Keratinocytes modulate the biosynthetic phenotype of dermal fibroblasts at a pretranslational level in a human skin equivalent

In this study we investigated the influence of keratinocytes on the phenotype of fibroblasts in an in vitro human skin equivalent. Keratinocytes were seeded at the surface of fibroblast-populated mechanically restrained type I collagen gels (lattices). Lattices without keratinocytes were handled in parallel as controls. After 2 and 4 days in culture, the keratinocyte layer was removed and the steady-state level of the mRNA for the main extracellular matrix macromolecules and interstitial collagenase produced by the fibroblasts was measured by Northern and dot blot analysis. A 50% decrease in the amount of procollagen type I and type III mRNAs was observed after 2 and 4 days of coculture while collagenase gene expression was upregulated by 300% when compared with control lattices. No significant modulation of type IV and type VI collagen, elastin or laminin B1 mRNA levels was found. Fibronectin mRNA levels in fibroblasts were significantly increased only on day 4. All the observed changes could be reproduced using a conditioned medium collected from a lattice covered with keratinocytes added to a lattice containing fibroblasts alone. These results indicate that in an in vitro reconstituted skin, keratinocytes are able to modulate the biosynthetic phenotype of fibroblasts at a pretranslational level through a paracrine signalling pathway.     

Biosynthesis of collagen and elastin by chick embryo skin during maturation

Summary The biosynthess of collagen and elastin by chick embryo skin of 11–16 days of development was studied. An increased uptake of (¹⁴C) proline and (¹⁴C) lysine on day 12 was observed in collagen peptides and in elastin extracted from the skin samples.(¹⁴C)hydroxyproline followed the same pattern as the uptake of its precursor into the collagenase-digested peptide fraction. The conversion of (¹⁴C)lysine into (¹⁴C)hydroxylysine was low. The conversion of (¹⁴C)glutamic acid and (¹⁴C)glutamine into (¹⁴C)proline and (¹⁴C)hydroxyproline by the skin of chicken embryos may be an important pathway.As far as (¹⁴C)proline and (¹⁴C)lysine is concerned elastin showed a similar pattern of changes during the maturation process.

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Zusammenfassung Die Kollagen- und Elastinbiosynthese in 11–16 Tage alter Hühnchenhaut wurde untersucht. Am 12. Tag zeigte sich eine erhöhte Aufnahme von¹⁴C-Prolin und C¹⁴-Lysin in Kollagenpeptide und Elastin, die aus den Hautpräparaten isoliert wurden. C¹⁴-Hydroxyprolin zeigte ein gleiches Muster des Einbaues in die kollagenase-verdauliche Peptidfraktion. Die Konversion von¹⁴C-Lysin in¹⁴C-Hydroxylysin war gering. Der Übergang von C¹⁴-Glutaminsäure und C¹⁴-Glutamin in¹⁴C-Prolin und¹⁴C-Hydroxyprolin in der Hühnchenhaut ist möglicherweise eine wichtige Route. Elastin zeigte hinsichtlich C¹⁴-Prolin und C¹⁴-Lysin während des Reifungsprozesses ein ähnliches Einbauverhalten.

     

角质形成细胞与皮肤免疫屏障

角质形成细胞(keratinocytes,KC)是皮肤免疫屏障中的重要一员.角质形成细胞可表达模式识别受体(pattern recognition receptor,PRR)识别病原体,启动固有免疫反应;角质形成细胞可作为抗原提呈细胞,参与抗原呈递,并分泌多种免疫活性分子参与皮肤免疫防御机制;还可表达抗菌肽直接杀灭或间接活化细胞因子防御病原体.烟酰胺腺嘌呤二核苷酸磷酸(nicotinamideadenine dinucleotide phosphate,NADPH)氧化酶参与上皮防御的机制则是新的研究热点,其在角质形成细胞的表达及影响因素尚待进一步研究.     

Development and characterization of a canine skin equivalent

The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio-matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).     

