Comparison of skin barrier function and sensory nerve electric current perception threshold between IgE-high extrinsic and IgE-normal intrinsic types of atopic dermatitis.

Background Two types of atopic dermatitis (AD) have been proposed, with different pathophysiological mechanisms underlying this seemingly heterogeneous disorder. The extrinsic type shows high IgE levels presumably as a consequence of skin barrier damage and feasible allergen permeation, whereas the intrinsic type exhibits normal IgE levels and is not mediated by allergen-specific IgE. Objectives To investigate the relationship between pruritus perception threshold and skin barrier function of patients with AD in a comparison between the extrinsic and intrinsic types. Methods Enrolled in this study were 32 patients with extrinsic AD, 17 with intrinsic AD and 24 healthy individuals. The barrier function of the stratum corneum was assessed by skin surface hydration and transepidermal water loss (TEWL), and pruritus perception was evaluated by the electric current perception threshold (CPT) of sensory nerves upon neuroselective transcutaneous electric stimulation. Results Skin surface hydration was significantly lower and TEWL was significantly higher in extrinsic AD than intrinsic AD or normal controls. Although there was no statistically significant difference in CPT among extrinsic AD, intrinsic AD and normal controls, CPT was significantly correlated with skin surface hydration and inversely with TEWL in intrinsic AD and normal controls, but not extrinsic AD. Finally, CPT was correlated with the visual analogue scale of itch in the nonlesional skin of patients with extrinsic but not intrinsic AD. Conclusions Patients with extrinsic AD have an impaired barrier, which increases the pre-existing pruritus but rather decreases sensitivity to external stimuli. In contrast, patients with intrinsic AD retain a normal barrier function and sensory reactivity to external pruritic stimuli.

Microanalysis of an antimicrobial peptide, beta-defensin-2, in the stratum corneum from patients with atopic dermatitis.

BACKGROUND: Antimicrobial peptides, such as defensin and cathelicidin, have recently been reported to play important roles in host defence and in cutaneous innate immunity. Although beta-defensin-2 has been reported to be downregulated in the skin of patients with atopic dermatitis (AD), little is known about its role in the colonization of Staphylococcus aureus in the stratum corneum of patients with AD. A precise evaluation of these peptides in the stratum corneum as an antimicrobial barrier against S. aureus colonization has not yet been performed. OBJECTIVES: To compare beta-defensin-2 levels in the skin of patients with AD and healthy controls. METHODS: We developed a microanalytical technique to measure beta-defensin-2 in the stratum corneum using a combination of immunoprecipitation and Western blotting. RESULTS: beta-Defensin-2 in the stratum corneum was significantly higher in AD lesional skin compared with healthy control skin. The beta-defensin-2 content in AD lesional skin also increased in proportion to the severity of the disease. Counting bacterial colonies revealed higher populations of S. aureus on lesional and nonlesional skin surfaces of patients with AD compared with healthy controls. Comparison of S. aureus colony numbers and beta-defensin-2 levels demonstrated a positive correlation (r = 0.342, P = 0.004, n = 67) between both factors. CONCLUSIONS: Collectively, these findings suggest that beta-defensin-2 is induced in response to bacteria, injury or inflammatory stimuli and is not associated with vulnerability to S. aureus colonization in the skin of patients with AD.

Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid-deficient rats and has an inhibitory effect on epidermal hyperplasia.

Pseudo-acylceramides with different acyl properties were investigated for their capacity to restore diminished barrier function in essential fatty acid-deficient rats. Daily topical applications of synthetic pseudo-acylceramides containing ester-linked linoleic acid caused a dose-dependent, significant reduction of transepidermal water loss (TEWL). Both other pseudo-acylceramides with ester-linked oleic acid or saturated alkyl chains and ordinary ceramides exhibited a poor effect on recovery of TEWL. Furthermore, pseudoceramide containing ether-linked linoleic acid, which is biologically inactive in terms of degradation by hydrolytic enzymes, also induced a significant and similar increase in the barrier function. This restoration of barrier function by pseudo-acylceramides with linoleic acid was accompanied by suppressed DNA synthesis in the EFAD rat epidermis. In UVB-irradiated guinea pig skin, topical applications of the pseudo-acylceramides with linoleic acid immediately after the exposure significantly reduced epidermal hyperplasia, secondary to markedly diminished barrier disruption, whereas linoleic acid itself did not. A comparison of both the anti-hyperplasia and the barrier recovery effects in the series of pseudo-ceramide derivatives examined revealed that the suppressive effect on the induced epidermal hyperplasia was paralleled by the recovery of the barrier defect in EFAD rats. These findings directly suggest that acylceramide with an ester-linked linoleic acid has an essential role in the epidermal permeability barrier.

