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Histamine Released from Epidermal Keratinocytes Plays a Role in α-Melanocyte–Stimulating Hormone-Induced Itching in Mice

2015; Elsevier BV; Volume: 185; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2015.07.015

1525-2191

Kyoko Shimizu, Tsugunobu Andoh, Yoko Yoshihisa, Tadamichi Shimizu,

Olfactory and Sensory Function Studies

Sunburn, wound repair, and chronic renal failure with hemodialysis are usually accompanied by both pigmentation and itching. Proopiomelanocortin-derived α-melanocyte–stimulating hormone (α-MSH) is produced in response to external stimuli, such as UV irradiation, and is involved in cutaneous pigmentation. However, it is unclear whether α-MSH is also involved in the itching. We therefore investigated whether α-MSH elicited itch-related responses in mice. We found that an intradermal injection of α-MSH induced hind-paw scratching, an itch-related response, in mice. The α-MSH–induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone and the H1 histamine receptor antagonist terfenadine. In mast cell-deficient mice, α-MSH also elicited scratching, which was inhibited by terfenadine. The immunoreactivity for l-histidine decarboxylase, a key enzyme required for the production of histamine, histamine, and the melanocortin 1 and 5 receptors were shown in not only mast cells but also keratinocytes in murine skin. In addition to the expression of l-histidine decarboxylase and melanocortin 1 and 5 receptors, the mouse keratinocyte cell lines (Pam212) also showed immunoreactivity for l-histidine decarboxylase, histamine, and melanocortin 1 and 5 receptors. The application of α-MSH induced the release of histamine from Pam212 cells. These findings indicate that α-MSH may play an important role in the itching associated with pigmented cutaneous lesions and that the histamine released from keratinocytes is involved in this α-MSH–induced itching. Sunburn, wound repair, and chronic renal failure with hemodialysis are usually accompanied by both pigmentation and itching. Proopiomelanocortin-derived α-melanocyte–stimulating hormone (α-MSH) is produced in response to external stimuli, such as UV irradiation, and is involved in cutaneous pigmentation. However, it is unclear whether α-MSH is also involved in the itching. We therefore investigated whether α-MSH elicited itch-related responses in mice. We found that an intradermal injection of α-MSH induced hind-paw scratching, an itch-related response, in mice. The α-MSH–induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone and the H1 histamine receptor antagonist terfenadine. In mast cell-deficient mice, α-MSH also elicited scratching, which was inhibited by terfenadine. The immunoreactivity for l-histidine decarboxylase, a key enzyme required for the production of histamine, histamine, and the melanocortin 1 and 5 receptors were shown in not only mast cells but also keratinocytes in murine skin. In addition to the expression of l-histidine decarboxylase and melanocortin 1 and 5 receptors, the mouse keratinocyte cell lines (Pam212) also showed immunoreactivity for l-histidine decarboxylase, histamine, and melanocortin 1 and 5 receptors. The application of α-MSH induced the release of histamine from Pam212 cells. These findings indicate that α-MSH may play an important role in the itching associated with pigmented cutaneous lesions and that the histamine released from keratinocytes is involved in this α-MSH–induced itching. Itch is an unpleasant sensation associated with the immediate desire to scratch, thereby making the cutaneous symptoms worse. Several skin and general conditions are associated with both pigmentation and itch. In addition, itch associated with sunburn is widely recognized to be the result of an inflammatory reaction to UV irradiation, which increases the amount of itch and erythema, after which the healing process results in later skin pigmentation.1Han A. Maibach H.I. Management of acute sunburn.Am J Clin Dermatol. 