Melanocortin Receptors 1 and 5 Might Mediate Inhibitory Effects of |[alpha]|-Melanocyte-Stimulating Hormone on Antigen-Induced Chronic Allergic Skin Inflammation in IgE Transgenic Mice

TO THE EDITOR Atopic dermatitis (AD) is a chronic allergic disease in skin with severe irritation and inflammation, and most patients with AD show elevated plasma levels of IgE against large numbers of allergens. It is known that IgE is involved in type I allergy, which is an immediate hypersensitivity through mast cells with IgE. However, it is not clear how IgE affects chronic allergic diseases such as AD. Anti-IgE antibody (omalizumab) has been shown to improve symptoms in not only allergic rhinitis, which is a typical type I allergy disease, but also AD and chronic asthma, which are chronic allergic diseases (Sheinkopf et al., 2008). We focused on elucidating the mechanism for IgE-mediated chronic inflammation to find and establish a original treatment for chronic allergic diseases. In 2,4,6-trinitrophenol-specific IgE transgenic mice (TNP-IgE Tg mice), a single subcutaneous injection of a multivalent antigen such as TNP-conjugated ovalbumin (TNP11-OVA) to the ear induces tri-phase ear swelling (Matsuoka et al., 1999; Sato et al., 2003). The immediate-phase (within 1 hour after antigen challenge) and late-phase (within 6–10 hours after antigen challenge) ear swelling are typical responses of type I allergy, whereas the third-phase ear swelling (began 2 days later and peaked on days 3–4 after antigen challenge) especially reflects chronic IgE-mediated severe inflammation, which is induced by the multivalent antigen (Sato et al., 2003). In the histological change of the third-phase ear swelling, massive infiltration of inflammatory cells such as eosinophils, neutrophils, and hyperplastic epidermis with hyperkeratosis were observed. Anti-histamine drugs did not show any significant effect on the third-phase swelling, whereas Cyclosporine A inhibited swelling and cellular infiltration (Sato et al., 2003). Moreover, we showed that a topical steroid strongly inhibited swelling and cellular infiltration (Supplementary Figure S1 online). The third-phase ear swelling in TNP-IgE Tg mice has hallmarks of chronic allergic inflammation in AD at both points of IgE-mediated intense chronic inflammation and effects of therapeutic drugs. To identify molecules that are responsible for IgE-mediated chronic inflammation, we investigated the gene expression profiles of antigen-administered ears in TNP-IgE Tg mice by Affymetrix GeneChip analysis (Santa Clara, CA). The thickness of TNP11-OVA-administered ears 24 hours after antigen challenge (just before the third-phase ear swelling) has the same degree as that of OVA-administered (control) ears (Sato et al., 2003). The histological inflammatory change was not observed in both ears; however, the TNP11-OVA-administered ears 24 hours after antigen challenge should change the expression of some genes, which may be responsible for inducing the third-phase inflammation. It is practicable to compare these gene expression profiles between the TNP11-OVA-administered and control ears within 24 hours after antigen challenge without the influence of inflammatory cells. We compared these gene expression profiles and found genes with different expression patterns. We selected genes that were expressed at higher levels in TNP11-OVA-administered ears than in control ears. Moreover, we focused on G protein–coupled receptors (GPCR), which have homology to those of humans. After these screening steps, we finally selected nine genes (Table 1). In this list, Mc1r and Mc5r, which are melanocortin receptors (MC-Rs), are expressed in both skin and immunocytes. Mc1r, Mc3r, Mc4r, and Mc5r have a common ligand, α-melanocyte-stimulating hormone (α-MSH), which has an anti-inflammatory effect on inflammatory animal models such as contact dermatitis (Grabbe et al., 1996; Brzoska et al., 2008). However, the mechanism of the anti-inflammatory effect of α-MSH is not clear because MC-Rs are widely distributed to various tissues, and selective agonists for each MC-Rs do not exist yet. To investigate the involvement of Mc1r and Mc5r in IgE-mediated chronic inflammation, we assessed the effect of α-MSH and the stable analog peptide [Nle4, D-Phe7]-α-MSH (NDP-α-MSH), which have several times higher agonist activity for Mc1r and Mc5r than α-MSH (Haskell-Luevano et al., 2001) on the third-phase ear swelling in TNP-IgE Tg mice. α-MSH and NDP-α-MSH suppressed the antigen-induced ear swelling in TNP-IgE Tg mice (Figure 1a). In particular, NDP-α-MSH significantly suppressed the third-phase ear swelling (31%) inhibitory rate in the area under the curve (ear thickness ( × 0.01 mm) × time (days 2–5)). In addition, α-MSH and prednisolone marginally suppressed the immediate-phase and third-phase ear swelling (21% and 20%, respectively, Figure 1b). α-MSH and NDP-α-MSH decreased to the nanomolar range in plasma within 2 hours after intravenous administration (data not shown). Therefore, if the agonists for Mc1r and Mc5r constantly exist with high concentration in plasma, they may show stronger inhibitory effects. Artuc et al. (2006) showed that human mast cells generate and secrete immunoreactive amounts of α-MSH, and that the generation and secretion of α-MSH were augmented by stimulation of the mast cells with an anti-IgE antibody (Artuc et al., 2006). TNP11-OVA administration in TNP-IgE Tg mice may strongly induce the generation and secretion of α-MSH from mast cells. In addition, NC/Nga mice, which develop AD-like dermatitis under conventional conditions, displayed an increase of the plasma levels of α-MSH and an increase of the expression of Mc1r and Mc5r in skin (Hiramoto et al., 2009). This report in NC/Nga mice indicated a similar result to ours, which is an increase of the expression of Mc1r and Mc5r on the ears in TNP-IgE Tg mice after TNP11-OVA administration. In conclusion, we identified the nine GPCR genes that are highly expressed just before IgE-mediated chronic inflammation in TNP-IgE Tg mice. The ligands for MC1R and MC5R showed inhibitory effects on IgE-mediated chronic allergic inflammation. This result indicates that MC1R and MC5R are involved, at least partially, in the regulation of IgE-mediated chronic inflammation. We previously showed that basophils are crucially involved in the third-phase ear swelling in TNP-IgE Tg mice (Mukai et al., 2005). To our knowledge, there is no available information about the expression of Mc1r and Mc5r on basophils; therefore, we assume that Mc1r and Mc5r indirectly inhibit IgE–basophil-mediated chronic inflammation. Further analysis of the mechanism of the other candidate genes could lead to the discovery of unknown mechanisms of IgE-mediated chronic allergic inflammation and a therapeutic target for treating chronic allergic inflammation. The authors state no conflict of interest. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper