Reconstructed human epidermis composed of keratinocytes, melanocytes and Langerhans cells

In addition to their basic biological interest, models of reconstructed epidermis provide useful tools for in vitro assessment of the toxicology and efficacy of new chemicals and drugs. The fact that the majority of these in vitro models are composed only of keratinocytes has excluded their use in the fields of skin pigmentation and immunology. After the successful introduction of functional melanocytes into the epidermal reconstruct, the integration of Langerhans cells remains an important challenge, particularly since after isolation of Langerhans cells from human epidermis, these cells cannot be subcultured and do not integrate into the reconstructing epidermis. The authors show that cord blood derived and CD34⁺ progenitors isolated from the peripheral blood give rise to residential Langerhans cells when co-seeded with normal human keratinocytes.     

Immunolocalization of protein C inhibitor in differentiation of human epidermal keratinocytes

Keratinocytes propagated in low calcium (0.05 mM) serum-free medium grow as monolayers and exhibit morphological and biosynthetic phenotypes similar to the keratinocytes of the basal layer in normal epidermis. When the calcium in the medium is increased to 1.5 mM, the keratinocytes start to stratify and differentiate. Such differentiation is important in the formation of an epidermal barrier. Proteolysis plays a crucial role in the process. The functions of most of the plasminogen activator cascade components in human skin have been studied, but little was known about the expression and role of protein C inhibitor in the differentiation of human epidermal keratinocytes. In the present study, we used immunohistochemistry and immunocytochemistry to examine the immunolocalization of protein C inhibitor in normal human skin and in cultured keratinocytes in serum-free medium with low and high calcium, respectively. The results indicated that protein C inhibitor is mainly localized in superficial and more differentiated keratinocytes in normal human epidermis. Keratinocytes positive for protein C inhibitor were detected in cultures containing both low and high calcium media, and the level of protein C inhibitor was increased in high calcium medium. This increase was accompanied by an altered intracellular distribution, from the perinuclear cytoplasm in undifferentiated keratinocytes to the whole cytoplasm in differentiated keratinocytes. Further study revealed that protein C inhibitor was incorporated into the cornified envelope in normal skin keratinocytes and cultured differentiated keratinocytes. Our results suggest that protein C inhibitor may be involved in the differentiation of keratinocytes.     

角质形成细胞对胞内金黄色葡萄球菌抗菌作用研究

目的:观察金黄色葡萄球菌在人角质形成细胞株HaCaT中生存的动态变化,了解金黄色葡萄球菌与皮肤角质形成细胞之间的相互关系。方法:用金黄色葡萄球菌标准株ATCC25923侵袭HaCaT细胞,分别于细菌进入细胞后的4、24、48、72h裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌。结果:金黄色葡萄球菌ATCC25923株在进入HaCaT细胞的24h有一定生长,但实验48h细胞内活菌数量明显减少。蛋白激酶C激活剂PMA和腺苷酸环化酶激活剂FSK可以促进HaCaT细胞清除胞内细菌。结论:皮肤角质形成细胞清除进入细胞内的金黄色葡萄球菌,可能是皮肤天然免疫的一种防御机制,而PMA和FSK增强细胞的抗菌作用,提示角质形成细胞抗菌活性与NADPH氧化酶相关。     

