Normal Adult Human Keratinocytes Research Articles

EPR Technology as Sensitive Method for Oxidative Stress Detection in Primary and Secondary Keratinocytes Induced by Two Selected Nanoparticles.

Exogenous factors can cause an imbalance in the redox state of biological systems, promoting the development of oxidative stress, especially reactive oxygen species (ROS). To monitor the intensity of ROS production in secondary keratinocytes (HaCaT) by diesel exhaust particles and thermoresponsive nanogels (tNG), electron paramagnetic resonance (EPR) spectroscopy after 1 and 24 h of incubation, respectively, was applied. Their cytotoxicity was analyzed by a cell viability assay (XTT). For tNG an increase in the cell viability and ROS production of 10% was visible after 24 h, whereas 1 h showed no effect. A ten times lower concentration of diesel exhaust particles exhibited no significant toxic effects on HaCaT cells for both incubation times, thus normal adult human keratinocytes (NHK) were additionally analyzed by XTT and EPR spectroscopy. Here, after 24 h a slight increase of 18% in metabolic activity was observed. However, this effect could not be explained by the ROS formation. A slight increase in the ROS production was only visible after 1 h of incubation time for HaCaT (9%) and NHK (14%).

Confluence-Induced Squamous Differentiation Is Not Accompanied by Changes in H3K27me3 Repressive Epigenetic Mark

Recent studies have reported that epigenetic mechanisms may regulate the initiation and progress of squamous differentiation in normal and transformed keratinocytes. In particular, the role of the repressive H3K27me3 mark in the regulation of squamous differentiation has been prominent. However, there is conflicting literature showing that squamous differentiation may be dependent upon or independent of changes in H3K27me3 status. In this study we have examined the binding of trimethylated H3K27 to the promoters of proliferation or differentiation genes in keratinocytes undergoing squamous differentiation in vitro and in vivo. Initially, we examined the expression levels for EZH1, EZH2, and H3K27me3 in differentiating keratinocytes in vitro and in vivo. We extended this to include H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq). Based on these studies, we could find no evidence for an association between widespread gain or loss of H3K27me3 on the promoters of proliferation-specific or differentiation-specific target genes, respectively, during squamous differentiation in adult human keratinocytes. These data suggest that squamous differentiation may occur independent of regulation by H3K27me3 on proliferation and differentiation genes of normal adult human keratinocytes.

Chemo-Enzymatic Synthesis of Silybin and 2,3-Dehydrosilybin Dimers

Divalent or multivalent molecules often show enhanced biological activity relative to the simple monomeric units. Here we present enzymatically and chemically prepared dimers of the flavonolignans silybin and 2,3-dehydrosilybin. Their electrochemical behavior was studied by in situ and ex situ square wave voltammetry. The oxidation of monomers and dimers was similar, but adsorption onto the electrode and cell surfaces was different. A 1,1-diphenyl-2-picrylhydrazyl (DPPH) and an inhibition of microsomal lipoperoxidation assay were performed with same trend of results for silybin and 2,3-dehydrosilybin dimers. Silybin dimer showed better activity than the monomer, while on the contrary 2,3-dehydrosilybin dimer presented weaker antioxidant/antilipoperoxidant activity than its monomer. Cytotoxicity was evaluated on human umbilical vein endothelial cells, normal human adult keratinocytes, mouse fibroblasts (BALB/c 3T3) and human liver hepatocellular carcinoma cell line (HepG2). Silybin dimer was more cytotoxic than the parent compound and in the case of 2,3-dehydrosilybin its dimer showed weaker cytotoxicity than the monomer.

A new in vitro protocol for assessing phototoxic effects of xenobiotics on human keratinocytes

