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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., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Originally, the neuropeptide α-melanocyte-stimulating hormone (α-MSH) has been described as a mediator of skin pigmentation. However, recent studies have shown that α-MSH is able to modulate inflammation in various tissues including the lung. So far, it is still not clear whether α-MSH also plays a role in allergic bronchial asthma., Objective: This study aimed at investigating the role and regulatory mechanisms of α-MSH in asthma pathogenesis., Methods: α-MSH levels were measured in bronchoalveolar lavage (BAL) fluid of asthmatic and non-asthmatic individuals as well as of healthy mice and mice with experimental asthma. Wild-type mice were sensitized to ovalbumin (OVA) and exposed to an OVA aerosol in order to induce experimental allergic asthma. α-MSH was administrated intratracheally, the α-MSH antibody intraperitoneally prior each OVA challenge. Airway inflammation, cytokine production, mucus production, airway hyperresponsiveness and receptor expression were assessed., Results: α-MSH levels in BAL of asthmatic individuals and mice were significantly higher compared to healthy controls. In a mouse model of experimental asthma, α-MSH neutralization increased airway inflammation and mucus production, whereas local administration of α-MSH significantly reduced inflammation of the airways. The beneficial effects were further associated with decreased levels of eosinophilic chemoattractant factors that are released by MC5R-positive T helper 2 and airway epithelial cells., Conclusion and Clinical Relevance: α-MSH acts as a regulatory factor to maintain local immune homeostasis in allergic bronchial asthma., (© 2019 John Wiley & Sons Ltd.)

Melanocortin 4 receptor (MC4R) plays a key role in regulation of appetite activated by its main ligand α-melanocyte-stimulating hormone (α-MSH) in both central and peripheral targets. α-MSH also binds to circulating immunoglobulins (Igs) but the functional significance of such immune complexes (ICs) in MC4R signaling in normal and pathological conditions of altered appetite has remained unknown. To address this question, we analyzed plasma levels, affinity kinetics, and binding epitopes of α-MSH-reactive IgG extracted from plasma samples of female patients with hyperphagic obesity, anorexia nervosa, bulimia nervosa, binge-eating disorder, and healthy controls. Ability of α-MSH/IgG IC to bind and activate human MC4R were studied in vitro and to influence feeding behavior in vivo in rodents. We found that α-MSH-reactive IgG were low in obese but increased in anorectic and bulimic patients and displayed different epitope and kinetics of IC formation. Importantly, while α-MSH/IgG IC from all subjects were binding and activating MC4R, the receptor binding affinity was decreased in obesity. Additionally, α-MSH/IgG IC had lower MC4R-mediated cAMP activation threshold as compared with α-MSH alone in all but not obese subjects. Furthermore, the cellular internalization rate of α-MSH/IgG IC by MC4R-expressing cells was decreased in obese but increased in patients with anorexia nervosa. Moreover, IgG from obese patients prevented central anorexigenic effect of α-MSH. These findings reveal that MC4R is physiologically activated by IC formed by α-MSH/IgG and that different levels and molecular properties of α-MSH-reactive IgG underlie biological activity of such IC relevant to altered appetite in obesity and eating disorders.

α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide derived from pro-opiomelanocortin that exhibits potent anti-inflammatory properties by regulating the production of inflammatory mediators. This peptide has been well established in several inflammatory models, including inflammatory bowel disease (IBD). However, its extremely short duration in vivo limits its clinical application. To address this limitation, Bifidobacterium was used here as a carrier to deliver α-MSH. We utilized α-MSH-engineered Bifidobacterium against IBD, which is closely linked to immune and intestinal microbiota dysfunction. First, we constructed a Bifidobacterium longum secreting α-MSH (B. longum-α-MSH). We then tested the recombinant α-MSH expression and determined its bioactivity in HT-29 cells. To assess its effectiveness, B. longum-α-MSH was used against an ulcerative colitis (UC) model in rats induced by dextran sulfate sodium. The data showed that α-MSH expression in B. longum-α-MSH was effective, and its biological activity was similar to the synthesized one. This UC model experiment indicated that B. longum-α-MSH successfully colonized the intestinal gut, expressed bioactive α-MSH and had a significant anti-inflammatory effect. The results demonstrate the feasibility of preventing IBD by using B. longum-α-MSH.

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1-8). Furthermore, ACTH(1-8) competed with [(125)I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1-8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1-8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

Objectives: The biological background of sex-related differences in the development of eating disorders (EDs) is unknown. Recent data showed that gut bacteria Escherichia coli induce autoantibodies against anorexigenic α-melanocyte-stimulating hormone (α-MSH) associated with psychopathology in ED. The aim of this study was to compare the effects of E. coli on feeding and autoantibodies against α-MSH and adrenocorticotropic hormone (ACTH), between female and male rats., Methods: Commensal E. coli K12 were given in a culture medium daily to adult Wistar rats by intragastric gavage over a 3-wk period; control rats received culture medium only., Results: Before gavage, E. coli K12 DNA was detected in feces of female but not male rats. E. coli provision was accompanied by an increase in body weight gain in females, but a decrease in body weight gain and food intake in males. Independent of E. coli treatment, plasma levels of anti-α-MSH and ACTH immunoglobulin (Ig)G were higher in female than male rats. Females responded to E. coli by increasing α-MSH IgG levels and affinity, but males by increasing α-MSH IgM levels. Affinity of IgG for ACTH was increased in both E. coli-treated females and males, although with different kinetics. IgG from females stimulated more efficiently α-MSH-induced cyclic adenosine monophosphate production by melanocortin 4 receptor-expressing cells compared with IgG from males., Discussion: Sex-related response to how E. coli affects feeding and anti-melanocortin hormone antibody production may depend on the presence of these bacteria in the gut before E. coli supplementation. These data suggest that sex-related presence of certain gut bacteria may represent a risk factor for ED development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Afamelanotide is an α-melanocyte-stimulating hormone (α-MSH) agonist with proven efficacy in photodermatoses such as erythropoietic protoporphyria (EPP). This peptide drug, repeatedly administered over prolonged time, may induce anti-drug antibodies (ADA). Here, we describe a new ELISA method developed to monitor the occurrence of ADA against afamelanotide as well as against α-MSH. Covalent binding instead of absorption of antigen onto the microtitre wells prevented antigen leakage and enabled extensive washings followed by lower background. The cut-off between antibody-negative and -positive sera was determined. Inhibition of the antigen-antibody reaction by excess soluble antigen tested for specificity. The sensitivity of the ELISA was 608 and 1,390 ng/ml of specific ADA against afamelanotide and α-MSH, respectively. This ELISA method enabled us to investigate the occurrence of ADA during long-term administration of afamelanotide. No immunoreactivity was found in 23 of the 26 EPP patients exposed to the drug for up to 6 years. Pre-existing immunoreactivity against afamelanotide as well as α-MSH was found in 3 patients, whose titres did not change during afamelanotide administration., Conclusion: The new ELISA is suitable to determine ADA against afamelanotide and α-MSH. Afamelanotide did not elicit ADA during long-term administration in patients with EPP., (© 2014 S. Karger AG, Basel.)