Loss of melanogenic properties in tyrosinases induced by glucosylation inhibitors within malignant melanoma cells.

Glycosylation inhibitors, glucosamine or tunicamycin, have been found to be specific inhibitory modulators for melanogenesis, which is accentuated generally in malignant melanoma cells. Exposure to glucosamine (1 mg/ml) or tunicamycin (0.2 to 0.4 micrograms/ml) induces a marked pigment loss within melanoma cells in vitro with a decrease in their grown curves. This melanogenic inhibition occurs without a substantial decrease in the synthesis of DNA, RNA, and protein in comparison with a specific, marked suppression of carbohydrate synthesis as revealed by suppressed mannose incorporation into these cells. Assay of tyrosinase of glucosamine- or tunicamycin-induced unpigmented melanoma cells has revealed a selective and marked decrease in the melanosome-rich large-granule fraction, but no substantial decrease in the total activity of remaining subcellular fractions. Electrophoresis of tyrosinase in the 30,000 X g supernatant fraction demonstrates an increase in the T1 form of soluble tyrosinase, while a disappearance of or marked decrease in membrane-bound tyrosinase, T3, is seen in the small- and large-granule fractions. Glycoprotein synthesis in the melanogenic subcellular compartments of pigment cells seems to play an integral role in melanogenesis which is principally enhanced in their carcinogenic status.

A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase.

We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.

Analysis of initial melanogenesis including tyrosinase transfer and melanosome differentiation through interrupted melanization by glutathione.

Because glycosylation-dependent melanization inhibition induced in cultured B-16 melanoma cells by glucosamine is reversible, producing synchronized initiation of melanogenesis after its removal, we have analyzed the possible dynamics of initial melanogenesis through their interruption by glutathione. The addition of glutathione at 0.2% concentration to the theophylline-stimulated recovery process completely interrupts the initiation of melanization for at least 72 h. At the electron microscopic level, theophylline-treated cells have many vacuolar melanosomes with distinct pigmentation which contain some vesicles (64% of total premelanosomes) or amorphous, filamentous, or granular materials within the interior which are suggestive of pheomelanotic melanosomes. The addition of glutathione induces a complete absence of melanization in the premelanosomes, within which a filamentous interior with periodicity is generally re-formed with almost complete disappearance of internal vesicles, providing dramatic changes to the size and shape characteristic of eumelanotic melanosome. Electron microscopic dopa reaction of glutathione-treated cells shows a predominant localization of tyrosinase activity in the Golgi-associated endoplasmic reticulum-lysosome and coated vesicles, but not in premelanosomes, in contrast to their dispersed distribution in all melanogenic organelles in the theophylline-treated control, suggesting a lack of tyrosinase translocation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of tyrosinase in the large granule fraction shows that in analogy with electron microscopic observations, glutathione blocks the reappearance of membrane-bound T3 tyrosinase which occurs in the theophylline-treated control during the recovery process, whereas the dynamics of T1 tyrosinase is almost the same as that of the control. These findings suggest that glutathione provides a new situation of interrupted melanogenesis in which melanization cannot proceed despite complete formation of melanosome matrix structure and a lack of inhibition of cellular metabolisms including protein glycosylation.

Analysis of carbohydrate properties essential for melanogenesis in tyrosinases of cultured malignant melanoma cells by differential carbohydrate processing inhibition.

