Immune studies in the depigmenting C57BL/Ler-vit/vit mice. An apparent isolated loss of contact hypersensitivity

Vitiligo is a human disorder which destroys pigment cells in the skin, ears, eyes, and meningeal tissues and has often been associated with a variety of autoimmune disorders. The C57BL/Ler-vit/vit mouse is a mutant strain that exhibits a loss of epidermal pigment cells and a selective cell-mediated immune deficiency to epicutaneous-administered allergens. This observation is consistent with that observed in humans with vitiligo, who also exhibit loss of contact hypersensitivity (CHS), that appears to be associated with loss of pigment cells from the epidermis. Other cellular immune parameters such as delayed type hypersensitivity and antibody generation to both particulate and soluble Ag are normal or even hyperimmune in the vit/vit mice compared with congenic C57BL/6 controls. Cyclophosphamide treatment could reconstitute CHS responsiveness of the vit/vit mice to the allergen dinitrofluorobenzene. Further, this loss of CHS responsiveness to dinitrofluorobenzene could be restored with skin transplants from normal pigmented C57BL/6 mice to vit/vit mice. Normal C57BL/6 mice bearing white skin grafts from vit/vit mice did not contact sensitize. We suggest that this vit/vit mouse strain may serve as an excellent system to investigate various aspects of other contact hypersensitivity reactions as well as vitiligo.     

Vitiligo: Where Do We Stand?

Vitiligo is a puzzling disorder characterized by a disappearance of epidermal and/or follicular melanocytes by unknown mechanisms. This very common disorder involving 1–4% of the world population is thus of great importance for the practicing dermatologist. The cellular and molecular mechanisms leading to the destruction of melanocytes in this disorder have not yet been elucidated, making it of major interest for the cell biologist involved in melanocyte research. Recent advances in this field, due largely to the availability of techniques for culturing normal human melanocytes, opened new perspectives in the understanding of vitiligo. Although vitiligo has long been considered a disorder confined to the skin, there is now good evidence that it also involves the extracutaneous compartment of the “melanocyte organ.” It is also clear that vitiligo is not only a melanocyte disorder, but that it also involves cells, such as keratinocytes and Langerhans cells, found in the epidermis and follicular epithelium. The three prevailing theories of the pathogenesis of vitiligo are the immune hypothesis, the neural hypothesis, and the self-destruct hypothesis. New hypotheses suggest that vitiligo may be due to (1) a deficiency in an unidentified melanocyte growth factor, (2) an intrinsic defect of the structure and function of the rough endoplasmic reticulum in vitiligo melanocytes, (3) abnormalities in a putative melatonin receptor on melanocytes and (4) a breakdown in free radical defense in the epidermis. None of these hypotheses has been demonstrated, and according to the available data, it is likely that the loss of epidermal and follicular melanocytes in vitiligo may be the result of several different pathogenetic mechanisms.     

Terminal migration and early differentiation of melanocytes in embryonic chick skin

The microenvironment is thought to play a key role in the control of neural crest cell diversification. To investigate its role in melanocyte differentiation we mapped the temporal and spatial distribution of pigmented melanocytes in embryonic chick skin and determined, by experimental means, the route taken by migrating melanocytes in the skin. We show that the New Hampshire Red/Black Australorp crossbreed exhibits melanization from 5 days of incubation (2 1/2 days earlier than is reported in other breeds). Contrary to previous reports our findings show that melanization is at first predominantly dermal. Both dermal and epidermal melanocyte numbers increase until Day 8, whereafter there is a dramatic decline in dermal melanocytes and by Day 10, melanocytes are almost exclusively located in the epidermis. Using homeotypic and heterotypic combinations of white and red/black dermis and epidermis we have demonstrated that premelanocytes arrive in the dermis of the trunk by Day 3 and begin to move into the epidermis from Day 4 onward. Results from these grafts and from tritium labeling studies strongly suggest that there is little or no reverse migration of premelanocytes from epidermis to dermis. Our findings indicate that overt melanocyte differentiation is not dependent on location in an epidermal environment, and that melanogenesis does not signify the end-stage in the migration process. Further, they suggest that the early dermal mesenchyme plays a key role in controlling melanogenesis.     

The cell biology of human hair follicle pigmentation

Although we have made significant progress in understanding the regulation of the UVR‐exposed epidermal‐melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR‐shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis‐follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub‐populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species‐specific issues involved which the general reader of the pigmentation literature (with its substantial mouse‐based data) may not fully appreciate.     

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.     

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.     

Maintenance of distinct melanocyte populations in the interfollicular epidermis

Hair follicles and sweat glands are recognized as reservoirs of melanocyte stem cells (MSCs). Unlike differentiated melanocytes, undifferentiated MSCs do not produce melanin. They serve as a source of differentiated melanocytes for the hair follicle and contribute to the interfollicular epidermis upon wounding, exposure to ultraviolet irradiation or in remission from vitiligo, where repigmentation often spreads outwards from the hair follicles. It is unknown whether these observations reflect the normal homoeostatic mechanism of melanocyte renewal or whether unperturbed interfollicular epidermis can maintain a melanocyte population that is independent of the skin's appendages. Here, we show that mouse tail skin lacking appendages does maintain a stable melanocyte number, including a low frequency of amelanotic melanocytes, into adult life. Furthermore, we show that actively cycling differentiated melanocytes are present in postnatal skin, indicating that amelanotic melanocytes are not uniquely relied on for melanocyte homoeostasis.     

