Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.     

The myofibroblast markers α-SM actin and β-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin

To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: -smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and -actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed -SM actin. Furthermore, similar amounts of -actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed -SM actin. They expressed -actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing -SM actin and overexpressing -actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts.     

Evidence for a direct role of α-MSH in morphological background adaptation of the skin in Sarotherodon mossambicus

Summary The skin colour of the cichlid teleost Sarotherodon mossambicus adapted rapidly to changes in background colour. The physiological adaptation was associated with morphological changes in the dermis. Differences in the dermis were found between fish adapted to a black or white background for 14 days. Number and size of the melanophores as well as the amount of pigment in the cytoplasm of the melanophores were significantly increased in fish adapted to a black background. Changes in the dermis parallelled changes in the state of activity of the two endocrine cell types in the pars intermedia of the pituitary. Both the PAS positive cells and the MSH producing cells were more active when the fish were exposed to a black rather than a white background. Fish continuously infused with -MSH, using an osmotic minipump, had more melanophore cytoplasm and pigment per dermis surface unit area than untreated fish. The activity of the MSH cells in MSH-infused fish exposed to a black background was reduced to a level comparable to the MSH cell activity of untreated fish on a white background. -MSH treated fish that were exposed to a white background had many disintegrating MSH cells. These findings point to inactivation of these cells by exogenous -MSH. The activity of the PAS positive cells was not influenced by treatment with -MSH.     

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal kerationocytes in immunodeficient mice

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.     

Histopathological and cellular studies of a case of cutaneous radiation syndrome after accidental chronic exposure to a cesium source.

This study was designed for the histopathological, cellular and biochemical characterization of a skin lesion removed surgically from a young male several months after accidental exposure to cesium-137, with an emphasis on expression of transforming growth factor beta1 (TGFB1) and tumor necrosis factor alpha (TNFA) and the occurrence of apoptosis. Under a hypertrophic epidermis, a highly inhomogeneous inflammatory dermis was observed, together with fibroblastic proliferation in necrotic areas. Immunostaining revealed overexpression of TGFB1 and TNFA inside the keratinocytes of the hypertrophic epidermis as well as in the cytoplasm of the fibroblasts and connective tissue of the mixed fibrotic and necrotic dermis. Inside this dermis, the TUNEL assay revealed areas containing numerous apoptotic fibroblasts next to areas of normal viable cells. Overexpression of TGFB1 was found in the conditioned medium and cellular fractions of both hypertrophic keratinocytes and fibrotic fibroblasts. This overexpression lasted for at least three passages in tissue culture. The present observations were consistent with the central role of TGFB1 in the determination of chronic radiation-induced damage to the skin and a significant involvement of TNFA. In addition, programmed cell death appeared to take place during the remodeling of the mixed fibrotic and necrotic tissue.     

Monoclonal antibody analysis of skin in chronic murine graft vs host disease produced across minor histocompatibility barriers

Chronic graft vs host disease (GVHD) across minor histocompatibility barriers was produced in BALB/c mice by the injection of spleen cells from B10.D2 mice. Changes in the skin were analyzed in frozen sections using a panel of monoclonal antibodies detected by immunoperoxidase methods. Compared to control animals, a number of changes occurred in the skin of animals with chronic GVHD. In the epidermis, there were increased numbers of Thy-1-positive dendritic cells; keratinocytes expressed Thy-1 and Ia antigens. T lymphocytes appeared in both dermis and epidermis. In the early stages, cells with "helper" and "suppressor" phenotypes were present, while at later times "helper" cells remained in the epidermis and "suppressor" cells remained in the dermis. Cells bearing markers of macrophages were prominent in both dermis and epidermis after the second week. Of great interest was the appearance of spindle-shaped cells in the dermis which expressed Thy-1 and Ia. These cells resembled fibroblasts which may be activated to produce the excess collagen seen in the skin of chronic GVHD.     

