Allergic contact dermatitis releases soluble factors that stimulate melanogenesis through activation of protein kinase C-related signal-transduction pathway.

Phenylazo-naphthol (PAN) allergy induces visibly well-defined and late-appearing hyperpigmentation of brownish yellow guinea pig skin in clear contrast to dinitrochlorobenzene (DNCB) allergy, which has very low incidence of hyperpigmentation. Skin extract from PAN allergy at 20-29 d post-challenge exhibited marked melanogenic stimulatory effects (3H2O release and 14C-thiouracil incorporation) when added to cultured guinea pig melanocytes. The time course in the appearance of melanogenic factor was definitely consistent with the induction pattern of visible pigmentation. By contrast, the addition of DNCB-challenged skin extract demonstrated no significant stimulating effect on melanogenesis in either assay system on any of the post-challenge days tested. Assay of intracellular inositol 1,4,5-trisphosphate formed through incubation with the melanocytes demonstrated that the PAN-allergy skin extract at day 28, which contains definite melanogenic factors, stimulated the formation of inositol 1,4,5-trisphosphate that occurs around 50 seconds in contrast to no or little increase with extracts obtained at days 0 and 1 post-challenge. Gel chromatographic analysis revealed that the PAN-allergy skin extract at day 28 contained a newly generated melanogenic fraction with a molecular weight of approximately 9000 Da which was also capable of stimulating DNA synthesis and activating the signal-transduction process (inositol trisphosphate formation) when added to guinea pig melanocytes. Both stimulations of melanogenesis and DNA synthesis by the 9000 Da fraction were completely abolished by the prior and simultaneous addition of protein kinase C (PKC) inhibitor (H-7) or its down-regulatory agent, phorbol 12,13-dibutyrate (PdBu). Taken together, these results suggest that PAN allergy provides a new mechanism of hypermelanization in which endogenous factors synthesized within skin induce the activation of signal-transduction pathways such as phosphoinositide turnover through ligands-receptor binding, resulting in the stimulation of melanocytes possibly through the activation of PKC.     

Epidermotropically metastatic malignant melanoma. Differentiating malignant melanoma metastatic to the epidermis from malignant melanoma primary in the epidermis

In four instances, metastases to epidermis from primary cutaneous malignant melanomas at different sites showed histological features similar to those of cutaneous malignant melanoma primary in the epidermis. In these metastases, atypical melanocytes were present within the epidermis and in the upper part of the dermis much as in primary cutaneous malignant melanoma. Therefore, the presence of atypical melanocytes within the epidermis is not in itself an absolute criterion of malignant melanoma primary in skin. Nor does that finding absolutely deny malignant melanoma metastastic to the skin. Features that may enable histologic differentiation of epidermotropically metastatic malignant melanoma from primary cutaneous malignant melanoma are emphasized.     

Comparison of alloimmunogenicity of Langerhans cells and keratinocytes from mouse epidermis

Among lymphoreticular cells, Langerhans cells and splenic dendritic cells stand alone in their capacity, when hapten-derivatized, to induce vigorous immune responses, irrespective of route of inoculation, including intravenous. We have examined the comparative efficiency of relatively purified populations of Langerhans cells and their epidermal companions, keratinocytes, to induce alloimmunity when injected intravenously into adult mice. It was found that as few as 100 BALB/c Langerhans cells injected intravenously into C3H mice are capable of inducing specific sensitization as evidenced by subsequent accelerated rejection of BALB/c skin grafts. By contrast, 10,000 BALB/c keratinocytes failed to immunize similarly injected C3H recipients. These results emphasize the unparalleled capacity of Langerhans cells to induce sensitization, and they point to Langerhans cells, among cells within the epidermal compartment, as dominant in the alloimmunogenic potential of skin grafts.     