Epidermal growth in the skin equivalent

Summary The skin equivalent (SE) has been validated as a model for studies on proliferation of keratinocytes. SEs were prepared from normal skin by implanting punch biopsies on dermal equivalents consisting of fibroblasts in a collagen matrix. The outgrowths were measured by planimetry. An immunohistochemical investigation with antibodies against markers associated with proliferation was performed on frozen sections from SEs during outgrowth at 3–6 days (SE6) as well as after completion of outgrowth at 21 days (SE21). Biopsies from normal controls and from uninvolved and involved skin in psoriatic patients were also studied. The antibodies used were Ki-67, cytokeratin 8.12, and antibodies against the receptors for epidermal growth factor (EGFr) and transferrin (TFr). The increase in area was linear during the first 7 days of culture and usually reached the edges of the dermal equivalent at this time. In SE6 TFr was expressed in the basal part of the outgrowth while the other markers were not observed. In SE21 and in psoriasis there was abundant epidermal staining of Ki-67-positive nuclei and cytokeratin 8.12 was detected in the suprabasal part of the epidermis. EGFr and TFr were seen in the basal layer in SE21. In the psoriatic lesions these receptors were found both in the basal and suprabasal layers. The lack of proliferation markers in SE6 indicates that the initial increase in the area of keratinocytes is due to migration from the punch biopsies. Increased cell proliferation is present in SE21, a finding in common with psoriasis and wound healing. The skin equivalents should therefore be an appropriate model for studies on these phenomena.     

Age changes of cross-links in rat skin elastin

Summary The content of elastin was determined in the skin of rats aged 2–33 months; it decreased with age of the skin, and the diameter of elastic fibers increased. In elastin isolated from the rat skin, the content of isodesmosine and desmosine was 3–10 times lower than in that from bovine ligamentum nuchae; both isomers increased with advancing age of the skin.

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Zusammenfassung Es wurde der Elastingehalt in der Haut der 2–33 Monate alten Ratten festgestellt. Der Elastingehalt sank entsprechend dem Alter, und die Dichte der elastischen Fasern erhöhte sich. Im isolierten Elastin sind Isodesmosin und Desmosin 3–10 mal niedriger als in dem vom Rind-Ligamentum nuchae, und steigen entsprechend dem fortschreitenden Alter der Haut.

     

Production of fibronectin by epithelium in a skin equivalent

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.     

皮肌炎与硬皮病皮损原位细胞凋亡研究

目的 :　探讨角质形成细胞凋亡在皮肌炎 (DM)和系统性硬皮病 (SSc)皮损发生发展中的作用。方法 :　采用原位细胞凋亡检测 (TUNEL)方法研究两病患者皮损角质形成细胞的凋亡情况。结果 :　 (1)性别、年龄、病程、疗前评分、疗后评分、住院天数、糖皮质激素总量等因素 ,对DM和SSc的凋亡指数的影响均无统计学意义 (P均 >0 .0 5 ) ;(2 )正常对照 8例表皮细胞凋亡指数均 <0 .0 0 5 ,DM与SSc凋亡指数均明显高于对照组 (P均 <0 .0 1) ;(3)角质形成细胞凋亡发生在整个皮损区的角质形成细胞。结论 :　角质形成细胞凋亡异常与DM和SSc皮损的发生可能有密切联系。     

Collagen and elastin changes in d-penicillamine-induced pseudoxanthoma elasticum-like skin

A patient with cystinuria who was treated with large doses of D-penicillamine for 19 years developed skin abnormalities resembling those seen in pseudoxanthoma elasticum. Biochemical and histological examination of the dermis showed that the collagen content, the ratio of the major genetic forms of collagen and the distribution of collagen types was normal. Light microscopy demonstrated the presence of vastly increased amounts of elastin in the dermis, and the individual elastin fibres were shown by electron microscopy to be abnormal; chemical analysis showed the elastin to be poorly cross-linked. Some of the collagen also appeared structurally abnormal, and biochemically resembled that seen in the dermis of a young child with respect to cross-linking and hexosyl-lysine content. The therapy led to an increased deposition of collagen and elastin fibres which appeared abnormal, and resulted in an increase in total skin surface area. These data indicate that D-penicillamine was not fully effective in inhibiting collagen and elastin cross-linking, and appeared to prevent or inhibit the natural maturation process of the collagen.     

Transfer of melanosomes in a skin equivalent model in vitro

The transfer of pigment granules from melanocytes to keratinocytes was studied using a new living skin equivalent (SE) model in vitro. The model was constructed by plating human neonatal melanocytes onto a dermal equivalent (DE) before it was overgrown with keratinocytes. The dermal component of the SE arises in vitro through the action of fibroblasts, which compact matrix proteins into a tissue. It becomes keratinized as keratinocytes migrate out of 2-mm punch biopsies of human neonatal skin embedded in the DE; keratinocytes from the biopsies covered the lattice in 14 days. A basal lamina develops at the dermal-epidermal junction in vitro. Exposure of some SEs to UVB irradiation for 14 days stimulated and enhanced pigment transfer. Pigment transfer from melanocytes to keratinocytes was documented in light and electron microscopic studies. Melanosomes, identified by their pigment as well as by dopa oxidase staining, were dispersed throughout the keratinocyte cytoplasm. We conclude that the SE model is valuable for studying the relationship between melanocytes and keratinocytes in vitro; since the SE has been shown to serve as a skin replacement, pigmenting it may be expected to increase its usefulness.     

Growth of melanocytes in a skin equivalent model in vitro

We have developed a pigmented human skin equivalent by inserting a punch biopsy of human infant foreskin as a source of epidermis into a collagen lattice (dermal equivalent). Using a conventional epidermal culture medium and stimulation with UVB irradiation or 8-MOP + UVA treatment, melanocytes were found to grow out from the biopsy with the epidermal sheet. In this newly formed epidermis, melanocytes and keratinocytes were maintained in an architectural relationship similar to that present in vivo and melanocyte outgrowth could be quantitatively evaluated. Consequently, this pigmented human skin equivalent is a useful model for investigating the biology and photobiology of human skin pigmentation.     

Expression of the 67-kDa elastin receptor in perforating skin disorders

BACKGROUND: Perforating skin dermatoses include elastosis perforans serpiginosa (EPS), reactive perforating collagenosis, Kyrle's disease and perforating folliculitis. In addition to these four diseases, an acquired form of perforating dermatosis associated with diabetes mellitus and/or chronic renal failure has been reported for which the term acquired perforating dermatosis (APD) was proposed. The molecular mechanism of transepidermal elimination of dermal components in perforating skin dermatoses remains unclear. We recently demonstrated that the 67-kDa elastin receptor can be detected in the epidermis eliminating altered elastic fibres in EPS, suggesting that the elastin-keratinocyte interaction may play a role in transepidermal elimination in EPS. OBJECTIVES: To determine whether the 67-kDa elastin receptor is involved in other perforating diseases. METHODS: Paraffin-embedded skin specimens from new cases of EPS (n = 2), APD (n = 15) and perforating granuloma annulare (PGA; n = 2) were studied immunohistochemically using a specific antibody to the 67-kDa elastin receptor. In one case of EPS, two different sites from a single lesion, a central atrophic area and a peripheral keratotic area, were studied. RESULTS: Expression of the elastin receptor was detected in the epidermis surrounding the elastic materials in both cases of EPS. The elastin receptor was not detected in the central inactive area, whereas it was expressed strongly in the peripheral keratotic active area. The elastin receptor was also detected in three of 15 cases of APD in which a few elastic fibres were found in the eliminated dermal materials. In one case of APD, the elastin receptor was not detected in spite of the presence of a few elastic fibres in the eliminated materials. The elastin receptor was not detected in either case of PGA. CONCLUSIONS: Expression of the elastin receptor in EPS was seen in both cases studied and was dependent on the stage of the lesion. Expression of the elastin receptor in APD appeared to be related to the amount of elastic fibres in the eliminated materials. Thus, expression of the elastin receptor in perforating skin disorders may depend on the stage of the lesion and/or the content of elastic fibres in the dermal materials being eliminated.     