Objectives: Many of peptides synthesized in gastrointestinal tract (GI) and adipose tissues, regulate growth and food intake. The GI microflora is an antigenic source. Based on the molecular mimicry hypothesis, intestinal microbe-derived antigens may trigger the production of autoantibodies cross-reacting with some neuropeptides., Design: The aim of the study was to assess whether in idiopathic short stature (ISS) children with Candida albicans (C.albicans) colonisation and/or Helicobacter pylori (H.pylori) infection the autoantibodies (in positive levels) against selected neuropeptides [anti-NP Abs(+)]: ghrelin, leptin, orexin A, αMSH are more prevalent than in Controls., Setting: The study group comprised 64 children with ISS and 36 children with normal height (Controls). In each child, IgG antibodies against H.pylori, ghrelin, leptin, orexin A and αMSH were assessed in serum, while presence of C.albicans - in stool samples., Results: The higher prevalence of anti-NP Abs(+) in ISS children with C.albicans and/or H.pylori than in normal height children with the colonization in question (34.4% vs 21.1%, p<0.01) was found. The prevalence of anti-NP Abs(+) in groups of children without C.albicans and H.pylori were low, anti-NP Abs(+) were detected in 9.4% of ISS children only, while in Controls they were not found., Conclusions: In short children with C.albicans and/or H.pylori the incidence of autoantibodies against selected neuropeptides is high. It probably is connected with molecular mimicry between antigens of these microbiota and the mentioned peptides. It is tempting to speculate that presence of cross-reacting autoantibodies against regulatory neuropeptides may results in worse growth velocity. However, further studies are necessary to elucidate this issue.

The molecular mechanisms at the origin of eating disorders (EDs), including anorexia nervosa (AN), bulimia and binge-eating disorder (BED), are currently unknown. Previous data indicated that immunoglobulins (Igs) or autoantibodies (auto-Abs) reactive with α-melanocyte-stimulating hormone (α-MSH) are involved in regulation of feeding and emotion; however, the origin of such auto-Abs is unknown. Here, using proteomics, we identified ClpB heat-shock disaggregation chaperone protein of commensal gut bacteria Escherichia coli as a conformational antigen mimetic of α-MSH. We show that ClpB-immunized mice produce anti-ClpB IgG crossreactive with α-MSH, influencing food intake, body weight, anxiety and melanocortin receptor 4 signaling. Furthermore, chronic intragastric delivery of E. coli in mice decreased food intake and stimulated formation of ClpB- and α-MSH-reactive antibodies, while ClpB-deficient E. coli did not affect food intake or antibody levels. Finally, we show that plasma levels of anti-ClpB IgG crossreactive with α-MSH are increased in patients with AN, bulimia and BED, and that the ED Inventory-2 scores in ED patients correlate with anti-ClpB IgG and IgM, which is similar to our previous findings for α-MSH auto-Abs. In conclusion, this work shows that the bacterial ClpB protein, which is present in several commensal and pathogenic microorganisms, can be responsible for the production of auto-Abs crossreactive with α-MSH, associated with altered feeding and emotion in humans with ED. Our data suggest that ClpB-expressing gut microorganisms might be involved in the etiology of EDs.

Anti-α-melanocyte-stimulating hormone (α-MSH) polyclonal antibodies have been used for α-MSH neutralization in functional studies, but the results are sometime inconsistent with the antibody expected blocking properties. The present study aimed to determine if rabbit (Rb) anti-α-MSH immunoglobulins (Ig) may inhibit or enhance α-MSH signaling on melanocortin receptor type 4 (MC4R) and α-MSH-induced anorexigenic effect if presented as immune complexes with α-MSH. Polyclonal Rb anti-α-MSH IgG were commercially available and their ability to bind α-MSH has been confirmed by the immunohistochemical detection of α-MSH neurons in the rat hypothalamus. In vitro assay of the cyclic-adenosine mono-phosphate (cAMP) secreted by cells transfected with MC4R was performed to analyze effect of Rb IgG on α-MSH-induced cAMP production. We found that adding Rb IgG to α-MSH resulted in stimulation of cAMP detected at lower peptide concentrations as compared to α-MSH alone. To determine effects of Rb IgG on food intake, rats were injected into the arcuate hypothalamic nucleus with either α-MSH, Rb IgG alone or Rb IgG preincubated with α-MSH. During 2 days after injections, food intake was increased in both groups of rats receiving Rb IgG. However, during following 4 days when food was restricted to 1h/day, only the Rb IgG group displayed higher food intake. Furthermore, after the refeeding, 24h food intake was lower in rats receiving Rb IgG - α-MSH immune complexes. This group of rats was also characterized by higher number of immunopositive neurons in the arcuate nucleus expressing α-MSH and agouti-related protein but not tyrosine hydroxylase. Taken together, these results show that Rb anti-α-MSH antisera, although efficient for immunohistochemical detection of α-MSH, does not always display α-MSH blocking properties but, in contrast, may enhance α-MSH binding to MC4R and increase α-MSH anorexigenic effects when presented as immune complexes with the peptide., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Within the immunosuppressive ocular microenvironment, there are constitutively present the immunomodulating neuropeptides alpha-melanocyte stimulating hormone (α-MSH) and neuropeptide Y (NPY) that promote suppressor functionality in macrophages. In this study, we examined the possibility that α-MSH and NPY modulate phagocytic activity in macrophages. The macrophages treated with α-MSH and NPY were significantly suppressed in their capacity to phagocytize unopsonized Escherichia coli and Staphylococcus aureus bioparticles, but not antibody-opsonized bioparticles. The neuropeptides significantly suppressed phagolysosome activation, and the FcR-associated generation of reactive oxidative species as well. This suppression corresponds to neuropeptide modulation of macrophage functionality within the ocular microenvironment to suppress the activation of immunogenic inflammation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We previously reported that increased expression of the endothelin (EDN)1/EDNB receptor (EDNBR) as well as the stem cell factor (SCF)/SCF receptor (c-KIT) linkages is mainly responsible for the activation of melanocytes in the epidermal hyperpigmentation of ultraviolet (UV)-B melanosis and lentigo senilis (LS). In this study, we characterized seborrheic keratosis (SK) to examine the paracrine cytokine mechanism(s) involved in its epidermal hyperpigmentation by reverse transcription polymerase chain reaction, immunohistochemistry and western blotting analyses. In contrast to our previous study which showed the upregulated expression of EDN1 and EDNBR at the transcriptional and translational levels in the epidermis of SK, we observed unexpectedly that the cytokine SCF and its receptor c-KIT are not upregulated, but are downregulated at both the gene and protein levels. We established SK cell lines to examine whether SK basaloid cells are less sensitive to SCF-inducible stimulation than are normal human keratinocytes (NHK). Comparison of the stimulatory effects of interleukin (IL)-1α or tumor necrosis factor (TNF)-α on SCF production between SK cells and NHK demonstrated that SK cells do not respond to IL-1α or TNF-α to stimulate production of SCF, whereas a significant stimulation of SCF is elicited by those same cytokines in NHK. These finding underscore a role of phenotypic changes in melanogenic cytokine production in the epidermis between SK and LS/UV-B melanosis., (© 2013 Japanese Dermatological Association.)

