Combined Dopa-premelanin Reaction Research Articles

Reduced Elastin Fibers and Melanocyte Loss in Vitiliginous Skin Are Restored after Repigmentation by Phototherapy and/or Autologous Minigraft Transplantation

Vitiligo is a hypopigmentation disease characterized by melanocyte death in the human epidermis. However, the mechanism of vitiligo development and repigmentation is largely unknown. Dermal fiber components might play an important role in vitiligo development and repigmentation. Indeed, our preliminary study demonstrated that elastin fibers were decreased in vitiliginous skin, suggesting that the elastin fiber is one of the factors involved in vitiligo development and repigmentation. To confirm our hypothesis, we investigated whether elastin fibers can be restored after treatment using phototherapy and/or autologous skin transplantation. Punch biopsies from 14 patients of stable nonsegmental vitiligo vulgaris were collected from nonlesional, lesional, and repigmented skin, and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. Moreover, elastin fibers were detected by Victoria blue staining. Numerous melanocytes and melanoblasts were observed in the epidermis of repigmented skin after the treatment. Moreover, in the dermis of repigmented skin, elastin fibers were completely recovered or even upregulated. These results suggest that melanocyte loss in the vitiliginous skin, as well as melanocyte differentiation in repigmented skin, may be at least in part regulated by elastin fibers in the dermis.

The human melanocyte and melanoblast populations per unit area of epidermis in the rete ridge are greater than in the inter‐rete ridge

In human skin, melanocytes and melanoblasts are mostly located in the epidermis in addition to hair follicles. In hairy skin such as mouse skin, the inter-follicular epidermis is generally flat. In contrast, in human skin, the epidermis is wavy and possesses well-developed rete ridges. It is not well understood what difference exists in cell function between melanocytes present in the inter-rete ridge and those in the rete ridge. To clarify this problem, we first tried to determine the melanocyte and melanoblast populations per unit area of epidermis both in the rete ridge and inter-rete ridge epidermis. Thirty-eight samples from normal skin sites of 28 patients (14 males and 14 females) aged from 5 to 76years were fixed and processed to dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa (melanocytes) and combined dopa-premelanin (melanoblasts and melanocytes) reactions were scored. The average melanocyte population/0.1mm2 of the inter-rete ridge was 74 cells, whereas that of the rete ridge was 147 cells. Moreover, the average melanoblast population/0.1mm2 in the inter-rete ridge was 43 cells, whereas that of the rete ridge was 131 cells. The melanoblast population in the rete ridge differed between female and male, but the melanocyte populations in the rete ridge and inter-rete ridge and the melanoblast population in the inter-rete ridge did not differ between female and male. However, no age difference in the melanocyte and melanoblast populations was observed between the rete ridge and inter-rete ridge. Human epidermal melanocytes and melanoblasts in the rete ridge exist more numerously than in the inter-rete ridge.

Histochemical study of the distribution of epidermal melanoblasts and melanocytes in Asian human skin.

The classical study revealed the melanocyte density in the epidermis of many skin sites of humans. However, these data were obtained by counting melanocytes using dihydroxyphenylalanine (dopa)-treated epidermal sheets. Since rete ridges are developed well in human epidermis, there is a concern about the accuracy of these data. The accurate counting of the melanocyte density in the epidermis including the rete ridges should be performed using histological sections after the dopa treatment. Moreover, it is not known well how many melanoblasts are present in Asian epidermis. The aim of this study was to count the accurate number of melanocytes and melanoblasts. Normal skin sites of 9- to 77-year-old patients were fixed with buffered formalin and processed to the dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa reaction (melanocytes) and to the combined dopa-premelanin reaction (melanoblasts and melanocytes) were scored. In the skin of arms, legs, back, and belly, similar density (approximately 110-120cells/0.1mm2 ) of melanocytes was observed, whereas in the skin of scalp, melanocyte density was much lower (approximately 70cells/0.1mm2 ). By contrast, the melanoblast density did not differ between skin sites (approximately 100cells/0.1mm2 ). These results suggest that the melanocyte density does not differ between skin sites except the scalp skin, and a certain number of melanoblasts are present in each skin site of Asian. Melanoblasts seem to be required for producing new melanocytes required to maintain epidermal homeostasis.

Activation of melanoblasts and melanocytes after treatment with monochromatic excimer light and narrowband-ultraviolet B of skin of vitiligo patients.

Vitiligo is a hypopigmentation disease characterized by achromatic or hypochromatic macules in several sites of human skin. Although monochromatic excimer light (MEL) and narrowband-ultraviolet B (NB-UVB) have recently been used for efficacious treatment of vitiligo, the repigmentation process and role of melanocytes/melanoblasts are not well known. White macules of 28 vitiligo patients were repeatedly exposed with MEL/NB-UVB. After the exposures, punch biopsies of the center and edge of the lesional skin in addition to normal skin were fixed and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. Two repigmentation patterns were observed; one was "marginal" and the other was "perifollicular." Although the frequency of the former pattern was much lower than that of the latter pattern, no differences were observed in average percentage of repigmentation and the number of melanocytes and melanoblasts observed at the center and edge of the lesional epidermis. These results suggest that exposures with MEL/NB-UVB induce the marginal or perifollicular repigmentation pattern in the white macules, though the two patterns produce no differences in the distribution and density of melanoblasts/melanocytes in the lesional epidermis.

Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro.

Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited. To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium. The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes. The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid. About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction. Eumelanin content in p/p was extremely reduced compared to P/P. The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit. However, the reactivities in p/p were lower than in P/P. Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them. These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs.

Effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci on the proliferation and differentiation of mouse epidermal melanocytes in serum-free culture

To examine the effects of coat-color genes on the proliferation and differentiation of mouse epidermal melanocytes, we cultured epidermal, cell suspensions derived from neonatal skins of C57BL/10JHir (black) and its congenic mice carrying agouti, brown, albino, dilute, and pink-eyed dilution genes in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate. The proliferative rates of agouti, brown and dilute black melanocytes were similar to that of black melanocytes, while those of albino and pink-eyed black melanocytes were about one-half of that of black melanocytes. The morphology of albino and pink-eyed black melanocytes, though nonpigmented, was similar to black melanocytes; namely, dendritic, polygonal or epithelioid. Dilute black melanocytes also possessed the similar morphology, whereas their melanosomes were accumulated in the perinuclear region. Dopa-melanin depositions after dopa reaction in brown and dilute black melanocytes were greater than in black and agouti melanocytes. Although dopa-melanin depositions were not observed in albino melanocytes, about 8% of pink-eyed black melanocytes were positive to dopa reaction. Silver depositions after combined dopa-premelanin reaction in agouti, brown and dilute black melanocytes were similar to that in black melanocytes. Although albino melanocytes were devoid of silver depositions, about 25% of pink-eyed black melanocytes were positive to the reaction. Pyrrole-2,3,5-tricarboxylic acid (PTCA, degradation product of eumelanin) contents in agouti and dilute black melanocytes were slightly lower than in black melanocytes, while that in brown melanocytes was reduced to one-third. In contrast, PTCA contents in albino and pink-eyed black melanocytes were reduced to less than 0.5%. Aminohydroxyphenylalanine (AHP, degradation product of pheomelanin) contents did not differ among these melanocytes. These results suggest that the coat-color genes exert their influences on the proliferation and differentiation of mouse epidermal melanocytes by affecting tyrosinase activity, melanosome maturation and transport, and eumelanin synthesis.

Genetic factors controlling the proliferative activity of mouse epidermal melanocytes during the healing of skin wounds.

A cut was made on the middorsal skin of newborn mice of strains C57BL/10J, C57BL/10J-A/A, and C3H/He using fine iridectomy scissors. In the epidermis within 1 mm of the wound edge in C57BL/10J and C57BL/10J-A/A, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically until the 3rd day, then gradually decreased. In contrast, the melanocyte population of C3H/He did not increase after wounding, despite that the melanoblast-melanocyte population increased. Pigment-producing melanocytes in mitosis were frequently found in C57BL/10J and C57BL/10J-A/A, but not in C3H/He. The F1, F2, and backcross matings were performed to get some information about the genetic basis of the difference between C57BL/10J and C3H/He. In the F1 generation the offspring from reciprocal crosses exhibited intermediate values in both populations on the 3rd day after wounding. The F2 generation included the C3H/He type, F1 type, and C57BL/10J type in a ratio of 1:2:1 in both populations. Moreover, both reciprocal backcrosses gave 1:1 ratios of parent type to F1 type in both populations. These results indicate that the proliferative activity of mouse epidermal melanocytes during the healing of skin wounds are controlled by semidominant genes.

Developmental changes of the proliferative response of mouse epidermal melanocytes to skin wounding.

A cut was made on the middorsal skin of mice of various ages of strain C57BL/0J using fine iridectomy scissors. Specimens from the wounded skins were fixed at various days after wounding and were subjected to the dopa reaction and to the combined dopa-premelanin reaction. When the dorsal skins of 1.5-day-old mice were wounded, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically in the epidermis adjacent to a skin wound. Pigment-producing melanocytes in mitosis were frequently found in the vicinity of a wound immediately after wounding. When the dorsal skins of 4.5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was smaller than that of 1.5-day-old mice. The increase in number of pigment-producing melanocytes in mitosis was reduced and delayed as compared to 1.5-day-old mice. When the dorsal skins of 8.5-, 20.5-, and 60.5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was much smaller than the newborn mice. Moreover, pigment-producing melanocytes in mitosis were never found. These results indicate that the proliferative response of mouse epidermal melanocytes to skin wounding becomes delayed and diminished with development.

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice.

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.

Effects of genic substitution at the brown locus on the differentiation of epidermal melanocytes in newborn mouse skin.

In the dorsal epidermis of both C57BL/10J (black, BB) and C57BR/cdJ (brown, bb) mice, the number of melanocytes positive to the dopa reaction (melanocyte population) increases from birth to day 3 or 4, and then gradually decreases. However, the number of melanoblasts plus melanocytes positive to the combined dopa-premelanin reaction (melanoblast-melanocyte population) remains constant until day 3 or 4 and then decreases in the two strains. Despite the similarity of the developmental dynamics in both black and brown mice, there is a significant difference in the number of differentiated melanocytes. Melanocytes are more numerous and more dopa-reactive in brown mice than in black. The maximal density of the melanoblast-melanocyte population on day 3 or 4 does not differ in brown and black mice. Moreover, the maximal density of the melanocyte population in brown epidermis does not differ from that of the melanoblast-melanocyte population of both brown and black. These results indicate that b allele, when homozygous, enhances the differentiation of epidermal melanoblasts by inducing high tyrosinase activity.

Fish Hereditary Melanoma Cell Lines of Different Degrees of Cell Differentiation: (fish/hereditary melanoma/melanoma cell line/pigment cell differentiation).

Four cell lines including two sublines were established from hereditary melanomas in interspecific hybrids between platyfish (Xiphophorus maculatus) carrying the Sp gene and swordtails (X. helleri) and maintained in vitro for more than 34 months. Cells in each cell line grew randomly across each other with an apparent lack of contact inhibition of growth and at a population doubling time of 50 to 72 hr. They retained the characteristics of young pigment cells in regard to ultrastructure, tyrosinase activity, the DOPA and combined DOPA-premelanin reactions. In the degree of differentiation, the cells of the three cell lines seemed comparable to early melanocytes close to melanoblasts, and those of the remaining one cell line seemed comparable to young melanocytes but were in a more differentiated state than the early melanocytes. Colony forming ability on plastic plates was at a level of 10% in the three cell lines but only 1% in the one cell line. All four cell lines failed to form colonies in soft agar. Chromosome analysis revealed that these four cell lines were heteroploid with many abnormal figures of chromosomes and double minute chromosomes. None of the cell lines showed transplantability to fish.

Proliferation of epidermal melanocytes during the healing of skin wounds in newborn mice.

A full-thickness cut 7 mm long was made on the middorsal skin of C57BL/10J mice using fine iridectomy scissors. Specimens from the wounded skins were fixed at various days after wounding and were subjected to the dopa reaction and to the combined dopa-premelanin reaction. In the epidermis within 1 mm from the wound edge, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically until the third day, then gradually decreased. In contrast, both populations in the regenerating wound epidermis appeared on the third day and increased until the seventh day, then gradually decreased. However, the maximal population density in the regenerating epidermis did not exceed the initial density. The size of the melanocyte population in both the epidermis within 1 mm from the wound edge and the regenerating epidermis did not differ from that of the melanoblast-melanocyte population in all stages of wound healing. Moreover, pigment-producing melanocytes in mitosis were found immediately after wounding in the epidermis within 1 mm from the wound edge, but not in the regenerating epidermis and control epidermis. These results indicate that the epidermal melanocytes in neonatal mouse skin can be stimulated to undergo mitosis immediately adjacent to a skin wound and, thereafter, to migrate into the regenerating epidermis.

マウス表皮メラノサイトの分化に対するアクチノマイシンDおよびシクロヘキシミドの効果

The increase in the number of epidermal melanocytes positive to the dopa reaction after birth was suppressed in C57BL/10J strain mice when injected with actinomycin D or cycloheximide. On the other hand, no change was observed in the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction. The initiation of melanogenesis in the skin of newborn mice, therefore, seems to require de novo transcription and translation. The increase in the number of melanocytes after treatment with alpha-MSH or DBc-AMP was also completely suppressed when mice were injected with actinomycin D or cycloheximide. The results suggest that normal and hormone-induced differentiation of epidermal melanocytes requires de novo transcription and translation.

Genes involved in regulating the melanocyte and melanoblast-melanocyte populations in the epidermis of newborn mouse skin.

In the dorsal epidermis of both C57BL/10J and C3H/He mice, the number of melanocytes positive to the dopa reaction increased from birth to 3 or 4 days of age, and then gradually decreased and disappeared by 30 days of age. However, the number of melanoblasts plus melanocytes positive to the combined dopa-premelanin reaction remained constant until 3 or 4 days of age and then decreased in both strains. Despite the similarity of the change in these populations from birth on, there was a significant difference in the number of such cells between the two strains. Both melanocytes and melanoblasts in C3H/He were fewer than in C57BL/10J mice. The F1, F2, and backcross matings were performed in order to gain some information about the genetic basis of the difference. In the F1 generation the offspring from reciprocal crosses exhibited intermediate values in both populations. The F2 generation included the C3H/He type, F1 type, and C57BL/10J type in a ratio of about 1:2:1 in both populations. Moreover, both reciprocal backcrosses gave ratios of parent type to F1 type, which did not differ significantly from 1:1 in either population. These results indicate that the melanocyte and melanoblast-melanocyte populations in the dorsal epidermis of newborn mouse skin are controlled by semidominant genes.

CLINICAL APPLICATION OF CYTOCHEMISTRY IN DERMATOLOGY WITH SPECIAL REFERENCE TO DISORDERS IN THE PIGMENTARY SYSTEM

The application of cytochemistry in clinical as well as basic medicine has been described with particular reference to pigmentary disorders which can be divided into three groups namely hyperfunction, hypofunction and neoplasm. For the hyperfunction of melanocytes which results in two types of hyperpigmentation, dermal and epidermal melanosome accumulation, the importance of cytochemical investigation of tyrosinase, and acid phosphatase at the cellular and subcellular level together with parallel biochemical enzyme assay has been presented. For the group of pigmentary disorders due to hypofunction of melanocytes the integral role of cytochemical findings such as dopa reaction, combined dopa-premelanin reactions, osmium iodide reaction and ATPase reaction has been described in relation to the differential diagnosis, prognosis and determination of therapeutic effects. For the third group of neoplastic pigmentary disorders, the clinical importance of dopa reaction and tyrosine reaction is mentioned in determining benign or malignant neo-plastic pigment cells. Furthermore the contribution of other enzyme cytochemistry such as butyryl and acetyl cholinesterase, development of combined dopa-acid phosphatase reaction, and thiamin pyrophosphatase reaction in the field of pigmentology from basic and clinical aspects has been presented with our recent findings at the cellular and subcellular level.

New Technic for Comprehensive Demonstration of Melanin, Premelanin, and Tyrosinase Sites: Combined Dopa-Premelanin Reaction

Bizzozero (1) (1908) pointed out that melanin granules reduce silver nitrate to the metallic state and become very dark. Miescher (2) (1922) described that the dark substance resulting from the silver reaction is a compound of silver and melanin. Reduction to organic silver compounds needs a much longer time than does the silver reaction for darkening melanin granules. Masson (3) (1911) introduced ammoniacal (reduced) silver nitrate, which proves to be more sensitive, and blackens all premelanin and melanin granules and those only. Bloch (4) (1917) found that the pigment-forming cells are darkened in their entirety when frozen sections of fresh tissue are placed in 3,4-dihydroxyphenylalanine. This darkening is due to the presence of dopa melanin which has been formed by the action of a tyrosinase on the dopa. Becker, Sr. (5, 6) (1935) discovered that the paraffin section method of performing the dopa reaction permits a more exact cytologic study than Bloch's frozen section method.