The beta-human chorionic gonadotropin-related peptide LQGV exerts anti-inflammatory effects through activation of the adrenal gland and glucocorticoid receptor in C57BL/6 mice.

The systemic inflammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin ß-chain-related tetrapeptide leucine-glutamine-glycine-valine (LQGV) reduces hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully understood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-α and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that LQGV stimulates corticosterone production. Moreover, by using specific pharmacological inhibitors of the adrenocorticotropic hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the ß-human chorionic gonadotropin-related tetrapeptide LQGV stimulates adrenal glucocorticosteroid production through activation of the ACTH receptor with consequent glucocorticoid receptor activation and immunosuppression in C57BL/6 mice.

ACTH receptor distribution and modulation among murine mononuclear leukocyte populations.

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.

Expression, candidate gene, and population studies of the melanocortin 5 receptor.

In mouse the melanocortin 5 receptor is known to regulate sebaceous gland function. To clarify its role in man, we have studied melanocortin 5 receptor expression in skin, and allelic variation at the melanocortin 5 receptor locus in diverse human populations and candidate disease groups. Melanocortin 5 receptor protein and mRNA expression were studied by immunohistochemistry and reverse transcriptase polymerase chain reaction. Melanocortin 5 receptor mRNA was detected in normal skin and cultured keratinocytes but not in cultured fibroblasts or melanocytes. Immunohistochemistry revealed melanocortin 5 receptor immunoreactivity in the epithelium and appendages, including the sebaceous gland, eccrine glands, and apocrine glands, as well as low level expression in the interfollciular epidermis. In order to screen for genetic diversity in the melanocortin 5 receptor that might be useful for allelic association studies we sequenced the entire melanocortin 5 receptor coding region in a range of human populations. One nonsynonymous change (Phe209Leu) and four synonymous changes (Ala81Ala, Asp108Asp, Ser125Ser, and Thr248Thr) were identified. Similar results were found in each of the populations except for the Inuit in which only the Asp108Asp variant was seen. The apparent "global distribution" of melanocortin 5 receptor variants may indicate that they are old in evolutionary terms. Variation of melanocortin 5 receptor was examined in patients with acne (n = 21), hidradenitis supprativa (n = 4), and sebaceous gland lesions comprising sebaceous nevi, adenomas, and hyperplasia (n = 13). No additional mutations were found. In order to determine the functional status of the Phe209Leu change, increase in cAMP in response to stimulation with alpha-melanocyte-stimulating hormone was measured in HEK-293 cells transfected with either wild-type or the Phe209Leu variant. The variant melanocortin 5 receptor was shown to act in a concentration-dependent manner, which did not differ from that of wild type. We have therefore found no evidence of a causative role for melanocortin 5 receptor in sebaceous gland dysfunction, and in the absence of any association between variation at the locus and disease group, the pathophysiologic role of the melanocortin 5 receptor in man requires further study.

Natural and synthetic agonists of the melanocortin receptor type 3 possess anti-inflammatory properties.

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3-R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with gamma2-melanocyte-stimulating hormone (gamma2-MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin-1 beta (IL-1beta), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL-1beta, was inhibited by gamma2-MSH and MTII. The mixed MC3/4-R antagonist SHU9119 prevented the inhibitory actions of gamma2-MSH and MTII in vitro and in vivo, whereas the selective MC4-R antagonist HS024 was without effect. Western blotting also showed the presence of MC3-R protein on murine peritoneal Mø. Furthermore, agonism at the MC3-R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long-acting compound MTII, activate MC3-R on peritoneal Mø to inhibit the experimental inflammatory response.

Specific detection of cell surface-displayed human melanocortin 4 receptors with antibodies generated in mice.

The melanocortin-4 receptor (MC-4R) is a 7-transmembrane protein, which is involved in the central regulation of appetite and obesity. Despite the great interest in this protein, tools for detecting this molecule (as expressed on the cell surface in its native state) have been unavailable. Radioactive- or otherwise labeled ligands showed low receptor specificity to this particular melanocortin receptor isotype. Also, the antibodies were only available for epitopes that were displayed in the cytoplasm. To produce antibodies that enable the detection of this receptor (as expressed on the cell surface without disruption of the target cells), a candidate epitope was selected from the extracellular domains by a computer-aided analysis of the IC-4R secondary structure. This particular region was then recombinantly expressed in E. coli. Immunization of BALB/c mice with the recombinant proteins induced a specific immune reaction, which resulted in the production of MC-4R-specific antibodies. Enzyme-linked immunosorbent assays confirmed the specificity of these antibodies. To examine whether this tool also reacts with native cell surface-displayed MC-4R, HEK-293 cells were transfected with the human MC-4R cDNA. They were analyzed with these antibodies using Western blot and flow cytometry. Specificity and exclusion of cross-reactivity of these antibodies to other MC receptors were further confirmed by an immunofluorescence analysis of the HEK-293 cells that were transfected with other MC receptor isotypes. It is evident that with the availability of this tool, studies on the cell- and tissue-specificity, as well as the regulation mechanism of the MC-4 receptor, will be largely facilitated.

New aspects on the melanocortins and their receptors.

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.

The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells.

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

The role of alpha-MSH as a modulator of cutaneous inflammation.

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.

