The antimicrobial properties of melanocytes, melanosomes and melanin and the evolution of black skin.

A biological issue that has not been satisfactorily resolved is the role of melanin in skin and other animal tissues. A hypothesis is outlined here to account for the evolution of black skin and the ubiquity of melanin in vertebrate tissues. Evidence is presented that melanization of skin and other tissues forms an important component of the innate immune defense system. A major function of melanocytes, melanosomes and melanin in skin is to inhibit the proliferation of bacterial, fungal and other parasitic infections of the dermis and epidermis. This function can potentially explain (a) the latitudinal gradient in melanization of human skin; (b) the fact that melanocyte and melanization patterns among different parts of the vertebrate body do not reflect exposure to radiation; (c) provide a theoretical framework for recent empirical findings concerning the antimicrobial activity of melanocytes and melanosomes and their regulation by known mediators of inflammatory responses.     

Organotypic Culture of Human Skin to Study Melanocyte Migration

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.     

Dermal melanocytosis in Japanese monkeys (Macaca fuscata)

The skin of Japanese monkeys (Macaca fuscata) shows diffuse discolorations resembling human dermal melanocytosis. Very few laboratory animals have melanocytes in the dermis. The purpose of this study was to clarify the dermatologic characteristics of Japanese monkeys in terms of gross appearance, skin color, and histopathologic findings. A colorimeter was used to record the skin colors of pigmented and nonpigmented sites. Tissue specimens obtained from both types of sites were examined histopathologically. All animals examined had pigmented sites on their bodies, and the discolorations extended over 25% to 33% of the body surface. The colorimeter could detect differences in skin color due to dermal melanocytosis. All parameters of the colorimetric systems used (Yxy, L*a*b*, and L*C*h* systems) demonstrated significant differences between pigmented and nonpigmented sites. In pigmented sites, the epidermis lacked melanocytes, but the dermis had numerous melanocytes with abundant melanin. Activated melanocytes with well-developed dendrites were distributed throughout the upper part of the dermal layer. Melanocytes were not arranged in clusters, and elastic and collagen fibers in the dermis showed no histological abnormalities. Nonpigmented sites lacked melanin granules in both the epidermis and dermis. This study revealed that gross dermal melanocytosis correlated well with colorimetric results and histopathologic findings. These findings suggest that the pigmentation of Japanese monkeys is equivalent to dermal melanocytosis in humans, to the end that Japanese monkeys may be a useful animal model for investigating dermal melanogenesis.     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

Effect of keratinocytes on regulation of melanogenesis in culture of melanocytes

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

Dermis‐derived stem cells: a source of epidermal melanocytes and melanoma?

Human multipotent dermal stem cells (DSCs) have been isolated and propagated from the dermal region of neonatal foreskin. DSCs can self-renew, express the neural crest stem cell markers NGFRp75 and nestin, and are capable of differentiating into a wide variety of cell types including mesenchymal and neuronal lineages and melanocytes, indicative of their neural crest origin. When placed in the context of reconstructed skin, DSCs migrate to the basement membrane zone and differentiate into melanocytes. These findings, combined with the identification of NGFRp75-positive cells in the dermis of human foreskin, which are devoid of hair, suggest that DSCs may be a self-renewing source of extrafollicular epidermal melanocytes. In this review, we discuss the properties of DSCs, the pathways required for melanocyte differentiation, and the value of 3D reconstructed skin to assess the behavior and contribution of DSCs in the naturalized environment of human skin. Potentially, DSCs provide a link to malignant melanoma by being a target of UVA-induced transformation.     

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.     

Induction of epidermal mucous metaplasia by culture of recombinants of undifferentiated epidermis and retinol-treated dermis in a chemically defined medium

Epidermal mucous metaplasia of cultured skin is known to be induced by excess retinol. Studies were made on whether retinol affects primarily the epidermis or the dermis during retinol-induced epidermal mucous metaplasia of 13-day-old chick embryonic skin in culture. When recombinants of 13-day-old normal epidermis and retinol-treated dermis were cultured for 7 days in chemically defined medium in the absence of retinol, hormones, and serum, they showed altered epidermal differentiation toward secretory epithelium (mucous metaplasia). Thus retinol acted primarily on dermal cells.     

