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Background CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. Objectives To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Methods Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. Results CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Conclusions Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease.

Cerebellar degeneration-related antigen 1 (CDR1) was described to be expressed in the nervous system and in different types of cancer tissues. In the present study, we demonstrate that CDR1 is in addition ubiquitously expressed in human epidermis, dermis and isolated skin cells. Both CDR1 mRNA and protein were detected in human skin-derived mast cells, melanocytes, fibroblasts and keratinocytes, suggesting that CDR1 does not have a neuron-specific function.

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

Antimicrobial peptides (AMPs), an important part of the innate immune system, are crucial for defense against invading microorganisms. Whereas AMPs have been extensively studied in adult skin, little is known about the impact of AMPs in the developing human skin. We therefore compared the expression and regulation of AMPs in fetal, neonatal, and adult keratinocytes (KCs) in vitro. The constitutive expression of human β-defensin-2 (HBD-2), HBD-3, S100 protein family members, and cathelicidin was significantly higher in KCs from fetal skin than in KCs from postnatal skin. The capacity to further increase AMP production was comparable between prenatal and postnatal KCs. Analysis of skin equivalents (SEs) revealed a strong constitutive expression of S100 proteins in fetal but not in neonatal and adult SEs. The elevated AMP levels correlated with reduced H3K27me3 (tri-methyl-lysine 27 on histone H3) levels and increased expression of the histone demethylase JMJD3. Knockdown of JMJD3 in fetal KCs elevated H3K27me3 levels and significantly downregulated the expression of HBD-3, S100A7, S100A8, S100A9, and cathelicidin. Our data indicate a crucial contribution of histone modifications in the regulation of AMP expression in the skin during ontogeny. The elevated AMP expression in prenatal skin might represent an essential defense strategy of the unborn.

Cornifelin (CNFN) has been identified as a protein component of epidermal corneocytes. Here, we investigated the tissue distribution of CNFN and potential consequences of CNFN deficiency on epithelial function in in vitro models of human skin and oral mucosa. Our detailed bioinformatics and immunostaining analysis revealed that CNFN is not only expressed in human epidermis but also in noncornifying oral mucosa. In normal epidermis, CNFN was confined to the upper granular layer and the stratum corneum. By contrast, in both partly cornifying and noncornifying oral mucosa, CNFN was expressed in a cell membrane-associated pattern over several suprabasal layers. Small interfering RNA-mediated knockdown of CNFN in epidermal keratinocytes (KCs) was associated with only subtle alterations of the overall epidermal architecture in skin models in vitro but led to altered morphology of corneodesmosomes, as detected by electron microscopy. Using dispase treatment followed by mechanical stress, epithelial sheets of CNFN-deficient epidermal KCs were easily disrupted, whereas their CNFN-competent counterparts remained intact. In contrast to the epidermal KCs, CNFN knockdown in oral KCs had a more severe effect and caused pronounced acantholysis in organotypic models of oral mucosa. Together, these findings indicate that CNFN is a structural component of the cell adhesion system of differentiated KCs in both epidermis and oral mucosa.

In normal skin, tightly connected keratinocytes in the stratum granulosum and terminally differentiated keratinocytes in the stratum corneum build an efficient barrier that inhibits extensive water loss while simultaneously preventing the entry of microbial pathogens and allergens into the skin (1). Dysregulation of keratinocyte differentiation together with pathologic changes in this natural barrier function is commonly associated with skin diseases such as atopic dermatitis, psoriasis, and ichthyosis vulgaris (2–4). Recently, loss-of-function mutations in the filaggrin gene have been identified (5, 6). These mutations not only impair the normal formation of the skin barrier, but also decrease the production of endogenous moisturizing molecules that lead to reduced stratum corneum hydration (4, 7). However, besides genetic defects of filaggrin production, other factors play a role in the pathogenesis of atopic dermatitis and the associated skin barrier defects as indicated by several observations: (i) The majority of individuals with atopic dermatitis do not exhibit null mutations in the filaggrin gene (8), (ii) individuals with atopic dermatitis without mutations in the filaggrin gene may also develop severe skin barrier defects (9), and (iii) a significant fraction of individuals carrying double-allele loss-of-function mutations in the filaggrin gene do not develop atopic dermatitis (4, 10). In line with this concept, it was shown recently that the pro-inflammatory cytokines interleukin-4 (IL-4), IL-31, and TNF-α compromise barrier function (11) and modulate the expression of differentiation-associated proteins in keratinocytes (12–14). Mast cells are present in normal skin, and increased numbers of mast cells are regularly observed in the skin of patients with atopic dermatitis even before the onset of inflammation (4, 15). Mast cells release a number of important signaling molecules, among which histamine has particularly potent pro-inflammatory activities (16). After mast cell degranulation, histamine concentrations within the tissue can rise to 10–1000 μM (17), and increased histamine levels have been reported for lesional and nonlesional skin of patients with atopic dermatitis (18). A role of endogenous histamine in the modulation of keratinocyte maturation has been suggested previously based on the observation that antihistamines have a beneficial effect on skin barrier recovery after tape striping in normal mouse skin (19). In the present study, we address the impact of histamine on the differentiation of human keratinocytes in different in vitro systems, among them a three-dimensional organotypic human skin model. This skin model has been shown previously to resemble native human skin, especially with regard to skin development and keratinocyte differentiation (20, 21). Our findings show that histamine prevents the expression of late differentiation antigens in keratinocytes and strongly decreases the expression of tight junction and desmosomal proteins, leading to the formation of a defective skin barrier.

