Tob is a potential marker gene for the basal layer of the epidermis and is stably expressed in human primary keratinocytes

BACKGROUND: Epidermis consists of multiple layers, from the proliferating basal layer to terminal differentiated cornified layers, and these layers are defined by differentiation status. Tob gene product is known to be a member of the BTG antiproliferative protein family. We investigated the expression pattern of Tob gene product to understand the possible role in differentiation of keratinocytes and epidermis. OBJECTIVES: In this study, we examined the expression of Tob gene product in the primary cultured human keratinocytes and in the in vivo epidermis. METHODS: The expression of Tob gene product was assessed by Western blotting analysis. Cellular localization of Tob was detected using the green fluorescent protein-tagged Tob cDNA expression construct. In vivo expression of Tob gene product in the epidermis was determined by immunohistochemistry with paraffin sections. RESULTS: Tob family members are degraded by the ubiquitine-proteasome system triggered by the growth signal. Tob is stably and abundantly expressed in primary cultured human keratinocytes. Furthermore, the expression of Tob in the keratinocytes persists during the differentiation induced by calcium; however, it was not detected in primary cultured fibroblasts. Also, the subcellular localization of Tob is mainly in the cellular membrane in the primary human keratinocytes. We evaluated Tob expression in normal skin, oral mucosa and different diseases, such as psoriasis, X-linked ichthyosis and squamous cell carcinoma (SCC). Using immunohistochemical analysis, we observed that Tob was selectively expressed in the basal layer of X-linked ichythyosis and the hyperproliferative basal layer of psoriasis and oral mucosa as well as in normal epidermis. In SCC, the expression of Tob gene product was relatively decreased. CONCLUSIONS: Tob is stably expressed in primary human keratinocytes and it is specifically expressed in the basal layer of in vivo epidermis.     

Reactive changes in human epidermis following simple occlusion with water

Reactive changes produced in human epidermis by occlusion with water for 24 or 48 h were studied. A focally widened intercellular space was common. Several, often pronounced, reactive events were observed in the Langerhans cell system, whereas the keratinocytes and the melanocytes seemed unaffected. The reactive events showed a scattered distribution and were revealed only by electron microscopic analysis of extensive section series. It seems that, among epidermal cells, the Langerhans cells are the most susceptible and most easily alerted by an exogenous challenge.     

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.     

Estrone-and dehydroepiandrosterone-sulfatase activities in human female epidermis

Summary Estrone (E₁)-sulfatase and dehydroepiandrosterone (DHEA)-sulfatase activities were studied in human female epidermis. Skin specimens were obtained by abdominal or plantar biopsies. The apparent Michaelis-Menten constants for E₁ and DHEA sulfatase were 35.2 M and 8.7 M, respectively. A substrate inhibition was only observed for DHEA sulfatase. Both sulfatases had an elevated temperature optimum (65°C). The effect of inorganic salts was also tested. In normal epidermis, E₁-sulfatase activity was constantly higher than DHEA-sulfatase activity, but no correlation between these activities was observed. On the other hand, E₁-and DHEA-sulfatase activities were lower in plantar than in abdominal epidermis. In plantar epidermis of palmoplantar keratoderma, large variations in E₁-sulfatase activity, but no significant variation in DHEA-sulfatase activity, were obseved. In human epidermis, the findings were consistent with the existence of two different sulfatases: E₁ sulfatase and DHEA sulfatase. It would also appear that sulfatase activities are not linked to the abnormal shedding of plantar stratum corneum in palmoplantar keratoderma.     

Distribution pattern of DNA polymerases in epidermis

DNA polymerases are known to play important roles in DNA replication and repair processes. The present study revealed that the DNA polymerase α and β participate in the cell differentiation of normal and n-hexadecane-induced hyperplastic epidermis of the guinea pigs. The epidermal cells were separated into three layers; high (HDCL), middle (MDCL), and low (LDCL) density cell layer, respectively, by Percoll gradient centrifugation. In epidermal homogenate, the activity of DNA polymerase β was higher than that of DNA polymerase α. DNA polymerase α activity was higher in the HDCL than in the other layers; however, DNA polymerase β was higher in the LDCL than in the other layers. In hyperplastic epidermis, distribution of DNA polymerase α activity was similar to the normal pattern, but DNA polymerase β was lower in the LDCL than in the other layers. The distribution of DNA polymerase α activity in epidermal nuclei was similar to that of the whole epidermal cell pattern; however, DNA polymerase β activity differed from the enzyme distribution of whole epidermal cell homogenate. Its activity was higher in the HDCL than in the other layers. In hyperplastic epidermis, both nuclear DNA polymerase α and β activities were similar to the distribution patterns of polymerase activities in hyperplastic epidermal cells. From these results, it is concluded that the distribution pattern of DNA polymerases in n-hexadecane induced-hyperplastic epidermis is not a simple augmentation of the distribution pattern in normal epidermis.     