In order to clarify the biologic significance of carbohydrate processing in tyrosinases for melanogenesis, we have studied the effect of differential carbohydrate processing inhibitors on the recovery process of interrupted melanization which occurs after exposure of cultured B-16 melanoma cells to the inhibitor of core carbohydrate synthesis, glucosamine (Glc). Treatment of this glycosylation-dependent repigmentation process with the early-stage carbohydrate processing inhibitors deoxynojirimycin (dNM), castanospermine (CS), and monensin (MS) at 0.8 mM, 40 micrograms/ml, and 30 nM, respectively, in the presence of 2 mM theophylline (Tp) almost completely inhibits the reappearance of the pigment 48-72 h after removal of Glc. In contrast, treatment with the later stage carbohydrate processing inhibitor swaisonine (SW) at 40-80 micrograms/ml does not interrupt the repigmentation process. Electrophoretic analysis of tyrosinases in the soluble fractions of these melanoma cells demonstrates that the alteration of soluble tyrosinase isozymes by all the processing inhibitors is associated with a dose-dependent loss of sialic acid-rich T1 tyrosinase and the concomitant appearance or increase of sialic acid-poor tyrosinases. In the large granule fraction, a recovery of membrane-bound tyrosinase (T3) is seen following both MS and SW treatments, whereas dNM treatment results in the substantial loss of T3 tyrosinase. At the electron microscopic level, a translocation of tyrosinase from GERL and coated vesicles to many unmelanized vacuolar premelanosomes occurs in MS-treated cells in contrast to its predominant distribution in the GERL-coated vesicle system of dNM-treated cells, which contain many unmelanized premelanosomes. The present evidence for differential effects on intracellular tyrosinase transfer and melanization by different stages of carbohydrate processing inhibition suggests that asparagine-linked oligosaccharides, relating to the first mannose-trimming stages, determine the function of tyrosinase transfer as well as melanization through a specific intracellular recognition process in pigment cells.

Differential hypermelanosis induced by allergic contact dermatitis.

In moderately colored guinea-pig skin, UVB, PUVA (psoralen plus UVA), and allergic contact dermatitis were shown to induce visibly well-defined hyperpigmentation that resembled the pigmentary changes observed in Mongoloid human skin. To clarify mechanisms of allergen-induced hyperpigmentation, we compared the effects of allergic contact dermatitis on pigmentation by using 6 different allergens: dinitrochlorobenzene (DNCB), 1-phenylazo-2-naphthol (PAN), benzyl salicylate (BS), jasmine oil (JO), hydroxycitronella (HC), and ylang ylang oil (YYO). The PAN-, JO-, HC-, and YYO-induced allergic reactions caused a definite visible hyperpigmentation that began to appear within 14 days, reaching maximum intensity about 40 days after the induction of the allergic reaction. These hyperpigmentations were accompanied by a significant increase in the population of dopa-positive melanocytes on day 24 following allergic reactions. In contrast, BS- and DNCB-induced allergic reactions did not give rise to visibly distinct hyperpigmentation in spite of the intensive allergic reactions following their challenge application. In a nonsensitized group, primary irritant reactions were induced by the application of 100% JO, but no distinctive hyperpigmentation was found 40 days after the last application. Quantitative analysis of the number of melanophages in the dermis showed that there was a marked increase in the number of melanophages in PAN, YYO, and HC allergy-induced hyperpigmented areas, with PAN showing a significant increase compared with those in non-treated areas of the same animals, whereas JO was associated with no such increase in hyperpigmented area, despite the stimulated pigmentation. In the case of the lack of induced hyperpigmentation, as seen in BS and DNCB allergy and JO irritation, there was also no substantial increase in the number of melanophages. Our findings indicate that allergic contact dermatitis is a unique melanogenic stimulant different from UV irradiation.

Importance of glycoproteins in the initiation of melanogenesis: an electron microscopic study of B-16 melanoma cells after release from inhibition of glycosylation.