Culture of human epidermal Langerhans cells in a skin equivalent

Langerhans cells (LCs) have been cultured in a skin equivalent (SE). Seventy-two SEs were produced by inserting skin biopsies from nine subjects into dermal equivalents consisting of fibroblasts in a collagen matrix. The SEs were cultured in a serum-free medium containing 2-mercaptoethanol with or without 5 ng/mL granulocyte–monocyte colony-stimulating factor (GM-CSF). The SEs were cultured for 12 or 15 days. In the latter case, 0, 1 or 10 μg/mL cyclosporin A (CyA) was added for the last 3 days. The SEs were then snap frozen for immunohistochemistry. The migration of LCs was evaluated by measuring the distances from the inserted skin biopsy in the SEs to the HLA-DR + and CD1a+ dendritic cells localized at the longest distance from the biopsy in the epidermal outgrowth on both sides of the biopsy. The density of these cells was estimated in 15-day-old SEs by counting them on both sides of the inserted skin biopsy and dividing the number of positive cells by the migrated distances. All epidermal outgrowths (range 0.6–3.7 mm) were well differentiated and displayed HLA-DR+, CD1a+ and Lag+ dendritic cells. Only occasionally were CD83+ cells observed. In the 15-day-old SEs cultured with GM-CSF, a few CD86+ cells were seen in the epidermal outgrowths and occasionally CD80+ cells. The median ( n   =  4) density of CD1a+ and HLA-DR+ cells in the epidermal outgrowths at day 15 was 5.2 and 9.1 cells/mm, respectively. GM-CSF did not influence migration in 12-day-old SEs, but there was a tendency to increased migration of HLA-DR+ dendritic cells in 15-day-old SEs. CyA did not affect migration or density. We conclude that LCs can be cultured with an in vivo -like density in a SE. They express the phenotype of immature antigen-presenting cells efficient in capturing and processing antigen. This model may be suitable for studies of the initial phase of contact allergic reactions.     

Increased elastin production by progeria skin fibroblasts is controlled by the steady-state levels of elastin mRNA

Hutchinson-Gilford progeria is a unique, rare disease with markedly accelerated aging. The average lifespan of affected individuals is 12 years. Although the biochemical basis of the syndrome is unknown, its influence appears to be primarily upon mesodermal tissues. Characteristics such as the altered appearance of the skin and the extensive and fatal involvement of the cardiovascular system led us to study elastin production in cultured skin fibroblasts from three progeroid individuals. We found tropoelastin production by progeroid cells was elevated six- to nine-fold at the protein and mRNA levels, while relative collagen synthesis was similar to control strains. There was little difference between progeroid and normal cells in expression of total protein or in total cellular mRNA content. Western blot analysis of tropoelastin from progeroid fibroblasts confirmed increased production of elastin but revealed no gross changes in the molecular mass. The significant increase in tropoelastin expression lends support to the concept that progeria results from a mesenchymal dysplasia, and offers a possible biochemical marker for the phenotype.     

Integration of Langerhans-like cells into a human skin equivalent

Studies regarding cellular interactions between Langerhans cells and other skin cells are somehow hampered by the difficult cultivation of these cells in vitro. Here, we show that the human MUTZ-3 cell line can be differentiated into Langerhans-like cells in the presence of a cytokine cocktail including GM-CSF, TGF-β1 and TNF-α. We used the expression of langerin, CD1a, CCR6 and the intracellular presence of Birbeck granules to identify the differentiated MUTZ-3 cells (MUTZ-3-LCs). The aim of this study was to integrate MUTZ-3-LCs into a three-dimensional full-thickness skin model. On top of fibroblast-containing collagen matrix a mixture of primary human keratinocytes and MUTZ-3-LCs were seeded and cultured for 24 h. Subsequently, the models were lifted up to the air-liquid interface. Histological evaluation featured a fully stratified epidermis with all characteristic epidermal strata. Langerin-positive cells were detected suprabasally within the epidermis indicating that keratinocytes provide environmental conditions for long-time maintenance of MUTZ-3-LCs. These skin models provide a tool to further investigate the interactions between Langerhans-like cells and other skin cells and particularly learn more about the cutaneous immune response.     

