The Melanocortin Agonist AP214 Exerts Anti-Inflammatory and Proresolving Properties
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.042
ISSN1525-2191
AutoresTrinidad Montero‐Melendez, Hetal Patel, Michael Seed, Søren Nielsen, Thomas E. N. Jonassen, Mauro Perretti,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoSynthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 μg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC1, mutant mice but lost in MC3 null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1β, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1β release was retained in MC1 mutant cells but was lost in MC3 null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC3 null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1β release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC3 is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists. Synthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 μg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC1, mutant mice but lost in MC3 null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1β, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1β release was retained in MC1 mutant cells but was lost in MC3 null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC3 null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1β release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC3 is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists. Inflammation is a localized defensive response of the body against pathogens and injury. Immune cells and soluble factors take part in this process to neutralize the injurious agent and initiate tissue repair to restore homeostasis. However, a loss of regulation of these mechanisms can prevent the final resolution of the inflammatory process, leading to a chronic pathologic status.1Serhan C.N. Savill J. Resolution of inflammation: the beginning programs the end.Nat Immunol. 2005; 6: 1191-1197Crossref PubMed Scopus (1794) Google Scholar, 2Nathan C. Ding A. Nonresolving inflammation.Cell. 2010; 140: 871-882Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar Chronic inflammation is extremely relevant in today's modern medicine; it is now recognized that chronic inflammation contributes significantly to the pathogenesis of the most important diseases of industrialized societies, including atherosclerosis, cancer, and asthma. Given that chronic inflammation requires long-term pharmacologic treatments, new therapies with a lower degree of adverse effects are preferred. We and others have proposed exploitation of endogenous anti-inflammatory pathways to develop new drugs, reasoning that this pharmacologic strategy, mimicking the natural course of resolving inflammation, would represent a novel therapeutic approach.3Getting S.J. Flower R.J. Perretti M. Inhibition of neutrophil and monocyte recruitment by endogenous and exogenous lipocortin 1.Br J Pharmacol. 1997; 120: 1075-1082Crossref PubMed Scopus (114) Google Scholar, 4Gonzalez-Rey E. Chorny A. Delgado M. Regulation of immune tolerance by anti-inflammatory neuropeptides.Nat Rev Immunol. 2007; 7: 52-63Crossref PubMed Scopus (196) Google Scholar, 5Levy B.D. Lukacs N.W. Berlin A.A. Schmidt B. Guilford W.J. Serhan C.N. Parkinson J.F. Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism.FASEB J. 2007; 21: 3877-3884Crossref PubMed Scopus (103) Google Scholar Among these natural anti-inflammatory and proresolving molecules are lipid mediators, such as lipoxins, resolvins, and protectins, as well as autacoids (eg, adenosine) and peptides, including annexin A1, galectin-1/9, and melanocortins (MCs). The role of the MC system in inflammation has been known for more than 2 decades.6Catania A. The melanocortin system in leukocyte biology.J Leukoc Biol. 2007; 81: 383-392Crossref PubMed Scopus (82) Google Scholar Scientific and clinical interest in MC has increased in recent years with the identification of