Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone

Type VII collagen is synthesized and secreted by both human keratinocytes and fibroblasts. Although both cell types can secrete type VII collagen, it is thought that keratinocytes account for type VII collagen at the dermal-epidermal junction (DEJ). In this study, we examined if type VII collagen secreted solely by dermal fibroblasts could be transported to the DEJ. We established organotypic, skin-equivalent cultures composed of keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB) and normal dermal fibroblasts. Immuno-labeling of skin equivalent sections with the anti-type VII collagen antibody revealed tight linear staining at the DEJ. RDEB fibroblasts, were gene-corrected to make type VII collagen and used to regenerate human skin on immune-deficient mice. The human skin generated by gene-corrected RDEB fibroblasts or normal human fibroblasts combined with RDEB keratinocytes restored type VII collagen expression at the DEJ in vivo. Further, intradermal injection of normal human or gene-corrected RDEB fibroblasts into mouse skin resulted in the stable expression of human type VII collagen at the mouse DEJ. These data demonstrate that human dermal fibroblasts alone are capable of producing type VII collagen at the DEJ, and it is possible to restore type VII collagen gene expression in RDEB skin in vivo by direct intradermal injection of fibroblasts.     

Heat shock proteins in cultured human keratinocytes and fibroblasts

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.     

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.     

Immunohistochemical Localization of Nitric Oxide Synthase in Normal Human Skin: Expression of Endothelial-type and Inducible-type Nitric Oxide Synthase in Keratinocytes

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.     

Detection of melanocortin-1 receptor antigenicity on human skin cells in culture and in situ

The proopiomelanocortin (POMC) products alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and In situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvascular endothelial cells and WM35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of sweat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated keratinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.     

白介素1α及白介素6对角朊细胞与真皮成纤维细胞相互作用的影响

为了探讨细胞因子在表皮与真皮相互作用中所起的作用,用体外细胞培养的方法研究了白介素1α(IL-1α)及白介素6(IL-6)对角朊细胞与真皮成纤维细胞之间相互作用的影响以及前列腺素E₁(PGE₁)的调节作用.IL-1α主要由角朊细胞产生,而IL-6则主要由成纤维细胞产生.来自成纤维细胞的培养上清液有较强的促进角朊细胞增殖能力.来自角朊细胞的培养上清液也有一定促进成纤维细胞增殖能力.IL-1α和PGE₁可促进成纤维细胞IL-6的产生.抗IL-6抗体可抑制成纤维细胞培养上清液对角朊细胞的促生长活性.以上结果说明IL-1α和IL-6可能是介导表皮-真皮相互作用的重要因子.     

The cellular origins of the linear IgA disease target antigens: an indirect immunofluorescence study using cultured human keratinocytes and fibroblasts

BACKGROUND: Linear IgA disease (LAD) is an IgA-mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology. Objectives To investigate to what extent the cellular origins of the target antigens account for the heterogeneity of the immune response in LAD. METHODS: Forty-nine adult and 33 childhood LAD sera were studied. Immunofluorescence was carried out to determine the expression of the LAD antigens by normal human keratinocytes, fibroblasts and mixed cultures of keratinocytes and fibroblasts. Immunoblotting was performed to determine the localization of the LAD target antigens in tissue extracts (48 adult and 31 childhood sera) and cell extracts (21 adult and 10 childhood sera). RESULTS: Thirty-one adult and 13 childhood LAD sera bound proteins expressed by human keratinocytes; of these sera, 15 adult and four childhood LAD sera also recognized proteins expressed by fibroblasts. A single adult serum was positive on fibroblasts alone. Seventeen adult and 20 childhood sera were negative on both cell types. There was a modest increase (9%) in the detection of the IgA autoantibodies on keratinocytes and fibroblasts grown together in mixed culture. Immunoblotting showed that the LAD target antigens could be detected in cell as well as in tissue extracts. CONCLUSIONS: Our results have shown that normal human keratinocytes and fibroblasts in culture express the LAD target antigens. LAD sera (with a single exception) bound antigens expressed by keratinocytes alone or by both keratinocytes and fibroblasts. The principal pattern of expression in keratinocytes was cytoplasmic, similar to that demonstrated by polyclonal antibodies to the 180-kDa bullous pemphigoid antigen (BP180). This reflects the pivotal role of BP180 in LAD. The finding that LAD antigens are expressed by both human keratinocytes and fibroblasts in culture may explain the heterogeneity of the target antigens, and may be a contributory factor in the immunopathology of the disease.     