人IL-31的克隆表达及对表皮角化细胞的影响

目的克隆人IL-31基因,构建真核表达载体,研究人IL-31对表皮角化细胞HaCaT的影响及其作用机制。方法PMA、PHA刺激正常人外周血单个核细胞,提取细胞总RNA,采用RT-PCR克隆人IL-31基因,并将其克隆到真核表达载体pcDNA3.1/myc-His（-）A,进行PCR和双酶切鉴定,并进行序列测定。将重组质粒转染CHO细胞,用RT-PCR和Western blot分析rhIL-31在CHO细胞中的表达。用Ni^2＋树脂纯化his融合蛋白,用不同剂量rhIL-31刺激HaCaT细胞,利用Transwell穿孔板检测HaCaT细胞培养上清对外周血单个核细胞的趋化作用,荧光定量PCR检测rhIL-31对HaCaT表达趋化因子的影响,Western blot检测STAT3磷酸化。结果成功获得全长人IL-31基因,测序正确,经双酶切、PCR和序列测定鉴定,真核表达质粒构建正确,可在CHO细胞中表达;该目的蛋白刺激人表皮角化细胞HaCaT,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞表达趋化因子MDC、TARC、I-309;细胞STAT3磷酸化增加。结论IL-31作用于正常人表皮角化细胞,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞趋化因子表达增加,细胞STAT3磷酸化增加,提示IL-31可通过STAT3发挥作用。     

Cloning and characterization of retinol dehydrogenase transcripts expressed in human epidermal keratinocytes.

The normal growth and differentiation of the epidermis require an adequate supply of vitamin A. The active form of vitamin A for normal epidermal homeostasis is retinoic acid (RA). Retinoic acid controls the expression of retinoid-responsive genes via interactions of the retinoic acid/nuclear receptor complexes at specific DNA sequences in their control regions. The message conveyed by RA is likely modulated by the concentration of the ligand available for binding to the receptors. Following the uptake of plasma retinol, epidermal keratinocytes synthesize retinoic acid via two sequential reactions with retinaldehyde as an intermediate. Several retinol dehydrogenase (RDH) enzymes, members of the short-chain dehydrogenase/reductase (SDR) gene superfamily, catalyze the first and rate-limiting step that generates retinaldehyde from retinol bound to cellular retinol-binding protein (holo-CRBP). However, little is known about these enzymes and their genes in the epidermal cells. Our work describes the first member of the RDH family found in epidermis. We show that this gene is expressed predominantly in the differentiating spinous layers and that it is under positive, feed-forward regulation by retinoic acid. It encodes a protein that, using NAD+ as a preferred cofactor, utilizes free and CRBP-bound all-trans-retinol and steroids as substrates.     

Common genes responsible for differentiation and senescence of human mucosal and epidermal keratinocytes

Serial subcultures of normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs) to the postmitotic stage result in terminal differentiation and replicative senescence. In order to investigate the common mode of differentiation and/or senescence between mucosal and epidermal keratinocytes, gene expression profiling on both NHOKs and NHEKs was performed by a cDNA microarray analysis. Primary NHOKs and NHEKs were serially subcultured, and the expression level of 3,063 genes was compared between the exponentially growing and senescent cultures. The senescent NHOKs and NHEKs highly expressed 55 and 37 genes, respectively. Among these genes, 16 genes were common in both NHOKs and NHEKs while the other genes were upregulated either in the NHOKs or in the NHEKs. Furthermore, the expression levels of the common genes did not change in the human diploid fibroblasts during the subcultures. These results suggest that subculture-induced differentiation and/or replicative senescence in NHOKs and NHEKs has similar characteristics, but that the pathways leading to these processes are distinct and keratinocyte specific.     

Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes.