Routine protocols of phototoxicity tests are based on cultured mouse fibroblasts, mainly because these cells are robust and easy to culture in vitro. However, in a real-life situation, phototoxic reactions take place primarily in the epidermis, comprised of keratinocytes – cells which differ substantially from fibroblasts with regard to structure and function. Therefore, keratinocyte cultures seem more appropriate for the phototoxicity testing of xenobiotics, such as cosmetic ingredients or drugs. Aim: To design and implement a test protocol for in vitro assessment of phototoxic properties of xenobiotics in normal human keratinocytes. Material and methods: As a starting point, we applied the EU-approved protocol for testing phototoxicity in mouse fibroblast cultures (3T3 Neutral Red Uptake Phototoxicity Assay, DB-ALM No. 78). The protocol was modified and adjusted in a series of experiments to the specific demands of cultured normal human keratinocytes. After obtaining a stable growth of keratinocytes in microcultures, the cells were exposed for 1 hour to model agents with phototoxic properties known from clinical observations: chlorpromazine, 8-methoxypsoralen, chloroquine, promethazine, etofenamate, ketoprofen, doxycycline, lymecycline, and isotretinoin in a series of concentrations of 0, 1, 3, 11, 33, and 100 μg/ml. Subsequently, the cultures were exposed to the cumulative dose of 5 J/cm2 of artificial sunlight using the EU-recommended solar simulator. The survival of keratinocytes was assessed by their uptake of neutral red (NR) dye. Results: Using the proposed test protocol, we were able to achieve stable growth of normal adult human keratinocytes in vitro. In the absence of phototoxic agents, no effects of light on cell viability were noticeable up to the dose of 10 J/cm2. The proposed system was capable of demonstrating phototoxicity of model phototoxic xenobiotics selected for the tests, which was in line with the clinical experience regarding phototoxic effects of these agents in humans. Conclusions: We have developed an in vitro protocol for assessment of the phototoxic potential of xenobiotics in normal human keratinocytes. Its functionality and reliability has been confirmed by tests results with known phototoxic agents. Although more difficult to culture than mouse fibroblasts, and therefore neglected in routine phototoxicity testing, human keratinocytes seem more appropriate for predicting in vitro phototoxic effects of xenobiotics in human skin, as phototoxic processes predominantly involve the epidermis which consists of keratinocytes.

Type 2 Helper T-Cell Cytokines Induce Morphologic and Molecular Characteristics of Atopic Dermatitis in Human Skin Equivalent

Both the immune system and the epidermis likely have an important role in the pathogenesis of atopic dermatitis (AD). The objective of the present study was to develop a human skin equivalent model exhibiting morphologic and molecular characteristics of AD in a controlled manner. Skin equivalents generated from normal adult human keratinocytes were stimulated with type 2 T-helper cell (Th2) cytokines IL-4 and IL-13, and morphologic features and gene expression of the epidermis were studied. Th2 cytokines induced intercellular edema similar to spongiotic changes observed in lesional AD as assessed at histopathologic analysis and electron microscopy. Furthermore, genes known to be specifically expressed in epidermis of patients with AD such as CAII and NELL2 were induced. In contrast, expression of psoriasis-associated genes such as elafin and hBD2 was not changed. Th2 cytokines caused DNA fragmentation in the keratinocytes, which could be inhibited by the caspase inhibitor Z-VAD, which suggests that apoptosis was induced. In addition, up-regulation of the death receptor Fas was observed in keratinocytes after Th2 cytokine stimulation. IL-4 and IL-13 induced phosphorylation of the signaling molecule STAT6. It was concluded that the skin equivalent model described herein may be useful in investigation of the epidermal aspects of AD and for study of drugs that act at the level of keratinocyte biology.

Development and Validation of Human Psoriatic Skin Equivalents

Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the keratinocytes or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult keratinocytes after air exposure and stimulation by keratinocyte growth factor and epidermal growth factor displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-2, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin keratinocytes were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult keratinocytes were stimulated with psoriasis-associated cytokines [tumor necrosis factor-alpha, interleukin (IL)-1alpha, IL-6, and IL-22] or combinations thereof, strong expression of hBD-2, SKALP/elafin, CK16, IL-8, and tumor necrosis factor-alpha was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.

Retinoid regulation of heparin-binding EGF-like growth factor gene expression in human keratinocytes and skin.

Retinoic acid (RA) has profound effects on epidermal homeostasis; however, the molecular mechanisms by which retinoids regulate keratinocyte cell proliferation and differentiation are not well understood. Here we report that mRNA expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family of growth factors, is induced by RA in human keratinocytes and skin, and is overexpressed in the context of epidermal hyperplasia in vivo. Treatment of normal adult human keratinocytes with micromolar concentrations of RA significantly induced the expression of HB-EGF. The response was efficiently blocked by specific inhibitors of ErbB tyrosine kinase activity, MAP kinase kinase (MEK), or p38 stress-activated protein kinase. RA also enhanced the induction of HB-EGF mRNA in human skin organ culture, an ex vivo model system displaying many similarities to wound healing in vivo. HB-EGF transcripts were markedly increased in human skin by topical treatment with RA under conditions known to provoke epidermal hyperplasia. HB-EGF transcripts were also markedly overexpressed in the hyperplastic epidermis of psoriatic lesions, relative to normal skin. These results support the hypothesis that the effects of RA on epidermal hyperplasia are mediated at least in part by HB-EGF, and suggest that signal transduction mechanisms other than or in addition to nuclear RA receptors contribute to this effect.