人表皮黑素细胞、毛囊无色素黑素细胞和鼠黑素瘤细胞的原子力显微镜观察

目的：观察培养的人表皮黑素细胞、毛囊无色素黑素细胞和S91鼠黑素瘤细胞的形态结构。方法：培养并纯化来自正常人包皮的黑素细胞以及来自毛囊的无色素黑素细胞，同时复苏S91细胞株，传代后接种到内置云母片的培养板中，细胞贴附到云母片上后固定，用原子力显微镜扫描观察。结果：正常人表皮黑素细胞有3级分支，在主干及分支的顶端和侧缘可见膨出的球形结构。鼠黑素瘤细胞仅有很短的2级分支，在2级树突近端可见黑素小体。毛囊无色素黑素细胞只有1级树突，并只在树突近端有少数黑素小体。结论：表皮黑素细胞在形态上比黑素瘤细胞、毛囊无色素黑素细胞更成熟，有更多的黑素颗粒从树突的顶端和侧缘以胞吐的形式被输出。     

Prostaglandin E2 and D2 but not MSH stimulate the proliferation of pigment cells in the pinnal epidermis of the DBA/2 mouse

Epidermal melanocytes proliferate following a variety of physical stimuli, for example, mechanical injury to the skin or exposure to UV radiation. We suggest that some transducer in the epidermis converts the physical modality into a biochemical signal which is responsible for initiation of mitosis. Melanocyte stimulating hormone, both alpha and beta variants, administered parenterally for periods up to 4 weeks do not alter the number of melanocytes per mm2 in several strains of neonatal or adult mice. Ultraviolet B and arachidonic acid both stimulate proliferation of pigment cells. Indomethacin which inhibits cyclooxygenase and the formation of prostaglandins (PGs) blocks the proliferation induced by both agents. We tested a wide variety of PGs. We observed that PGD2 applied daily to the skin of a mouse causes a small increase in melanocyte density (cells/mm2). PGE2 in similar doses applied topically caused a large increase. PGE2 caused an increase in the uptake of tritiated thymidine by dopa-positive dendritic cells. This indicates that PGE2 stimulated some melanocytes to proliferate. Histologic studies indicate that PGE2 also enhances melanogenesis. PGE2 is synthesized in the skin and affects keratinocytes and Langerhans cells as well as pigment cells. We postulate that it is one compound that can modulate the interaction of these 3 main cells of the epidermis.     

PPARgamma activators stimulate aquaporin 3 expression in keratinocytes/epidermis

Aquaporin 3 (AQP3), a member of the aquaglyceroporin family, which transports water and glycerol, is robustly expressed in epidermis and plays an important role in stratum corneum hydration, permeability barrier function and wound healing. PPAR and LXR activation regulates the expression of many proteins in the epidermis and thereby can affect epidermal function. Here, we report that PPARgamma activators markedly stimulate AQP3 mRNA expression in both undifferentiated and differentiated cultured human keratinocytes (CHKs). The increase in AQP3 mRNA by PPARgamma activator occurs in a dose- and time-dependent fashion. Increased AQP3 mRNA levels are accompanied by an increase in AQP3 protein in undifferentiated keratinocytes and a significant increase in glycerol uptake. Activation of LXR, RAR and RXR also increases AQP3 mRNA levels in undifferentiated and differentiated CHKs, but to a lesser extent. PPARdelta activation stimulates AQP3 expression in undifferentiated CHKs but decreases expression in differentiated CHKs. In contrast, PPARalpha activators do not alter AQP3 expression. AQP9 and AQP10, other members of aquaglyceroporin family, are less abundantly expressed in CHKs, and their expression levels are not significantly altered by treatment with LXR, PPAR, RAR or RXR activators. Finally, when topically applied, the PPARgamma activator, ciglitazone, induces AQP3 but not AQP9 gene expression in mouse epidermis. Our data demonstrate that PPAR and LXR activators stimulate AQP3 expression, providing an additional mechanism by which PPAR and LXR activators regulate epidermal function.     

Three-dimensional reconstruction and stereometric analysis of Langerhans cells in mouse epidermis.