Specific inhibition of core carbohydrate synthesis has been found to induce selective aberration and melanin loss in melanosomes, accompanied by alteration of the carbohydrate moiety in functioning glycoproteins, tyrosinases. In order to further clarify the biologic significance of glycoproteins in the initiation of melanization, initial melanogenesis which occurs during the recovery period following interrupted melanogenesis induced by glycosylation inhibitors, has been electron microscopically investigated. Changes in the tyrosinase activity of the corresponding melanogenic subcellular compartments have also been studied electron cytochemically. Removal of glycosylation inhibition was carried out after exposure of B-16 melanotic melanoma cells to the inhibitors for 10-20 culture days resulting in the loss of their melanization. Recovery of melanization begins visibly 48 h later, thereafter almost attaining the previous normal level by 72 h. At the electron microscopic level, re-formation of melanosomal matrix with periodicity is observed within premelanosomes 48 h after removal of glucosamine, soon followed by deposition of apparent melanin particles along their periodicity by 72 h. In tunicamycin experiments, melanization within premelanosomes starts after the concentration of the fine threadlike interior becomes less distinct followed by a prominence of multiple accumulation of microvesicles within the interior. Electron microscopic dopa reaction shows that the deposition of dopa melanin is prominent in Golgi-associated endoplasmic reticulum of lysosome and coated vesicles up to 24 h after the removal. Thereafter, predominant localization of dopa melanin in premelanosomes gradually increases and finally becomes almost uniform. These observations suggest 2 possible mechanisms for the involvement of glycosylation in melanogenesis: first, the translocation of tyrosinases may be regulated by the presence of specific carbohydrate moieties; second, melanosomal matrix proteins contain carbohydrates which may contribute to the tyrosinase-accepting function or in vivo melanizing function of tyrosinases after forming particle-bound T3 tyrosinase.

Skin organ culture model for examining epidermal melanization.

To clarify the regulatory mechanism of skin pigmentation through epidermal proliferation and differentiation, organ cultures of pigmented guinea pig skins have been studied for their melanogenic responses to exogenous stimuli. Melanogenic activity was measured by both tyrosinase activity assessed by observing release of 3H2O from tissue after incubation with 3H-tyrosine and melanin synthesis, indicated by the incorporation of 14C-2-thiouracil into tissue. Those skin explants that were maintained in serum-free media under air conditions equilibrated with 5% CO2, 50% O2, and 45% N2 formed new, chemically analyzable pigment in the tissue within 4 d of culture. This melanization was accompanied by an increased number of melanocytes as well as by enhanced tyrosinase activity and elevated melanin synthesis. This organ culture system responded well to known tyrosinase inhibitors such as phenylthiourea, which decreased melanogenic activity. Of the arachidonic acid metabolites tested, PGE2, LTC4, LTB4, and 5-HETE were found to significantly stimulate melanogenic activity as indicated by tyrosinase activity, whereas PGF2-alpha, 12-HETE, and 15-HETE did not. Among several known growth factors, only bFGF significantly stimulated melanogenesis in the organ cultured melanocytes. TGF-alpha, which increased DNA synthesis, had a slightly stimulating effect on melanogenesis, whereas TGF beta, which inhibited DNA synthesis, did not stimulate melanogenesis, but rather slightly decreased it. 8-methoxypsoralen+ultraviolet A-treated skin that underwent a marked pigmentation within 14 d in vivo demonstrated enhanced melanogenesis in the organ culture system in proportion to its in vivo pigmentation. Our organ culture system might provide an opportunity to examine the mechanism of action of epidermal melanization.

A possible role of prostaglandins in PUVA-induced inflammation: implication by organ cultured skin.

The role of arachidonate metabolites in UV-induced inflammation was analyzed by measuring the amounts of prostaglandins or leukotrienes released into a culture medium in the organ culture of biopsied guinea pig skin following UV exposure. Increased concentrations of prostaglandin E2, F2 alpha, and 6-oxo-F1 alpha were found in the medium subjected to UVB radiation followed by 24 h incubation in the organ culture, compared to medium from non-exposed skin, whereas LTC4 was at an undetectable level in both. This release occurred in UVB dose-dependent fashion and was completely inhibited by the addition of indomethacin to the medium. Exposure to monochromatic UV light of 250, 300, and 350 nm released prostaglandin E2, F2 alpha, and 6-oxo-F1 alpha, in increasing order of 250, 300, and 350 nm, but not LTC4. This order of amount released almost paralleled the intensity of inflammation that occurred with each wavelength. Exposure of skin to UVA following intraperitoneal injection of 8-methoxypsoralen (PUVA) was also found to stimulate the synthesis of prostaglandin E2 in UVA dose-dependent fashion, and the stimulation through an increasing dose of UVA occurred more significantly than UVB exposure. The time course of prostaglandin release following UVB and PUVA exposures was found to have a pattern similar to their dynamics in association with erythema and edema, conditions which could be significantly diminished by the topical application of indomethacin. The present studies demonstrate that prostaglandins play an important role in the production of inflammation by UVB as well as UVA and PUVA.