Comparative caustic and biological activity of trichloroacetic and glycolic acids on keratinocytes and fibroblasts in vitro

OBJECTIVES: Beside their causticity, the biological mechanism by which trichloroacetic acid (TCA) and glycolic acid (GA), two agents extensively used for chemical peeling, might act remains unknown. The purpose of this study was to examine in vitro the effect of TCA and GA on human keratinocytes and the influence of the released epithelial mediators on collagen and matrix metalloproteinases (MMPs) production by human dermal fibroblasts. METHOD: Cultured keratinocytes were treated by TCA and GA at 10 mg/ml brought to pH 3, 5 and 7, and the conditioned media neutralized to pH 7 were added to human normal skin fibroblasts. RESULTS: TCA was cytotoxic for keratinocytes at each tested pH. The conditioned medium depressed protein and collagen synthesis and the expression of MMPs when added to fibroblasts as did also TCA when added directly to fibroblasts. GA was not cytotoxic for keratinocytes at neutral pH and the conditioned medium obtained at each pH applied to fibroblasts did not alter protein, collagen nor MMPs production while causing an elevated secretion of IL-6. CONCLUSION: TCA exerts a toxic effect on keratinocytes and fibroblasts while GA does not alter the metabolism of fibroblasts but induces the secretion of IL-6.     

Keratinocyte conditioned medium stimulates type IV collagenase synthesis in cultured human keratinocytes and fibroblasts

We have previously shown that conditioned medium from cultured human keratinocytes stimulates proliferation of a variety of cell types involved in wound healing. as well as re-epithelization of wounds in human skin in vitro. We now present evidence for an autocrine/paracrine control of the synthesis of type IV collagenases in human keratinocytes and fibroblasts. During wound healing, keratinocytes migrate over the wound bed, an activity coupled with lysis of basement membranes, and hence requiring the presence of collagenases. Collagenases are also needed for the production and remodelling of the granulation tissue. In order to study the autocrine/paracrine control of collagenase production in keratinocytes and fibroblasts, we stimulated these cells in culture with conditioned medium from cultured keratinocytes. Protease synthesis was determined by affinity labelling with ³H-diisopropylfluorophosphoridate (DFP) and by zymography. Keratinocyte-conditioned medium was found to increase the expression of 72 and 92 kDa type IV collagenase in human keratinocytes, and the 72 kDa collagenase in human fibroblasts. indicating that an autocrine/paracrine control mechanism is involved in collagenase production in these cell types during wound healing. This increased expression of collagenases could he partly responsible for the stimulated healing seen in wounds treated with sheets of cultured keratinocytes.     

A membrane protein preferentially expressed by a subpopulation of immature lymphoid cells, epidermal basal keratinocytes, and other epithelial cells

A murine monoclonal antibody, designated EL-1, was raised by immunization with a human malignant T cell line. It reacted specifically with a membrane antigen expressed on T and B lymphoblastoid cell lines, a subpopulation of normal thymocytes and bone marrow lymphocytes, lymphocytes from a subset of patients with non-B, non-T cell acute lymphoblastic leukemia or T cell acute lymphoblastic leukemia and epithelial stem cells. The latter reactivity was especially striking in the skin, where only basal epidermal keratinocytes and epidermal appendages, including eccrine sweat glands, sebaceous glands and hair follicles, stained positively. A human epidermoid carcinoma cell line was also stained by EL-1. Suprabasilar keratinocytes and acellular keratin did not stain. However, in vitro proliferating fetal lung fibroblasts stained positively. Membrane immunoprecipitation analysis demonstrated that the antigen recognized by antibody EL-1 is a single protein of molecular weight 105 kilodaltons which did not change with exhaustive chemical reduction. Metabolic radiolabeling studies demonstrated that this protein is synthesized by the cell and not merely taken up from the culture medium. This antibody can be useful in studying keratinocyte differentiation in epidermal malignancies and normal skin.     

UV-induced DNA damage initiates release of MMP-1 in human skin

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.     

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.     

Direct role of human dermal fibroblasts and indirect participation of epidermal keratinocytes in MMP-1 production after UV-B irradiation

In vivo, matrix metalloproteinases are produced in response to ultraviolet B (UV-B) irradiation and are considered to be involved in connective tissue alterations observed in photoaging. The respective roles of keratinocytes and fibroblasts in UV-B-induced MMP-1 production were investigated in monolayer cultures of keratinocytes and fibroblasts as well as in an epidermis model reconstructed in vitro. In contrast to fibroblasts, which secreted MMP-1 in response to UV-B irradiation, no accumulation of MMP-1 was observed after UV-B irradiation of keratinocytes. However, culture medium from UV-B-irradiated keratinocytes, which showed an increase in IL-1alpha and IL-6, induced MMP-1 production by human fibroblasts, suggesting that UV-B irradiation modulates MMP-1 production via both direct and indirect mechanisms.     