Autoantibodies reacting with alpha-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are involved in regulation of feeding. In this work we studied if intestinal inflammation (mucositis) may influence α-MSH autoantibodies production relevant to food intake and body weight. Mucositis and anorexia were produced in Sprague-Dawley rats by methotrexate (MTX, 2.5mg/kg/day, for three days, subcutaneously). Plasma levels of total IgG and of α-MSH autoantibodies were measured during and after MTX-induced mucositis and were compared with pair-fed and ad libitum-fed controls. Effects of intraperitoneal injections of rabbit anti-α-MSH IgG (3 or 10 μg/day/rat) on MTX-induced anorexia and on plasma α-MSH peptide concentration were separately studied. Here we show that in MTX rats, intestinal mucositis and anorexia were accompanied by decreased plasma levels of both total IgG and of α-MSH autoantibodies while refeeding was characterized by their elevated levels. In spite of similar food intake in MTX and pair-fed rats, recovery of body weight was delayed by at least 1 week in the MTX group. During refeeding and body weight deficit in MTX rats, α-MSH IgG autoantibody levels correlated negatively with food to water intake ratios. Injections of anti-α-MSH IgG induced a dose-dependent attenuation of food intake and body weight regain in MTX-treated rats accompanied by increased concentrations of α-MSH peptide which correlated positively with plasma levels of α-MSH autoantibodies. These data show that intestinal inflammation, independently from food restriction, affects general humoral immune response which may influence food intake and body weight control via modulation of α-MSH plasma concentration by α-MSH reactive autoantibodies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Neuropeptides galanin and α-melanocyte-stimulating hormone (α-MSH) are involved in the regulation of memory and appetite. Increased galanin and decreased α-MSH levels were reported in postmortem brains of patients with Alzheimer's disease (AD) but the underlying mechanisms are uncertain. Here we studied if autoantibodies (autoAbs) reacting with galanin and α-MSH are altered in AD., Methods: Levels of free and total IgG autoAbs reacting with galanin and α-MSH were measured in sera and cerebrospinal fluid (CSF) of 18 subjects with AD and in 15 age-matched non-demented controls. Values were correlated with Mini-Mental State Examination (MMSE) score, body mass index (BMI) and CSF levels of AD biomarkers., Results: CSF levels of total but not free IgG autoAbs against galanin were increased in AD, resulting in increased percentage of galanin autoAbs present as immune complexes. CSF levels of galanin total autoAbs and α-MSH free autoAbs correlated negatively with the severity of cognitive impairment as measured by MMSE. Both total and free autoAbs against galanin and α-MSH in CSF correlated negatively with age in AD patients but not in controls. CSF levels of galanin autoAbs and free α-MSH AutoAbs negatively correlated with CSF levels of t-Tau, p-Tau and ratios of t-Tau/Aβ42 or p-Tau/Aβ42 in AD patients but not in controls., Conclusions: AutoAbs reacting with galanin and α-MSH are present in CSF and are associated with clinical characteristics of AD patients. The functional significance and therapeutic potential of these autoAbs should be further clarified., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The functionality of immune cells is manipulated within the ocular microenvironment to protect the sensitive and non-regenerating light-gathering tissue from the collateral damage of inflammation. This is mediated partly by the constitutive presence of immunomodulating neuropeptides. Treating primary resting macrophages with soluble factors produced by the posterior eye induced co-expression of Arginase1 and NOS2. The neuropeptides alpha-melanocyte stimulating hormone and Neuropeptide Y alternatively activated the macrophages to co-express Arginase1 and NOS2 like myeloid suppressor cells. Similar co-expressing cells were found within healthy, but not in wounded retinas. Therefore, the healthy retina regulates macrophage functionality to the benefit of ocular immune privilege., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: To determine the spectrum of personality and psychopathology features of patients with primary SS (pSS) and explore whether they are linked to disease characteristics as well as the presence of autoantibodies (autoAbs) against neuropeptides., Methods: Personality and psychopathological variables were determined in 103 pSS patients and 110 healthy controls (HCs). AutoAbs against hypothalamic and pituitary neuropeptides were measured by ELISA in 25 pSS patients and 25 HCs. Data analysis was performed by univariate and multivariate logistic regression models and by comparison with regression models., Results: A higher number of pSS patients reported distinct personality traits (neuroticism, psychoticism and obsessiveness) and psychological distress compared with HCs. After adjustment for personality characteristics and demographics, only hypochondriasis was the main psychopathology feature associated with pSS, suggesting that psychopathological manifestations in the setting of pSS are primarily dependent on premorbid personality characteristics. Although no differences were detected between serum levels of neuropeptide autoAbs in pSS cases and controls, levels of autoAbs against alpha-melanocyte-stimulating hormone (alpha-MSH) correlated with anxiety scores in both groups examined but with higher intercept in pSS subjects. Significant correlations between anxiety score and autoAbs directed against oxytocin and vasopressin were also detected in the pSS patients., Conclusions: pSS patients exhibit a distinct pattern of personality traits and high levels of psychological distress compared with HCs, which seems to be determined by premorbid personality characteristics. Correlations between anxiety and alpha-MSH autoAbs suggest their potential involvement in anxiety development in both pSS and HCs.

Background: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor, pro-opiomelanocortin (POMC). Previous research has shown that MC receptor (MCR) agonists reduce, and MCR antagonists increase, ethanol consumption in rats and mice. Consistently, genetic deletion of the endogenous MCR antagonist, agouti-related protein (AgRP), causes reductions of ethanol-reinforced lever pressing and binge-like ethanol drinking in C57BL/6J mice. Ethanol also has direct effects on the central MC system, as chronic exposure to an ethanol-containing diet causes significant reductions of alpha-melanocyte stimulating hormone (alpha-MSH) immunoreactivity in specific brain regions of Sprague-Dawley rats. Together, these observations suggest that the central MC system modulates neurobiological responses to ethanol. To further characterize the role of the MC system in responses to ethanol, here we compared AgRP and alpha-MSH immunoreactivity in response to an acute injection of saline or ethanol between high ethanol drinking C57BL/6J mice and moderate ethanol drinking 129/SvJ mice., Methods: Mice received an intraperitoneal (i.p.) injection of ethanol (1.5 g/kg or 3.5 g/kg; mixed in 0.9% saline) or an equivolume of 0.9% saline. Two hours after injection, animals were sacrificed and their brains were processed for AgRP and alpha-MSH immunoreactivity., Results: Results indicated that acute ethanol administration triggered a dose-dependent increase in AgRP immunoreactivity in the arcuate (ARC) of C57BL/6J mice, an effect that was not evident in the 129/SvJ strain. Although acute administration of ethanol did not influence alpha-MSH immunoreactivity, C57BL/6J mice had significantly greater overall alpha-MSH immunoreactivity in the ARC, dorsomedial, and lateral regions of the hypothalamus relative to the 129/SvJ strain. In contrast, C57BL/6J mice displayed significantly lower alpha-MSH immunoreactivity in the medial amygdala., Conclusions: The results show that acute ethanol exposure has direct effects on endogenous AgRP activity in ethanol preferring C57BL/6J mice. It is suggested that ethanol-induced increases in AgRP may be part of a positive feedback system that stimulates excessive binge-like ethanol drinking in C57BL/6J mice. Inherent differences in alpha-MSH immunoreactivity may contribute to differences in neurobiological responses to ethanol that are characteristically observed between the C57BL/6J and 129/SvJ inbred strains of mice.

Purpose: The neuropeptide, alpha-melanocyte stimulating hormone (alpha-MSH), is an endogenous antagonist of inflammation. Injections of alpha-MSH peptide into inflamed tissues have been found to be very effective in suppressing autoimmune and endotoxin mediated diseases. We evaluated the potential to suppress ocular autoimmune disease (uveitis) by augmenting the expression of alpha-MSH through subconjunctival injections of naked adrenocorticotropic hormone amino acids 1-17 (ACTH1-17) plasmid., Methods: We clinically scored the uveitis over time in B10.RIII, C57BL/6, and melanocortin 5 receptor knock-out (MC5r((-/-))) mice with experimental autoimmune uveitis (EAU) that were conjunctively injected with a naked DNA plasmid encoding ACTH1-17 at the time of EAU onset and three days later. The post-EAU retina histology of plasmid injected eyes was examined, and post-EAU concentrations of alpha-MSH in aqueous humor was assayed by ELISA., Results: The subconjunctival injection of ACTH1-17 plasmid augmented the concentration of alpha-MSH in the aqueous humor of all post-EAU mice. The injection of ACTH1-17 suppressed the severity of EAU in the B10.RIII and C57BL/6 mice but the MC5r((-/-)) mice. In all the models of EAU, the ACTH1-17 injection helped to preserve the structural integrity of the retina; however, post-EAU aqueous humor was not immunosuppressive., Conclusions: The subconjunctival injection of the alpha-MSH expression vector ACTH1-17 plasmid is effective in suppressing EAU. The suppressive activity is dependent on MC5r expression, and possibly works though alpha-MSH antagonism of inflammation than on alpha-MSH directly modulating immune cells. The results suggest that an effective therapy for uveitis could include a gene therapy approach based on delivering alpha-MSH.

In the present study we made an attempt to understand the skin irritation cascade of selected aliphatic hydrocarbons using microdialysis technique. Microdialysis probes were inserted into dermis in the dorsal skin of hairless rats. After 2h of probes insertion, occlusive dermal exposure (2h) was carried out with 230 microl of nonane, dodecane and tetradecane, using Hill top chambers((R)). Inflammatory biomarkers such as substance P (SP), alpha-melanocyte stimulating hormone (alpha-MSH) Interleukin 6 (IL-6) and prostaglandin E2 (PGE(2)) were analyzed in the dialysis samples by enzyme immunoassay (EIA). SP, alpha-MSH and IL6 were released in significant amounts following the dermal exposure of nonane and dodecane, whereas tetradecane did not induce any of these markers in significant amounts compared to control. Nonane increased the PGE(2) levels in significant amounts within 2h of chemical exposure compared to dodecane and tetradecane. IL-6 response was found to be slow and 2-3-fold increase in IL-6 levels was observed after 5h following nonane and dodecane application. The magnitude of skin irritation exerted by all three chemicals was in the order of nonane>or=dodecane>or=tetradecane. The results demonstrate that microdialysis can be used to measure the inflammatory biomarkers in the skin irritation studies and irritation response of chemicals was quantifiable by this method. In conclusion, microdialysis was found to be an excellent tool to measure several inflammatory biomarkers as a function of time after dermal exposures with irritant chemicals.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a stress-related neuropeptide involved in the regulation of motivated behavior, appetite and emotion including stimulation of satiety and anxiety. Although autoantibodies (autoAbs) reactive with alpha-MSH have been identified in human subjects and in rats, it remained unknown if these autoAbs are involved in the regulation of feeding and anxiety and if their production is related to stress. Here we show that repeated exposure of rats to anxiolytic mild stress by handling increases the levels and affinity of alpha-MSH reactive IgG autoAbs and that these changes are associated with adaptive feeding and anxiety responses during exposure of rats to a strong stress by food restriction. Importantly, an increase in affinity of alpha-MSH reactive autoAbs was associated with changes of their functional roles from stimulation to inhibition of alpha-MSH-mediated behavioural responses, suggesting that these autoAbs can be a carrier or a neutralizing molecule of alpha-MSH peptide, respectively. Using a model of passive transfer into the brain, we show that alpha-MSH autoAbs affinity purified from blood of rats exposed to repeated mild stress, but not from control rats, are able to increase acutely food intake, suppress anxiety and modify gene expression of hypothalamic neuropeptides in naïve rats. These data provide the first evidence that autoAbs reactive with alpha-MSH are involved in the physiological regulation of feeding and mood, supporting a further role of the immune system in the control of motivated behavior and adaptation to stress.

Objective: Recent findings of autoantibodies directed against melanocortin peptides suggest that these autoantibodies may represent a source of variability in peptidergic signaling that can be responsible for altered appetite and emotion in eating disorders. However, it is still unknown if autoantibodies directed against some other appetite-regulating neuropeptides and peptide hormones exist in healthy human subjects and if these autoantibodies can regulate appetite and emotion., Methods: We determined the presence of autoantibodies against some key appetite-regulating neuropeptides and peptide hormones in sera of human subjects and in rats, and used animal models to study the role of alpha-melanocyte-stimulating hormone autoantibodies in food intake and anxiety., Results: Immunoglobulin G and A autoantibodies against alpha-melanocyte-stimulating hormone, neuropeptide Y, agouti-related protein, ghrelin, leptin, and some other neuropeptides or peptide hormones involved in appetite control were present in healthy humans and rats. Animal models including active and passive transfer showed that alpha-melanocyte-stimulating hormone autoantibodies are involved in the regulation of feeding and anxiety. Sequence homology was found between neuropeptides and proteins from some members of intestinal microflora, whereas germ-free rats showed altered levels of autoantibodies directed against several neuropeptides., Conclusion: Autoantibodies directed against appetite-regulating neuropeptides and peptide hormones are emerging as important participants in the peptidergic mechanisms controlling motivated behavior. Furthermore, these autoantibodies could provide a link in the gut-brain axis and may represent new biological targets for the diagnosis and treatment of eating disorders.

Purpose of Review: Anorexia nervosa remains a disease of unknown etiology. This situation explains the failure to develop effective therapy and emphasizes the fact that the neurobiological mechanisms of appetite and emotion are still incompletely understood. The present review is the first summary of recent research assigning to the immune system a new role in energy and emotional regulation by the production of autoantibodies directed against neuropeptides. The results of this research are promising to shed light on the etiology of eating disorders and open new fields for biological diagnosis and follow-up as well as designing new therapeutic strategies., Recent Findings: Following the initial identification of autoantibodies against alpha-melanocyte-stimulating hormone, a key neuropeptide involved in the regulation of satiety and mood, in the plasma of patients with anorexia and bulimia nervosa, it has been further found that the serum levels of these autoantibodies correlated with psychopathological traits in individuals with eating disorders. Furthermore, recent findings show that autoantibodies against alpha-melanocyte-stimulating hormone and against some other appetite-regulating peptide hormones are normally present in the blood of humans and rats and their production may be influenced by stress and the gut microflora., Summary: Novel data provide evidence that autoantibodies against neuropeptides can be involved in the regulation of appetite and emotion and that alteration in autoantibody-mediated signaling pathways may be responsible for the development of eating disorders.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a tridecapeptide derived from the proopiomelanocortin by post-translational processing. In addition to its effects on melanocytes, alpha-MSH has potent anti-inflammatory effects when administered systemically or locally. The anti-inflammatory effects of alpha-MSH are mediated by direct effects on cells of the immune system as well as indirectly by affecting the function of resident non-immune cells. alpha-MSH affects several pathways implicated in regulation of inflammatory responses such as NF-kappaB activation, expression of adhesion molecules and chemokine receptors, production of pro-inflammatory cytokines and other mediators. Thus alpha-MSH may modulate inflammatory cell proliferation, activity and migration. The anti-inflammatory effects of alpha-MSH have been confirmed by means of animal models of inflammation such as irritant and allergic contact dermatitis, cutaneous vasculitis, asthma, inflammatory bowel disease, rheumatoid arthritis, ocular and brain inflammation. Most of the anti-inflammatory activities of alpha-MSH can be attributed to its C-terminal tripeptide KPV. K(D)PT, a derivative of KPV corresponding to the amino acid 193-195 of IL-1beta, is currently emerging as another tripeptide with potent anti-inflammatory effects. The anti-inflammatory potential together with the favourable physiochemical properties most likely will allow these agents to be developed for the treatment of inflammatory skin, eye and bowel diseases, allergic asthma and arthritis.