Keratinocytes regulate melanocyte number in human fetal and neonatal skin equivalents

To determine if keratinocytes influence melanocyte number and position in the developing epidermis we have experimentally recombined keratinocytes and melanocytes from epidermis of different stages of differentiation in the skin equivalent (SE) system. Previously we showed that developmental differences in the position and number of melanocytes characteristic of the epidermis in vivo were preserved in fetal and neonatal skin equivalents. In the present study we have combined cultured fetal or neonatal keratinocytes with age-matched or non-age-matched cultured melanocytes on the dermal equivalent. The ratio of basal keratinocytes to melanocytes (BK/M) present in multiple high-power fields was determined after localization of melanocytes by staining with the melanocyte-specific monoclonal antibody, HMB-45. The BK/M ratio in SE composed of neonatal keratinocytes and either fetal (n = 4) or neonatal (n = 5) melanocytes was 26.2 and 21.5, respectively. The BK/M ratio in SE composed of fetal keratinocytes and either fetal (n = 8) or neonatal (n = 5) melanocytes was 9.2 and 7.7, respectively. In each case, the BK/M ratio was dependent on the keratinocytes rather than the melanocytes. With either type of melanocyte, ratios in SE composed of neonatal keratinocytes were significantly greater than those with fetal keratinocytes. These results establish that keratinocytes regulate the BK/M ratio in this model and suggest that developmental differences between fetal and neonatal keratinocytes may be responsible for determining melanocyte numbers in the epidermal-melanin unit in vivo. The precise mechanisms that control the organization and number of melanocytes in the epidermis are unknown although keratinocytes may interact with melanocytes via growth factors, cell surface molecules, or other factors related to proliferation and differentiation of the epidermis.     

Stimulation of elastin expression by minoxidil in chick skin fibroblasts

Minoxidil inhibited the proliferation of embryonic skin fibroblasts during the growth phase but not during the stationary phase. Minoxidil stimulated elastin synthesis two-fold in a dose-dependent manner at a concentration of 1 mM during the stationary phase. The stimulation of elastin synthesis paralleled a comparable increase in elastin mRNA level. These results suggest that the stimulation of elastin expression by minoxidil in skin fibroblasts was controlled at the elastin mRNA level and also suggest that its elastinstimulating effect is not related to the suppressive effect on cell proliferation. Minoxidil appears to be a potent stimulator for elastin expression in skin fibroblasts.     

The growth of ichthyotic epidermal cells in a 3-dimensional reconstruction of human skin, the skin equivalent

We have studied the differentiation of ichthyotic epidermis in vitro using the skin equivalent model. The morphology of these ichthyotic cultures has been investigated using histopathological and histometric techniques including epidermal and stratum corneum thickness measurements. The skin equivalents have also been investigated for the presence of markers of differentiation using immunolocalization techniques. These markers include the 65·5 and 67 kDa keratins, desmoplakin, involuerin, laminin and filaggrin. It has been shown that the ichthyotic epidermis develops a fully differentiated epidermis and stratum corneum, equivalent to those seen in normal skin equivalents.     

Keratinocytes in lesional skin of atopic eczema bear HLA-DR, CD1a and IgE molecules

Apparently normal, and lesional skin from patients with atopic eczema were investigated immunohistochemically with anti-HLA-DR, -CD1a and -IgE antisera. A CD1a+ intercellular pattern was observed in uninvolved skin in the majority of the patients whereas an HLA-DR+/CD1a+ network, mostly localized in basal and supra-basal areas, was shown in lesional skin of virtually all of them. Moreover, an HLA-DR+/CD1a+IgE+ intercellular pattern was observed in some of the patients only and was predominantly localized in those areas characterized by lymphocyte exocytosis, spongiosis or vesicle formation. Whether keratinocytes are able to synthesize CD1a antigen and Fc epsilon R or if these molecules are only produced and shed by CD1a+/IgE+ epidermal dendritic cells remains unclear.