We established an in vitro adherence model with primarily cultured human keratinocytes as target cells which allows for the investigation of the molecular mechanisms that are responsible for Candida albicans host cell attachment in the initiation of cutaneous candidosis. The extent of C. albicans binding to cultured human keratinocytes was dependent on the yeast inoculum size and the incubation temperature. Heat and paraform-aldehyde treatment of yeasts completely abolished the binding activity of C. albicans. Of the different Candida species tested, C. albicans was by far the most adhesive species. C. albicans adherence was blocked by the acid protease inhibitor pepstatin A and the metabolic inhibitor sodium azide. The latter, however, was much less effective when yeasts were preincubated, suggesting that sodium azide was mainly acting on the keratinocytes. The extracellular matrix protein fibronectin was slightly inhibitory, whereas the fibronectin-derived peptides RGD and RGDS were not able to prevent attachment. PepTite-2000, another RGD-containing synthetic peptide, reduced C. albicans adherence by a margin of 25% (P < 0.005). CDPGYIGSR-NH2, which is a synthetic adhesive peptide derived from the laminin B chain, was much more efficient in its inhibitory activity than the RGD peptides and reduced C. albicans adherence to cultured human keratinocytes up to 76% (P < 0.001). Laminin itself and the synthetic pentapeptide YIGSR were less active. A dose-dependent reduction in adherence was also observed with collagen type III. Additionally, saccharides were tested for their potential to inhibit C. albicans attachment to keratinocytes. The most potent competitive saccharide inhibitors of C. albicans adherence to human keratinocytes were the amino sugars D-(+)-glucosamine and D-(+)-galactosamine with one isolate of C. albicans (4918) and D-(+)-glucosamine and alpha-D-(+)-fucose with another C. albicans isolate (Sp-1). Collectively, our data suggest the existence of multiple molecular mechanisms such as protein-protein, lectin-carbohydrate, and yeast-yeast coaggregational interactions that are responsible for optimal C. albicans attachment to cultured human keratinocytes.     

A chemically defined surface for the co-culture of melanocytes and keratinocytes

Patients with stable vitiligo can be helped surgically using transplantation of autologous cultured melanocytes, but there is a need for a culture methodology that is free from xenobiotic agents and for a simple way of delivering cultured melanocytes to the patient to achieve pigmentation with good wound healing. The aim of this study was to develop a chemically defined surface, suitable for the co-culture of melanocytes and keratinocytes which could be used in the future for the treatment vitiligo patients to achieve both restoration of pigmentation and good wound healing. Two keratinocyte growth media and two melanocyte growth media were compared; two of these were serum free. Cells were seeded on a range of chemically defined substrates (produced by plasma polymerisation of acrylic acid, allylamine or a mixture of these monomers) either as mono- or co-cultures. Melanocytes and keratinocytes attached and proliferated on both acid and amine substrates (without significant preferences), and co-cultures of cells proliferated more successfully than individual cultures. One media, M2, which is serum free, supported expansion of melanocytes and to a lesser extent keratinocytes on several plasma polymer substrates. In conclusion, these data indicate that a combination of a chemically defined substrate with M2 media allows serum-free co-culture of melanocytes and keratinocytes.     

异欧前胡素抑制人表皮黑素细胞c-Kit蛋白的表达

目的探索异欧前胡素对体外人表皮黑素细胞c-kit蛋白表达的作用。方法体外分离、纯化培养人表皮黑素细胞,随机分为1组(对照组)、2组(10 nmol/L SCF组)、3组(25μmol/L异欧前胡素组)和4组(25μmol/L异欧前胡素+10nmol/L SCF组),各组黑素细胞按设定条件培养48 h,倒置显微镜观察黑素细胞的形态,免疫荧光显微镜观察黑素细胞的干细胞因子受体c-Kit蛋白表达和分布,NaOH裂解法测定黑素细胞黑素的含量。结果与1组比较,2组黑素细胞胞体增大,3组黑素细胞胞体变小,树状突起变长,4组黑素细胞胞体小于2组。免疫荧光显微镜观察可见1组黑素细胞c-kit蛋白表达呈红色荧光,分布于胞膜,2组黑素细胞c-Kit蛋白由于SCF诱导而表达增强,3组、4组黑素细胞c-Kit蛋白因异欧前胡素的抑制而表达减弱,各组黑素细胞c-Kit蛋白表达的结果与1组比较,差异有统计学意义(P0.01)。黑素含量检测结果表明,异欧前胡素对SCF诱导的黑素细胞黑素的合成有抑制作用,2组、3组、4组与1组之间黑素含量的差异、4组与2组之间黑素含量的差异均具有统计学意义(P0.05,P0.01)。结论 25μmol/L异欧前胡素可抑制体外培养人表皮黑素细胞c-Kit蛋白的表达,调节SCF/c-Kit信号传导而降低黑素细胞的黑素含量。     