Purpose: The authors demonstrated that in vitro-generated alpha-melanocyte stimulated hormone (MSH)-induced Treg cells specific to ocular autoantigen suppress ocular autoimmune disease in vivo when adoptively transferred. They examined the possibility of using these ocular autoantigen-specific Treg cells to promote the survival of a retinal allograft placed in the mouse vitreous., Methods: Enhanced green fluorescent protein (eGFP)-C57BL/6 neonatal retinal microaggregates were injected into the vitreous of B10-RIII mice before the adoptive transfer of interphotoreceptor retinoid-binding protein (IRBP; an ocular antigen) or ovalbumin (OVA)-specific alpha-MSH-induced Treg cells. GFP transplants were imaged in vivo on days 7 and 12. In addition, on day 12, the eyes were cryosectioned and immunostained with a panel of neuronal and immune cell markers., Results: GFP allografts underwent no detectable changes in size on days 7 and 12 in the B10-RIII mice injected with IRBP-specific Treg cells; however, mice that received OVA-specific Treg cells or no Treg cells experienced remarkable reductions in graft size on day 12. Only one quarter of the original size was seen. Using neuronal-specific markers, immunohistochemistry showed that the architecture of the retinal allografts in the IRBP Treg cell-injected group had intact rosettes and neuronal cells on the outermost layer, whereas the allografts in the OVA Treg cell-injected mice were disorganized. Immune cell-specific markers demonstrated that Treg cells and activated microglial cells were found in the retinal allografts of the mice injected with IRBP Treg cells, but not in the retinal allografts of the OVA Treg-injected mice., Conclusions: These results demonstrate that adoptive transfer of alpha-MSH-generated IRBP-specific Treg cells promotes retinal allograft survival and development.

Eating disorders constitute major medical health problems in the western world. Even though little is known about the molecular mechanisms behind abnormal eating behavior, it has become clear that the central nervous system (CNS), particularly the hypothalamus, plays a significant role. The anorexic anx/anx mouse is a unique model for studying food intake and energy expenditure. The anx mutation is linked to marked alterations in hypothalamic distributions of signal substances known to have potent regulatory roles in the control of food intake. Another mouse model that displays an anorectic phenotype similar to the anx/anx mouse is the Contactin KO mouse. This model displays very similar hypothalamic alterations as seen in the anx/anx mouse, arguing for a role of these specific hypothalamic changes in an anorectic phenotype. In human eating disorders, hypothalamic systems corresponding to those defective in mouse models could be compromised since autoantibodies against melanocortin peptides have been detected in anorectic and bulimic patients. These findings represent research avenues that may lead to a better understanding of eating disorders and development of targeted therapeutic approaches.

Interstitial inflammation has emerged as a key event in the development of acute renal failure. To gain better insight into the nature of these inflammatory processes, the interplay between tubular epithelial cells, endothelial cells, and neutrophils (PMN) was investigated. A coculture transmigration model was developed, composed of human dermal microvascular endothelial (HDMEC) and human renal proximal tubular cells (HK-2) cultured on opposite sides of Transwell growth supports. Correct formation of an endoepithelial bilayer was verified by light and electron microscopy. The model was used to study the effects of endotoxin (LPS), tumor necrosis factor (TNF)-alpha, and alpha-melanocyte-stimulating hormone (alpha-MSH) by measuring PMN migration and cytokine release. To distinguish between individual roles of microvascular endothelial and epithelial cells in transmigration processes, migration of PMN was investigated separately in HK-2 and HDMEC monolayers. Sequential migration of PMN through endothelium and epithelium could be observed and was significantly increased after proinflammatory stimulation with either TNF-alpha or LPS (3.5 +/- 0.58 and 2.76 +/- 0.64-fold vs. control, respectively). Coincubation with alpha-MSH inhibited the transmigration of PMN through the bilayer after proinflammatory stimulation with LPS but not after TNF-alpha. The bilayers produced significant amounts of IL-8 and IL-6 mostly released from the epithelial cells. Furthermore, alpha-MSH decreased LPS-induced IL-6 secretion by 30% but had no significant effect on IL-8 secretion. We established a transmigration model showing sequential migration of PMN across microvascular endothelial and renal tubular epithelial cells stimulated by TNF-alpha and LPS. Anti-inflammatory effects of alpha-MSH in this bilayer model are demonstrated by inhibition on PMN transmigration and IL-6 secretion.

Background: Altered stress response is characteristic for subjects with abnormal aggressive and antisocial behavior, but the underlying biological mechanisms are unclear. We hypothesized that autoantibodies (autoAbs) directed against several stress-related neurohormones may exist in aggressive subjects., Methods: Using enzyme-linked immunosorbent assay, we studied whether autoAbs directed against corticotropin (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), oxytocin, and vasopressin are present in serum of male subjects with conduct disorder and prisoners with history of violence. Healthy blood donors served as control subjects., Results: Both conduct disorder and prisoners groups displayed strongly increased levels of ACTH-reactive immunoglobulin G (IgG) and immunoglobulin M (IgM) autoAbs compared with control subjects. Levels of oxytocin-reactive IgM autoAbs were slightly increased in both groups of aggressive subjects, whereas levels of vasopressin-reactive IgG and IgM autoAbs were lower only in conduct disorder. No differences in the levels of alpha-MSH-reactive autoAbs were found between aggressive and control subjects., Conclusions: High levels of ACTH-reactive autoAbs as well as altered levels of oxytocin- and vasopressin-reactive autoAbs found in aggressive subjects may interfere with the neuroendocrine mechanisms of stress and motivated behavior. Our data suggest a new biological mechanism of human aggressive behavior that involves autoAbs directed against several stress-related neurohormones.

Although recent reports underscore a close association between the ethanol consumption and the central melanocortin (MC) system in rats, neurobehavioral component of this association has not been explored. In this study, we investigated the role of alpha-melanocyte stimulating hormone (alpha-MSH) in ethanol (1.5-2 g/kg, i.p.) induced anxiolysis and anxiety-like behavior following withdrawal from prolonged ethanol (9% v/v ethanol, 15 days) consumption, using elevated plus maze (EPM) test in rats. While alpha-MSH (1-5 microg/rat, i.c.v.) showed dose-dependent anxiogenic-like effect, the MC4 receptor antagonist HS014 (1-10 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:500-1:50 dilution, 5 microl/rat, i.c.v.) failed to produce any effect in the EPM test. The anxiolytic-like effect of ethanol was suppressed by central administration of alpha-MSH (0.5 microg/rat, i.c.v.). On the other hand, pretreatment with either HS014 (5 nM/rat, i.c.v.) or antiserum against alpha-MSH (1:100 dilution, 5 microl/rat, i.c.v.) enhanced anxiolytic action of ethanol. Moreover, ethanol withdrawal anxiety was markedly blocked by HS014 (1-10 nM/rat, i.c.v.). These results suggest that alpha-MSH may be implicated in ethanol-induced anxiolysis and withdrawal anxiety. These findings also suggest MC4 receptors as possible therapeutic target for development of drugs to address the ethanol withdrawal-related conditions.

In recent years, scientific interest in melanocortins (MC) has progressively increased due to their wide range of effects and expression on various tissues. Primarily discovered as mediators of skin pigmentation, recent research has shown their important roles in various body functions, such as energy homeostasis, sexual function, and inflammation. The anti-inflammatory and immunomodulatory properties discovered so far have led to the hypothesis that alpha-melanocyte-stimulating hormone (MSH) and its cognate receptors might present potential anti-inflammatory treatment options.

In the last years the neuropeptide a-melanocyte-stimulating hormone has emerged as a regulator of various biological processes far beyond the initially described pigment-inducing action. Expression of melanocortin-receptors (MC-Rs), mainly MC-1R, has been identified in several non-pigmentary human skin cell types. Moreover, expression of MC-5R has been detected in sebocytes and skin mast cells while MC-4R has been reported in dermal papilla cells, a specialized myofibroblast cell type regulating hair follicle activity. In accordance with early observations in the rat preputial gland alpha-melanocyte-stimulating and related peptides have lipogenic activity in the human system. The immunomodulatory actions of alpha-melanocyte-stimulating include regulation of expression and secretion of chemokines, downregulation of proinflammatory signal-induced NF-kappaB activation and adhesion molecule expression, prostaglandin E2 synthesis, as well as induction of interleukin-10. Depending on the cell type studied and the experimental conditions alpha-melanocyte-stimulating however may also have weak proinflammatory actions. In dermal fibroblasts alpha-melanocyte-stimulating was further reported to modulate collagen metabolism via upregulating interstitial collagenase as well as by attenuating the inductive effect of transforming growth factor B on 1 collagen synthesis and fibrosis, the latter pointing towards a potential role of a-melanocyte-stimulating during chronic inflammatory skin responses. The immunomudulatory effects, the established melanotropic action and the recently identified cytoprotective activity of a-melanocyte-stimulating on UVB-induced apoptosis and DNA damage may be part of the body's host defence in which this neuropeptide--typically induced by proinflammatory signals--maintains tissue homeostasis and prevents genotoxicity during inflammatory responses.

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.

Objective: There is evidence that brain death has detrimental effects on peripheral organs. Clinical and experimental studies on organ donors showed marked inflammation in tissue samples of livers and kidneys collected during brain death. The inflammatory reaction is characterized by release of cytokines and inflammatory cell infiltration. Because melanocortins and their receptors are significant modulators of inflammation, we hypothesized that downregulation of melanocortin receptors during brain death could contribute to enhance inflammation., Methods: Using real-time polymerase chain reaction (PCR) analysis, we determined expression of melanocortin receptors in liver biopsies obtained from brain-dead organ donors before cold ischemia and in normal liver tissue during resection of benign focal lesions of the liver. Tissue biopsies were also analyzed for expression of intercellular adhesion molecule-1 (ICAM-1), which has a central function in inflammatory cell migration., Results: Expression of melanocortin-1 receptor (MC1R) mRNA was markedly reduced in liver samples obtained from brain-dead organ donors compared to hepatic tissue collected during resection of benign focal lesions of the liver. Conversely, expression of the adhesion molecule ICAM-1 was significantly increased in livers of brain-dead organ donors., Conclusions: Disruption of the endogenous anti-inflammatory circuit based on MC1R could contribute to tissue damage during brain death.

Many neuropeptides and peptide hormones are very similar to antimicrobial peptides in their amino acid composition, amphipathic design, cationic charge and size. Their antimicrobial activities suggest they may also be directly involved in innate defense. Here we discuss recent advances in understanding how the mammalian nervous system, equipped with neuropeptides and peptide hormones with potent antimicrobial properties, might directly defend the host from microbial assault. As examples, we have chosen five well described, locally produced neuropeptides that may serve a previously unrecognized, direct antimicrobial, innate immune function in four anatomical sites.

There is a substantial body of evidence that the tridecapeptide alpha-melanocyte-stimulating hormone (alpha-MSH) functions as a mediator of immunity and inflammation. The immunomodulating capacity of alpha-MSH is primarily because of its effects on melanocortin receptor (MC-1R)-expressing monocytes, macrophages, and dendritic cells (DCs). alpha-MSH down-regulates the production of proinflammatory and immunomodulating cytokines (IL-1, IL-6, TNF-alpha, IL-2, IFN-gamma, IL-4, IL-13) as well as the expression of costimulatory molecules (CD86, CD40, ICAM-1) on antigen-presenting DCs. In contrast, the production of the cytokine synthesis inhibitor IL-10 is up-regulated by alpha-MSH. At the molecular level, these effects of alpha-MSH are mediated via the inhibition of the activation of transcription factors such as NFkappaB. Not only alpha-MSH but also its C-terminal tripeptide (alpha-MSH 11-13, KPV) was able to bind to MC-1R and to modulate the function of APCs. In vivo, using a mouse model of contact hypersensitivity (CHS) systemic and topical application of alpha-MSH or KPV inhibited the sensitization and the elicitation phase of CHS and was able to induce hapten-specific tolerance. To investigate the underlying mechanisms of tolerance induction, we have performed in vivo transfer experiments. Treatment of naive mice with bone marrow-derived immature haptenized and alpha-MSH-pulsed DCs resulted in a significant inhibition of CHS. Furthermore, tolerance induction was found to be mediated by the generation of CTLA4(+) and IL-10-producing T lymphocytes. The potent capacity of alpha-MSH to modulate the function of antigen-presenting cells (APCs) has been further supported in another experimental approach. In vitro, by activating APCs, alpha-MSH has been shown to modulate IgE production by IL-4 and anti-CD40 stimulated B lymphocytes. Moreover, in a murine model of allergic airway inflammation, systemic treatment with alpha-MSH resulted in a significant reduction of allergen-specific IgE production, eosinophil influx, and IL-4 production. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to alpha-MSH treatment. Therefore, therapeutic application of alpha-MSH or related peptides (KPVs) as well as alpha-MSH/KPV-pulsed DCs may be a useful approach for the treatment of inflammatory, autoimmune, and allergic diseases in the future.

Although many immunosuppressive factors have been identified in the eye, one of these factors, alpha-melanocyte stimulating hormone (alpha-MSH), both suppresses the activation of inflammatory activity by primed T cells and induces the activation of regulatory T cells (Treg cells). This neuropeptide alone at its ocular physiological concentration can account for most of the immunosuppressive activity of aqueous humor (the fluid filing the anterior chamber of the eye). Aqueous humor made devoid of alpha-MSH no longer suppresses IFN-gamma production by Th1 cells. It is alpha-MSH that mediates aqueous humor induction of regulatory T cells. What we have found is that alpha-MSH mediates the induction of C4+ CD25+ Treg cells, and that if the alpha-MSH Treg cells are specific to an autoantigen they can be used to suppress autoimmune disease. It is the objective of this review to demonstrate how we came to discover that alpha-MSH could have such an important role in the extreme regional immunity of the immune privileged eye and how this discovery could be applied to create or reestablish tolerance to prevent autoimmune disease.

