed) when only two groups were compared. A (Fig. 1b). Electron microscopy was then used to highlight MC3-R threshold value of p Ͻ 0.05 was taken as significant. gold immunolabeling which was predominantly located on the plasma membrane with a particular higher density in correspon- Results dence of membrane protrusions in wild-type (Fig. 1c) and reces- ␾ Identification and functionality of the MC-R on peritoneal M sive yellow (e/e) mice (Fig. 1d). This immunostaining was specific RT-PCR, Western blotting, and electron microscopy analyses were for MC3-R insofar as it was absent when the primary Ab was used to monitor MC-R expression in resident peritoneal M␾ and as preabsorbed with the blocking peptide in wild-type (Fig. 1e)or a positive control spleen (Western blotting only) from both wild- recessive yellow (e/e) mice (Fig. 1f). As a final control, cells type and recessive yellow (e/e) mice. RNA extracted from murine stained with nonimmune goat IgG did not display immuno-gold Downloaded from http://www.jimmunol.org/

FIGURE 1. Expression of MC3-R on wild- type and recessive yellow (e/e) mice peritoneal M␾ as detected by RT-PCR and electron microscopy. a, RT-PCR analysis demonstrates the presence of speciﬁc products for MC3-R (820 bp) in M␾ taken from wild-type and recessive yellow

(e/e) mice. Genomic DNA (Gen) was used as a by guest on September 27, 2021 positive control. The arrows indicate the presence of MC3-R message and 18s RNA detected as control. Gel depicts a representative of four distinct experiments with identical results. M, markers shown as base pair. (b) Western blot analysis demonstrates protein expression of MC3-R in peritoneal M␾ and spleen from wild- type and recessive yellow (e/e) mice. c—h, Elec- tron microscopy analysis: expression of MC3-R on murine peritoneal M¯ taken from wild-type (c) and recessive yellow (e/e) (d) mice; immu- noreactivity was predominantly localized on the plasma membrane, with a minor degree of staining also detected in the cytosol; arrowheads, characteristic clusters of gold particles on plasma membrane protrusions. Lack of immuno-gold labeling on murine peritoneal M␾ when the anti- MC3-R Ab was preabsorbed with the blocking peptide in wild-type (e) and recessive yellow (e/e) (f) cells. Similar negative results were obtained following nonimmune goat IgG staining of M␾ from wild-type (g) and recessive yellow (e/e) (h) mice. Microphotographs are representative of 10 distinct cells. n, nucleus; magnification ϫ16,000. 3326 MELANOCORTINS’ ANTI-INFLAMMATORY EFFECTS IN MC1-R-DEFICIENT MICE labeling on their plasma membrane in wild-type (Fig. 1g)orre- the cell emigration at 6 and 24 h in recessive yellow (e/e) mice cessive yellow (e/e) mice (Fig. 1h). compared with wild type following injection of urate crystals into Receptor functionality was determined quantifying cAMP ac- the peritoneal cavity. Very few resident lymphocytes or M␾ could cumulation in peritoneal M␾. In C57 wild-type mice the natural be detected in the lavage fluids following challenge (data not ␥ and synthetic MC3-R agonists, 2-MSH and MTII, caused signif- shown). The maximal PMN accumulation occurred at the 6-h time icant increases in cAMP accumulation with a 450% and 420% point and was preceded by a marked release of the proinflamma- increase above basal levels (73 Ϯ 12 fmol/well). A similar obser- tory CXC chemokine KC, maximal at 2 h (Fig. 3b). Again no vation was noted with respect to the recessive yellow (e/e) mice in difference in the release of KC was measured between wild-type which a 436% and 400% increase above basal levels (80 Ϯ 11 and recessive yellow (e/e) mice. ␥ fmol/well) was measured for 2-MSH and MTII, respectively (Fig. 2a). These increases in both wild-type and recessive yellow (e/e) Anti-inflammatory effect of nonselective MC-R agonists in mice were blocked in the presence of the MC3/4-R antagonist experimental peritonitis SHU9119 (Fig. 2b) but not the selective MC4-R antagonist HS024 Nonselective MC-R agonists, the synthetic peptide HP228, and the (Fig. 2c). The effect of forskolin was retained in the presence of naturally occurring hormone, ␣-MSH, modulated the inflamma- either antagonist. The selective MC1-R agonist MS05 failed to tory response in wild-type mice, with inhibitions in PMN migra- elicit any increase in cAMP in either type of M␾ (Fig. 2, aÐc). tion of 35% and 47%, for HP228 and ␣-MSH, respectively (Fig. 4a). The peptides were then administered to recessive yellow (e/e) MSU crystal-induced peritonitis mice and similar inhibitory responses (ϳ50%) were observed (Fig. Intraperitoneal injection of MSU crystals produced a time depen- 4a). HP228 and ␣-MSH caused a reduction in IL-1␤ and KC levels Downloaded from dent accumulation of PMN with a similar profile in wild-type and measured in peritoneal lavages collected from both wild-type and recessive yellow (e/e) mice. MSU crystal-induced PMN accumu- recessive yellow (e/e) mice (Fig. 4, b and c). In the absence of lation was maximal at 6 h postinjection (with an approximate in- MSU crystal injection, the number of PMN and levels of IL-1␤ flux rate of 1.1 ϫ 106 PMN per hour in the 2- to 6-h time interval) and KC were below the detection limits in either mouse strain (Fig. 3a). PMN recruitment plateaued between 6 and 24 h, remain- (data not shown). ing at the same level at the later time point in wild-type mice http://www.jimmunol.org/ (Ͼ6 ϫ 106 PMN per mouse). There appears to be no difference in Anti-inflammatory effect of selective agonists MC-R in experimental peritonitis Next we evaluated the effect of more selective melanocortin pep- ␥ ␮ tides in this model of peritonitis. The natural ( 2-MSH, 30 g/ mouse) and synthetic (MTII, 10 ␮g/mouse) MC3-R agonists inhibited both the cellular and humoral response produced by urate by guest on September 27, 2021