Selective aberration and pigment loss in melanosomes of malignant melanoma cells in vitro by glycosylation inhibitors: premelanosomes as glycoprotein.

We have found that glucosamine (1 mg/ml) or tunicamycin (0.2-0.4 micrograms/ml), specific inhibitors of lipid carrier-dependent glycosylation of protein, when added to cultured B-16 melanoma cells produce a marked loss of pigmentation, accompanied by distinctive biochemical as well as ultrastructural aberrations in their melanogenic compartments. Electron microscopic analysis shows that these newly induced unpigmented cells form uniquely altered melanosomes containing little or no melanin, although their population is not substantially reduced. Within the melanogenic compartments, selective aberration of melanosomes is seen, that is, deformity, bulging, and segregation of their interior membrane, as well as the intramelanosomal formation of irregularly concentric lamellar structure. No apparent structural deformity of Golgi apparatus, Golgi-associated endoplasmic reticulum of lysosome (GERL), and coated vesicles has been observed. Further, no substantial alteration is seen in mitochondria or other cellular structures. Quantitative analysis of altered and nonaltered melanosomes has revealed that the ratio of altered premelanosomes to the total number increases to 44% in glucosamine-treated cells and to 99.5% in tunicamycin-treated cels. Compared to 13% in the control. Electron microscopic dopa reaction has also revealed that these altered melanosomes seem to exhibit a weakly positive or a negative dopa reaction in both glucosamine- and tunicamycin-treated melanoma cells although a number of dopa-positive altered melanosomes are still seen. However, GERL and coated vesicles show no apparent decrease in dopa reaction. It may be concluded that glycoprotein synthesis is integral to the formation of normal melanosomes and to their specific melanizing function, which could be impaired by inhibition of the synthesis of asparagin-linked mannose-containing sugar chains. This results in retrogressive changes in the premelanosomal tyrosinase during its maturation process.

A possible function of structural lipids in the water-holding properties of the stratum corneum.

In order to clarify the possible role of lipids in the water-holding property of stratum corneum, the forearm skin of 6 healthy male volunteers was treated with acetone/ether (1/1) for 1, 5, 10, and 20 min. A prolonged treatment period of 5-20 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a considerable and selective loss of intercellular lipids such as cholesterol, cholesterol esters, and phospholipids. These impairments persisted until day 4 after treatment. Electron microscopic observation of the altered stratum corneum revealed that naturally occurring intercellular materials were absent, leaving the area with the appearance of a vacant space. These findings suggest an additional and essential role of the specific structural lipids for the water-holding properties of the stratum corneum.

Analysis of tyrosinases as asparagin-linked oligosaccharides by concanavalin A lectin chromatography: appearance of new segment of tyrosinases in melanoma cells following interrupted melanogenesis induced by glycosylation inhibitors.

The structural alteration of carbohydrate moieties of tyrosinases associated with the depigmentation process induced by glycosylation inhibition has been investigated by using concanavalin A (Con A) affinity chromatography. Con A affinity chromatography of deoxycholate-solubilized large and small granule fractions shows that while all tyrosinases found in control B-16 cells exhibit affinity to Con A lectin, there is an emergence on non-Con A binding tyrosinases in the unpigmented cells induced by glycosylation inhibitors, such as tunicamycin and glucosamine. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, control tyrosinase activity forms 2 distinct bands consisting of T1 and T3. But tyrosinases from the unpigmented cells lose T3 tyrosinase and are resolved into a few different molecular weight components, one of which is Con A affinitive T1 tyrosinase and the others are non-Con A affinitive tyrosinases with smaller molecular weights than the T1 tyrosinase. These findings suggest that altered structures of carbohydrate moiety in tyrosinase molecules play a role in the induction of loss of membrane-binding capacity of tyrosinases, resulting in the loss of melanization in pigment cells.

Functional analysis of tyrosinase isozymes of cultured malignant melanoma cells during the recovery period following interrupted melanogenesis induced by glycosylation inhibitors.