The hypothalamic arcuate nucleus is involved in the control of energy intake and expenditure and may participate in the pathogenesis of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). Two systems are of particular interest in this respect, synthesizing alpha-melanocyte-stimulating hormone (alpha-MSH) and synthesizing neuropeptide Y, respectively. We report here that 42 of 57 (74%) AN andor BN patients studied had in their plasma Abs that bind to melanotropes andor corticotropes in the rat pituitary. Among these sera, 8 were found to bind selectively to alpha-MSH-positive neurons and their hypothalamic and extrahypothalamic projections as revealed with immunostaining on rat brain sections. Adsorption of these sera with alpha-MSH peptide abolished this immunostaining. In the pituitary, the immunostaining was blocked by adsorption with alpha-MSH or adrenocorticotropic hormone. Additionally, 3 ANBN sera bound to luteinizing hormone-releasing hormone (LHRH)-positive terminals in the rat median eminence, but only 2 of them were adsorbed with LHRH. In the control subjects, 2 of 13 sera (16%) displayed similar to ANBN staining. These data provide evidence that a significant subpopulation of ANBN patients have autoantibodies that bind to alpha-MSH or adrenocorticotropic hormone, a finding pointing also to involvement of the stress axis. It remains to be established whether these Abs interfere with normal signal transduction in the brain melanocortin circuitryLHRH system andor in other central and peripheral sites relevant to food intake regulation, to what extent such effects are related to andor could be involved in the pathophysiology or clinical presentation of ANBN, and to what extent increased stress is an important factor for production of these autoantibodies.

Application of alpha-MSH immunohistochemistry to the brain of elasmobranchs (Scyliorhinus torazame and Etmopterus brachyurus) demonstrated a marked species difference concerning the distribution of the alpha-MSH-like molecule in the brain. In S. torazame, alpha-MSH-like immunoreactive cells were present in the hypothalamus, mainly in the tuberculum posterius and the nucleus lateralis tuberis, and also in the distal and neurointermediate lobes of the hypophysis. Labeled varicose fibers were densely distributed in the hypothalamus, but they were sparse or absent in other portions of the brain. In striking contrast to the results for S. torazame, the immunoreactivity in the E. brachyurus brain was associated exclusively with the glial system, represented by astrocytes and tanycytes, throughout the central nervous system; no immunoreactivity was found in the neuronal elements. In the E. brachyurus hypophysis, the labeled cells were present in the distal and intermediate lobes, similarly to their presence in S. torazame, but in the intermediate lobe the immunoreactivity was confined to the peripheral cell cord closely adjacent to the neural lobe. The present findings are the first as regards the occurrence of alpha-MSH-like immunoreactivity in the glial system of the central nervous system of vertebrates and suggest diversity of expression and/or processing of proopiomelanocortin in the brain of elasmobranchs., (Copyright 2001 Academic Press.)

Knowledge of melanocortins and their receptors has increased tremendously over the last few years. The cloning of five melanocortin receptors, and the discovery of two endogenous antagonists for these receptors, agouti and agouti-related peptide, have sparked intense interest in the field. Here we give a comprehensive review of the pharmacology, physiology and molecular biology of the melanocortins and their receptors. In particular, we review the roles of the melanocortins in the immune system, behaviour, feeding, the cardiovascular system and melanoma. Moreover, evidence is discussed suggesting that while many of the actions of the melanocortins are mediated via melanocortin receptors, some appear to be mediated via mechanisms distinct from melanocortin receptors., (Copyright 2000 Academic Press.)

The distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures was studied in the brainstem of the cat using an indirect immunoperoxidase technique. Immunoreactivity was observed in several brainstem nuclei of the cat in which no immunoreactivity had been previously reported. Immunoreactive fibres were observed in the following; the inferior central nucleus; the pontine gray nuclei; the Kölliker-Fuse nucleus; the motor trigeminal nucleus, the anteroventral cochlear nucleus; the abducens nucleus; the retrofacial nucleus; the superior, lateral, inferior, and medial vestibular nuclei; the lateral nucleus of the superior olive; the external cuneate nucleus; the nucleus of the trapezoid body; the postpyramidal nucleus of the raphe; the medial accessory inferior olive; the dorsal accessory nucleus of the inferior olive; the nucleus ambiguus; the principal nucleus of the inferior olive; the preolivary nucleus; the nucleus ruber; the substantia nigra; and in the area postrema. Our results point to a more widespread distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures in the cat brainstem than that reported in previous studies carried out in the same region of the cat, rat and humans.

Alpha-melanocyte-stimulating hormone (alpha-MSH), a neuroimmunomodulatory peptide of ancient origin, is known to be involved in the control of host responses. In inflammatory cells, in the periphery and within the central nervous system, alpha-MSH modulates the production and action of proinflammatory cytokines. This broad influence occurs via endogenous alpha-MSH (melanocortin) receptors. The key to this anti-inflammatory influence is inhibition of NF-kappa B. Indeed alpha-MSH inhibits activation of this nuclear factor through preservation of I kappa B alpha, which binds to NF-kappa B and prevents its migration to the nucleus. Cells transfected with alpha-MSH plasmid vector are resistant to challenge with bacterial lipopolysaccharide. The peptide also act on central melanocortin receptors to modulate inflammation in the periphery. In brief, alpha-MSH and certain of its fragments such as alpha-MSH [11-13] KPV modulate inflammation via three general actions: direct actions on peripheral host cells; actions on inflammatory cells within the brain to modulate local reactions; and descending neural anti-inflammatory pathways that control inflammation in peripheral tissues.

Among various neuropeptides such as substance P, calcitonin gene-related peptide and others, alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC-1R), which is specific for alpha-MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells alpha-MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-2, IFN-gamma, TNF-alpha and IL-1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL-10. On endothelial cells alpha-MSH is capable of downregulating the LPS-induced expression of adhesion molecules such as vascular cell adhesion molecule (VCAM) and E-selectin. Moreover, the LPS-induced activation of transcription factors such as NF kappa B is downregulated by alpha-MSH. In a mouse model i.v. or topical application of alpha-MSH was found to inhibit the induction phase as well as the effector phase of contact hypersensitivity (CHS) reactions and to induce hapten-specific tolerance. These findings indicate that the production of immunosuppressing neuropeptides such as alpha-MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

The ocular microenvironment is an extreme example of regional immunity. Within its microenvironment, expression of delayed type hypersensitivity (DTH) is suppressed. This immunosuppression is mediated in part by the constitutive expression of alpha-MSH. Previously we have found that alpha-MSH suppresses the production of IFN-gamma by activated effector T cells. Recently we have found that alpha-MSH can mediate induction of TGF-beta-producing T cells that act as regulatory T cells. This has encouraged us to further examine the potential for alpha-MSH to suppress T cell-mediated inflammation (autoimmune disease) and to regulate lymphokine production by effector T cells. When alpha-MSH was injected i.v. into mice at the time of peak retinal inflammation, the severity of experimental autoimmune uveitis (EAU) was significantly suppressed. Effector T cells activated in vitro in the presence of alpha-MSH proliferated and produced IL-4 and enhanced levels of TGF-beta while their IFN-gamma and IL-10 production was suppressed. The alpha-MSH-treated T cells functioned as regulatory T cells by suppressing in vitro IFN-gamma production by other inflammatory T cells. This regulatory activity was the function of alpha-MSH-treated CD4+ CD25+ T cells. Therefore, alpha-MSH mediates immunosuppression by inducing a differential expression of lymphokine production and by inducing activation of regulatory functions in T cells. This implies that alpha-MSH may take part in regional mechanisms of immunosuppression and possibly peripheral tolerance. Thus, alpha-MSH can be used to suppress autoimmune disease and possibly reestablish tolerance to autoantigens.