2004; 5: 39-47Crossref PubMed Scopus (42) Google Scholar Gilchrest et al2Gilchrest B.A. Soter N.A. Stoff J.S. Mihm Jr., M.C. The human sunburn reaction: histologic and biological studies.Am Acad Dermatol. 1981; 5: 411-422Abstract Full Text PDF PubMed Scopus (250) Google Scholar reported that the histamine level rises immediately after the onset of UV-induced erythema. Itch and cutaneous pigmentation also occur in association with chronic wounds and hypertrophic scars. Paul3Paul J. Characteristics of chronic wounds that itch.Adv Skin Wound Care. 2013; 26: 320-332Crossref PubMed Scopus (10) Google Scholar noted that wound-related itch is more frequently observed in patients with severe wounds. Itch may also be caused by dry skin or serious internal diseases, such as chronic renal failure that requires hemodialysis, which is usually accompanied by cutaneous pigmentation. Taken together, it is still unclear if the underlying mechanisms of itch in these pruritic diseases involve the interaction between cutaneous pigmentation and itch. α-Melanocyte–stimulating hormone (α-MSH) is one of the neuropeptides that is generated through the cleavage of a precursor protein called proopiomelanocortin, which is produced through the production of corticotropin-releasing hormone, also known as corticotropin-releasing factor, after various stressors, such as UV irradiation.4Slominski A. Tobin D.J. Shibahara S. Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation.Physiol Rev. 2004; 84: 1155-1228Crossref PubMed Scopus (1471) Google Scholar, 5Slominski A. Wortsman J. Luger T. Paus R. Solomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress.Physiol Rev. 2000; 80: 979-1020Crossref PubMed Scopus (634) Google Scholar, 6Slominski A.T. Zmijewski M.A. Zbytek B. Tobin D.J. Theoharides T.C. Rivier J. Key role of CRF in the skin stress response system.Endocr Rev. 2013; 34: 827-884Crossref PubMed Scopus (284) Google Scholar, 7Slominski A.T. Zmijewski M.A. Skobowiat C. Zbytek B. Slominski R.M. Steketee J.D. Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system.Adv Anat Embryol Cell Biol. 2012; 212 (vii, 1-115): vPubMed Google Scholar α-MSH receptors are known as melanocortin receptors (MC1R, MC3R, MC4R, and MC5R).4Slominski A. Tobin D.J. Shibahara S. Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation.Physiol Rev. 2004; 84: 1155-1228Crossref PubMed Scopus (1471) Google Scholar These receptors belong to the G protein-coupled receptor family, and the activation of these receptors increases the production of cAMP.8Cooray S.N. Clark A.J. Melanocortin receptors and their accessory proteins.Mol Cell Endocrinol. 2011; 331: 215-221Crossref PubMed Scopus (61) Google Scholar In addition, it was also reported that α-MSH increases intracellular-free Ca2+ concentration in human embryonic kidney cells that express these melanocortin receptors.9Mountjoy K.G. Kong P.L. Taylor J.A. Willard D.H. Wilkison W.O. Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.Physiol Genomics. 2011; 5: 11-19Google Scholar In the skin, MC1R and MC5R are mainly expressed.10Böhm M. Luger T.A. Tobin D.J. García-Borrón J.C. Melanocortin receptor ligands: new horizons for skin biology and clinical dermatology.J Invest Dermatol. 2006; 126: 1966-1975Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar α-MSH produced by external stimuli, such as UV irradiation, induces cutaneous pigmentation through the activation of MC1R.4Slominski A. Tobin D.J. Shibahara S. Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation.Physiol Rev. 2004; 84: 1155-1228Crossref PubMed Scopus (1471) Google Scholar, 6Slominski A.T. Zmijewski M.A. Zbytek B. Tobin D.J. Theoharides T.C. Rivier J. Key role of CRF in the skin stress response system.Endocr Rev. 2013; 34: 827-884Crossref PubMed Scopus (284) Google Scholar, 7Slominski A.T. Zmijewski M.A. Skobowiat C. Zbytek B. Slominski R.M. Steketee J.D. Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system.Adv Anat Embryol Cell Biol. 2012; 212 (vii, 1-115): vPubMed Google Scholar, 10Böhm M. Luger T.A. Tobin D.J. García-Borrón J.C. Melanocortin receptor ligands: new horizons for skin biology and clinical dermatology.J Invest Dermatol. 