Multiple forms of tyrosinase, T1, T2, T3, have been shown to differ with respect to carbohydrate moieties of these isozymes. We demonstrated that, in cultured B-16 melanoma cells, melanization can be completely interrupted by glycosylation inhibitors, such as glucosamine and tunicamycin, and that these inhibitors cause a selective loss of membrane-bound T3. It is further found that inhibition of melanization induced by glucosamine occurs even in the presence of protease inhibitors, such as phenylmethylsulfonyl fluoride and leupeptin, and that melanization inhibition is reversible upon removal of the inhibitor. In this report we have also examined the process of development and recovery of the tyrosinase isozymes in cells in which the interruption of melanogenesis has been released by the removal of these glycosylation inhibitors. The recovery process, which occurs during the period after interrupted melanogenesis and is a process of remelanization, has been biochemically followed. Tyrosinases obtained from the deoxycholate-solubilized large-granule fraction of these melanoma cells have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immediately after removal (0 h recovery) of the glycosylation inhibitor, loss of melanization and T3 is accompanied by T1 heterogeneity which is visualized as two electrophoretically distinct species, T1' and T1''. At this time, T1' and T1'' do not have a concanavalin A affinitive carbohydrate moiety but do possess in vitro dopa reactivity. When recovery of melanization begins visibly 24 h later, T3 is re-formed with disappearance of T1 heterogeneity. By 48 h, the previous normal level of melanization is almost attained. These results suggest that maturation of tyrosinase may occur via T1' and T1'' as precursors of T3, or possibly T1 through the addition of N-glycosydically linked oligosaccharide moieties which can be interrupted by glucosamine and tunicamycin.

Stratum corneum lipids serve as a bound-water modulator.

Stratum corneum lipids have been found to be an important determinant involved in the water-holding function of the stratum corneum. In order to further elucidate the significance of stratum corneum lipids in the water-holding properties, the hydration behavior of the stratum corneum following depletion and replenishment of the lipids has been studied, using an isolated human forearm stratum corneum sheet, by differential scanning calorimetry parallel to ultrastructural changes of intercellular lipids in the stratum corneum. Extraction of the stratum corneum sheet with acetone/ether (1/1) decreased the bound-water content from 33.3% to 19.7%, where the melting temperature of ice remained constant. Further extraction with water, which released a large amount of water-soluble materials such as amino acids, did not change the bound-water content, but definitely raised the melting temperature of ice. The application of the stratum corneum lipids, which were solubilized in squalane containing 1% alpha-monomethyl heptadecyl glyceryl ether to the lipid-depleted stratum corneum sheet, caused a significant recovery of bound-water content to the previous, almost normal level. In accordance with the hydration behavior, electron microscopic analysis of the acetone/ether-treated stratum corneum sheet revealed selective depletion of lipids from the intercellular spaces, accompanied by a marked disruption of multiple lamellar structures. In contrast, the application of stratum corneum lipids into the lipid-depleted stratum corneum sheet resulted in the restoration of the lamellar structure between the stratum corneum cells. These findings strongly suggest that stratum corneum lipids serve a water-holding function through the formation of lamellar structures within the stratum corneum.

Induction of melanization within hair bulb melanocytes in chinchilla mutant by melanogenic stimulants.

In an attempt to clarify the mechanisms underlying the lack of melanin formation in hair bulb melanocytes of chinchilla mice (genotype a/a, cch/cch, strain PW), we studied the effect of exogenous melanogenic stimulants such as theophylline (Tp), dibutyryl cyclic AMP (db-cAMP), and alpha-melanocyte-stimulating hormone (alpha-MSH) on the induction of melanization. Skin explants excised from the dorsa of chinchilla or lethal yellow C57BL/6J, Ay/a) mice at 7 to 9 days of age were cultured in the presence of Tp (2 mM), db-cAMP (2 mM), or alpha-MSH (1.0 microgram/ml). After 2 to 5 days, melanin formation was induced in hair bulb melanocytes of chinchilla mutant in response to both Tp and db-cAMP, but alpha-MSH did not produce new melanin formation. In contrast, yellow mutant increased the melanin formation in response to all stimulants. Electron microscopic studies demonstrated that while non-treated hair bulb melanocytes of chinchilla mutant contain a large number of stage II-III melanosomes without melanin deposition, a hair bulb treated with Tp exhibits the new formation of melanin within melanosomes that appears both as typical eumelanosomes with striated longitudinal matrices and as pheomelanosomes with vacuolar melanization. Quantitative analysis of melanin has revealed that in chinchilla mutant, Tp and db-cAMP induce a severalfold increase in the formation of both eumelanin [pyrrole-2,3,5-tricarboxylic acid (PTCA)] and pheomelanin (aminohydroxyphenylalanine), whereas alpha-MSH does not stimulate production of either melanin. In yellow mutant, db-cAMP induced a remarkable increase in eumelanin (PTCA), in contrast to the fewfold increase induced by alpha-MSH and Tp. All stimulants induced a slight increase in pheomelanin to a similar extent. These different reactions to melanogenic stimulation suggest a possible defect in the tyrosinase activation system within hair bulb melanocytes in chinchilla mutants.

Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.

We previously demonstrated that human keratinocytes produce and secrete endothelins (ET), which can be strong mitogens for human melanocytes. Ultraviolet B (UVB) exposure highly stimulated the paracrine linkage of endothelins between keratinocytes and melanocytes, indicating that they are keratinocyte-derived intrinsic mitogens in UVB-induced pigmentation. In this study, the role of ET-1 as a melanogen in UVB melanogenesis was investigated in vitro and in vivo. In the conditioned medium of keratinocytes exposed to UVB, melanin synthesis by human melanocytes, as measured by 14C-thiouracil incorporation, was significantly accentuated. This stimulatory effect was reduced by anti-ET-1 to the level of that in the non-UVB-exposed control, suggesting an essential role of ET-1 as an intrinsic melanogen in UVB-induced melanogenesis. In a parallel study, the addition of 10 nM ET-1 induced an increase in tyrosinase activity in cultured human melanocytes and was accompanied by elevated levels of tyrosinase and tyrosinase-related protein-1 mRNA expression as shown by Northern blotting. Reverse transcription-polymerase chain reaction of RNA isolated from the epidermis of human skin exposed to UVB revealed that, whereas in non-exposed sites ET-1, IL-1 alpha, and tyrosinase mRNA signals were scarcely detected, UVB-irradiation, with a dose of twice the minimal erythema dose, caused a significant increase in the expressions of the three genes 5 d after irradiation. These findings suggest that ET-1 is an important mediator for UVB-induced pigmentation in the epidermis in vivo.

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.

Selective recovery of deranged water-holding properties by stratum corneum lipids.

Selective removal of stratum corneum lipids following applications of acetone/ether to the human forearm for extremely prolonged periods of 5-20 min induced an enduring (more than 4 days), chapped and scaly appearance of the skin which was accompanied by a significant decrease in the water-holding properties of the stratum corneum. In order to further elucidate the significance of lipids in the water-holding properties, lipids, which were extracted as sebaceous-rich lipids (SL) for the first 10-min acetone/ether treatment and as stratum corneum lipids (SCL) for the additional 30-min treatment, were topically applied daily on lipid-depleted forearm skin which had been pretreated with acetone/ether for 40 min. Two daily applications of the SCL which were solubilized in squalane containing 1% alpha-monomethyl heptadecyl glyceryl ether (GE) caused a significant increase of conductance, accompanied by a marked improvement in the level of scaling as compared with nontreatment or GE/squalane base, whereas the SL in the GE/squalane base did not exhibit any significant recovery in either conductance value or scaling. To clarify which components of the SCL are primarily responsible for the observed recovery of the water-holding properties, chromatographically separated fractions of the SCL were also topically applied in the same manner for 2 successive days. Out of the following separated fractions: cholesterol, cholesterol ester, free fatty acid, glycolipids, and ceramide, 2 daily topical applications of ceramide fraction induced a significant and the highest increase in the conductance value as compared with GE/squalane base. Furthermore, glycolipids and cholesterol fractions also exhibited a significant recovery when compared with no application at all. In contrast, free fatty acid and cholesterol ester fractions did not indicate any significant increase in the conductance value. These findings strengthen the hypothesis that structural lipids present in the intercellular spaces of the stratum corneum, especially ceramide, play a critical role in the water-holding properties of the stratum corneum.