FIGURE 2. MC3-R activation in peritoneal M␾ collected from wild- type and recessive yellow (e/e) mice. Adherent peritoneal M␾ were incu- FIGURE 3. Time dependency of MSU crystal-induced PMN accumu- ␥ ␮ ␮ ␮ bated with 2-MSH (95 M), MTII (9.3 M), MS05 (22 M), forskolin (3 lation and KC release in the mouse peritoneal cavity. Mice received an ␮M), alone or together with the given MC-R antagonist, for 30 min before injection of MSU crystals (3 mg in 0.5 ml sterile PBS i.p.) at time 0. determination of intracellular cAMP. a, Melanocortin agonists alone; b, in Peritoneal cavities were washed at the reported time points before mea- the presence of 9 ␮M SHU9119; c, in the presence of 9 ␮M HS024. Data surement of PMN inﬂux (a) or KC protein in the cell-free exudates (b). .p Ͻ 0.05 vs time 0 group ,ء .p Ͻ 0.05 vs vehicle control. Data are mean Ϯ SE of eight mice per group ,ء .are mean Ϯ SE of four mice per group The Journal of Immunology 3327 Downloaded from http://www.jimmunol.org/

␥ FIGURE 5. Effect of 2-MSH, MTII, and MS05 on MSU crystal-induced inﬂammation in wild-type and recessive yellow (e/e) mice. Mice ␮ ␮ ␥ ␮ were pretreated s.c. with sterile PBS (100 l), 30 g 2-MSH, 10 g MTII, ␮ FIGURE 4. Effect of HP228 and ␣-MSH on MSU crystal-induced in- or with 1Ð100 g MS05 30 min before i.p. injection of MSU crystals (3 mg in 0.5 ml sterile PBS). PMN accumulation (a) and IL-1␤ levels in cell-free