In the presented studies stereometric analysis and spatial reconstruction was performed on two Langerhans cell (LC) types. One was free of LC-I and the other contained LC-II Birbeck granules in the perinuclear space. The presented stereometric analysis demonstrated significant differences between the so-distinguished two cell types. Differences were observed not only in the number and distribution of Birbeck's granules but also in the areas of smooth and rough endoplasmic reticulum, in the area of vesicles surrounding Golgi apparatus, in the volume of cisterns of the apparatus, and in the ratio of cell nucleus area to its volume. Differences noted between the two cell types were of quantitative character. They might result from different stages of differentiation of the cells from their precursors in the epidermis or from distinct functional stages of the cells.     

Cytochemical identification of ATPase-positive Langerhans cells in EDTA-separated sheets of mouse epidermis

Sheets of epidermis for incubation to demonstrate ATPase activity were obtained from specimens of mouse footpad using EDTA as the separation medium. The use of EDTA in place of the NaBr method previously described, resulted in a greatly reduced incubation time, precise localization of reaction product and preservation of ultrastructural detail. A population of closely and regularly spaced ATPase-positive dendritic cells was demonstrated by light microscopy. Electron microscopy demonstrated that, with short incubation times, reaction product was found only in the extracellular space adjacent to dendritic cells, the majority of which possessed the typical ultrastructural features of Langerhans cells.     

DNA synthesis in mouse epidermis: S phase cells that remain unlabeled after pulse labeling with DNA precursors progress slowly through S

Epidermal basal cells from hairless mice were isolated after pulse labeling with tritiated DNA precursors and subjected to DNA flow cytometry combined with cell sorting. Cells were sorted from a window in the middle of the S phase, collected on glass slides, and subjected to autoradiography. Unlabeled cells in the middle of the S phase were found in normal mouse epidermis after optimal pulse labeling with tritiated thymidine [( 3H]dThd), in accordance with previous results. The proportion of unlabeled S phase cells was considerably increased among basal cells from mice treated with growth-inhibitory epidermal extracts. Reanalysis and re-sorting of cells previously sorted from mid S showed that unlabeled cells could not be accounted for by G1 contamination. Furthermore, labeling with precursors incorporated into DNA by "de novo" metabolic pathway [( 3H]Urd) did not reduce the proportion of unlabeled S phase cells, either when given alone or when given in combination with the precursor for DNA incorporated by the "salvage" pathway [( 3H]dThd). This strongly indicates that the unlabeled S phase cells do not synthesize DNA continuously, or are synthesizing DNA at a rate below the level of detection. A reduced proportion of unlabeled S phase cells was found in regenerating epidermis. This may be explained by a dilution effect caused by the 3-fold increase in the total number of cells within S phase at this condition. The observation that essentially all cells in mid S phase were labeled during 4 days of continuous labeling with [3H]dThd, indicates that cells in S phase that remain unlabeled after optimal pulse labeling are cycling, albeit slowly. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells are larger than the average. These cells may represent a subpopulation of basal cells going through their last division cycle before differentiation.     

Consumption of the epidermis in acral lentiginous melanoma

Background: Consumption of the epidermis (hereafter, consumption), namely thinning of the epidermis with attenuation of basal and suprabasal layers and loss of rete ridges adjacent to collections of melanocytes, has been used to differentiate invasive melanoma from Spitz nevi. Evaluation of 213 invasive melanomas, including only two cases of acral lentiginous melanoma (ALM), showed that the frequency of consumption increases with increasing tumor thickness. Methods: We evaluated consumption in 52 acral melanomas relative to age, gender, Breslow depth, tumor thickness (based on the 2010 American Joint Commission on Cancer guidelines), Clark level, mitoses, ulceration, vertical‐growth phase, regression, tumor‐infiltrating lymphocytes and anatomical site. Results: Consumption was more frequent in ALM with increasing Breslow depth (p = 0.01), and in the presence of ulceration (p = 0.0078); in all cases with ulcer, consumption was found adjacent to the ulceration. There was no statistically significant difference in consumption in nail melanomas in comparison to melanomas of acral skin other than the nail. Conclusions: These results support the hypothesis that epidermal thinning in consumption represents an early phase of ulceration. No statistically significant difference in consumption was found between nail melanomas and melanomas of acral skin other than the nail, probably because of similar tumor thickness in both groups. Ohata C, Nakai C, Kasugai T, Katayama I. Consumption of the epidermis in acral lentiginous melanoma.     