specific receptors and the development of new analogues with improved properties over those of the natural agonist α-melanocyte–stimulating hormone (α-MSH). Natural peptides, especially corticotropin, α-MSH, and γ-MSH, and synthetic analogues are anti-inflammatory in several in vivo models. Getting et al reported the anti-inflammatory effects of the analogue D-Trp8–γ-MSH in a model of crystal-induced inflammation7Getting S.J. Lam C.W. Chen A.S. Grieco P. Perretti M. Melanocortin 3 receptors control crystal-induced inflammation.FASEB J. 2006; 20: 2234-2241Crossref PubMed Scopus (61) Google Scholar and, more recently, in an model of allergic inflammation.8Getting S.J. Riffo-Vasquez Y. Pitchford S. Kaneva M. Grieco P. Page C.P. Perretti M. Spina D. A role for MC3R in modulating lung inflammation.Pulm Pharmacol Ther. 2008; 21: 866-873Crossref PubMed Scopus (50) Google Scholar The same compound was also active in a model of ischemia-reperfusion injury9Leoni G. Patel H.B. Sampaio A.L. Gavins F.N. Murray J.F. Grieco P. Getting S.J. Perretti M. Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion.FASEB J. 2008; 22: 4228-4238Crossref PubMed Scopus (38) Google Scholar and in inflammatory arthritis.10Patel H.B. Bombardieri M. Sampaio A.L. D'Acquisto F. Gray M. Grieco P. Getting S.J. Pitzalis C. Perretti M. Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis.FASEB J. 2010; 24: 4835-4843Crossref PubMed Scopus (43) Google Scholar The α-MSH–derived C-terminal tripeptide KPV is anti-inflammatory in crystal-induced peritonitis11Getting S.J. Schioth H.B. Perretti M. Dissection of the anti-inflammatory effect of the core and C-terminal (KPV) α-melanocyte-stimulating hormone peptides.J Pharmacol Exp Ther. 2003; 306: 631-637Crossref PubMed Scopus (37) Google Scholar and in two models of inflammatory bowel disease.12Kannengiesser K. Maaser C. Heidemann J. Luegering A. Ross M. Brzoska T. Bohm M. Luger T.A. Domschke W. Kucharzik T. Melanocortin-derived tripeptide KPV has anti-inflammatory potential in murine models of inflammatory bowel disease.Inflamm Bowel Dis. 2008; 14: 324-331Crossref PubMed Scopus (40) Google Scholar New therapeutic strategies based on this line of research have also explored α-MSH gene therapy in a model of thioacetamide-induced hepatic fibrosis,13Wang C.H. Lee T.H. Lu C.N. Chou W.Y. Hung K.S. Concejero A.M. Jawan B. Electroporative α-MSH gene transfer attenuates thioacetamide-induced murine hepatic fibrosis by MMP and TIMP modulation.Gene Ther. 2006; 13: 1000-1009Crossref PubMed Scopus (20) Google Scholar affording tissue protection, and the oral administration of recombinant Lactobacillus casei, which secretes α-MSH, which reduced body weight loss, survival, clinical score, and myeloperoxidase (MPO) activity in a model of ulcerative colitis.14Yoon S.W. Lee C.H. Kim J.Y. Sung M.H. Poo H. Lactobacillus casei secreting α-MSH induces the therapeutic effect on DSS-induced acute colitis in Balb/c mice.J Microbiol Biotechnol. 2008; 18: 1975-1983PubMed Google Scholar In in vitro settings, MC agonists display anticytokine effects on macrophages15Sarkar A. Sreenivasan Y. Manna S.K. α-Melanocyte-stimulating hormone inhibits lipopolysaccharide-induced biological responses by downregulating CD14 from macrophages.FEBS Lett. 2003; 553: 286-294Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar and can induce the development of cytotoxic CD8+ T cells16Loser K. Brzoska T. Oji V. Auriemma M. Voskort M. Kupas V. Klenner L. Mensing C. Hauschild A. Beissert S. Luger T.A. The neuropeptide α-melanocyte-stimulating hormone is critically involved in the development of cytotoxic CD8+ T cells in mice and humans.PLoS One. 2010; 5: e8958Crossref PubMed Scopus (34) Google Scholar and CD25+CD4+ regulatory T cells.17Taylor A. Namba K. In vitro induction of CD25+ CD4+ regulatory T cells by the neuropeptide α-melanocyte stimulating hormone (α-MSH).Immunol Cell Biol. 2001; 79: 358-367Crossref PubMed Scopus (111) Google Scholar These anti-inflammatory effects are attributed to the interaction of MC peptides with the MC receptors MC1 and MC3, which are expressed in several immune cells and tissues.6Catania A. The melanocortin system in leukocyte biology.J Leukoc Biol. 2007; 81: 383-392Crossref PubMed Scopus (82) Google Scholar In fact, of the five MC receptors cloned,18Gantz I. Fong T.M. The melanocortin system.Am J Physiol Endocrinol Metab. 