Low-molecular-weight proteinase inhibitor of human epidermis inhibiting papain and trypsin activity

Summary A low-molecular-weight proteinase inhibitor was isolated from human epidermal extracts by means of ultrafiltration, gel filtration and ion-exchange chromatography. On polyacrylamide gel electrophoresis (PAGE), the isolated fraction exhibited a single band of activity at pH 9.4, and inhibitory activity against papain and trypsin was detected in this band. Using sodium-dodecyl-sulphate PAGE, the molecular mass of the inhibitor was estimated to be 2,200 daltons.     

1,25-dihydroxyvitamin D3 and the vitamin D analogue KH1060 induce hyperproliferation in normal mouse epidermis

Abstract 1,25-dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08 2.0 μg/ml) and of a new vitamin D analogue, the epi-20-analogue KH1060 (0.4 –2.0 μg/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single-dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariale bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse-labelling with BrdUrd. Both calcitriol and KM 1060 induced a dose- and time-dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G₂ phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti-proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.     

Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation

Four techniques for dissociation of skin biopsies were compared to identify the method of choice for optimal expansion of isolated keratinocytes. Equivalent biopsies were obtained from 4 healthy human subjects and each divided into four parts. One part was minced and placed in a trypsinizing flask containing 0.05% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA). Released cells were harvested hourly. With the other parts, the epidermis was separated from the dermis after treatment with 0.5 mg/nml thermolysin, 2.5 mg/ml Dispase, or 0.17% trypsin and the epidermal portions were minced and incubated for 1 h in trypsin:EDTA. The cells were cocultivated with irradiated 3T3 fibroblasts to study the keratinocytes proliferative capacity. Freshly isolated cells were immunostained with anti-vimentin antibodies or grown in fibroblast-supportive conditions to detect the presence of human dermal fibroblasts. The mean number of cells dissociated per cm2 biopsy was higher after trypsin:EDTA digestion of a dermis-containing biopsy using a trypsinizing flask (4.0x 10(6) cells/cm2) compared to a biopsy where dermis-epidermis had been separated by thermolysin (2.8x 10(6) cells/cm2), Dispase (2.3x 10(6) cells/cm2) or trypsin (1.1 x 10(6) cells/cm2). Between 0.5% and 4% of the cells dissociated from a dermis-containing biopsy were human fibroblasts. This comprised more than twice the number of fibroblasts obtained by using epidermal/dermal split techniques. The proliferative capacity in primary and secondary culture was higher in cells isolated by trypsin:EDTA incubation in the trypsinizing flask or after epidermal-dermal separation using thermolysin, suggesting that Dispase or trypsin may have a more detrimental effect on the isolated keratinocytes. Our results show that dissociating the cells by trypsin:EDTA incubation in a trypsinizing flask or after epidermal-dermal separation using thermolysin, are preferable methods for isolating keratinocytes from human skin.     