Camptothecin induces differentiation, tissue transglutaminase and apoptosis in cultured keratinocytes.

Cultured normal human adult keratinocytes were exposed to (S)-(+)- camptothecin over the concentration range 10(-5) to 10(-10) M. The dose-dependent inhibition of growth was recorded using cell counting. The induction of terminal differentiation was demonstrated by the relative increase in squamous and cornified cells, and the concomitant decrease in small, proliferative cells, with an overall decrease in total cell numbers on going from 10(-10) to 10(-6) M concentration of the drug. The induction of apoptosis was studied by assay of two types of transglutaminase, "tissue" and "keratinocyte", and by assay of histone-linked mono- and oligonucleosomes. Induction of apoptosis was accompanied with increase in "tissue" transglutaminase and in the amount of nucleosomes, the latter being indicative of endonuclease activity. This activity was sharply increased at a camptothecin concentration of 10(-5) M, and may have been facilitated by "tissue" transglutaminase at lower concentrations. The data suggest that camptothecin restricts keratinocyte growth by several mechanisms including apoptosis and emphasize its possible use in topical therapy for psoriasis.

Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model

Composites of human de-epidermised acellular dermis and normal adult human keratinocytes and fibroblasts were examined for the ability of cells to contract these composites. Image analysis of the outline of the composites showed that, in this model, keratinocytes alone or in the presence of fibroblasts caused highly significant contraction (of the order of 25% by day. 12). There was no significant contraction of the dermis with fibroblasts alone or in the absence of cells. The presence or absence of basement membrane antigens did not influence the effect of keratinocytes on dermal contraction. Analysis of the conditioned media from these composites showed that the greatest fibronectin production was seen with fibroblasts alone in the presence of basement membrane. Keratinocytes alone produced little fibronectin irrespective of the presence of the basement membrane. If keratinocytes were present with fibroblasts, however, then fibronectin production was significantly reduced both in the presence and absence of the basement membrane, indicating that keratinocytes modify dermal fibroblast extracellular matrix production. This study shows that while keratinocytes and fibroblasts are clearly influencing each other's activity in this human skin composite model, under the circumstances we describe it is the keratinocyte and not the fibroblast which causes contraction of the human de-epidermised acellular dermis.

Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses.

Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes.

Cultured keratinocytes are frequently employed for studies of epidermal lipid metabolism. Interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime and variations between passages, problems that are not encountered to the same extent with immortalized cell lines. The present study was undertaken to compare the lipid composition and synthesis of normal human adult keratinocytes (NHAK) with HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line, in relation to proliferation and differentiation. No differences between the two cell types were observed: a) in total lipid content; b) in the distribution of major lipid classes during growth at 50%, 75% and 100% confluence; c) in cultures grown at 0.6 mM calcium, at which differentiation is retarded, or at 1.6 mM calcium, at which some differentiation takes place; d) in the incorporation of [14C] acetate into cellular lipids at confluence, or e) in the fatty acid composition of major cellular lipid classes. At 100% confluence NHAK and HaCaT cells differ in their cholesterol metabolism. At all stages of growth, cholesterol synthesis in HaCaT cells is more LDL-dependent than in NHAK. Furthermore, NHAK become less LDL-dependent at confluence whereas HaCaT cells do not. HaCaT cells also revealed a significantly larger fraction of phosphatidyl-ethanolamine, -serine and -inositol at 0.6 mM calcium concentration than NHAK. These findings suggest that HaCaT cells do not differentiate as well as NHAK in vitro and may therefore serve as a model for the study of lipid metabolism in cells defective in terminal differentiation.