Accelerated proliferation and abnormal differentiation of epidermal keratinocytes in endo-beta-galactosidase C transgenic mice

Transgenic (TG) mice that have systemically expressed endo-beta-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Galalpha1-3Gal disaccharide (alphaGal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the alphaGal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1-2 layers to 4-5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the alphaGal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

Migration and differentiation of Langerhans cell precursors

Epidermal Langerhans cells (LC) are the first sentinels of the skin immune system. To study immigration of human LC precursor cells into the skin, we established a two-compartmental skin model consisting of a dermal matrix and an epidermal sheet of keratinocytes. We tested the individual components of the skin model for their influence on phenotype and function of LC precursors. A time window at day 5/6 of differentiation was determined, during which in vitro generated LC precursors expressed adhesion molecules and chemokine receptors required for transmigration across endothelial cell layers and the dermis towards the epidermis. They expressed L-selectin, integrins, platelet endothelial cell adhesion molecule-1, E-cadherin and CC-chemokine receptor 6 and were thus fitted out for transendothelial migration and immigration into the dermis. In a transwell system, these LC precursors migrated towards the chemokine MIP3alpha, demonstrating functional integrity of chemokine receptor 6. For the in vitro reconstituted skin, keratinocytes were grown on a de-epidermized dermis for one to three weeks and formed an epidermal sheet. We allowed LC precursor cells to migrate into this two-compartmental model from the dermal side and examined the presence of CD1alpha--positive cells. LC precursors migrated through the dermal matrix towards the layer of keratinocytes representing the epidermis and could be identified by immunohistology. Experiments designed to investigate the influence of signals provided by both the skin components and by the LC precursors on LC immigration into the skin are in progress.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

T-2 toxin-induced acute skin lesions in Wistar-derived hypotrichotic WBN/ILA-Ht rats

Acute lesions in the dorsal skin topically applied with T-2 toxin (10 microliters of 0.5 mg/ml-solution to 1 cm2) were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats up to 24 hours after treatment (24HAT). In the epidermis, depression of basal cell proliferating activity was detected at 3HAT by immunostaining for proliferating cell nuclear antigen (PCNA), and the percentage of PCNA-positive basal cells decreased thereafter. At 12HAT, in addition to intracytoplasmic edema of spinous cells, acidophilic degeneration of basal cells characterized by shrinkage of cell body with acidophilic cytoplasm and pyknotic or karyorrhectic nuclei became prominent. Most of these nuclei were positive for TUNEL which is a widely used immunostaining for the in situ detection of fragmented DNA, i.e. apoptosis, and the percentage of TUNEL-positive basal cells increased thereafter. The nuclei of these basal cells also showed ultrastructural changes characteristic for apoptosis. On the other hand, in the dermis, infiltration of inflammatory cells including mast cells started at 3HAT and increased thereafter. In addition, capillary and small vessel endothelial degeneration developed at 6HAT and progressed thereafter. These results suggest that T-2 toxin directly affects the epidermis and produces apoptosis in basal cells.     

Immunolocalization of a p53/p63‐like protein in the regenerating tail of the wall lizard (Podarcis muralis) suggests it is involved in the differentiation of the epidermis

During tail regeneration in lizards, the epidermis forms new scales comprising a hard beta‐layer and a softer alpha‐layer. Regenerated scales derive from a controlled folding process of the wound epidermis that gives rise to epidermal pegs where keratinocytes do not invade the dermis. Basal keratinocytes of pegs give rise to suprabasal cells that initially differentiate into a corneous wound epidermis and later in corneous layers of the regenerated scales. The immunodetection of a putative p53/63 protein in the regenerating tail of lizards shows that immunoreactivity is present in the nuclei of basal cells of the epidermis but becomes mainly cytoplasmic in suprabasal and in differentiating keratinocytes. Sparse labelled cells are present in the regenerating blastema, muscles, cartilage, ependyma and nerves of the growing tail. Ultrastructural observations on basal and suprabasal keratinocytes show that the labelling is mainly present in the euchromatin and nucleolus while labelling is more diffuse in the cytoplasm. These observations indicate that the nuclear protein in basal keratinocytes might control their proliferation avoiding an uncontrolled spreading into other tissues of the regenerating tail but that in suprabasal keratinocytes the protein moves from the nucleus to the cytoplasm, a process that might be associated to keratinocyte differentiation.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity- purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down- regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.