Structural diversity of mast cell granules in black and white skin

BACKGROUND: There are conflicting reports of structural differences between black and white skin, other than pigmentary differences. OBJECTIVES: To evaluate differences in mast cells between black and white skin. METHODS: Biopsies of normal buttock skin were obtained from four African-American males (29.2 +/- 3.0 years old) and four Caucasian males (29.4 +/- 1.2 years old) and processed routinely for electron microscopy. For the quantitative assessment of mast cell granules, five electron micrographs at a final magnification of x 53,700 were analysed for each individual, using a computer-assisted image analyser. More than 10 granules per cell, and a total of 1210 granules, were evaluated for their internal structures. RESULTS: Mast cells in black skin contained larger granules than those in white skin (P < 0.0001). In black skin, fusion of granules seemed to account for the larger sizes. The percentage of granule matrix occupied by curved lamellae was higher in white skin, whereas parallel-linear striations were more frequent in black skin (P < 0.05). The subgranular distribution of the mast cell proteases, tryptase and cathepsin G, were evaluated by immunoelectron microscopy. Tryptase reactivity was localized preferentially over the parallel-linear striations and partially over the dark amorphous subregions within granules of black skin, whereas it was confined to the peripheral area of granules, including curved lamellae, in white skin. Cathepsin G reactivity was more intense over the electron-dense amorphous areas in both groups, while parallel-linear striations in black skin and curved lamellae in white skin were negative. CONCLUSIONS: This study has confirmed ultrastructural differences in mast cell granules between black and white skin, which may be of functional importance.     

Correlation between hyperproliferation and suprabasal integrin expression in human epidermis reconstituted in culture

Abstract In normal epidermis integrin expression is largely confined to the basal layer. However, during wound healing and in psoriatic lesions suprabasal expression is observed. Although the potential importance of suprabasal integrin expression in the pathogenesis of psoriasis has been established, the cause of suprabasal expression is unknown. We now describe changes in integrin expression that occur with time when normal human keratinocytes are grown on two types of dermal equivalent, de-epidermized dermis and collagen gels containing fibroblasts. We show that suprabasal integrin expression is correlated with suprabasal expression of the EGF receptor, but not with expression of keratin 10 or keratin 16. By quantitating the proportion of basal keratinocytes expressing the proliferation marker Ki-67 we could show that suprabasal integrin expression is correlated with high proliferative activity within the basal layer. Taken together with our earlier work, these results suggest that suprabasal integrin expression is linked to hyperproliferation and not to abnormal terminal differentiation or to inflammation; they also establish dermal equivalent cultures as useful experimental models with which to manipulate keratinocyte integrin expression.     

Expression of involucrin in normal, hyperproliferative and neoplastic mouse keratinocytes

Involucrin is a protein precursor of the epidermal cornified envelope. Although expression of the human protein has been documented extensively, studies in the mouse have been hampered by a shortage of good antibodies. We describe the production of recombinant mouse involucrin and preparation of rabbit antisera to the protein that work well by immunohistochemistry and Western blotting. We confirm that in normal mouse epidermis the onset of involucrin expression is in the upper spinous layers and inner root sheath of the hair follicle. Involucrin was also detected in the differentiating epithelial cells of normal tongue, oesophagus and bladder. Involucrin was expressed in a subpopulation of mouse keratinocytes cultured in standard or low calcium medium and the proportion of involucrin-positive cells increased during suspension-induced terminal differentiation. Western blotting of keratinocytes from several inbred mouse strains revealed a remarkable heterogeneity in the electrophoretic mobility of involucrin, reflecting inter-strain variation in the number of tandem repeats in the protein. In the hyperproliferative epidermis of healing wounds involucrin was expressed in most of the suprabasal layers. In epidermal papillomas and carcinomas involucrin expression correlated well with degree of histological differentiation. The sites of expression of the mouse protein were thus the same as those previously reported for human involucrin. With the development of the new antibodies we anticipate that involucrin will become as widely used a marker of keratinocyte differentiation in the mouse as it is in the human.     

Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis

Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model.     

Topical ceramides neither enhance UVB‐induced apoptosis in normal human keratinocytes nor affect viability in UVB‐irradiated reconstructed human epidermis

Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water‐holding properties for decades. In vitro studies have indicated cytotoxic potential for cell‐permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB‐induced apoptosis. Phytosphingosine, N‐hexanoyl‐phytosphingosine and N‐stearoylphytosphingosine (ceramide III) in concentrations ≤5 μm have been used for co‐stimulation with low (160 J/m²) or high (600 J/m²) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase‐3 activity was observed in basal keratinocytes irradiated with 600 J/m² UVB and in differentiating keratinocytes with both UVB doses. Co‐stimulation with the named ceramides did not further increase (i) caspase‐3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co‐stimulation with 1‐mm ceramides did not further affect viability/lactate dehydrogenase release in UVB‐irradiated reconstructed human epidermis corroborating the safety of these ceramides.