2006; 126: 1966-1975Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Oren M. Bartek J. The sunny side of p53.Cell. 2007; 128: 826-828Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar α-MSH is also increased in plasma of patients with chronic hemodialysis12Airaghi L. Garofalo L. Cutuli M.G. Delgado R. Carlin A. Demitri M.T. Badalamenti S. Graziani G. Lipton J.M. Catania A. Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.Nephrol Dial Transplant. 2000; 15: 1212-1216Crossref PubMed Scopus (28) Google Scholar and in epidermal keratinocytes during cutaneous wound repair.13Muffley L.A. Zhu K.Q. Engrav L.H. Gibran N.S. Hocking A.M. Spatial and temporal localization of the melanocortin 1 receptor and its ligand α-melanocyte-stimulating hormone during cutaneous wound repair.J Histochem Cytochem. 2011; 59: 278-288Crossref PubMed Scopus (41) Google Scholar However, no previous reports indicate whether α-MSH is also involved in the itching that results from these factors. Here, we investigated whether α-MSH elicited itch in mice. Furthermore, we performed a series of experiments to elucidate the mechanism underlying the development of α-MSH–induced itch, focusing on the involvement of histamine, which is known to play an important role in the pathogenesis of itch. Male ICR mice (4 to 9 weeks old), mast cell-deficient WBB6F1 W/Wv (6 to 9 weeks old), and the normal littermates (WBB6F1+/+, 6 to 9 weeks old) were used in this study. These mice were purchased from Japan SLC (Hamamatsu, Japan). They were housed in a room with controlled temperature (21°C to 23°C), humidity (45% to 65%), and light (7:00 AM to 7:00 PM) conditions. Food and water were available ad libitum. Procedures for animal experiments were approved by the Committee for Animal Experiments at the University of Toyama. The murine epidermal cell line (Pam212) was cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin under standard cell culture conditions (37°C, 5% CO2 in a humidified incubator). In a part of the experiment, siRNA-treated cells were used. siRNAs as a control, MC1R and MC5R, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These siRNAs were transfected with Lipofectamin RNAi MAX reagent (Life Technologies, Carlsbad, CA). The transfection was performed according to the manufacturer's protocol (Life Technologies). α-MSH was purchased from Peptide Institute, Inc. (Osaka, Japan). For in vivo experiments, α-MSH was dissolved in physiologic saline and was injected intradermally in a volume of 50 μL into the rostral skin of mice. For in vitro experiments, α-MSH was dissolved in Opti-MEM (Thermo Fisher Scientific Inc., Waltham, MA). Naltrexone hydrochloride (Sigma-Aldrich, St. Louis, MO) was dissolved in physiologic saline and injected subcutaneously 15 minutes before α-MSH injection. Terfenadine (Sigma-Aldrich) was suspended in 0.5% sodium carboxy methylcellulose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and was administered 30 minutes before α-MSH injection. SQ 22,536 (Tocris Bioscience, Bristol, UK) and EGTA (Dojindo Laboratories, Kumamoto, Japan) were dissolved in dimethyl sulfoxide and diluted with Opti-MEM (final concentration dimethyl sulfoxide, 0.1%). These agents were treated 30 minutes before the application of α-MSH. The day before the experiment, the hair was clipped over the rostral part of the mouse back. Before behavioral observation, the animals were placed individually in an acrylic cage composed of four compartments (13 × 9 × 35 cm) for at least 1 hour for acclimation. Immediately after intradermal injection, mice were put back into the same cells, and their behaviors were recorded with the use of a digital video camera (HDC-TM25; Panasonic Co., Osaka, Japan) for 1 hour with personnel kept out of the observation room. Playback of the digital recording allowed for counting of injection site scratching by the hind paw. The series of movements scratched several times for about 1 second were considered as one bout of scratching.14Kuraishi Y. Nagasawa T. Hayashi K. Satoh M. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice.Eur J Pharmacol. 