Ultrastructure of normals and castrates and the effects of testosterone and ultraviolet (UVL-B) irradiation on scrotal skin of rats.

The ultrastructure of the testosterone dependent epidermal melanocyte system of the scrotal skin of normals and castrates, with and without testosterone replacement therapy, and UVL-B (280-315 nm) radiation in black Long Evans rats is reported. UVL-B increases melanocyte activity, melanosome forming apparatus, (size of Golgi zone and RER, and quantity of cytoplasmic vesicles, dendrites, and stages of melanosomes) in normals and in castrates. Testosterone replacement therapy to castrates is not a prerequisite for stimulation by UVL-B, but it enhances the effects of UVL-B without restoring normalcy as melanosome packaging into complexes predominates. After UVL-B stimulation of normals or castrates, melanocyte dendrites are observed more often. Melanocyte dendrites of skin of castrated rats are observed less often than in normals, but with testosterone replacement therapy, the dendrites become more numerous. Melanosomes donated to keratinocytes are mostly located as singles in normals and as complexes in castrates. After UVL-B, castration, or testosterone replacement therapy, the melanosomes are packaged in keratinocytes in complexes larger than in normals. In the epidermis of long term castrates (9-109 days), non-specific clear cells are observed and Langerhans cells containing melanosomes; we did not observe them in normals. Melanocytes of castrates have a reduced melanosome forming apparatus. The dermis of castrates contains many dermal melanocytes in the superficial dermis with melanosomes in several stages of formation. These cells are not apparent in normals at this location in the dermis. Testosterone replacement therapy and/or UVL-B administered to castrates does not restore the epidermal melanocyte system nor the dermis to precastration ultrastructural appearance; castration has a permanent altering effect as melanosomes are packaged into complexes.     

Melanocyte growth factor in normal human skin

Normal human skin is shown to contain melanocyte growth factor (MeGF). We found MeGF activity in extracts of both the epidermal portion of skin and the dermal portion. This activity was completely adsorbed onto heparin beads and eluted by 2.5 M NaCl. In addition, the activity of both extracts was completely blocked by antibodies directed against basic fibroblast growth factor (bFGF). It is suggested that melanocytes in epidermis are supported by bFGF-like MeGF in normal human skin.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.     

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.     

HGF/SF Increases Number of Skin Melanocytes but Does Not Alter Quality or Quantity of Follicular Melanogenesis

Melanins are an important factor determining the vulnerability of mammalian skin to UV radiation and thus to UV-induced skin cancers. Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) have extra-follicular dermal melanocytes, notably in the papillary upper dermis, and are susceptible to UV-induced melanoma. Pigmented HGF/SF neonatal mice are more susceptible than albino HGF/SF animals to UVA -induced melanoma, indicating an involvement of melanin in melanoma formation. This raises the question of the effect of transgenic HGF/SF on melanization. We developed a methodology to accurately quantitate both the production of melanin and the efficiency of melanogenesis in normal, and HGF/SF transgenic mice in vivo. Skin and hair shafts of 5 day old and adult (3 week old) C57BL/6-HGF/SF and corresponding C57BL/6 wild type mice were investigated by electron paramagnetic resonance spectroscopy (EPR) to quantitate melanin, by transmission electron microscopy (TEM) for the presence of melanosomes, and by standard histology and by Western blotting and zymography to determine the expression and activity of melanogenesis-related proteins. Eumelanin but no phaeomelanin was detected in transgenic C57BL/6-HGF and C57BL/6 wild type mice. Transgenic HGF/SF overexpression did not change the type of melanin produced in the skin or hair, did not affect the terminal content of melanin production in standard samples of hair and did not influence hair cycle/morphogenesis-related changes in skin thickness. No melanocytes were found in the epidermis and no melanosomes were found in epidermal keratinocytes. HGF/SF transgenic mice thus lack the epidermal melanin UV-protection found in constitutively dark human skin. We conclude that melanocytes in the HGF/SF transgenic mouse, particularly in the papillary dermis, are vulnerable to UVA which interacts with eumelanin but not phaeomelanin to induce melanoma.     