2003; 284: E468-E474Crossref PubMed Scopus (380) Google Scholar these two have been associated with the anti-inflammatory and tissue protective properties of MC peptides. In some experimental settings, α-MSH seems to control peripheral inflammation through activation of MC receptors in the brain.6Catania A. The melanocortin system in leukocyte biology.J Leukoc Biol. 2007; 81: 383-392Crossref PubMed Scopus (82) Google Scholar In any case, the main issue of which real MC receptor to target for anti-inflammatory drug discovery programs is still unsolved, possibly indicating the existence of regional and disease-specific expression patterns. In the present study, we investigated the anti-inflammatory properties of AP214, an α-MSH analogue with the addition of a long N-terminal lysine sequence yielding high affinity for MC1 and MC3 over MC5. AP214 displays tissue-protective effects against renal postreperfusion injury,19Doi K. Hu X. Yuen P.S. Leelahavanichkul A. Yasuda H. Kim S.M. Schnermann J. Jonassen T.E. Frokiaer J. Nielsen S. Star R.A. AP214, an analogue of α-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.Kidney Int. 2008; 73: 1266-1274Crossref PubMed Scopus (93) Google Scholar, 20Simmons M.N. Subramanian V. Crouzet S. Haber G.P. Colombo Jr, J.r. Ukimura O. Nielsen S. Gill I.S. α-Melanocyte stimulating hormone analogue AP214 protects against ischemia induced acute kidney injury in a porcine surgical model.J Urol. 2010; 183: 1625-1629Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar but the mechanisms behind these tissue-protective actions have not been elucidated. Besides studying the anti-inflammatory and anticytokine effects of AP214 in mice and in cells bearing inactive MC1 or nullified for MC3, we describe the proresolving nature of AP214 on the process of phagocytosis and efferocytosis, a new aspect of MC-based therapy. Male mice (age, 7 to 8 weeks; body weight, ∼30 g) were maintained on a standard chow pellet diet and had free access to water, with a 12-hour light-dark cycle. C57BL/6J wild-type (WT) mice were purchased from Charles River Laboratories (Wilmington, MA). MC1−/− mice (bearing an inactive mutant MC1)21Robbins L.S. Nadeau J.H. Johnson K.R. Kelly M.A. Roselli-Rehfuss L. Baack E. Mountjoy K.G. Cone R.D. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function.Cell. 1993; 72: 827-834Abstract Full Text PDF PubMed Scopus (774) Google Scholar and MC3−/− mice22Chen A.S. Marsh D.J. Trumbauer M.E. Frazier E.G. Guan X.M. Yu H. Rosenblum C.I. Vongs A. Feng Y. Cao L. Metzger J.M. Strack A.M. Camacho R.E. Mellin T.N. Nunes C.N. Min W. Fisher J. Gopal-Truter S. MacIntyre D.E. Chen H.Y. Van der Ploeg L.H. Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.Nat Genet. 2000; 26: 97-102Crossref PubMed Scopus (783) Google Scholar were kindly donated by Dr. Nancy Levin (Trega Biosciences, San Diego, CA) and Dr. Howard Y. Chen (Merck, Whitehouse Station, NJ), respectively. All the animal studies were approved by and performed under the guidelines of the Ethical Committee for the Use of Animals, Barts and The London School of Medicine and Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). AP214 has been produced by standard solid phase peptide synthesis by Bachem AG (Bubendorf, Switzerland). This MC agonist displays higher affinity for human MC1 and MC3 over MC5, for instance, with inhibition constants calculated from binding assays performed with transfected cells of 2.9 and 1.9 nmol/L, respectively.19Doi K. Hu X. Yuen