The presence of the ancient peptide alpha-melanocyte-stimulating hormone (alpha-MSH) in barrier organs such as gut and skin suggests that this potent anti-inflammatory molecule may be a component of the innate host defense. In tests of antimicrobial activities, alpha-MSH and its fragment KPV showed inhibitory influences against the gram-positive bacterium Staphylococcus aureus and the yeast Candida albicans. Anti-tumor necrosis factor and antimicrobial effects of alpha-MSH suggest that the peptide might likewise reduce replication of human immunodeficiency virus (HIV). Treatment with alpha-MSH reduced HIV replication in chronically and acutely infected human monocytes. At the molecular level, alpha-MSH inhibited activation of the transcription factor NF-kappa B known to enhance HIV expression. alpha-MSH that combines antipyretic, anti-inflammatory, and antimicrobial effects could be useful in the treatment of disorders in which infection and inflammation coexist.

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP's modulate opioid receptor levels. In this study, we investigated the effects of chronically infused alpha-melanocyte stimulating hormone (alpha-MSH), dynorphin(1-8) (DYN(1-8)), dynorphin A (DYNA), and NPFF antibodies on delta-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against alpha-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of delta-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.

alpha-Melanocyte stimulating hormone (alpha-MSH) modulates all forms of inflammation by acting on peripheral inflammatory cells, glial inflammatory cells, and on CNS receptors that activate descending antiinflammatory neural pathways. The multiple actions of this ancient peptide suggest that there is no singular biochemical mechanism through which it exerts its antiinflammatory activity. However, research on IL-10 deficient and Agouti protein hypersecreting mice provide new insights into the actions of the peptide in living animals. Studies of cultured human astrocytes, whole murine brain, and human monocyte/macrophages indicate that a primary effect of the peptide is modulation of activation of the nuclear transcription factor kappa B. The latter influence may underlie the established reduction of gene expression and production of proinflammatory peptides and inducible nitric oxide by alpha-MSH peptides.

We previously reported that a sex steroid-thyroid hormone (Metharmon-F; MF, 2 tablets daily) was a potent drug for treatment of vitiligo. Using five patients with generalized vitiligo who were successfully treated with oral administration of MF, we performed an immunohistochemical analysis to elucidate its action mechanism at the cellular level. Histopathologically, the repigmented skin after the treatment showed increased numbers of melanocytes and melanin granules. Immunohistochemically, there was little significant difference between the depigmented lesions before treatment and the repigmented lesion after treatment in terms of the reactivity to adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in keratinocytes. The immunoreactivity to ACTH in melanocytes both before and after the treatment was minimal, but alpha-MSH in melanocytes became much stronger after the treatment, than before the treatment. The efficacy of MF in treatment of vitiligo was proven to be due to the stimulatory effect of melanocyte proliferation and melanin production via alpha-MSH.

We and others have previously shown that exogenous alpha-MSH antagonizes the stimulatory effects of the cytokine interleukin (IL)-1 on the hypothalamic-pituitary-adrenal (HPA) axis. It is currently unknown, however, if endogenous alpha-MSH plays a physiological role in regulating the HPA response to IL-1. We have therefore examined the HPA response to IL-1beta in rats pretreated with an affinity purified alpha-MSH antiserum (AS) infused intracerebroventricularly to neutralize endogenous alpha-MSH within the brain. alpha-MSH AS or a similarly purified fraction of normal rabbit serum (NRS) was injected intracerebroventricularly at 16 h and at 1 h prior to the i.c.v. injection of IL-1beta (2 ng or 20 ng) and blood samples were collected through an indwelling atrial catheter. After 2 ng IL-1beta, the adrenocorticotropic hormone (ACTH) response was significantly greater in the alpha-MSH AS treated rats (n = 7) compared to the NRS treated rats (n = 7) (P <0.01); the mean ACTH level rose to a peak of 594+208 pg/ml in the alpha-MSH AS treated rats vs 274+/-122 pg/ml in the NRS treated rats. The area under the ACTH response curve in the alpha-MSH AS treated animals was 181% of that in the NRS treated animals (P<0.05). A significant effect of alpha-MSH AS on the corticosterone response to i.c.v. IL-1beta was also noted during the first 3 h of the study (P<0.05). The mean area under the corticosterone response curve for the first 3 h in the alpha-MSH AS treated animals was 144% of that in the NRS treated animals (P <0.05). After 20 ng IL-1beta, the ACTH response over time was again significantly greater in the alpha-MSH AS treated rats (n=8) compared to the NRS treated rats (n=9) (P<0.02); the mean ACTH level rose to a peak of 673+/-190 pg/ml after alpha-MSH AS vs 490+/-115 pg/ml after NRS. Corticosterone levels rose to a peak of 42+/-3.9 microg/dl in the alpha-MSH AS treated rats vs 37+/-4.6 microg/dl in the NRS treated rats; this difference was not significant. We conclude that the IL-1beta induced stimulation of ACTH is significantly enhanced by antagonizing the activity of alpha-MSH. These results support a physiological role for endogenous alpha-MSH in limiting the HPA response to this inflammatory cytokine.

There is accumulating evidence for a strong interaction between components of the nervous system and the immune system. Accordingly, specific receptors for neuropeptides were found to be expressed on immunocompetent cells and several neuropeptides were recognized as potent regulators of immune and inflammatory reactions. Among various neuropeptides such as substance P, calcitonin gene-related peptide and others alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 which is specific for alpha-MSH and ACTH is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells alpha-MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-2, IFNgamma and IL-1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL-10. On endothelial cells alpha-MSH is capable of downregulating the LPS-induced expression of adhesion molecules such as vascular cellular adhesion molecules and E-selectin. Moreover, the LPS-induced activation of transcription factors such as NFkappaB is downregulated by alpha-MSH. In a mouse model intravenous or topical application of alpha-MSH was found to inhibit the induction as well as the effector phase of a contact hypersensitivity reaction and to induce hapten-specific tolerance. Moreover, there is evidence that the N-terminal tripeptide of alpha-MSH is sufficient for its in vitro and in vivo immunomodulatory effects. These findings indicate that the production of immunosuppressing neuropeptides such as alpha-MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin., (Copyright 2000 S. Karger AG, Basel.)

It is clear that inflammatory processes contribute to neurodegenerative disease, stroke, closed head injury, encephalitis, and other CNS disorders. These inflammatory processes are marked by local increases in cytokines, in particular tumor necrosis factor-alpha (TNF-alpha). It is important to control such CNS inflammation in order to preserve neural function. The neuroimmunomodulatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to modulate peripheral inflammation by acting on melanocortin receptors in host cells (macrophages, neutrophils) to inhibit production of such proinflammatory agents. Our results indicate that alpha-MSH likewise acts directly within the brain to modulate local inflammation. To determine if microglia are involved in anti-inflammatory responses to alpha-MSH within the brain, murine cells were tested; they produced TNF-alpha and nitric oxide in response to challenge, and production of both was reduced by alpha-MSH. In tests on human astrocytes, both alpha-MSH (1-13) and alpha-MSH (11-13) reduced TNF-alpha. Ischemia/reperfusion in the posterior circulation in dogs causes inflammatory reactions and disturbance of function, estimated from decreases in auditory-evoked potentials. These deficits were reduced by administering alpha-MSH systemically during reperfusion, moreso when the peptide was given during both ischemia and reperfusion. The results indicate that, much as for inflammation in the periphery, alpha-MSH modulates brain inflammatory responses mediated by proinflammatory agents.