flammation in wild-type and recessive yellow (e/e) mice. Mice were pre- by guest on September 27, 2021 Ϯ treated s.c. with sterile PBS (100 ␮l), 15 ␮g HP228, or 10 ␮g ␣-MSH 30 exudates (b) were measured 6 h later. Data are mean SE of eight mice Ͻ ء min before i.p. injection of MSU crystals (3 mg in 0.5 ml sterile PBS). per group. , p 0.05 vs PBS group. PMN accumulation (a) and IL-1␤ and KC levels in cell-free exudates (b and c) were measured 6 h later. Data are mean Ϯ SE of eight mice per p Ͻ 0.05 vs PBS group. yellow (e/e) mice and this action of the peptide was again blocked ,ء .group in the presence of the antagonist SHU9119 (Fig. 6b). ␥ ␾ crystals. Fig. 5a shows the data for C57 wild-type mice, in which In vitro effects of 2-MSH on cytokine release from cultured M ␥ Ͻ ␾ MTII and 2-MSH significantly ( p 0.05) inhibited MSU crystal- Cytokine and chemokine release from adherent M in vitro was induced PMN recruitment by 54% and 45%, respectively. Similar evaluated as marker cell activation. C57 wild-type M␾ incubation ␥ degrees of inhibition were observed in the recessive yellow (e/e) with 2-MSH significantly reduced MSU crystal-elicited KC and ␥ ␤ ␾ mice with MTII and 2-MSH causing a 63% and 42% reduction in IL-1 release (Table I). A similar profile was observed in M PMN migration ( p Ͻ 0.05). The selective synthetic MC1-R ago- taken from recessive yellow (e/e) mice. In line with the in vivo ␮ ␥ ␤ ␾ nist MS05 (1Ð100 g) did not modify the inflammatory response data, 2-MSH inhibition of KC and IL-1 release by M collected in either wild-type or recessive yellow (e/e) mice (Fig. 5a). from either wild-type and recessive yellow (e/e) mice was abol- In view of our previous studies with intact mice (16), exudate ished by coincubation with 9 ␮M SHU9119 (Table I). IL-1␤ levels were measured in recessive yellow (e/e) mice. Treat- ␥ ment of these mice with MTII or 2-MSH caused a significant Discussion reduction in IL-1␤ release with 62% and 40% of inhibition, re- In this study we have used an integrated approach with ultrastruc- spectively (Fig. 5b). tural in vitro and in vivo analyses to demonstrate that a functional MC1-R is not necessary for the anti-inflammatory efficacy of mela- Effect of MC-R antagonists nocortin peptides. This does not appear to be the case for MC3-R, ␥ The attenuation of MSU crystal-induced inflammation by 2-MSH thus possibly explaining why MC1-R deficiency or lack of func- was prevented by the MC3/4-R antagonist SHU9119 but not by the tion does not cause clear immunological defects in rodents or selective MC4-R antagonist HS024, both antagonist being essen- humans. tially inactive when administered on their own (Fig. 6a). In this set This study was prompted by the unclear role that distinct MC-Rs ␥ of experiments, 2-MSH inhibited MSU crystal-induced PMN re- might play in mediating the anti-inflammatory properties of mela- cruitment by 33% and 31% in wild-type and recessive yellow (e/e) nocortin peptides. We have previously shown that MC3-R is mice, respectively; this effect was abrogated by SHU9119 but not present on rodent resident peritoneal M␾ by Western blotting and ␥ HS024 (Fig. 6a). 2-MSH inhibition of PMN migration was asso- RT-PCR, and proposed that its activation down-regulated the ex- ciated with lower IL-1␤ levels in both wild-type and recessive perimental inflammatory response (13, 16). However, it has been 3328 MELANOCORTINS’ ANTI-INFLAMMATORY EFFECTS IN MC1-R-DEFICIENT MICE

