Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.
The dermis contains two distinct layers: the papillary and the reticular layers. In vitro cultures of the fibroblasts from these layers show that they are different. However, no molecular markers to differentiate between the two subtypes of fibroblasts are known. We performed gene expression analysis on cultured fibroblasts isolated from the papillary and reticular dermis. In all, 116 genes were found to be expressed differentially. Of these, 13 were validated by quantitative reverse transcriptase-PCR analysis and two markers could be validated at the protein level in monolayer cultures. Three markers showed differential expression in in vivo skin sections. The identified, characteristic markers of the two fibroblast subpopulations provide useful tools to perform functional studies on reticular and papillary fibroblasts.
Human skin equivalents (HSEs) are three-dimensional culture models that are used as a model for native human skin. In this study the barrier properties of two novel HSEs, the fibroblast-derived matrix model (FDM) and the Leiden epidermal model (LEM), were compared with the full-thickness collagen model (FTM) and human skin. Since the main skin barrier is located in the lipid regions of the upper layer of the skin, the stratum corneum (SC), we investigated the epidermal morphology, expression of differentiation markers, SC permeability, lipid composition, and lipid organization of all HSEs and native human skin. Our results demonstrate that the barrier function of the FDM and LEM improved compared with that of the FTM, but all HSEs are more permeable than human skin. Further, the FDM and LEM have a relatively lower free fatty acid content than the FTM and human skin. Several similarities between the FDM, LEM and FTM were observed: (1) the morphology and the expression of the investigated differentiation markers were similar to those observed in native human skin, except for the observed expression of keratin 16 and premature expression of involucrin that were detected in all HSEs, (2) the lipids in the SC of all HSEs were arranged in lipid lamellae, similar to human skin, but show an increase in the number of lipid lamellae in the intercellular regions and (3) the SC lipids of all HSEs show a less densely packed lateral lipid organization compared with human SC. These findings indicate that the HSEs mimic many aspects of native human skin, but differ in their barrier properties.
Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.
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