Relationship between Merkel cells and nerve endings during embryogenesis in the mouse epidermis

Close relationships between Merkel cells (MC) and nerve endings (NE) exist in the adult mouse. Because MC may serve as targets for the ingrowth of NE during embryogenesis, the purpose of the present study was to analyze the relationship between MC and NE during embryogenesis. Frozen tissue from whisker pads and backs of NMRI mouse embryos (12-17 d gestational age) were studied by double-labeling indirect immunofluorescence (IIF) with a cytokeratin monoclonal antibody that recognizes MC and with a neurofilament anti-serum. Such an approach allowed the analysis of a large number of MC (up to 5000), thus yielding quantitative data. At day 12 of gestational age, no MC were observed by IIF. From day 13 to 17, the number of MC, as well as their association with NE, progressively increased. On day 13, only 57% of whisker pad MC were NE associated, whereas by day 17, 95% were NE associated. These results were confirmed by electron microscopic (EM) observations. On the back, the same chronologic relationship between MC and NE was observed, but was later in the course of embryogenesis. There was also a time- and zone-dependent increase in MC association with NE in the epidermal zones studied (isthmic, parafollicular, interfollicular). These observations 1) establish the time course of MC and NE contacts during embryogenesis in the mouse epidermis, 2) show that MC are present in the epidermis and appendages before NE reach the epithelium, and 3) support the hypothesis that MC could act as targets for the growing NE.     

Cholesterol sulfotransferase of newborn mouse epidermis

Recent studies of the epidermis in patients with recessive X-linked ichthyosis indicate that cholesterol sulfate is an important endogenous substrate for steroid sulfatase in the stratum corneum. We report here that cholesterol sulfotransferase, which converts cholesterol to cholesterol sulfate, is present in the lower living epidermis. Epidermal cytosol also sulfates phenols and some steroids, and such reactions may be important in defending against compounds absorbed percutaneously and in modulating the pharmacologic activity of topical medicaments. Sodium salicylate and sodium citrate inhibit the sulfotransferase activity noncompetitively, and inhibition of cholesterol sulfate formation may be important in the desquamative action of these topical "keratolytics."     

Ultraviolet A exposure alters adhesive properties of mouse melanoma cells

BACKGROUND: We have examined whether ultraviolet A (UVA) irradiation could alter adhesive properties of melanoma cells. As an experimental in vitro model, we have used C57BL/6 mouse-derived B16- F1 and B16-F10 melanoma cell lines and the syngeneic MS-1 endothelial cell line. METHOD/RESULT: The melanoma cells were exposed to different doses of UVA irradiation. We have determined that a single dose of UVA at 8 and 12 J/cm(2) causes an 88% (P<0.001) and a 32% (P<0.05) increase in B16-F1 melanoma cell adhesiveness to the non-irradiated endothelial monolayer, respectively. The peak of the response was 24 h after the irradiation. The UVA dose of 8 J/cm(2) delivered in four doses separated by 1 h intervals (4 x 2 J/cm(2)) had led to a caused 149% (P<0.001) increase of B16-F1 melanoma adhesiveness already at 1 h after the last dose of UVA. Besides the induction of increase in the melanoma-endothelial cell adhesion, UVA exposure has induced a rapid decline (1 h after exposure) in homotypic melanoma-melanoma cell adhesion (clustering). The clustering decline of B16-F1 cells with a single dose of UVA at 8 J/cm(2) was by 61% (P<0.05) and by 35% (P<0.05) with 4 x 2 J/cm(2). Pilot experiments have shown that the changes of the adhesive properties of melanoma cells were accompanied by an increase in N-cadherin expression and a decline in E-cadherin expression. Such a change in cadherin expression profile has been shown to be an indicator of the increased metastatic potential. CONCLUSION: Our results suggest that UVA radiation appears to alter the adhesive properties of melanoma cells in vitro, by diminishing the melanoma-melanoma adhesion and by increasing melanoma adhesion to the endothelium. This suggests that UVA exposure might increase the metastatic capability of the melanoma cells.     