M␾ 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 M␾ and, as a positive control, spleens. We then used electron microscopy to visualize a punctuate distribution of the receptor on the M␾ 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 M␾ 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 dif- Downloaded from ference 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 http://www.jimmunol.org/ regards to the maximal responses, but also for the time-course ␥ FIGURE 6. SHU9119 prevents 2-MSH inhibition of MSU crystal peri- profiles. Together these data indicate that an alteration in MC1-R tonitis in wild-type and recessive yellow (e/e) mice. Mice received PBS functionality does not lead to subtle changes in animal homeostasis ␮ ␥ ␮ (100 l s.c.) or 2-MSH (30 g s.c.) with or without 9 nmol i.p. SHU9119 as well as in the host response to crystal injury. or HS024 30 min before MSU crystals (3 mg in 0.5 ml sterile PBS i.p.). Satisfied with the background conditions, we then tested the Peritoneal cavities were washed 6 h later, and the number of accumulated PMN (a) or the content of IL-1␤ in cell-free exudates (b) were measured effect of several melanocortin peptides in these two mouse strains. p Ͻ 0.05 vs Systemic administration of the nonselective melanocortin agonists ,ء .h later. Data are mean Ϯ SE of eight mice per group 6 ␣ appropriate PBS group. -MSH (7Ð10) and HP228 (22) inhibited MSU crystal-induced by guest on September 27, 2021 PMN migration and this was associated with a reduction in IL-1␤ and KC in the inflammatory exudates. These peptides were equally suggested by other studies (3, 25) that MC1-R could be responsible active in wild-type and recessive yellow (e/e) mice. Another study for the anti-inflammatory effects displayed by ␣-MSH and other has reported the anti-inflammatory effect of ␣-MSH in a model of melanocortin peptides. Here we sought to address this discrepancy LPS induced brain inflammation in recessive yellow (e/e) mice (9), using a panel of more selective melanocortin agonists and antag- strongly indicating that involvement of another MC-R also in this onists and, more importantly, mice with a defected MC1-R. The ␣ recessive yellow (e/e) mouse has a frameshift mutation that leads experimental condition. Whereas -MSH anti-inflammatory ac- to the expression of a nonfunctional MC1-R (18). tions have been reported in several studies (13, 25Ð28) this is the Initially, we monitored the basal conditions in the recessive yel- first study that the nonselective peptide HP228 has been shown to low (e/e) mouse. We have previously shown that MC3-R is ex- inhibit PMN migration, adding HP288 to the growing list of mela- pressed on mouse resident peritoneal M␾ by RT-PCR and Western nocortins able to down-regulate experimental inflammation. Sim- blotting (13, 16) and on rat knee joint M␾ by electron microscopy ilarly to ␣-MSH, HP228 fully retained its anti-inflammatory effi- (17). Thus, we confirmed first the presence of MC3-R mRNA in cacy in recessive yellow (e/e) mice.

␥ ␤ a Table I. Effect of 2-MSH on MSU crystal stimulated KC and IL-1 release in vitro

Mice Agonist Antagonist KC (pg/ml) IL-1␤ (pg/ml)

Wild type PBS PBS 390 Ϯ 92 355 Ϯ 24 ء Ϯ ء Ϯ ␥ 2-MSH PBS 188 12 142 11 PBS SHU9119 438 Ϯ 94 307 Ϯ 14 ␥ Ϯ Ϯ 2-MSH SHU9119 494 143 339 23

Recessive yellow (e/e) PBS PBS 109 Ϯ 11 398 Ϯ 34 ء Ϯ Ϯ ␥ 2-MSH PBS 81 6 203 12 PBS SHU9119 210 Ϯ 63 394 Ϯ 27 ␥ Ϯ Ϯ 2-MSH SHU9119 137 23 343 24 a ␮ ␾ ϫ 6 ␮ ␥ ␾ PBS or SHU9119 (9 M) were added to adherent M (5 10 ) prepared from wild-type or recessive yellow (e/e) mice, 10 min prior to PBS or 95 M 2-MSH. M were stimulated 15 min later with 1 mg/ml MSU crystals. Supernatants were removed 2 h later and cell-free aliquots were analyzed for chemokine and cytokine content using speciﬁc ELISA. Data are mean Ϯ SE of three or four determinations. .p Ͻ 0.05 vs relevant PBS control ,ء The Journal of Immunology 3329

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