1995; 275: 229-233Crossref PubMed Scopus (360) Google Scholar Under anesthesia with pentobarbital (80 mg/kg, intraperitoneal), mice were transcardially perfused with phosphate-buffered saline (PBS) and then 4% paraformaldehyde. The skin of the rostral back was isolated, postfixed with 4% paraformaldehyde, and immersed in 30% sucrose solution for 2 days. The tissue was embedded in Tissue-Tek O.C.T. Compound (Sakura Fineteck Co., Ltd., Tokyo, Japan) and kept at −80°C until use. The frozen samples were sectioned at 20 μm with a cryostat (Leica, Wetzlar, Germany). In the keratinocyte cell line Pam212, cells cultured on glass-bottomed dishes were washed twice with PBS and fixed with 4% paraformaldehyde. After being washed three times with PBS, the sections or cells were treated with 0.3% Triton X-100 in PBS and then with 0.25% fetal bovine serum to block nonspecific immunoglobulin binding. The sections or cells were treated with the first antibodies at a dilution of 1:100 at 4°C overnight; the antibodies used were rabbit antibodies against histidine decarboxylase (HDC; Santa Cruz Biotechnology Inc.) and histamine (Abcam, Cambridge, UK), and goat antibodies against MC1R, MC5R, and mast cell protease 5 (Santa Cruz Biotechnology Inc.). After washing, the preparations were incubated with Alexa Fluor 555-conjugated anti-goat IgG (Life Technologies) for 1 hour at room temperature. The sections and cells were rinsed in PBS after each treatment. Finally, the sections and cells were counterstained with DAPI. Immunofluorescence was visualized with the use of a laser scanning confocal microscope (Leica Microsystems, Tokyo, Japan) or a fluorescent microscope (Olympus Co., Tokyo, Japan). After scanning, the slide glass was washed with PBS. The skin section was stained with toluidine blue and observed with the use of light microscope (Olympus Co.). For a portion of the immunostaining, we used the antibody preabsorbed with the antigen peptides as a negative control. The antigen peptides for HDC, MC1R, and MC5R were purchased from Santa Cruz Biotechnology, Inc. The preparation of the antibody preabsorbed with the antigen peptides was performed according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc.). Total RNA was extracted from cultured Pam212 cells. The cell samples were lyzed with the TRIzol reagent (Invitrogen) for RNA preparation. Total RNA (0.4 μg/sample) was used for cDNA synthesis with oligo (dT)16 primers and reverse transcriptase (Reverscript III; Wako Pure Chemical Industries Ltd.). cDNA was amplified with the use of the following primers: MC1R, 5′-GCCCACATGTTCACGAGAGC-3′ (forward) and 5′-AGTTACCCTTTCTCCTGGCCC-3′ (reverse); MC5R, 5′-AAATCCGATGCCAAGAAGTG-3′ (forward) and 5′-GGTAGCGCAAGGCATAGAAG-3′ (reverse); HDC, 5′-AGCACAAGCTGTCGTCCTTT-3′ (forward) and 5′-GTGGATCACGAAGACCCTGT-3′ (reverse). Glyceraldehyde 3-phosphate dehydrogenase was used as a positive control. The primers used for glyceraldehyde 3-phosphate dehydrogenase were 5′-ACCCAGAAGACTGTGGAT-3′ (forward) and 5′-TCGTTGAGGGCAATGCCA-3′ (reverse). The cycling conditions were 5 minutes at 94°C, 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 30 seconds at 72°C, and 7 minutes at 72°C. After PCR, the amplified products were analyzed by 2% agarose gel electrophoresis. Proteins were extracted from cultured Pam212 cells with a lysis buffer [20 mm Tris-HCl (pH 7.5), 137 mm NaCl, 1% NP-40, 10% glycerol, 1 mm phenylmethyl sulfonyl fluoride, 10 μg/mL aprotinin, and 1 μg/mL leupeptin]. The protein lysates were denatured at 95°C for 5 minutes and were applied to an SDS-polyacrylamide gel for electrophoresis and transferred to nitrocellulose membranes. After blocking with 1% skim milk in PBS that contained 0.1% Tween 20, the membrane was reacted with goat polyclonal anti-HDC, anti-MC1R, anti-MC5R, and anti–β-actin antibodies (dilution 1:1000 each), respectively, overnight at 4°C. After washing with PBS that contained 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-labeled donkey anti-goat IgG antibody (dilution 1:1000; Bethyl Laboratories, Inc., Montgomery, TX) for 2 hours at room temperature. These membranes were then scanned with the lumino