The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

Corneal transplantation is the most common form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain tissue transparency by pumping out excess water from the cornea. After transplantation, the rate of CEnC loss far exceeds that seen with normal aging, which can threaten sight. The underlying mechanisms are poorly understood. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide that is constitutively found in the aqueous humor with both cytoprotective and immunomodulatory effects. The curent study found high expression of melanocortin 1 receptor (MC1R), the receptor for α-MSH, on CEnCs. The effect of α-MSH/MC1R signaling on endothelial function and allograft survival in vitro and in vivo was investigated using MC1R signaling-deficient mice (Mc1re/e mice with a nonfunctional MC1R). Herein, the results indicate that in addition to its well-known immunomodulatory effect, α-MSH has cytoprotective effects on CEnCs after corneal transplantation, and the loss of MC1R signaling significantly decreases long-term graft survival in vivo. In conclusion, α-MSH/MC1R signaling is critical for CEnC function and graft survival after corneal transplantation.

MC1R gene polymorphisms are associated with dysfunctional immune responses and wound infection after burn injury.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is associated with organ failure and infectious complications after major burn injury. Recent evidence has linked melanocortin signaling to anti-inflammatory and wound-repair functions, with mutations in the melanocortin 1 receptor (MC1R) gene leading to increased inflammatory responses. Our group has previously demonstrated that MC1R gene polymorphisms are associated with postburn hypertrophic scarring. Thus, we hypothesized that MC1R single nucleotide polymorphisms (SNPs) would be associated with increased burn-induced SIRS and increased infectious complications. METHODS: We performed a retrospective cohort study of adults (>18 y of age) admitted to our burn center with >20% total body surface area (TBSA) partial/full thickness burns between 2006 and 2013. We screened for five MC1R SNPs (V60L, V92M, R151C, R163Q, T314T) by polymerase chain reaction from genomic DNA isolated from blood samples. We performed a detailed review of each patient chart to identify age, sex, race, ethnicity, %TBSA burned, burn wound infections (BWIs), and 72-hr intravenous fluid volume, the latter a surrogate for a dysfunctional inflammatory response to injury. Association testing was based on multivariable regression. RESULTS: Of 106 subjects enrolled, 82 had complete data for analysis. Of these, 64 (78%) were male, with a median age of 39 and median burn size of 30% TBSA. A total of 36 (44%) subjects developed BWIs. The median total administered IV crystalloid in first 72h was 24.6 L. In multivariate analysis, the R151C variant allele was a significant independent risk factor for BWI (adjusted prevalence ratio 2.03; 95% CI: 1.21-3.39; P = 0.007), and the V60L variant allele was independently associated with increased resuscitation fluid volume (P = 0.021). CONCLUSIONS: This is the first study to demonstrate a significant association between genetic polymorphisms and a nonfatal burn-induced SIRS complication. Our findings suggest that MC1R polymorphisms contribute to dysfunctional responses to burn injury that may predict infectious and inflammatory complications.

An electrochemical immunosensing method for detecting melanoma cells.

An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6](3-)), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50-7500 cells/2.5 mL. The immunosensor is highly sensitive (20 cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.

Melanocortin 1 Receptor-derived peptides are efficiently recognized by cytotoxic T lymphocytes from melanoma patients.

BACKGROUND: Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R291-298, MC1R244-252 and MC1R283-291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown. METHODS: The presence of anti-MC1R specific CD8(+) T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2(+) melanoma patients. RESULTS: CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2(+)MC1R(+) melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2(+) melanoma cell lines. Finally, HLA-A2/MC1R244-specific CD8(+) T cell clones derived from TILs and a subset of MC1R291 specific TILs were identified using HLA-A2/MC1R tetramers. CONCLUSION: Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy.

Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update.

Since the identification of S100 protein as an immunohistochemical marker that could be useful in the diagnosis of melanoma in the early 1980s, a large number of other melanocytic-associated markers that could potentially be used to assist in the differential diagnosis of these tumors have also been investigated. A great variation exists, however, among these markers, not only in their expression in some subtypes of melanoma, particularly desmoplastic melanoma, but also in their specificity because some of them can also be expressed in nonmelanocytic neoplasms, including various types of soft tissue tumors and carcinomas. This article reviews the information that is currently available on the practical value of some of the markers that have more often been recommended for assisting in the diagnosis of melanomas, including those that have only recently become available.

The synthetic melanocortin (CKPV)2 exerts anti-fungal and anti-inflammatory effects against Candida albicans vaginitis via inducing macrophage M2 polarization.

In this study, we examined anti-fungal and anti-inflammatory effects of the synthetic melanocortin peptide (Ac-Cys-Lys-Pro-Val-NH2)2 or (CKPV)2 against Candida albicans vaginitis. Our in vitro results showed that (CKPV)2 dose-dependently inhibited Candida albicans colonies formation. In a rat Candida albicans vaginitis model, (CKPV)2 significantly inhibited vaginal Candida albicans survival and macrophages sub-epithelial mucosa infiltration. For mechanisms study, we observed that (CKPV)2 inhibited macrophages phagocytosis of Candida albicans. Meanwhile, (CKPV)2 administration inhibited macrophage pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) release, while increasing the arginase activity and anti-inflammatory cytokine IL-10 production, suggesting macrophages M1 to M2 polarization. Cyclic AMP (cAMP) production was also induced by (CKPV)2 administration in macrophages. These above effects on macrophages by (CKPV)2 were almost reversed by melanocortin receptor-1(MC1R) siRNA knockdown, indicating the requirement of MC1R in the process. Altogether, our results suggest that (CKPV)2 exerted anti-fungal and anti-inflammatory activities against Candida albicans vaginitis probably through inducing macrophages M1 to M2 polarization and MC1R activation.

Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response.

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1ß and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.

MC1R-dependent, melanin-based colour polymorphism is associated with cell-mediated response in the Eleonora's falcon.

Colour polymorphism in vertebrates is usually under genetic control and may be associated with variation in physiological traits. The melanocortin 1 receptor (Mc1r) has been involved repeatedly in melanin-based pigmentation but it was thought to have few other physiological effects. However, recent pharmacological studies suggest that MC1R could regulate the aspects of immunity. We investigated whether variation at Mc1r underpins plumage colouration in the Eleonora's falcon. We also examined whether nestlings of the different morphs differed in their inflammatory response induced by phytohemagglutinin (PHA). Variation in colouration was due to a deletion of four amino acids at the Mc1r gene. Cellular immune response was morph specific. In males, but not in females, dark nestling mounted a lower PHA response than pale ones. Although correlative, our results raise the neglected possibility that MC1R has pleiotropic effects, suggesting a potential role of immune capacity and pathogen pressure on the maintenance of colour polymorphism in this species.

Involvement of the melanocortin-1 receptor in acute pain and pain of inflammatory but not neuropathic origin.

BACKGROUND: Response to painful stimuli is susceptible to genetic variation. Numerous loci have been identified which contribute to this variation, one of which, MC1R, is better known as a gene involved in mammalian hair colour. MC1R is a G protein-coupled receptor expressed in melanocytes and elsewhere and mice lacking MC1R have yellow hair, whilst humans with variant MC1R protein have red hair. Previous work has found differences in acute pain perception, and response to analgesia in mice and humans with mutations or variants in MC1R. METHODOLOGY AND PRINCIPAL FINDINGS: We have tested responses to noxious and non-noxious stimuli in mutant mice which lack MC1R, or which overexpress an endogenous antagonist of the receptor, as well as controls. We have also examined the response of these mice to inflammatory pain, assessing the hyperalgesia and allodynia associated with persistent inflammation, and their response to neuropathic pain. Finally we tested by a paired preference paradigm their aversion to oral administration of capsaicin, which activates the noxious heat receptor TRPV1. Female mice lacking MC1R showed increased tolerance to noxious heat and no alteration in their response to non-noxious mechanical stimuli. MC1R mutant females, and females overexpressing the endogenous MC1R antagonist, agouti signalling protein, had a reduced formalin-induced inflammatory pain response, and a delayed development of inflammation-induced hyperalgesia and allodynia. In addition they had a decreased aversion to capsaicin at moderate concentrations. Male mutant mice showed no difference from their respective controls. Mice of either sex did not show any effect of mutant genotype on neuropathic pain. CONCLUSIONS: We demonstrate a sex-specific role for MC1R in acute noxious thermal responses and pain of inflammatory origin.

Patients affected by vitiligo and autoimmune diseases do not show antibodies interfering with the activity of the melanocortin 1 receptor.

BACKGROUND: Vitiligo is an acquired depigmenting disorder characterized by the loss of melanocytes from the epidermis with the development of white patches in various distribution. The pathogenesis of vitiligo is still unknown, but the association with autoimmune disorders and organ specific autoantibodies, supports the hypothesis of an autoimmune pathogenesis. AIM: The aim of the present study was to investigate if autoantibodies present in sera of patients affected by vitiligo may be able to interfere with the activity of the αMSH on the melanocortin 1 receptor (MC1R). MATERIALS/ SUBJECTS AND METHODS: IgG from the sera of 41 patients with vitiligo associated or not with thyroid autoimmune diseases or other autoimmune pathologies were incubated with HBL20 cells (human malignant melanocytes expressing the MC1R) in the presence of a sub-maximal dose of αMSH. A normal IgG range was determined by using IgG extracted from 30 control sera of normal subjects. RESULTS: None of the IgG from vitiligo patients was able to inhibit αMSH-stimulated cAMP production in HBL20 cells. CONCLUSIONS: Autoantibodies against MC1R are rare or absent in sera of vitiligo patients.

Accumulation of low-avidity anti-melanocortin receptor 1 (anti-MC1R) CD8+ T cells in the lesional skin of a patient with melanoma-related depigmentation.

Spontaneous or therapy-induced depigmentation in patients with melanoma has long been considered a favourable prognostic indicator. In this report, we isolated T cells infiltrating the depigmented skin of an HLA-A2+/DR4+ patient with melanoma, and detected a very high frequency of CD8+ T cells specific for melanocortin receptor 1 (MC1R), a hormone receptor involved in cutaneous pigmentation. In particular, tissue-infiltrating CD8+ T cells dominantly recognized the novel MC1R52-60 peptide epitope in an HLA-A2-restricted manner, and peptide-reactive CD8+ T cells were also detected in freshly isolated peripheral blood from this patient. Although type 1 CD4+ T-cell responses against MC1R were not detected in fresh tissue isolates, short-term in-vitro stimulation of peripheral blood lymphocytes resulted in the rapid expansion of CD4+ T cells reactive against novel HLA-DR4-presented epitopes derived from the MC1R protein (i.e. MC1R82-95, MC1R105-118 and MC1R149-161). MC1R peptide-specific CD8+ T-cell clones isolated from the depigmented skin of this patient were characterized by comparatively low functional avidity for specific major histocompatibility complex-peptide complexes and were poorly lytic; however, these effector cells were capable of secreting both interferon-gamma and granzyme B against relevant target cells in vitro, and may have played an important role in the induction of leucoderma in situ in this patient.

Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress.