Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots.

BACKGROUND: Repeated exposures to ultraviolet B radiation (UVB) induce pigmented spots on dorsal skin of (HR-1 x HR/De) F(1) hairless mouse. We showed previously that this mouse is suitable for studies of melanocyte function. OBJECTIVE: To clarify the mechanism of development of pigmented spots induced by chronic UVB exposure. METHODS: We used light and fluorescence microscopy to quantify changes in the numbers of differentiated melanocytes containing melanin pigments (MM) and melanoblasts/melanocytes immunohistochemically positive for tyrosinase-related protein (TRP)-1, TRP-2 (dopachrome tautomerase), and c-kit in epidermis during the development of pigmented spots in hairless mice chronically exposed to UVB (99 mJ/cm(2), 3 times/week, 8 weeks). RESULTS: The change in the number of TRP-1-positive cells during chronic UVB exposure was similar to that of MM: both increased dramatically during the stage of acute pigmentation, then decreased sharply after cessation of UVB, concomitantly with depigmentation; subsequently they increased gradually with the development of pigmented spots. In contrast, after two UVB exposures, no c-kit-positive cells were detected, then the number gradually increased during UVB irradiation, and continued to increase after cessation of irradiation; TRP-2-positive cells showed a rather similar pattern, except that they did not disappear initially. CONCLUSION: Our results indicate that chronic UVB irradiation induces differentiation and proliferation of melanoblasts, followed by an increase of differentiated melanocytes, leading to the development of pigmented spots. The sequence of expression of markers appeared to be c-kit, TRP-2, TRP-1, and finally melanin, as it is during normal melanocyte differentiation.

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.

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.

Stress augmented ultraviolet-irradiation-induced pigmentation.

It was reported that adrenocorticotropic hormone stimulates melanogenesis in cultured melanocytes. Stress (high population density and restraint stress) induced a significant increase in adrenocorticotropic hormone levels in plasma and skin compared to control. The serum obtained from HR-1 x HR/De F1 female mice subjected to stress showed significantly increased tyrosinase activity in human melanocytes compared to that from nonstressed mice. The increase in tyrosinase activity was inhibited in the presence of 10 nM corticostatin, an adrenocorticotropic hormone inhibitor. The aim of this study was to examine whether adrenocorticotropic hormone released into the circulation under stressful conditions is associated with the regulation of ultraviolet-induced pigmentation. Mice divided into three groups were housed for 22 d under the following conditions: five mice per cage (control); 10 mice per cage (high population density); restraint stress 4 h per d. The animals were exposed to ultraviolet-B irradiation (72 mJ per cm2, thrice per wk). After ultraviolet-B irradiation, delayed tanning was marked in stressed mice. The number of dihydroxyphenylalanine-positive melanocytes also significantly increased in stressed animals. Pretreatment with 100 microg of corticostatin inhibited the augmentation of the stress-induced pigmentary response and the increase in dihydroxyphenylalanine-positive melanocytes after ultraviolet irradiation. Adrenocorticotropic hormone released by stress may activate tyrosinase in melanocytes, resulting in the augmentation of ultraviolet-induced pigmentation. These results suggest that adrenocorticotropic hormone is at least partly responsible for the sensitivity of the pigmentary response after ultraviolet irradiation under stressful conditions.

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.

Cultured human dermal papilla cells secrete a chemotactic factor for melanocytes.

Large numbers of pigmented melanocytes are located in human hair follicles, predominantly around the dermal papillae, and the number of melanocytes and the melanogenic activity of the hair follicles are closely related to the hair cycle. We found that cultured human dermal papilla cells secreted a melanocyte chemoattractant into the medium. Skin fibroblasts also showed weak chemoattraction of melanocytes, while skin keratinocytes and melanocytes did not. Since this chemotactic activity was heat-and protease-sensitive and was present in the relatively high molecular weight fraction (130-200 kDa), it may be due to extracellular matrix (ECM) that proteins secreted from the cultured dermal papilla cells. This chemotactic signal between dermal papillae and melanocytes may control the localization and migration of hair melanocytes in vivo.