Testosterone and UVL-B stimulation of epidermal melanocytes in rat scrotal skin

The effects of UVL-B and/or testosterone replacement therapy are compared in normal and castrated rats in order to determine whether testosterone is required for UVL-B (280-315 nm) stimulation of melanogenesis in the testosterone-dependent epidermal melanocyte system of the scrotal skin of black Long Evans rats. Testosterone is not a prerequisite for UVL-B stimulation of melanocytes as in both castrates and normal animals the melanocytes respond to UVL-B by increases in size, length and number of dendrites (dendriticness), and tyrosinase activity (intensity of Dopa reaction). Addition of testosterone to castrates does enhance the effects of UVL-B. However, UVL-B with or without testosterone cannot maintain normal melanogenesis in rats irradiated immediately after castration nor can it restore normal melanogenesis following long term castration. Both the amount of UVL energy/exposure and the number of exposures are important variables in stimulation of the epidermal melanocytes. Administration of a dose of UVL-B to castrates in a single exposure is ineffective, while the same overall dose spread over several exposures increases the size and dendriticness of melanocytes. Testosterone and UVL-B act synergistically in affecting melanogenesis although neither singly nor in combination are they able to fully restore normal melanogenesis.     

Expression of HLA-DR (Ia like) antigen on epidermal keratinocytes in human dermatoses

Ia antigen (HLA-DR in man) has been demonstrated in keratinocytes in graft versus host disease. This study investigates the occurrence of HLA-DR in keratinocytes in the following dermatoses: eczematous dermatitis, discoid lupus erythematosus, with immunoglobulin and non-exposed skin from cases of systemic lupus erythematosus with immunoglobulin deposits, lichen planus, lichen simplex, bullous pemphigoid, pemphigus vulgaris, 'toxic erthema', tuberculid and chillblain. Keratinocyte staining was found in a variety of conditions. The unifying features of the instances of its occurrence was lymphoid infiltration and usually some focal evidence of keratinocyte damage. Thus in eczema the staining was mid-epidermal, while in discoid lupus erythematosus and lichen planus it was basal. HLA-DR staining was absent in bullous pemphigoid and pemphigus vulgaris, which is consistent with the hypothesis that in these conditions the damage is mediated by autoantibodies and complement in the absence of cellular immune attack.     

Plasma-polymerized surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model

The aim of this study was to develop plasma-polymerized surfaces suitable for the attachment and culture of human keratinocytes and that would allow their subsequent transfer to a wound-bed model. Keratinocyte attachment has been assessed on a carrier polymer, either untreated or treated with a hydrocarbon plasma polymer, collagen I, or carboxylic-acid-containing plasma copolymers. Cell attachment was poor on the "bare" carrier polymer and hydrocarbon plasma polymer (PP) surfaces. Cell attachment was good and comparable on collagen I-coated carrier polymer and carrier polymer plasma coated with carboxylic acid functionalities. After 24 h of cell culture, surfaces were inverted so that cells were adjacent to a de-epidermalized dermis (DED) for 4 days. After 4 days in contact with DED, the surfaces were removed and the level of residual cells and cells transferred to DED were assessed using a cell viability assay. Cell transfer from the collagen I-coated surface was on the order of 90%. Transfer from the carrier polymer surface and the hydrocarbon-coated surface was poor while cells cultured on acid-containing surfaces showed high levels of transfer. Cell transfer was greatest from those surfaces containing the highest level of acid functionality (ca. 21%). Cell transfer was not significantly affected by the choice of carrier polymer material although some sample-to-sample variation was seen. To determine that plasma-polymerized surfaces could be used clinically, selected samples were sterilized with ethylene oxide. Subsequent analysis and cell culture indicated that the surface chemistry and cell-transfer capability of these plasma-polymerized surfaces were unaffected by the sterilization procedure. Plasma-polymerized carboxylic-acid-containing surfaces show great promise in the field of wound healing, encouraging keratinocyte attachment and permitting keratinocyte transfer to a wound bed.     

