Melanosome complexes and melanin macroglobules in normal human skin.

The presence of melanin macroglobules, and sometimes that of melanosome complexes also, in epidermal melanocytes has been considered a feature of various skin diseases. Opinions differ as to whether these structures can occur in normal skin. We have studied these melanin inclusions in normal Caucasian skin in the entire soma of 116 melanocytes and the occurrence of melanosomes in phagosomes of 77 Langerhans' cells obtained in different seasons. During winter the melanocytes contained few melanosomes but many melanosome complexes and melanin macroglobules. These melanosome inclusions were in 86%, localized in the most basal part of the melanocytes, particularly in the dermal protrusions. It is suggested that these structures can be transferred from epidermal melanocytes to dermal cells and that melanin macroglobules derive from melanosome complexes. Irrespective of the season, most of the Langerhans' cells contained melanosomes in their phagosomes, which suggests a phagocytic capacity of these cells and a role in the elimination of the melanin.     

Cellular localization of tyrosinase in human malignant melanoma cell lines.

Human malignant melanocytes show characteristic morphologic modifications which are particularly evident in their specific organelles: melanosomes. These modifications are conserved in cell culture. The ultrastructural localization of tyrosinase, the enzyme which converts tyrosine and dopa into melanin, was determined in 13 human melanoma cell lines. The different cell lines possess 4 distribution patterns of melanin synthesis based on dopa oxidase activity. The two first pathways, which involve the Golgi apparatus, seem to differ by the amount of enzyme within this organelle. The third pathway mainly involves the smooth endoplasmic reticulum, whereas tyrosinase is visible only in vesicles in the fourth. Some cells synthesize the enzyme in the manner observed in very early embryos.     

Tyrosine-induced melanogenesis shows differences in morphologic and melanogenic preferences of melanosomes from light and dark skin types

The quality, quantity and distribution of melanosomes in epidermis play a crucial role in the determination of skin color and its sensitivity to UV radiation. Melanocyte cultures originating from individuals with light and dark skin types were grown in media with varying concentration of L-tyrosine. Melanosomal melanin content and the size of the organelles were measured after subcellular fractionation. In light-skin type cells, increased melanin production resulted in a more elliptical shape of melanosomes. In melanosomes that constitutively produce more melanin, the tyrosine-induced melanogenesis caused enlargement in all dimensions. X-ray microanalysis provided evidence that the increase in sulfur content induced by high tyrosine concentration was more prominent in the melanosomes from light skin types. A ratio between pheomelanin and eumelanin found in light-skin type melanosomes by HPLC was increased more markedly than that in melanosomes from dark skin melanocytes. These findings suggest that the melanocytes of light-skinned individuals exhibit a preference for pheomelanogenesis. Pheomelanin production is a thiol-consuming process and that might increase the risk of oxidation stress in these cells. This fact, together with the limited ability of pheomelanin to absorb UV radiation may lead to an elevated skin cancer risk among light-skinned individuals.     

皮肤黑素转运的调节机制

黑素是决定皮肤颜色的主要色素，它由黑素细胞合成。黑素细胞主要存在于皮肤和毛囊中，黑素在黑素小体内合成后，随其迁移到树突末端，树突朝邻近的角质形成细胞生长并释放。然后，黑素小体在多种分子的调节下，进入邻近的角质形成细胞，形成皮肤色素并起到有效的光防护作用。     

Kinesin participates in melanosomal movement along melanocyte dendrites

Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.     

The color of skin: white diseases of the skin,nails, and mucosa

White diseases are a heterogenous group characterized by hypopigmentation or depigmentation. Skin and eye color are determined by the number and size of melanosomes present. Melanin is produced by melanosomes in the melanocytes present within the epidermis of the skin, uvea, and retinal pigmented epithelium (RPE). Conditions altering the number of melanocytes or concentration of melanin result in a lack of pigmentation, appearing as “white diseases” ranging from the well-known albinism and vitiligo to more esoteric white hand syndrome and Degos disease.     