image analyzer Image Quant LAS-4000 (Fujifilm, Tokyo, Japan). α-MSH (200 μmol/L) was dissolved in Opti-MEM, as a reaction medium, and applied to the cells. Five and 10 minutes later, the reaction medium was collected. In the experiments that used the cells treated with siRNA, SQ 22,536, or EGTA, the reaction medium was collected 5 minutes after α-MSH application. The concentration of the released histamine was measured with the histamine enzyme immunoassay kit (Bertin Pharma, Montigny-le-Bretonneux, France) according to the manufacturer's recommendation. The protein of Pam212 cells was extracted by the application of 1% Triton X-100, and the concentration was measured by using protein assay reagent (Bio-Rad, Hercules, CA). The released histamine concentration in the reaction medium was normalized to the protein concentration of the Pam212 cells. All values are expressed as the means ± SEM of the respective test or control group. Statistical significance was evaluated with either Student's t-test or Holm-Šidák multiple comparisons. P < 0.05 was considered significant. An intradermal injection of α-MSH (100 nmol/L per site) induced marked scratching of the injected site by the hind paws compared with the vehicle. The effect peaked in the initial 10-minute period and almost completely subsided within 60 minutes (Figure 1A). The administration of α-MSH at intradermal doses of 10 to 100 nmol per site elicited scratching in a dose-dependent manner (Figure 1B). In the following in vivo experiments, a dose of 100 nmol per site of α-MSH was used. Subcutaneous pretreatment with 1 mg/kg selective μ-opioid receptor antagonist, naltrexone hydrochloride,15Andoh T. Kuwazono T. Lee J.B. Kuraishi Y. Gastrin-releasing peptide induces itch-related responses through mast cell degranulation in mice.Peptides. 2011; 32: 2098-2103Crossref PubMed Scopus (36) Google Scholar inhibited the α-MSH–induced scratching (Figure 1C). Oral pretreatment with 30 mg/kg H1 histamine receptor antagonist, terfenadine,16Ohtsuka E. Kawai S. Ichikawa T. Nojima H. Kitagawa K. Shirai Y. Kamimura K. Kuraishi Y. Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.Jpn J Pharmacol. 2001; 86: 97-105Crossref PubMed Scopus (64) Google Scholar also suppressed the α-MSH–induced scratching (Figure 2A). An intradermal injection of α-MSH elicited significant scratching in both mast cell-deficient mice (WBB6F1 W/Wv) and their normal littermates (WBB6F1+/+), compared with vehicle-injected mice (Figure 2B). The number of scratches was almost the same between these mice. Interestingly, the administration of 30 mg/kg H1 histamine receptor antagonist terfenadine significantly inhibited the α-MSH–induced scratching in both mast cell-deficient mice (WBB6F1 W/Wv) and their normal littermates (WBB6F1+/+), compared with vehicle-treated mice (Figure 2C). Immunohistochemical staining showed that HDC, MC1R, and MC5R were mainly expressed in both epidermal keratinocytes and dermal cells stained with toluidine blue (Figure 3A). Histamine was similarly expressed in both epidermal keratinocytes and dermal cells seen by the immunoreactivity of mast cell protease 5 (Figure 3B). Pam212 cells showed immunoreactivity for HDC, MC1R, MC5R, and histamine (Figure 4A). In addition, RT-PCR and Western blot analysis also showed the expression of HDC, MC1R, and MC5R in Pam212 cells (Figure 4, B and C). In the present in vivo study, an intradermal injection of α-MSH (100 nmol/50 μL = 2 mmol/L) elicited scratching (Figure 1A). However, α-MSH (10 nmol/50 μL = 200 μmol/L) led to a slight, but not significant, increase in scratching, compared with the vehicle-injected group (Figure 1B). Because α-MSH acts directly on the cells in vitro, α-MSH was administered at a final concentration of 200 μmol/L in these cell culture studies. Because α-MSH–induced scratching was observed mainly during the initial 10-minute period, the release of histamine was measured for 10 minutes after α-MSH stimulation. Treatment with 200 μmol/L α-MSH significantly increased the concentration of histamine in the culture medium 5 and 10 minutes after the application of α-MSH, compared with the medium of cells treated without α-MSH (Figure 4D). The effect peaked after the initial 5-minute period (Figure 4D). α-MSH did not elicit the release of histamine in the cells treated with MC1R or MC5R siRNA (Figure 5A and Supplemental Figure S1). In addition, 100 μmol/L adenylyl cyclase inhibitor SQ 22,53617Turcato S. Clapp L.H. Effects of the adenylyl cyclase inhibitor SQ22536 on iloprost-induced vasorelaxation and cyclic AMP elevation in isolated guinea-pig aorta.Br J Pharmacol. 