Human epidermal keratinocytes accumulate superoxide due to low activity of Mn-SOD, leading to mitochondrial functional impairment

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.     

Influences of keratinocyte–fibroblast interaction on the expression of epimorphin by fibroblasts in vitro

Epimorphin was demonstrated to be a mesenchymal signal factor modulating epithelial morphogenesis of skin, lung and liver in vitro. Most of the previous studies were performed biochemically and functionally. In the present study, expression of epimorphin was immunohistochemically compared between cultured fibroblasts and cocultured fibroblasts with keratinocytes obtained from normal skin. Cultured fibroblasts revealed a low level of epimorphin expression. In contrast, the expression by fibroblasts was greatly enhanced in skin explant culture where both fibroblasts and keratinocytes were present. In three-dimensional coculture of fibroblasts and keratinocytes, the expression of epimorphin was enhanced. The staining pattern of epimorphin in three-dimensional coculture was similar to that in human skin. These results suggest that dermal fibroblasts are manufacturers of epimorphin, and keratinocyte–fibroblast interaction may play important roles in the expression of epimorphin in vitro.     

Human dermal fibroblasts synthesize laminin

Laminin, a glycoprotein of approximately 900,000 daltons, is a major component of the basement membrane that separates the epidermis from dermis in human skin. Previous studies have shown that keratinocytes and other epithelial cells synthesize laminin and utilize it for attachment to other extracellular matrices such as heparan sulfate proteoglycan and basement membrane collagen. The relationships between phenotypically normal mesenchymal cells and laminin have been much less emphasized in the literature. In this study, we have used antibodies that specifically label the A and B chains of laminin (but not fibronectin or other unrelated proteins) by Western blot analysis to immunoprecipitate biosynthetically derived laminin from [35S] methionine labeled cultures of neonatal and adult human skin fibroblasts. To be sure that the precipitated bands were laminin and not fibronectin, which has a molecular size very close to that of the laminin B chains, experiments were performed in which fibronectin was removed from the radiolabeled proteins by first immunoprecipitating with antifibronectin antibody and then sequentially immunoprecipitating laminin from the fibronectin-depleted supernates with antilaminin antibody. These experiments definitively demonstrate that human dermal fibroblasts synthesize and secrete laminin.     

The occurrence of profilaggrin and its processing in cultured keratinocytes

An affinity-purified antibody to rat filaggrin detects filaggrin and profilaggrin in extracts of newborn rat epidermis, and a monoclonal antibody to human filaggrin, HF-1, detects the two proteins in extracts of human epidermis. Immunohistologic studies show that HF-1 reacts with keratohyaline granules of human epidermis and those seen in cultured human keratinocytes. Immunoblotting studies have demonstrated that profilaggrin is synthesized in both cultured human keratinocytes and in a long-lived line of cultured rat keratinocytes, but only in the latter is the protein processed to a product of the molecular weight of filaggrin.     

Dermcidin is constitutively produced by eccrine sweat glands and is not induced in epidermal cells under inflammatory skin conditions

BACKGROUND: Antimicrobial peptides (AMPs) are important effector molecules of innate immunity, protecting epithelial surfaces of multicellular organisms. In human skin two classes of AMPs-the beta-defensins and the cathelicidins-are produced by keratinocytes primarily under inflammatory conditions. In contrast, dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is expressed in eccrine sweat glands and transported via sweat to the epidermal surface. OBJECTIVES: To investigate whether DCD expression is induced under inflammatory conditions in epidermal keratinocytes. METHODS: Lesional skin of the inflammatory skin diseases atopic dermatitis, psoriasis and lichen planus was analysed by immunohistochemistry using a polyclonal anti-DCD antiserum. We also examined whether DCD RNA expression is induced in cultured human keratinocytes, fibroblasts, melanocytes and melanoma cells. RESULTS: Whereas DCD was constitutively expressed in eccrine sweat glands of all skin biopsies, we found that, independent of the type of the inflammatory skin lesion, DCD protein expression was not induced in human epidermal keratinocytes. In contrast, beta-defensin 2 was expressed in epidermal keratinocytes of inflammatory human skin, but not in keratinocytes of healthy human skin. Upon stimulation of the cultured cells with 12-O-tetradecanoyl-phorbol-13-acetate, tumour necrosis factor-alpha, lipopolysaccharide or H2O2, DCD mRNA expression was not detected in primary keratinocytes, fibroblasts and melanocytes, but was detected in MeWo and SKMEL28 melanoma cells. CONCLUSIONS: These results indicate that, unlike human cathelicidins and beta-defensins which are inducible peptides that primarily function in response to injury and inflammation, DCD is exclusively part of the constitutive innate defence of human skin. By modulating surface colonization, DCD may help to prevent local and systemic invasion of pathogens.