Sub-populations of melanocytes in pigmented basal cell carcinoma: a quantitative, ultrastructural investigation

BACKGROUND: Pigmentation is a characteristic clinical feature of basal cell carcinomas (BCCs) in Japanese patients. The pathogenesis of melanin pigment in pigmented BCCs is poorly understood. METHODS: We have combined the techniques of morphometric analysis and electron microscopy to assess accurately the morphologic aspects of melanocytes that occurred in pigmented and non-pigmented areas of pigmented BCCs. RESULTS: In the pigmented areas melanocytes were not only located along the basal membrane but also interspersed between tumor cells in the central parts of the tumor nest, and had large and numerous dendrites. Those in a supra-basal location displayed some degree of degeneration due to mitochondrion and melanosome swelling. In the non-pigmented areas melanocytes were only basally located, showed fewer dendrites, and frequently showed abortive melanosomes. However, melanocytes in these two different portions were in the active state of melanogenesis and proliferation. Ultrastructural cytomorphometric analysis also showed significant differences in most of the nuclear and cell parameters including nuclear and cell area, the nuclear/cell area ratio, cell perimeter and cell form factor between these two types of melanocytes. Particularly melanocytes in the pigmented areas were twice the cell size of the latter. In addition, the melanosomes remained almost completely in the apoptotic tumor cells, and the phagocytosis of the melanosome-containing apoptotic cells by the neighboring tumor cells appeared to be followed by the formation of the melanosome complexes. CONCLUSIONS: These findings suggest that different populations of melanocytes are probably present in pigmented BCCs, and repeated cycles of phagocytosis of melanosome-containing apoptotic cells may represent the predominant way of forming large melanosome complexes. The present morphological observation and quantitative analysis provide a morphological basis for further studies to interpret other pathologic changes in pigmented BCCs.     

ULTRASTRUCTURAL STUDIES ON PIGMENTED MACULES OF PEUTZ-JEGHERS SYNDROME

Light and electron microscopic studies were carried out on pigmented macules on the lips, fingers and toes of the patients with Peutz-Jeghers syndrome. Our studies indicated basal pigmentation was noticed in the basal cell layer of the epidermis, but no differences were recognized between the lesions and normal skin in either the number of melanocyte or the length of the dendrite of melanocyte in the lips. In fingers and toes, few melanin granules or melanosomes could be found in the basal cells. Numerous dendrites of melanocyte filled with melanin granules or melanosomes were seen in the intercellular spaces of the keratinocytes. The dendrites of melanocytes in the lesions were much longer and more branched than those in the normal skin. However, the population of melanocytes in the both areas was almost identical. Some melanosomes in the dendrites of the upper epidermis seemed to be degraded and exhibited a positive acid phosphatase reaction. We may assume that melanosome transfer from melanocytes or melanosome receptivity of the keratinocytes remarkably decreased in the lesions on the fingers and toes of Peutz-Jeghers syndrome and then melanosomes had accumulated in the dendrites and some of them are being degraded.     

Proceedings of the 2nd Annual Meeting of the Japanese Photoaging Research Society. July, 2001. Kobe, Japan

Abstract Complexion coloration in humans is primarily regulated by the amount and type of melanin synthesized by the epidermal melanocyte. However, additional and equally contributing factors consist of (1) efficient transfer of melanin from the melanocytes to the neighboring keratinocytes and (2) distribution and degradation of the transferred melanosomes by the recipient keratinocytes. Once synthesized in the cell body of the epidermal melanocyte, pigmented melanosomes are translocated down the dendrites and captured at the dendritic tips via various cytoskeletal elements. Molecules recently identified that participate in this process consist of Rab27a, myosin‐Va and melanophilin. Eventually, these peripherally localized melanosomes are transferred to keratinocytes by a presently undefined mechanism. The protease‐activated receptor‐2 (PAR‐2) and unidentified surface lectins and glycoproteins facilitate this transfer process. Once incorporated into the keratinocytes, melanosomes are distributed individually or as clusters, aggregated towards the apical pole of the nucleus, and degraded as the keratinocytes undergo terminal differentiation and desquamation. Ultraviolet irradiation (UVR) can modulate the process of melanosome transfer from the melanocytes to the keratinocytes. UVR can upregulate expression of PAR‐2 and lectin‐binding receptors and increase phagocytic activity of cultured keratinocytes. Therefore, many cellular and molecular events that occur after melanogenesis contribute to skin color.     

Behavioral differences between donor site-matched adult and neonatal melanocytes in culture

Abstract Little is known about the biologic behaviors of cultured melanocytes in relation to donor age. To investigate age-dependent differences, neonatal and adult melanocytes were isolated from the same anatomical site, the foreskin, and cultured in the same growth medium supplemented with cAMP inducers (choleratoxin and 3-isobutyl-methylxanthine). The morphology, melanin content, pattern of melanosome distribution, and growth rate were then compared. Neonatal melanocytes were bipolar in appearance, whereas adult melanocytes were highly dendritic in appearance. Image analysis showed that adult melanocytes were larger and longer, and had a greater number of dendrites than neonatal melanocytes. When the growth medium was replaced by a medium without cAMP inducers, adult melanocytes showed a change in their morphology from dendritic to spindle-shaped, while the morphology of neonatal melanocytes remained unchanged. Melanosomes of adult melanocytes were distributed singly along the dendrites, and extracellular secretion of melanosomes was also found. In contrast, melanosomes of neonatal melanocytes were aggregated near the nuclei. No age-dependent differences in melanin content and growth rate were noted in the donor site-matched cultured melanocytes. These results suggest that donor age is one of the factors involved in determining melanocyte dendricity and melanosome distribution, and that increased dendricity of adult melanocytes is due to increased sensitivity to cAMP inducers. In addition, the adult melanocytes established in our culture system, which resembled dendritic melanocytes in vivo, could be considered a desirable model for studying the mechanisms of adult-onset hyperpigmentary disorders and melanogenesis. Received: 2 June 1998 / Revised: 29 November 1999 / Accepted: 10 December 1999     

THE ROLE OF EPIDERMAL LYSOSOMES IN MELANIN PHYSIOLOGY

SUMMARY.— Melanosomes are formed within melanocytes and transferred via dendrites to keratinocytes. In caucasoids, most melanosomes are aggregated in membrane-bound organelles (melanosome complexes). These aggregates have phagocytic capacity, stain positively with aryl sulphatase and acid phosphatase, contain melanosomes apparently undergoing degradation, and are lysosomes. In addition to melanosome complexes, individual melanosomes, both in melanocytes and keratinocytes, also exhibit lysosomal properties of phagocytosis and hydrolytic enzyme activity. In contrast, negro melanosomes are mostly singly dispersed and are rarely aggregated in complexes. Melanosome dispersal contributes to the darker colour and superior sunlight protection of negro skin. Degradation by lysosomal enzymes explains the limitation of melanin principally to the basal layer.
In skin with severe solar degeneration, particularly in xeroderma pigmentosum, pigment production is often apparently adequate since hypertrophic melanocytes and dendrites are conspicuous. However, pigment transfer from melanocytes to keratinocytes does not occur normally. Some keratinocytes contain little melanin while others show an increase. Melanosomes within keratinocytes of actinically damaged skin tend to be concentrated within larger than normal complexes. Skin colour and sunlight protection appear to depend not only upon the rate of melanin formation but also upon distribution and rate of degradation.     

Melanocyte-lymphocyte interaction in human graft-versus-host disease

Pigmentary disorders in chronic human GvHD comprise reticular or diffuse hyper-and hypopigmentation and seem to result from an interaction between lymphocytes and melanocytes. This interaction could be visualized by standard microscopy with a specific staining for melanin. The main ultrastructural findings were, in hyperpigmented areas: broad contact between melanocytes and lymphocytes, numerous and packed melanosomes, presence of cytoplasmic lipid droplets; in hypopigmented areas: disintegrating melanocytes with large intercellular spaces, small melanosomes, numerous lipid droplets, vacuolated organelles, as well as condensed and convoluted nuclei. Different hypotheses to explain the dysfunction of melanocytes are presented.     

Morphologic alterations of epidermal melanocytes and melanosomes in PUVA lentigines: a comparative ultrastructural investigation of lentigines induced by PUVA and sunlight

Ultrastructural studies were conducted in order to determine morphologic and functional differences in melanocytes and melanosomes in PUVA lentigines and solar lentigines, and light-protected buttock skin. Compared to melanocytes in solar lentigines from 7 subjects and light-protected buttock skin from 5 subjects (none of these subjects had received UV radiation therapy), melanocytes in PUVA lentigines from 6 subjects generally had longer and more numerous dendrites, and showed more active melanogenesis. Basal keratinocytes in PUVA lentigines had a significantly increased frequency of large, single melanosomes, and revealed significantly larger individual melanosomes within compound melanosomes. Other findings in some PUVA lentigines included the close apposition of Langerhans cells to melanocytes, and atypical nuclear, cytoplasmic and melanosomal alterations, including melanosomal pleomorphism and melanin macroglobules. The presence of relatively large and predominantly single melanosomes in basal keratinocytes of PUVA lentigines suggests more active melanogenesis and/or an irreversible somatic alteration. It will be important to determine the clinical course and ultrastructural findings of PUVA lentigines that persist long after PUVA is discontinued.     

Dyschromatosis Universalis Hereditaria: An Electron Microscopic Examination

We report electron microscopic findings from both hyperchromic and achromic macules of dyschromatosis universalis hereditaria (DUH). The keratinocytes of the hyperchromic macules contained numerous, fully melanized melanosomes; almost all of them were aggregated to form the melanosome complex. In contrast, the melanosomes were absent from both keratinocytes and melanocytes of achromic macules, even though intact melanocytes were found. Our results suggest that DUH may be a disorder of melanosome production and distribution in epidermal melanin units rather than a disorder of melanocyte number.     

Pigmentation-associated glycoprotein of human melanomas and melanocytes: definition with a mouse monoclonal antibody

Pigmented melanoma cells and cultured melanocytes express a differentiation-related glycoprotein designated as pigmentation-associated antigen (PAA) of Mr 70,000-80,000. As described previously, PAA was initially defined by reactivity with antibodies in the serum of a patient with melanoma. Here we describe the production and characterization of a mouse monoclonal antibody to PAA. This antibody (TA99, an IgG2a) was shown by sequential immunoprecipitation experiments to react with the same component as the human antibody. Ab TA99 immunoprecipitated PAA from lysates of cells radiolabeled with [35S]methionine, [3H]glucosamine, [3H]fucose, and [3H]mannose as well as 125I. Using Ab TA99, the distribution of PAA was examined in frozen sections of 19 normal tissues and quantitatively in 68 tissue culture cell lines. In frozen sections, only melanin-containing cells were positive, including epithelial cells in the basal layer of the epidermis, in which pigment originates from melanocytes by transfer of melanosomes, and pigmented cells of the eye. In tissue culture cell lines, only pigmented melanoma cells were positive. PAA is an intracellular antigen, with a distribution very similar to that of melanosomes. This evidence confirms the close association of PAA with melanin production, and suggests that PAA may be a melanosome component. PAA was shown to be different from tyrosinase, the enzyme involved in melanin synthesis, but it was found to be identical to the previously recognized glycoprotein, gp75, characteristic of pigmented melanomas and melanocytes.     