1999; 126: 845-847Crossref PubMed Scopus (64) Google Scholar (Supplemental Figure S218Andoh T. Kuraishi Y. Antipruritic mechanisms of topical E6005, a phosphodiesterase 4 inhibitor: inhibition of responses to proteinase-activated receptor 2 stimulation mediated by increase in intracellular cyclic AMP.J Dermatol Sci. 2014; 76: 206-213Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and 1 mmol/L calcium chelator EGTA19Sun F.C. Shyu H.Y. Lee M.S. Lee M.S. Lai Y.K. Involvement of calcium-mediated reactive oxygen species in inductive GRP78 expression by geldanamycin in 9L rat brain tumor cells.Int J Mol Sci. 2013; 14: 19169-19185Crossref PubMed Scopus (6) Google Scholar also inhibited the release of histamine induced by α-MSH (Figure 5B). Intradermal injections of α-MSH into the rostral part of the skin elicited hind-paw scratching of the injection site in mice. The α-MSH–induced scratching was inhibited by treatment with the μ-opioid receptor antagonist naltrexone. It was reported that μ-opioid receptor antagonists inhibit scratching induced by pruritogens15Andoh T. 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A controlled trial of naloxone infusions for the pruritus of chronic cholestasis.Gastroenterology. 1992; 102: 544-549PubMed Google Scholar However, μ-opioid receptor antagonists attenuate itch-related but not pain-related behavior.30Maekawa T. Yamaguchi-Miyamoto T. Nojima H. Kuraishi Y. Effects of naltrexone on spontaneous itch-associated responses in NC mice with chronic dermatitis.Jpn J Pharmacol. 2002; 90: 193-196Crossref PubMed Scopus (41) Google Scholar, 31Nojima H. Simons C.T. Cuellar J.M. Carstens M.I. Moore J.A. Carstens E. Opioid modulation of scratching and spinal c-fos expression evoked by intradermal serotonin.J Neurosci. 2003; 23: 10784-10790PubMed Google Scholar, 32Akiyama T. Carstens M.I. Carstens E. Differential itch- and pain-related behavioral responses and μ-opoid modulation in mice.Acta Derm Venereol. 2010; 90: 575-581Crossref PubMed Scopus (65) Google Scholar, 33Gotoh Y. Andoh T. Kuraishi Y. Noradrenergic regulation of itch transmission in the spinal cord mediated by α-adrenoceptors.Neuropharmacology. 2011; 61: 825-831Crossref PubMed Scopus (39) Google Scholar Taking into account these findings in humans and rodents, our results showing that the action of α-MSH was inhibited by μ-opioid receptor antagonists are consistent with the idea that α-MSH–induced scratching is because of pruritogenic, but not algesiogenic, stimulation of the treated skin. In this study, we also found that α-MSH–induced scratching was inhibited partially, but significantly, by treatment with a H1 histamine receptor antagonist terfenadine at a dose that almost completely inhibited the histamine-induced scratching,16Ohtsuka E. Kawai S. Ichikawa T. Nojima H. Kitagawa K. Shirai Y. Kamimura K. Kuraishi Y. Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.Jpn J Pharmacol. 2001; 86: 97-105Crossref PubMed Scopus (64) Google Scholar suggesting that histamine is involved in α-MSH–induced scratching. α-MSH is involved in the pigmentation due to sunburn,10Böhm M. Luger T.A. Tobin D.J. García-Borrón J.C. Melanocortin receptor ligands: new horizons for skin biology and clinical dermatology.J Invest Dermatol. 2006; 126: 1966-1975Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Oren M. Bartek J. The sunny side of p53.Cell. 2007; 128: 826-828Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar hemodialysis,12Airaghi L. Garofalo L. Cutuli M.G. Delgado R. Carlin A. Demitri M.T. Badalamenti S. Graziani G. Lipton J.M. Catania A. Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.Nephrol Dial Transplant. 2000; 15: 1212-1216Crossref PubMed Scopus (28) Google Scholar and wound repair.13Muffley L.A. Zhu K.Q. Engrav L.H. Gibran N.S. Hocking A.M. Spatial and temporal localization of the melanocortin 1 receptor and its ligand α-melanocyte-stimulating hormone during cutaneous wound repair.J Histochem Cytochem. 2011; 59: 278-288Crossref PubMed Scopus (41) Google Scholar Antihistamines are effective for treating pruritus in the patients with the above-mentioned causes of pigmentation.1Han A. Maibach H.I. Management of acute sunburn.Am J Clin Dermatol. 2004; 5: 39-47Crossref PubMed Scopus (42) Google Scholar, 34Matsui C. Ida M. Hamada M. Morohashi M. Hasegawa M. Effects of azelastine on pruritus and plasma histamine levels in hemodialysis patients.Int J Dermatol. 1994; 33: 868-871Crossref PubMed Scopus (22) Google Scholar, 35Matheson J.D. Clayton J. Muller M.J. The reduction of itch during burn wound healing.J Burn Care Rehabil. 2001; 22: 76-81Crossref PubMed Scopus (64) Google Scholar Therefore, these reports support our findings. It is well known that histamine is mainly produced by the mast cells in skin. However, in this study, α-MSH elicited scratching in both mast cell-deficient mice and their normal littermates. Interestingly, terfenadine inhibited the α-MSH–induced scratching in both the mast cell-deficient mice and normal littermates. Taken together, these findings suggest that histamine is involved in the α-MSH–induced scratching, and mast cells may not contribute to the release of histamine involved in this scratching. HDC is a key enzyme in the biosynthesis of histamine.36Ichikawa A. Sugimoto Y. Tanaka S. Molecular biology of histidine decarboxylase and prostaglandin receptors.Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86: 848-866Crossref PubMed Scopus (25) Google Scholar In this study, we observed the immunoreactivity to HDC and histamine not only in mast cells but also keratinocytes in mouse skin. In addition, the mouse keratinocyte cell line, Pam212, also showed the immunoreactivity to HDC and histamine. Human epidermal cells and keratinocytes also express HDC.37Inami Y. Andoh T. Sasaki A. Kuraishi Y. Topical surfactant-induced pruritus: involvement of histamine released from epidermal keratinocytes.J Pharmacol Exp Ther. 2013; 344: 459-466Crossref PubMed Scopus (26) Google Scholar, 38Gutowska-Owsiak D. Greenwald L. Watson C. Selvakumar T.A. Wang X. Ogg G.S. The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin.Br J Dermatol. 2014; 171: 771-778Crossref PubMed Scopus (20) Google Scholar We also detected low (54 kDa)- and high (74 kDa)-molecular weight protein bands of HDC in the Pam212 cells; 74-kDa HDC is a precursor protein that exhibits a low enzyme activity. Furthermore, 74-kDa HDC is post-translationally cleaved to a 53- to 55-kDa species, and histamine is synthesized mainly by a 53- to 55-kDa HDC.36Ichikawa A. Sugimoto Y. Tanaka S. Molecular biology of histidine decarboxylase and prostaglandin receptors.Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86: 848-866Crossref PubMed Scopus (25) Google Scholar Treatment with α-MSH induced histamine release from Pam212 cells. Thus, keratinocytes may play an important role in the production of histamine, and this appears to be related to the α-MSH–induced scratching. In mast cells, histamine is stored in granules and is released by several types of stimuli (such as immune reactions).36Ichikawa A. Sugimoto Y. Tanaka S. Molecular biology of histidine decarboxylase and prostaglandin receptors.Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86: 848-866Crossref PubMed Scopus (25) Google Scholar Macrophages also express HDC and spontaneously release histamine without it being stored intracellularly (these cells do not contain histamine-storing granules).36Ichikawa A. Sugimoto Y. Tanaka S. Molecular biology of histidine decarboxylase and prostaglandin receptors.Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86: 848-866Crossref PubMed Scopus (25) Google Scholar, 39Hirasawa N. Murakami A. Ohuchi K. Expression of 74-kDa histidine decarboxylase protein in a macrophage-like cell line RAW 264.7 and inhibition by dexamethasone.Eur J Pharmacol. 