MicroRNA-205对人皮肤上皮细胞迁移能力的调控

目的 观察microRNA-205 (miR-205)在人皮肤上皮细胞中的表达抑制和过量表达对于该类细胞迁移能力的影响。 方法 使用体外合成的miR-205抑制剂（antagomir-205）处理人皮肤上皮细胞并检测抑制效果,观察其对皮肤上皮细胞迁移的影响。在293TN细胞中包装并收获能过表达成熟miR-205的重组慢病毒颗粒,感染人皮肤上皮细胞并检测过表达效果,进一步观察其对皮肤细胞迁移的影响。 结果 成功使用miR-205抑制剂实现对人皮肤上皮细胞内源性miR-205的抑制,证实其抑制了皮肤上皮细胞的迁移。成功获得过表达miR-205的重组慢病毒颗粒,证明miR-205可促进皮肤上皮细胞的迁移。 结论 miR-205可以促进皮肤细胞的迁移。     

Cultivation,frozen storage,and clonal growth of normal human epidermal keratinocytes in serum-free media

Summary Methods are described for serum-free culture of human epidermal keratinocytes derived from neonatal foreskin tissue. Cultures are initiated, stored frozen, and returned to active growth, all with bovine pituitary extract as the only undefined supplement. Clonal growth assays are then performed in a biochemically defined medium. The degree of stratification and differentiation in the defined medium (and also with pituitary extract) is controlled by the extracellular calcium ion concentration.     

Structure of a collagen-GAG dermal skin substitute optimized for cultured human epidermal keratinocytes

Collagen and glycosaminoglycan (GAG) dermal skin substitutes (membranes) were studied as substrates for cultured human epidermal keratinocytes. Structure of dermal substitutes was optimized for pore size to promote ingrowth of fibrovascular tissue from the wound bed and for culture of human keratinocytes of the membrane's surface. Pore size of the freeze-dried material was regulated by control of the temperature of freezing between -50 degrees C and -20 degrees C and by concentration of starting materials between 0.17% and 1.62% wt/vol. A nonporous surface of collagen-GAG was laminated to the membranes to provide a planar substrate for cultured epidermal keratinocytes. Thickness of dermal substitutes was regulated by control of the volume and concentration of starting materials. Biotin was conjugated to solubilized collagen for binding with avidin of specific quantities of biologically active molecules. The optimized membranes are suitable substrates for the culture of human epidermal keratinocytes, and together with the cells yield a composite material that is histologically similar to skin.     

The role of Langerhans cells and keratinocytes in epidermal immunity

The immunology of the epidermis has received considerable study over recent years. After the antigen-presenting capacity of epidermal Langerhans cells was confirmed, subsequent studies suggested that keratinocytes could modulate certain immunologic events through production of a cytokine, epidermal cell-derived thymocyte-activating factor (ETAF). Most recently, a murine epidermal cell population, the dendritic Thy-1-positive cell, has been shown to possess natural killer-cell-like activity. In this review, the biology of these cell types are discussed. A discussion of allergic contact hypersensitivity and its alteration by ultraviolet light is used to illustrate some of the complex control mechanisms that continue to be the subject of ongoing study.     

Induction of hyaluronan cables and monocyte adherence in epidermal keratinocytes

Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.     

Interleukin-8 and tumor necrosis factor alpha production in human epidermal keratinocytes induced by Trichophyton mentagrophytes

Production of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) was confirmed by enzyme-linked immunosorbent assay in a medium where human epidermal keratinocytes were cocultured with Trichophyton mentagrophytes for 1 to 12 h. IL-8 and TNF-alpha mRNAs were also detected in the keratinocytes cocultured with T. mentagrophytes.