Release of inflammatory mediators in human keratinocyte cultures following exposure to a skin irritant

The most common pathological response of the skin, associated with chemical-induced primary irritation, is inflammation. Release of inflammatory mediators occurs in primary irritant dermatitis in both laboratory animals and humans. Inflammatory mediators (prostaglandin E 2, leukotrienes C 4, D 4, E 4 and 15-hydroxyeicosatetranoic acid) were determined by radioimmunoassay, and leukotriene B 4 and the cytokine, interleukin 1α, were measured by enzyme-linked immunosorbent assay, in cultures of normal adult human keratinocytes. Release of inflammatory mediators into the culture medium was assessed at various time points following treatment of the cultures with the anionic surfactant sodium dodecyl sulphate (SDS), a known human skin irritant. A cytotoxic response was confirmed by the neutral red uptake assay after a 24-hr exposure to SDS. A dose-related release of inflammatory mediators was observed. The magnitude of the response varied between different mediators and as a function of time. The results show that release of inflammatory mediators occurs in human epidermal keratinocyte cultures, following chemical insult. Large inter-experimental variations in release of the various mediators probably preclude their use in a routine assay to determine irritation potential. In the case of SDS, release of inflammatory mediators is not a sensitive indicator of cytotoxicity. However, such techniques may provide important mechanistic data on the role of inflammatory mediators in the irritative response to certain chemicals, or on the possible role and interaction of certain mediators in the irritative process.

Insulin-Like Growth Factors are Mitogenic for Human Keratinocytes and a Squamous Cell Carcinoma

Normal adult human keratinocytes in monolayer culture and SCL-1, a skin-derived squamous-cell carcinoma cell line, were investigated for the expression of receptors for insulin-like growth factors (IGF) and insulin. As demonstrated by affinity crosslinking, radiolabeled IGF-1, IGF-2, and insulin bound specifically to both cell types. Each cell expressed type I IGF receptors, with affinity for IGF-1 greater than IGF-2 much greater than insulin. Insulin receptors, with highest affinity for insulin, were also present on both cells. However, keratinocytes and SCL-1 cells differed in 125I-IGF-2 binding. 125I-IGF-2-bound to both type I and type II IGF receptors in normal keratinocytes, but bound predominantly to membrane-associated IGF binding proteins in SCL-1. IGF-1 was slightly more potent than IGF-2 in stimulating growth of both keratinocytes and SCL-1 cells. In keratinocytes, concentrations of IGF-1 ranging from 5-100 ng/ml, and of IGF-2 from 50-100 ng/ml, resulted in a significant increase in cell number. At the maximum dose of 100 ng/ml, either IGF-1 or IGF-2 caused a 2.3-times increase in cell number. In SCL-1 cells, IGF-1 was more potent than IGF-2 or insulin at lower concentrations, but either IGF-1 or IGF-2 at the maximal concentration of 333 ng/ml stimulated a 4.7-times increase in thymidine incorporation. The stimulatory effect of insulin in SCL-1 was 10-50 times less potent than that of the IGF. The effect of either IGF on SCL-1 was completely inhibited by the type I IGF receptor antibody alpha IR-3, suggesting that both IGFs are mitogenic through the type I IGF receptor. Insulin action was partially blocked by alpha IR-3, suggesting that insulin can act through both the insulin and type I IGF receptors. It thus appears that IGF-1 and IGF-2 are mitogens for normal and transformed human keratinocytes and that their actions are primarily mediated through the type I IGF receptor, whereas insulin is a mitogen through both the IGF-1 receptor and the insulin receptor.

Detection of chemical-induced unscheduled DNA synthesis in cultures of normal adult human keratinocytes

Human keratinocytes are the most appropriate target cells to employ in an in vitro model to study the potential ability of a chemical to react with the genome of human skin cells. The resulting damage to DNA may be a key initiating factor in the subsequent development of squamous epithelial carcinoma, which is a common form of cancer in humans. Keratinocytes were routinely established from adult human skin samples and maintained in culture for at least three passages. Genotoxic damage was assessed by a quantitative autoradiographic technique which enables detection of unscheduled DNA synthesis without prior treatment of the cultures with agents, such as hydroxyurea, to suppress S-phase synthesis. Tertiary cultures from two individuals were exposed to the experimental carcinogens, methyl methane sulphonate (direct-acting genotoxin) and benzo[ a]pyrene (requiring metabolic activation). Both chemicals induced unscheduled DNA synthesis in human keratinocytes. This study indicates that human keratinocyte cultures are a suitable model for the detection of genotoxic potential in human epidermis.