Detection of melanocortin-1 receptor antigenicity on human skin cells in culture and in situ.

The proopiomelanocortin (POMC) products alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and In situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvascular endothelial cells and WM35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of sweat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated keratinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.

Inhibition of peripheral NF-kappaB activation by central action of alpha-melanocyte-stimulating hormone.

With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central alpha-MSH operates by inhibiting activation of the nuclear factor kappa B (NF-kappaB) that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-alpha demonstrated that centrally administered alpha-MSH does inhibit NF-kappaB activation. Western blot analysis revealed that this inhibition was linked to central alpha-MSH-induced preservation of expression of IkappaBalpha protein in the peripheral tissue. The NF-kappaB and IkappaBalpha effects were inhibited in mice with spinal cord transection. Intraperitoneal (i.p.) injection of the nonspecific beta-adrenergic receptor blocker propranolol, and of a specific beta2-adrenergic receptor antagonist, likewise prevented these effects of central alpha-MSH; blockade of cholinergic, alpha-adrenergic, or beta1-adrenergic receptors did not. Centrally administered alpha-MSH inhibited peripheral NF-kappaB activation and IkappaBalpha degradation even in mice with nonfunctional melanocortin 1 receptors (MC1R). These findings indicate that alpha-MSH can act centrally to inhibit NF-kappaB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves beta2-adrenergic receptors, but does not require activation of MC1R within the brain.

Characterization of a polyclonal antibody raised against the human melanocortin-1 receptor.

We have generated a polyclonal antibody raised against a synthetic peptide corresponding to the amino acids 2-18 of the extracellular, N-terminal domain of the human melanocortin-1 receptor (MC-1R). Specificity of the affinity-purified anti-MC-1R antibody was confirmed by dot blot analysis with the antigenic peptide. The antibody detected MC-1R antigenicity on the surface of normal human melanocytes and WM35 melanoma cells, as shown by FACS and immunofluorescence analysis. The antibody was suitable for immunoperoxidase staining of deparaffinized skin sections, revealing prominent MC-1R staining of a cutaneous melanoma as opposed to undiseased skin in which normal melanocytes were only occasionally immunoreactive. Distinct adnexal structures in normal skin also displayed MC-1R immunostaining. Specificity of the MC-1R immunoreactivity in each technique was confirmed by preabsorption with the immunogenic peptide, omission, or substitution of the primary antibody with preimmune serum. Our results provide a baseline for future studies on MC-1R expression in diseased human skin.

HLA-independent heterogeneity of CD8+ T cell responses to MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 in vaccine-treated melanoma patients.

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients.

Immunohistochemical detection of the melanocortin 1 receptor in human testis, ovary and placenta using specific monoclonal antibody.

We describe the immunohistochemical detection of the melanocortin 1 receptor (MC1R) protein in human gonadal tissues using a specific monoclonal antibody. The MC1R was found to be present in Leydig's cells in testis, in lutein cells in the corpus luteum and in the nucleus of the trophoblastic cells of the placenta. Though it has been speculated earlier that MC1R is present in gonadal tissues, this is the first report demonstrating the presence of MC1R protein in these cells.

Receptor-binding properties of the peptides corresponding to the ACTH-like sequence of human pro-interleukin-1 alpha.

The octapeptide Gly-Lys-Val-Leu-Lys-Lys-Arg-Arg (termed leukocorticotropin, LCT) corresponding to the ACTH-like sequence 81-88 of human pro-interleukin-1 alpha and its derivative Tyr-Gly-Lys-Val-Leu-Lys-Lys-Arg-Arg were synthesized. The 125I-labeled Tyr-LCT specifically interacts with one type of receptor on the surface of murine splenocytes (Kd = (1.45 +/- 0.04) x 10(-8) M, the number of binding sites is equal to 4500) and with two types of receptors on the surface of murine peritoneal macrophages (Kd1 = (5.9 +/- 1.0) x 10(-9) M and Kd2 = (2.6 +/- 2.2) x 10(-7) M). LCT and Tyr-LCT significantly increase the adenylate cyclase activity of murine peritoneal macrophages. The receptor binding and adenylate cyclase stimulation activity of LCT and Tyr-LCT are inhibited by ACTH (13-24).

Polyclonal antibodies against human melanocortin MC1 receptor: preliminary immunohistochemical localisation of melanocortin MC1 receptor to malignant melanoma cells.

Peptides of 11 and 15 residue lengths were synthesised according to the sequence of the N-terminal region of the human MC1 melanocyte stimulating hormone receptor. The peptides were conjugated to thyroglobulin and used for preparation of antisera in the rabbit. Each of the conjugates raised antisera which showed high titre and specificity for its respective peptide antigen when evaluated in an ELISA test. Both types of antisera immunostained MC1 receptor expressing COS-7 cells. By contrast, the sera did not stain control COS-7 cells not expressing the MC1 receptor. Moreover, preimmune sera or antiserum preadsorbed with its respective peptide did not stain the MC1 receptor expressing cells. The antisera were used to immunostain sections of normal human skin, as well as samples of cutaneous malignant melanoma tumours obtained from a patient. The cells of the melanoma tumours were very strongly immunostained with the MC1 receptor antisera. By contrast, melanocytes which were present in the normal skin could not be visualised with our antisera.