Genetic control over the determination and proliferation of melanocyte stem cells in mammals]

Data obtained in mutant mouse strains provide evidence for multilocus control of determination and proliferation of melanocyte stem cells. Mice are known to have five loci (mi, Sp, s, Ls, Dom) controlling melanoblast determination. Locus mi is expressed in pluripotent cells of the neural crest from which melanocyte and neuron clones are formed; it is also expressed in a strain of ectomesenchyme cells. Loci Sp, s, ls and Dom are expressed somewhat later, probably during one of the last quantal cell cycles leading to the determination of unipotent melanocyte stem cells. Mutant genes of these loci impair the development of pigment cells as well as of ganglial neurons. Three loci (W, vs, Sl) control the proliferation of melanocyte stem cells. Mutations of locus W present in a single copy inhibit the proliferative activity of melanoblasts, whereas when present at the double dose they completely block their proliferation. Locus Sl is not expressed in melanocytes but acts in another cell system, which is very important for the proliferation of melanocyte stem cells. Mutant genes Ga, si and vit decrease the lifespan of stem cells for epidermal melanocytes.     

Understanding the melanocyte distribution in human epidermis: an agent-based computational model approach

The strikingly even color of human skin is maintained by the uniform distribution of melanocytes among keratinocytes in the basal layer of the human epidermis. In this work, we investigated three possible hypotheses on the mechanism by which the melanocytes and keratinocytes organize themselves to generate this pattern. We let the melanocyte migration be aided by (1) negative chemotaxis due to a substance produced by the melanocytes themselves, or (2) positive chemotaxis due to a substance produced by keratinocytes lacking direct physical contact with a melanocyte, or (3) positive chemotaxis due to a substance produced by keratinocytes in a distance-to-melanocytes dependent manner. The three hypotheses were implemented in an agent-based computational model of cellular interactions in the basal layer of the human epidermis. We found that they generate mutually exclusive predictions that can be tested by existing experimental protocols. This model forms a basis for further understanding of the communication between melanocytes and other skin cells in skin homeostasis.     

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.     

Immune responses in a mouse model of vitiligo with spontaneous epidermal de‐ and repigmentation

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human‐derived, tyrosinase‐reactive T‐cell receptor on T cells and the matching HLA‐A2 transgene, were crossed to keratin 14‐promoter driven, stem cell factor transgenic (K14‐SCF) mice with intra‐epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor‐related orphan receptor gamma (RORγt)⁺ T‐cell compartment, these cells displayed markedly increased IL‐17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14‐SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin‐infiltrating, melanocyte‐reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.     

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.     

RADIATION DEPIGMENTATION OF MOUSE HAIR: A STUDY OF FOLLICULAR MELANOCYTE POPULATIONS

The radiation depigmentation of mouse hair has been studied by a technique enabling melanocyte per follicle counts to be made. Distributions for normal skin show a large peak corresponding to the zigzag hair type. Changes in the frequency distributions of melanocytes per follicle after irradiation are presented for Strong F and DBA-1 mice irradiated in anagen or telogen stages of hair growth. These distributions clearly suggest the existence of some precursor cells, and the dose-response curves obtained by defining radiation survivors as follicles containing more than ten melanocytes gives the sensitivity of these cells to inactivation. D₀ values are 180–220 rads. A melanocyte-melanoblast model is proposed for the follicular melanocyte cycle which can be outlined as follows: The telogen follicle contains a small number of amelanotic melanocytes that survived through catagen. These cells possess the ability to repopulate the follicle with melanocytes. In catagen functional and/or amelanotic melanocytes are lost at random. Genes for dilution (possibly only when modified by other coat colour genes) and radiation both increase the chance of melanocyte loss at catagen by altering the melanocyte-dermal papilla relationship. One way in which this is affected is by a shortening of the dendrites. A feedback may operate in the follicle so that the full complement of melanocytes is achieved whatever number of melanocytes persists in telogen.     

Corticotropin Expression by Human Melanocytes in the Skin

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G₂-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.     

Melanocytes in conditional Rb–/– mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro

The function of the retinoblastoma tumour suppressor (Rb1), and the pocket protein family in general, has been implicated as an important focal point for deregulation in many of the molecular pathways mutated in melanoma. We have focused on the role of Rb1 in mouse melanocyte homeostasis using gene targeting and Cre/loxP mediated tissue‐specific deletion. We show that constitutive Cre‐mediated ablation of Rb1 exon 2 prevents the production of Rb1 and recapitulates the phenotype encountered in other Rb1 knockout mouse models. Mice with conditional melanocyte‐specific ablation of Rb1 manifest overtly normal pigmentation and are bereft of melanocytic hyperproliferative defects or apoptosis‐induced depigmentation. Histologically, these mice have melanocyte morphology and distribution comparable with control littermates. In contrast, Rb1‐null melanocytes removed from their in vivo micro‐environment and cultured in vitro display some of the characteristics associated with a transformed phenotype. They proliferate at a heightened rate when compared with control melanocytes and have a decreased requirement for mitogens. With progressive culture the cells depigment at relatively early passage and display a gross morphology which, whilst reminiscent of early passage melanocytes, is generally different to equivalent passage control cells. These results indicate that Rb1 is dispensable for in vivo melanocyte homeostasis when its ablation is targeted from the melanoblast stage onwards, however, when cultured in vitro, Rb1 loss increases melanocyte growth but the cells are not fully transformed.     

Evidence for nerve growth factor-mediated paracrine effects in human epidermis

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.