P.S. Leelahavanichkul A. Yasuda H. Kim S.M. Schnermann J. Jonassen T.E. Frokiaer J. Nielsen S. Star R.A. AP214, an analogue of α-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.Kidney Int. 2008; 73: 1266-1274Crossref PubMed Scopus (93) Google Scholar, 20Simmons M.N. Subramanian V. Crouzet S. Haber G.P. Colombo Jr, J.r. Ukimura O. Nielsen S. Gill I.S. α-Melanocyte stimulating hormone analogue AP214 protects against ischemia induced acute kidney injury in a porcine surgical model.J Urol. 2010; 183: 1625-1629Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Herein, we quantify the ability of AP214 to activate the five MC receptors. Murine B16-F1 cells that express MC1, Cloudman M3 cells that express MC2, and CHO cells that express MC3, MC4, or MC5 were used to determine agonistic activity, testing AP214 in a concentration range of 10−10 to 10−4 mol/L. Incubation periods were optimized in preliminary experiments, selecting 10 minutes at 22°C for MC1 and MC2 transfected cells, 30 minutes at 27°C for MC3 and MC4 cells, and 30 minutes at 22°C for MC5 cells. Accumulation of cAMP was expressed as a percentage of the maximal control specific agonist response [(measured specific response/control specific agonist response) × 100]. [Nle4, D-Phe7]–α-MSH was used as control specific agonist for MC1, MC3, and MC4 cells; corticotropin was used as control specific agonist for MC2 transfected cells; and α-MSH was used as control specific agonist for MC5 cells. All the experiments were performed in triplicate. EC50 values were determined by nonlinear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting as follows: Y = D+[(A–D)/(1+[C/C50]nH)], where Y indicates specific response; D, minimum specific response; A, maximum specific response; C, compound concentration; C50, EC50; and nH, slope factor. Preliminary pharmacokinetics analyses in the rat reveal that AP214 (1 mg/kg given i.v.) displays a short half-life (∼15 minutes) with a relative large volume of distribution, suggesting distribution to tissues and/or extravascular sites. Indications for lower clearance in renal and hepatic blood flow would be consequent to restricted clearance; hence, the compound is likely bound to receptors and/or plasma proteins in a tight manner. Peritonitis was induced by the injection of 1 mg of zymosan (Zymosan A; Sigma-Aldrich, Poole, UK) i.p. in 0.5 mL of sterile PBS. Animals (six per group) were pretreated with compounds or vehicle (PBS) administered i.p. 30 minutes before zymosan injection. AP214 initially was studied with a large dose range (100–1600 μg/kg), and doses of 400 and 800 μg/kg were chosen for the experiments reported herein. Four hours later, mice were sacrificed by CO2 exposure, and the peritoneal cavities were washed with 4 mL of ice-cold PBS containing 3 mmol/L EDTA and 25 U/mL of heparin. Aliquots of lavage fluids (100 μL) were stained with Turk's solution (0.01% crystal violet in 3% acetic acid), and cells were counted using a Neubauer hemocytometer or were stained with fluorescein isothiocyanate–conjugated monoclonal antibody for Ly-6G/Gr1+ (clone RB6-8C5; 10 μg/mL final concentration) before flow cytometry using a BD FACSCalibur platform (BD Biosciences, Franklin Lakes, NJ). Isotype control fluorescein isothiocyanate rat IgG2b (clone eBR2a) and blocking antibody anti-mouse CD16/32 were used. All the antibodies were purchased from eBioscience (Hatfield, UK). Arthritis was induced by the i.p. injection of mice with 100 μL of serum from K/BxN arthritic mice at day 0 (a gift from Dr. Mohini Gray, University of Edinburgh, Edinburgh, Scotland). Mice (n = 6) were treated i.p. with compounds or vehicle (PBS) twice daily for 10 days. The development of the disease was monitored for 2 weeks by assessing paw volume using a plethysmometer (Ugo Basile, Comerio, Italy), body weight, disease incidence, and disease score (1 point was given for each digit or wrist joint that presented with erythema plus swelling; a maximum of 22 points could be scored per animal).10Patel H.B. Bombardieri M. Sampaio A.L. D'Acquisto F. Gray M. Grieco P. Getting S.J. Pitzalis C. Perretti M. Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis.FASEB J. 2010; 24: 4835-4843Crossref PubMed Scopus (43) Google Scholar Mice were injected with 1 mL of 2% Bio-Gel (Bio-Rad, Hemel Hempstead, UK) i.p., and 4 days later cells were collected by peritoneal lavage using 4 mL of 3 mmol/L EDTA in PBS and were plated in 24-well plates at a density of 0.5 × 106 cells per well in RPMI 1640 medium containing 10% fetal calf serum and 50 mg/mL of gentamicin. After 2 hours of incubation, nonadherent cells were removed, and 1% fetal calf serum medium containing compounds or vehicle was added for 30 minutes before stimulation with 25 μg/mL of zymosan. Supernatants [for enzyme-linked immunosorbent assay (ELISA) measurements] or cells (stored in RLT buffer for RNA extraction) were collected after 6 hours and were kept frozen at −80°C until use. Experiments using healthy volunteers were approved by the local research ethics committee (P/00/029 East London and The City Local Research Ethics Committee 1). Informed written consent was provided according to the Declaration of Helsinki. Blood was collected into 3.2% sodium citrate and was diluted 1:1 in RPMI 1640 medium before separation through a double-density gradient using Histopaque 10771 and 11191 (Sigma-Aldrich). After polymorphonuclear cell isolation and washing, contaminating erythrocytes were removed by hypotonic lysis. After a final washing step with RPMI 1640 medium, cells were resuspended at a concentration of 4 × 106 cells/mL in 10% fetal calf serum containing medium and were incubated overnight at 37°C, 5% CO2, to allow neutrophils to undergo spontaneous apoptosis (range, 40% to 60%). Phagocytosis of zymosan particles was studied in Bio-Gel–elicited murine macrophages. Macrophages were plated at a density of 1.5 × 106 cells per well in 48-well plates in a final volume of 0.5 mL. After pretreatment for 30 minutes with 10 nmol/L AP214 (in 1% fetal calf serum medium) or vehicle, zymosan was added at an approximate ratio of 1:5 (macrophage/zymosan, as established in preliminary analyses). At the reported time points, cells were washed three times with PBS to remove free particles and were analyzed by light microscopy, with three random fields per well (n = 3 wells per treatment) and counting an average of 250 cells per condition per time point. For in vitro studies, a plate-based MPO assay was performed as previously described.23Hart S.P. Dransfield I. Rossi A.G. Phagocytosis of apoptotic cells.Methods. 2008; 44: 280-285Crossref PubMed Scopus (49) Google Scholar Bio-Gel–elicited peritoneal macrophages were collected and plated at a density of 0.5 × 106 cells per well in 24-well plates. Cells were pretreated with AP214 or vehicle for 30 minutes in a final volume of 0.5 mL; apoptotic neutrophils were then added at a ratio of 1:2 (macrophage/neutrophil). After 1-hour incubation, cells were washed with cold PBS and were fixed for 30 minutes in 2.5% glutaraldehyde. Cells were then rinsed with PBS, and the MPO assay was performed by adding 0.1 mg/mL of dimethoxybenzidine (Sigma-Aldrich) and 0.03% (v/v) hydrogen peroxide. Cells were washed with PBS 1 hour later and were analyzed by light microscopy, with three random fields being acquired per well (n = 3 wells per treatment). More than 400 cells were counted per treatment point. To study in vivo phagocytosis by resident peritoneal macrophages, mice were injected i.p. with apoptotic CFSE-labeled human neutrophils (3 × 106 cells per mouse) 30 minutes after i.p. dosage with AP214 (400 or 800 μg/kg) or vehicle (PBS, 250 μL). Neutrophils were stained by incubation in CFSE (5 μmol/L in PBS) for 5 minutes. Mice were sacrificed 30 minutes later, and peritoneal cells were collected by lavage with 3 mL of ice-cold PBS containing 3 mmol/L EDTA. Phagocytosis was assessed by flow cytometry as previously described24Taylor P.R. Carugati A. Fadok V.A. Cook H.T. Andrews M. Carroll M.C. Savill J.S. Henson P.M. Botto M. Walport M.J. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.J Exp Med. 2000; 192: 359-366Crossref PubMed Scopus (620) Google Scholar using a BD FACSCalibur platform. Levels of IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 were quantified in the supernatants from cultured macrophages according to the manufacturer's instructions using ELISA Ready-SET-Go! (eBioscience) and a 1:5 dilution for IL-1β and a 1:10 dilution for TNF-α and IL-6. Intracellular cAMP was measured in macrophages treated for 30 minutes with α-MSH using the cAMP Biotrak enzyme immunoassay system (Amersham, Abingdon, UK). Total RNA was extracted using the RNeasy mini kit and genomic DNA was removed by on-column digestion using the RNase-Free DNase set, following the manufacturer's instructions (QIAGEN Ltd, Crawley, UK). cDNA was synthesized using 1 μg of pooled RNA from three or more replicates using SuperScript III Reverse Transcriptase (Invitrogen, Paisley, UK). Real-time PCR was performed in duplicate or triplicate, with 200 ng of cDNA per well, 1 μL of primers, and Power SYBR Green PCR master mix (Applied Biosystems, Warrington, UK), using the ABI Prism 7900HT sequence detection system (Applied Biosystems). The following QuantiTect primers (QIAGEN Ltd) were used: Gapdh (QT01658692), Anxa1 (QT00145915), Il1b (QT01048355), Il6 (QT00098875), Tnf (QT00104006), Tlr2 (QT00129752), Tlr4 (QT00259042), Ptgs2 (QT00165347), and Nos2 (QT00100275). A dissociation step was always included to confirm the absence of unspecific products. TaqMan gene expression assays (Applied Biosystems) were used to quantify MC receptor genes (Mc1r: Mm00434851_s1, Mc3r: Mm00434876_s1, Mc5r: Mm00442970_m1, and Gapdh: Mm99999915_g1). PCR reaction was performed with 100 ng of cDNA, 1 μL of primers, and 10 μL of 2X TaqMan gene expression master mix (Applied Biosystems). Fold change was calculated as 2-ΔΔCT using Gapdh as endogenous control. All the experiments were performed at least in triplicate and were repeated two to five times. The t-test, one-way analysis of variance followed by Dunnett's correction, two-way analysis of variance followed by Bonferroni correction, or the log-rank (Mantel-Cox) test was applied as appropriate, with P ≤ 0.05 considered statistically significant. The data obtained in the in vitro MC receptor functional assays revealed that AP214 is a full pan MC receptor agonist (ie, a full agonist against MC1, MC3, MC4, and MC5) and, similar to many other MCs, lacks agonist activity against MC2 (Table 1).Table 1Determination of the Functional Variables for AP214 Activation of Human MC ReceptorsMC1MC2MC3MC4MC5EC50 (mol/L)Max (%)EC50 (mol/L)Max (%)EC50 (mol/L)Max (%)EC50 (mol/L)Max (%)EC50 (mol/L)Max (%)6.6 × 10−8109 ± 32.4 × 10−438 ± 36.0 × 10−8104 ± 181.8 × 10−7116 ± 19.0 × 10−7102 ± 7Data report calculated EC50 values and maximal responses (Max) as determined using cells transfected with the indicated human MC receptors. AP214 was added for up to 30 minutes (see Materials and Methods), and accumulation of intracellular cAMP was determined by enzyme immunoassay. See Materials and Methods for details on cell types used, incubation periods, and temperatures. Maximal responses are given as mean ± SD. Values are representative of three separate analyses. Open table in a new tab Data report calculated EC50 values and maximal responses (Max) as determined using cells transfected with the indicated human MC receptors. AP214 was added for up to 30 minutes (see Materials and Methods), and accumulation of intracellular cAMP was determined by enzyme immunoassay. See Materials and Methods for details on cell types used, incubation periods, and temperatures. Maximal responses are given as mean ± SD. Values are representative of three separate analyses. This study began by testing the effect of AP214 in a model of sterile peritonitis,25Ajuebor M.N. Das A.M. Virag L. Flower R.J. Szabo C. Perretti M. Role of resident peritoneal macrophages and mast cells in chemokine production and neutrophil migration in acute inflammation: evidence for an inhibitory loop involving endogenous IL-10.J Immunol. 