Melanogenesis in dermal melanocytes of Japanese Silky chicken embryos

The Japanese Silky chicken (SK) shows dermal and visceral hyperpigmentation. This study characterizes ultrastructurally the melanin granules developing in dermal melanocytes of the dorsal skin of SK, in an attempt to better understand the processes of melanogenesis in these permanently ectopic cells. The steps of melanogenesis are similar to those described for epidermal melanocytes, with melanosomes going from stage I to IV but, in SK, the maturation occurs in the cell body, as well as in the cytoplasmic processes. At stage III, the deposition of melanin is cumulative and can aggregate in rounded structures, which combine to turn into the mature granule. The final destiny of mature melanosomes is still unclear, although it was observed that dermal macrophages can accumulate melanin granules in their phagosomes. Even with the close proximity between melanocytes and other dermal cells, the transference of melanosomes was not observed. Our findings indicate that melanogenesis in dermal melanocytes in SK has the same morphological characteristics found in epidermal melanocytes, but the functional aspect still remains to be elucidated.     

THE FINE STRUCTURE OF NORMAL AND NEOPLASTIC MELANOCYTES IN THE SYRIAN HAMSTER, WITH PARTICULAR REFERENCE TO CARCINOGEN-INDUCED MELANOTIC TUMORS

The dermal melanocyte system of the Syrian hamster is particularly responsive to the melanogenetic and tumor-inducing effects of 7,12-dimethylbenz(a)anthracene (DMBA). The melanocytes of the hair follicles appear to be susceptible to the melanogenetic effect of DMBA but not to its tumor-inducing effect. The epidermal melanocytes are non-pigmented and are unresponsive to both melanogenetic and carcinogenic effects of DMBA. The pigmented granules of the dermal melanocytes of both the golden and the white hamster have an identical substructure and pattern of melanization which occurs in an orderly fashion on a delicate fibrillar component. The hair melanocytes have larger pigment granules with a more complicated fibrillar substructure. The epidermal melanocytes do not possess pigment granules but are recognized by their dendritic shape, the absence of desmosomes and tonofilaments, and the presence of racket-shaped or rod-shaped organelles. The melanin granules in neoplastic melanocytes of the golden hamster differ from corresponding normal melanocytes only in their larger size. In the white hamster, however, the melanin granules in tumors produced under identical experimental conditions are so bizarre and atypical that consideration was given to the possibility that a genetic difference in the melanization pattern between the two varieties becomes apparent in carcinogen-induced melanotic tumors. No definite conclusions could be reached as to the precise origin of the melanin granules in either normal or neoplastic melanocytes.     

Effect of bromocriptine on the larval skin of the green toad, Bufo viridis viridis leurenti

In the premetamorphic larval green toad, B. viridis viridis, as in other anurans, the skin is made up of a fibrous dermis and an epidermis of stratified epithelium. The effects of bromocriptine, an antiprolactin drug, on the premetamorphic skin of B. viridis viridis was examined. Bromocriptine, dissolved in rearing water at four different concentrations, induced a number of changes in the skin of treated tadpoles. In rough sequence of appearance, these changes include: retraction ofthe melanocyte dendrites, synchronous burst ofthe apical vesicles of the superficial epithelial cells, gradual disappearance of the melanosomes from the epithelial cells and widening of the intercellular spaces. In addition, macrophages appeared in the superficial dermis amongst the retracted melanocytes. White crystals were observed on the skin surface and similar crystals were ingested by the macrophages. Prolonged treatment with bromocriptine resulted in hypertrophy and extraction of some epidermal cells. Deep melanocytes of the mesenteries were not affected by bromocriptine-treatment indicating that the drug did not penetrate deep into the tadpole tissue. Whether the macrophages observed in the dermis were recruited from deeper tissues or were converted melanocytes is another issue in need of study.     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.