2001; 418: 23-28Crossref PubMed Scopus (23) Google Scholar Because keratinocytes also do not have histamine-storing granules, the histamine would be released immediately after its biosynthesis. In this study, keratinocytes slightly produced histamine. A recent report has shown histamine induces proliferation in keratinocytes through H4 histamine receptors.40Glatzer F. Gschwandtner M. Ehling S. Rossbach K. Janik K. Klos A. Bäumer W. Kietzmann M. Werfel T. Gutzmer R. Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.J Allergy Clin Immunol. 2013; 132: 1358-1367Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Thus, in a normal skin condition, histamine is involved in the proliferation in keratinocyte as one of the actions. However, overproduction of histamine induced by several stimulations, such as α-MSH (present study) and surfactant,37Inami Y. Andoh T. Sasaki A. Kuraishi Y. Topical surfactant-induced pruritus: involvement of histamine released from epidermal keratinocytes.J Pharmacol Exp Ther. 2013; 344: 459-466Crossref PubMed Scopus (26) Google Scholar may be involved in the induction of itching. The application of α-MSH induced the release of histamine from the mouse keratinocytes cell line Pam212 in this study. Pam212 cells also expressed MC1R and MC5R receptors. The cells treated with siRNA for MC1R and MC5R showed no release of histamine after stimulation with α-MSH, suggesting that at least MC1R and MC5R are involved in α-MSH–induced histamine release. The mechanism underlying the production of histamine after the stimulation with α-MSH is still unclear. Miyazaki et al41Miyazaki T. Ohgoh M. Ohmori E. Yamamoto J. Emoto S. Yatsunami K. Ichikawa A. Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.Biochim Biophys Acta. 1992; 1133: 179-186Crossref PubMed Scopus (14) Google Scholar have shown that the activity of HDC was increased by N6,O2-dibutyryl cAMP plus Ca2+ ionophore A23187. The activation of MC1R and MC5R receptors by α-MSH increases both the production of cAMP8Cooray S.N. Clark A.J. Melanocortin receptors and their accessory proteins.Mol Cell Endocrinol. 2011; 331: 215-221Crossref PubMed Scopus (61) Google Scholar and the intracellular-free Ca2+ concentration.9Mountjoy K.G. Kong P.L. Taylor J.A. Willard D.H. Wilkison W.O. Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.Physiol Genomics. 2011; 5: 11-19Google Scholar Here, α-MSH–induced histamine release was inhibited by the adenylyl cyclase inhibitor, which suppressed cAMP production and a calcium chelator. Therefore, our findings suggest that increased cAMP (Supplemental Figure S2) and intracellular calcium levels may be involved in the production of histamine through melanocortin receptors. In the present study, the treatment with a H1 histamine receptor antagonist did not completely inhibit the α-MSH–induced scratching. The cutaneous distribution of melanocortin receptors (MC1R, MC3R, MC4R, and MC5R) for α-MSH is not fully understood. It is known that in the mouse dorsal root ganglia, MC1R, MC3R, and MC4R, but not MC5R, are expressed,42Tanabe K. Gamo K. Aoki S. Wada K. Kiyama H. Melanocortin receptor 4 is induced in nerve-injured motor and sensory neurons of mouse.J Neurochem. 2007; 101: 1145-1152Crossref PubMed Scopus (21) Google Scholar suggesting that MC1R, MC3R, and MC4R are presented on primary afferents. Thus, α-MSH may act directly via primary afferents to induce itch. Recently, it was reported that there are two main types of itch-related primary afferents; H1 histamine receptor-expressing neurons and mas-related G-coupled protein receptor A3-expressing neurons.43Akiyama T. Carstens E. Neural processing of itch.Neuroscience. 2013; 250: 697-714Crossref PubMed Scopus (188) Google Scholar A future study should be performed to determine the distribution of melanocortin receptors in these primary afferents. In conclusion, α-MSH is an itch mediator, and the histamine released from keratinocytes, but not mast cells, may be involved in this α-MSH–induced itching.