Adenosine receptors as mediators of both cell proliferation and cell death of cultured human melanoma cells

Adenosine displays contradictory effects on cell growth: it improves cell proliferation, but it may also induce apoptosis and impair cell survival. Following the pharmacologic characterization of adenosine receptor expression on the human melanoma cell line A375, we chose A375 as our cellular model to define how the extracellular adenosine signals are conveyed from each receptor. By using selective adenosine receptor agonists or antagonists, we found that A2A stimulation reduced cell viability and cell clone formation, whereas, at the same time, it improved cell proliferation. In support of this finding we demonstrated that the stimulation of A2A adenosine receptors stably expressed in Chinese hamster ovary cell clone reproduced deleterious effects observed in human melanoma cells. A3 stimulation counteracted A2A-induced cell death but also reduced cell proliferation. Furthermore, we found that A3 stimulation ensures cell survival. We demonstrated that adenosine triggers a survival signal via A3 receptor activation and it kills the cell through A2A receptor inducing a signaling pathway that involves protein kinase C and mitogen-activated protein kinases.     

Long-term effects of local ionizing radiation treatment on Langerhans cells in mouse footpad epidermis

Langerhans cells (LC) were studied with ADPase histochemistry in sheets of hind footpad epidermis from groups of CBA/H mice. Single, local 20-Gy doses of 250-kV x-rays were administered to the right hind feet of the mice when they were 3-4 months old, and LC were counted at intervals ranging from 2 to 24 months later. In unirradiated mice, aged 5-19 months, the mean density of LC in footpad was 1521-1617 cells/mm2. It dropped to 1137 +/- 86 cells/mm2 (mean +/- SE) in untreated 28-month-old mice. At times from 2-15 months after irradiation, normal mean densities of LC were present in footpad epidermis. On average, LC numbers were subsequently reduced to 1078 +/- 65/mm2 by 19 months after irradiation (71% of the cells in age-matched controls) and to 789 +/- 53/mm2 by 24 months (59% of the cells in age-matched controls). Loss of cells was focal. Chronic radiation-induced fibrosis and damage to circulatory function in skin may have contributed to impaired replacement of LC from bone marrow precursors. The possibility that late radiation-related depletion of the LC population permits development of skin tumors as a delayed consequence of exposure to ionizing radiation is discussed.     

Stem cells in the epidermis

The epidermis consists of three actively proliferating units, the interfollicular epidermis, the hair follicle, and the sebaceous gland. Stem cells in the epidermis have the capacity to produce all three of these units. The fate of the epidermal stem cells and some of their progeny can be altered, dependent on the environment in which they reside and the genes they express. In this review, we describe the major experiments that have contributed to the understanding of the epidermal stem cells and the control of their fate.     

Neurotrophins and their receptors stimulate melanoma cell proliferation and migration

Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.     

Immunohistochemical identification of Skn antigens in mouse epidermis

Rejection of murine skin grafts by hematopoietic chimeras that are fully compatible genetically with the skin-graft donor has been attributed to a disparity in skin-selective alloantigens between the irradiated host and the skin graft donor. Monoclonal antibodies recognizing Skna alloantigen were produced and used in indirect immunofluorescent and immunoperoxidase tests with sections of skin to demonstrate and confirm the expression of Skna alloantigen on epidermal cells of Skna genotype and absence of Skna alloantigen from epidermal cells of non-Skna genotype.