Penile lentiginosis. An ultrastructural study.

This study on five patients has revealed more extensive alterations to melanocytes than previously reported, and emphasizes the fact that depigmentation is an essential element of the condition. In hyperpigmented areas, melanocytes were increased in number along the basal layer of the epithelium, were hyperactive, and in some cases contained bizarre melanosomes. In two cases there was suggestion of a defect in melanosome transfer to keratinocytes. Lymphocytes were closely apposed to melanocytes, and, in hypopigmented areas, were clearly involved in their disintegration. In depigmented areas, there was complete absence of melanocytes and of melanosomes in keratinocytes, and lymphocytes were present in the basal layer. In general, the appearances did not resemble melanoma in situ with spontaneous regression, although a second biopsy of one patient after one year did reveal features of melanocytes suggestive of an early stage of this condition. The study has provided no clear information as to the initial cause of the condition, but the manner of destruction of melanocytes suggests an immune reaction. Neither has it been of assistance in suggesting a more precise name for it.     

Penile Lentiginosis. An Ultrastructural Study

This study on five patients has revealed more extensive alterations to melanocytes than previously reported, and emphasizes the fact that depigmentation is an essential element of the condition. In hyperpigmented areas, melanocytes were increased in number along the basal layer of the epithelium, were hyperactive, and in some cases contained bizarre melanosomes. In two cases there was suggestion of a defect in melanosome transfer to keratinocytes. Lymphocytes were closely apposed to melanocytes, and, in hypopigmented areas, were clearly involved in their disintegration. In depigmented areas, there was complete absence of melanocytes and of melanosomes in keratinocytes, and lymphocytes were present in the basal layer. In general, the appearances did not resemble melanoma in situ with spontaneous regression, although a second biopsy of one patient after one year did reveal features of melanocytes suggestive of an early stage of this condition. The study has provided no clear information as to the initial cause of the condition, but the manner of destruction of melanocytes suggests an immune reaction. Neither has it been of assistance in suggesting a more precise name for it.     

Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.     

Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour

Vitiligo depigmentation is considered a consequence of either melanocyte disappearance or loss of functioning melanocytes in the involved areas. However, it has been reported that keratinocytes in involved vitiligo skin are damaged too. Based on this evidence, we evaluated the in vitro behaviour, in life span cultures, of involved and uninvolved vitiligo keratinocytes and their expression of proliferation, differentiation and senescence markers. An additional purpose was to investigate whether vitiligo keratinocytes from depigmented skin are able to sustain survival and growth of normal melanocytes (when added in co-culture experiments), as normal human keratinocytes manage to do. Our results demonstrate that almost all involved vitiligo keratinocytes have a shorter life span in vitro than the uninvolved cells and all of them do not maintain melanocytes in culture in a physiological ratio. Modification of proliferation and senescence marker expression also occurs. Indeed, we detected low initial expression levels of the senescence marker p16 in involved vitiligo keratinocytes, despite their shorter in vitro life span, and increased expression of proliferating cell nuclear antigen and p53. This preliminary analysis of a small number of in vitro cultured vitiligo keratinocytes suggests an impaired senescence process in lesional vitiligo keratinocytes and attempts to regulate it.     

The specificity of UVA-induced DNA damage in human melanocytes

Exposure to solar UV radiation is the origin of most skin cancers, including deadly melanomas. Melanomas are quite different from keratinocyte-derived tumours and exhibit a different mutation spectrum in the activated oncogenes, possibly arising from a different class of DNA damage. In addition, some data suggest a role for UVA radiation in melanomagenesis. To get further insight into the molecular mechanisms underlying induction of melanoma, we quantified a series of UV-induced DNA damage in primary cultures of normal human melanocytes. The results were compared with those obtained in keratinocytes from the same donors. In the UVB range, the frequency and the distribution of pyrimidine dimers was the same in melanocytes and keratinocytes. UVA was also found to produce thymine cyclobutane dimer as the major DNA lesion with an equal efficiency in both cell types. In contrast, following UVA-irradiation a large difference was found for the yield of 8-oxo-7,8-dihydroguanine; the level of this product was 2.2-fold higher in melanocytes than in keratinocytes. The comet assay showed that the induction of strand breaks was equally efficient in both cell types but that the yield of Fpg-sensitive sites was larger in melanocytes. Our data show that, upon UVA irradiation, oxidative lesions contribute to a larger extent to DNA damage in melanocytes than in keratinocytes. We also observed that the basal level of oxidative lesions was higher in the melanocytes, in agreement with a higher oxidative stress that may be due to the production of melanin. The bulk of these results, combined with qPCR and cell survival data, may explain some of the differences in mutation spectrum and target genes between melanomas and carcinomas arising from keratinocytes.     

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin‐containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross‐talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus‐mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication‐Competent Avian sarcoma‐leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back‐transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)⁺Neo^r+‐electroporated melanocytes from Hypeco nera were selectively grown in G418‐supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS‐mOrange, mOrange⁺ keratinocytes and transplanted EGFP⁺ melanocytes were frequently juxtaposed each other. High‐resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.     

Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome.

To determine the survival of cultured allogeneic keratinocytes transplanted to a deep dermal bed 24 tattoos that had been removed by deep shave excision in 19 patients were grafted with sheets of cultured allogeneic keratinocytes from donors of the opposite sex. Cells carrying the Y chromosome were identified in biopsy specimens taken from the graft site by in situ DNA hybridisation with a biotinylated Y probe (pHY 2.1) and visualised with a technique using immunoperoxidase. The cultured allograft sites were biopsied one, two, and three weeks after transplantation. No male cells were identified in any biopsy specimen from female patients who were given transplants of male cultured keratinocytes, and all biopsy specimens from male patients, who received female cultured keratinocytes, showed percentages of male cells within the normal range for male skin. The beneficial effects of cultivated allogeneic keratinocytes result from effects on wound healing other than forming a successful graft that "takes."     

A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures. Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice. Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice. The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice. These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.     

The melanocytorrhagic hypothesis of vitiligo tested on pigmented, stressed, reconstructed epidermis

Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and hair follicles. We previously proposed a new theory that vitiligo involves the chronic detachment and transepidermal loss of melanocytes caused by autoimmune, neural and impaired redox mechanisms associated with mechanical trauma. In this study, we reconstructed epidermis on dead de-epidermized dermis with normal and/or non-segmental non-lesional vitiligo (NSV) cells and tested catecholamines or sera or hydrogen peroxide. Under unstressed conditions, the number of melanocytes located in the basal layer was significantly lower in reconstructs made with melanocytes from non-lesional NSV skin and normal keratinocytes compared with controls made with autologous normal melanocytes. The number of non-lesional NSV melanocytes was even lower in reconstructs made with keratinocytes from non-lesional NSV skin. Epinephrine and H(2)O(2) could trigger the transepidermal loss of normal and vitiligo melanocytes. Some sera induced melanocyte detachment but without any clear correlation with disease activity in the donors. In conclusion, our results are the first step to obtaining a reproducible melanocytorrhagic model in vitro with some of the stressors investigated. They support the hypothesis that NSV melanocytes have an intrinsic defect, which limits their adhesion in a reconstructed epidermis, with an enhancer effect of the vitiligo keratinocyte milieu.     

Reconstruction of a tissue-engineered skin containing melanocytes

The objective of this study was to establish a new method for reconstruction of a tissue-engineered skin containing melanocytes by employing tissue engineering. The keratinocytes, melanocytes and dermal fibroblasts were isolated and purified from human foreskin biopsies. Then the cells were used to construct a tissue-engineered skin containing melanocytes. The localization of melanocytes in the tissue-engineered skin was detected by DOPA staining, S-100 immunohistochemical staining and transmission electron microscope (TEM). The results showed that the melanocytes could be detected in the basal layer of the constructed skin and the melanocytes showed dendritic morphology. Moreover the constructed skins were used to repair the athymic mice skin defects. Animal experiment results indicated that the skin equivalents could successfully repair full thickness skin defects in athymic mice and generated black skins by 6weeks after grafting. Melanocytes located in the basal layer of the athymic mice skin could also be detected by using the S-100 immunohistochemical staining. Our established method is useful to repair the full-thickness skin defects.     

Deposition of Basic Fibroblast Growth Factor on Surface of Epidermal Melanocytes Suggesting the Stromal Control of Epidermal Pigmentation

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.     

Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.     

Activating mutation of GS alpha in McCune-Albright syndrome causes skin pigmentation by tyrosinase gene activation on affected melanocytes

McCune-Albright syndrome (MAS) is a sporadic disease characterized by café-au-lait spots, polyostotic fibrous dysplasia and hyperfunctional endocrinopathies. To elucidate the mechanism of skin pigmentation, melanocytes, keratinocytes and fibroblasts were primary cultured from the café-au-lait spot of a MAS patient. Then, mutational analysis and morphologic evaluation were performed. Also, cAMP level and tyrosinase gene expression in cultured cells were determined. Only Gsalpha mutation was found in affected melanocytes and the cAMP level in affected melanocytes was higher than that of normal melanocytes. The mRNA expression of tyrosinase gene was increased in the affected melanocytes. This study suggests that skin pigmentation of MAS results from activating mutation of Gsalpha in melanocytes and the mechanism involves the c-AMP-mediated tyrosinase gene activation.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Genetic and epigenetic control of the proliferation and differentiation of mouse epidermal melanocytes in culture.

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that alpha-melanocyte-stimulating hormone (alpha-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.     

Genetic and Epigenetic Control of the Proliferation and Differentiation of Mouse Epidermal Melanocytes in Culture

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that α-melanocyte-stimulating hormone (α-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3′:5′-cyclic monophosphate (DB-cAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.