1999; 162: 1685-1691PubMed Google Scholar testing two different doses (400 and 800 μg/kg) in WT MC1−/− and MC3−/− mice. Injection of zymosan elicited a marked accumulation of neutrophils at 4 hours, ranging from 8 to 12 × 106 cells per mouse, which did not differ significantly across genotype. In WT mice, administration of AP214 produced a dose-dependent inhibitory effect (a 30% to 45% reduction) (Figure 1). Of interest, AP214 retained its full anti-inflammatory capacity in mice bearing an inactive MC1 (MC1−/−), producing essentially a similar degree of inhibition on cell trafficking as in WT animals, whereas its effects were lost in the absence of MC3 receptor (Figure 1). Table 2 reports the original values of neutrophil trafficking as measured in these experiments. On these bases, we then tested the effects AP214 on macrophages collected from these mice, using cytokine production as readouts.Table 2AP214 Inhibits Neutrophil Recruitment in the Zymosan Peritonitis ModelWTMC1−/−WTMC3−/−Vehicle23.29 ± 2.7224.5 ± 5.1613.29 ± 1.0910.10 ± 1.40AP214, 400 μg/kg15.09 ± 2.9116.25 ± 1.219.99 ± 1.5111.10 ± 1.61AP214, 800 μg/kg13.57 ± 3.5315.63 ± 3.376.54 ± 4.4710.80 ± 1.38Reported are the numbers of neutrophils infiltrating the peritoneal cavity 4 hours after injection of zymosan (1 mg). AP214 was given i.p. 30 minutes before zymosan. Data, expressed as 106 cells per cavity, are the mean ± SEM of six mice per group. See Figure 1 for analysis as percentage of inhibition. Open table in a new tab Reported are the numbers of neutrophils infiltrating the peritoneal cavity 4 hours after injection of zymosan (1 mg). AP214 was given i.p. 30 minutes before zymosan. Data, expressed as 106 cells per cavity, are the mean ± SEM of six mice per group. See Figure 1 for analysis as percentage of inhibition. To study the anti-inflammatory properties of AP214 at the cellular level, including assessing the potential role of the two MC receptors, Bio-Gel–elicited peritoneal macrophages were used. Thus, we initially validated the cells with respect to the MC system. RT-PCR demonstrated that these primary cells express the products of the mouse genes Mc1r, Mc3r, and Mc5r (Figure 2A). These receptors are functional because cell incubation with α-MSH led to intracellular accumulation of cAMP (Figure 2B). We then assessed the properties of two validated MC peptides on cytokine production. Figure 2C shows these data, with α-MSH and D-Trp8–γ-MSH abolishing zymosan-induced IL-1β release and marginally, yet significantly, reducing TNF-α release after stimulation with zymosan. In the control samples, we determined cell expression of mRNA for three MC receptors, Mc1r, Mc3r, and Mc5r, noting that on treatment with zymosan, there was a modest or null modulation in WT and MC1−/− macrophages (Figure 2D). In contrast, when MC3−/− cells were used, the “inflammatory status” produced by zymosan led to marked gene activation for Mc1r and Mc5r (Figure 2D). Once the model was characterized, Bio-Gel–elicited macrophages obtained from WT, MC1−/−, and MC3−/− mice were used to study whether MC stimulation by AP214 could affect zymosan-induced cytokine release. The release of IL-6, TNF-α, and IL-1β was significantly reduced by AP214 in WT cells and in MC1−/− macrophages (Figure 3). The effect on IL-1β reduction was particularly prominent in both cases. However, this potent inhibition of IL-1β release was lost in MC3−/− cells. In the latter cells, AP214 was still active in reducing IL-6 and TNF-α, suggesting additional mechanisms and/or
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