BUSULFAN HYPERPIGMENTATION: LIGHT AND ELECTRON MICROSCOPIC STUDIES

Skin with busulfan hyperpigmentation was studied using light and electron microscopy. There were both toxic effects on the melanocytes and stimulatory effects on the production of melanin. The mitochondria appeared damaged, and reduced in number. Cytoplasmic vacuoles, myelin figures and conglomerate masses of melanosomes were present. The melanosomes were normal in size but appeared increased. 'Ghost melanocytes' were found in the dermis. The basal lamina was damaged with evidence of duplication. Incontinence of melanin was seen. This hyperpigmentation may be similar to that in fixed drug eruption.     

Light microscopic, immunohistochemical, and ultrastructural alterations in patients with melasma

Despite new technologies, few studies have assessed the histologic alterations in patients with melasma. Using current technologies, the present study was designed to re-evaluate the light microscopic, immunohistochemical, and ultrastructural changes of the hyperpigmented and adjacent normal skin of patients with melasma. Twenty-one patients were included in this study. Two millimeter punch biopsies were taken from the hyperpigmented and adjacent normal skin of the face. The integrity of the epidermis and dermis was assessed by light microscopy, computer-assisted image analysis, immunohistochemistry, and electron microscopy. Stains included hematoxylin-eosin and Fontana-Masson for melanin detection. Immunostaining was performed using Mel-5 antibody and CD1a antibody as markers for melanin and Langerhans cells, respectively. However, mild lymphohistiocytic infiltrates were present in 75% of the hyperpigmented areas. The areas of hyperpigmentation showed increased deposition of melanin in the epidermis and dermis of all cases. There was a statistically significant increase in the content of epidermal melanin. There were no quantitative increases in melanocytes in the hyperpigmented areas of skin. However, the melanocytes in the hyperpigmented areas were larger, intensely stained cells with very prominent dendrites. Electron microscopy revealed more melanosomes in keratinocytes, melanocytes, and dendrites in the involved skin in comparison to the uninvolved skin. The results of this study suggest that melasma is a consequence of specific hyperfunctional melanocytes that cause excessive melanin deposition in the epidermis and dermis.     

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.     

Ebselen is a new skin depigmenting agent that inhibits melanin biosynthesis and melanosomal transfer

We assessed the ability of ebselen, a glutathione peroxidase mimic, to reduce pigmentation in various models. In murine B16 melanocytes, 25 μm ebselen inhibited melanogenesis and induced a depolymerisation of actin filaments. In co-cultures of B16 melanocytes with BDVII keratinocytes, a pretreatment of melanocytes with ebselen resulted in a strong inhibition of melanosome transfer to keratinocytes, as shown under optical and electron microscopy. In reconstructed epidermis, topical 0.5% ebselen led to a twofold decrease of melanin without affecting the density of active melanocytes. A similar result was obtained with topical 0.5% ebselen in black guinea pig ears. Ebselen induced a decrease of epidermal melanin parallel to a localisation of melanin and melanosomes in the basal layer. Ebselen appears as a new depigmenting compound that inhibits melanin synthesis and melanosome transfer to keratinocytes.     

Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata

It is well recognized that alopecia areata (Aa) may preferentially affect pigmented hair and may spare white hair, and that regrowing hair in the disease is often initially white. In addition, there is an association with vitiligo and ocular depigmentation. To date, the pathomechanisms of the melanocyte effects are unclear. We have studied 10 patients with untreated acute alopecia areata, and three normal patients without hair loss. Morphologic changes, studied by conventional light and electron microscopy, in the cytoplasm of affected melanocytes often predated nuclear hyperchromatism. Increased numbers of bizarre melanosomes were found in affected melanocytes compared with normal ones; such melanosomes had incomplete or "aborted" melanization, resulting in poor pigment deposition, and were disrupted, enlarged and rounded, with loss of normal ellipsoidal shape. An unusual outer root sheath (ORS) distribution of hair bulb melanocytes was seen. Other atypical melanosome effects included marked pigment displacement into peribulbar and DP melanophages. In the DP clumped melanin granules formed giant spherical complexes without discernible limiting membranes, which were sometimes associated with lymphocytes. These morphologic changes indicate an active involvement of hair bulb melanocytes in alopecia areata.