The pathology of atopic dermatitis is orchestrated on the cellular level by several different cell types in the characteristic skin lesions. In such lesions, histamine as a mediator of many biological functions is also present in high concentrations. Most of the cells involved in the inflammatory responses express the histamine H1 and H2 receptors, but drugs targeting these receptors are not clinically effective. The discovery of the fourth histamine receptor, which is differentially expressed on immune and nonimmune cells, has shed new light on the actions of histamine in the complexity of atopic dermatitis. In this review, we describe a possible genetic impact on the expression level of the histamine H4 receptor and summarize the current data regarding the activity of the histamine H4 receptor on the key effector cells in atopic dermatitis. We do so in the context of whether the histamine H4 receptor offers a novel target for effective treatments of inflammatory skin diseases.

Skin inflammation and the migration of cells at the site of the immune response play an important role in allergic skin diseases. It has already been described that matrix metalloproteinase 9 (MMP-9) influences tissue remodeling and facilitates cell migration by proteolytic degradation of basal membrane components. The aim of this study was to investigate MMP-9 expression on human primary keratinocytes (KCs) upon stimulation with histamine, a potent mediator in allergic responses. With ELISA and zymography, we could show that histamine induced dose-dependent upregulation of MMP-9 in cultured KCs and in punch biopsies of human skin. The histamine H(1) receptor (H(1)R) agonist beta-histine-but not agonists for H(2)R, H(3)R, and H(4)R-induced MMP-9, whereas the H(1)R antagonist clemastine blocked the effect in a dose-dependent manner. Immunohistological staining showed that histamine-induced MMP-9 led to destruction of type IV collagen at the basement membrane in healthy skin. In a coculture system of KCs and T cells, migration of T cells through an artificial basement membrane was increased after histamine stimulation of KCs. Our findings demonstrate enhanced MMP-9 production and cell migration after histamine stimulation and may represent a new mechanism by which KCs contribute to the pathology of skin diseases.

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

Background Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. Objective Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. Methods The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. Results We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. Conclusion H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.

Histamine down-regulates IL-27 production in monocytes, and stimulation of keratinocytes with supernatants from histamine-treated monocytes down-regulates CXCL10 secretion. Histamine is a potent mediator in allergic inflammation with immunomodulatory properties. Since histamine was described to inhibit IL-12 production in human APCs, we hypothesized that also the expression of IL-27, a newly described member of the IL-12 family, which is present in inflammatory skin lesions, is modulated by histamine. Stimulation of human monocytes with histamine resulted in significant reduction of TLR ligand-induced IL-27 production in human monocytes. IL-27 subunits, p28 and EBI3, were down-regulated at the mRNA and protein level, whereas other cytokines, such as IL-6, IL-10, and TNF-α, were not influenced. Studies with histamine receptor-specific agonists and antagonists showed that the down-regulation of IL-27 was mediated via H2R and H4R but not H1R and H3R. Human KCs treated with supernatants of histamine-prestimulated monocytes induced significantly less CXCL10 than supernatants containing high levels of IL-27. DCs from H4R−/− mice responded to TLR simulation with higher IL-27 production as compared with WT mice. The down-regulation of IL-27 by histamine might be a new mechanism in the pathogenesis of inflammatory skin diseases, in particular, if increased concentrations of histamine are present at sites of inflammation, such as in chronic eczema and psoriasis.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.