Investigation of irradiation by different nonablative lasers on primary cultured skin fibroblasts

Background. A variety of lasers with different wavelengths and biological effects are widely used for nonablative skin rejuvenation, but the underlying mechanisms have not been fully investigated. Aim. To investigate the effects of irradiation by different nonablative lasers on collagen synthesis and the antioxidant status of cultured fibroblasts to identify a possible mechanism for laser photorejuvenation. Methods. Cultured skin fibroblasts were irradiated with three different lasers: 532 nm potassium–titanyl phosphate (KTP), 1064 nm Q‐switched neodymium:yttrium–aluminium–garnet (Nd:Yag) and 1064 nm long‐pulse Nd:YAG, and production of collagen and changes in lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) were assayed. Results. Irradiation by all three lasers led to a marked increase in collagen production. Two major antioxidant enzymes, SOD and GSH, were significantly increased, whereas MDA was markedly reduced after laser irradiation. No change in LDH activity was found between nonirradiated and irradiated fibroblasts. Conclusion. This study indicates that the increased collagen synthesis by fibroblasts after laser treatment may be partly due to improved antioxidant capacity, which reduces oxidative stress and thus stimulates new collagen production.     

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.     

Epidermal interleukin 1 alpha functional activity and interleukin 8 immunoreactivity are increased in patients with cutaneous T-cell lymphoma

Previous studies have suggested that epidermal-derived interleukin-1 is involved in the pathogenesis of cutaneous T-cell lymphoma (CTCL); however, the findings are conflicting and studies that combine immunohistochemistry and functional activity have not been performed. We investigated the interleukin-1 level in epidermis of patients with cutaneous T-cell lymphoma using both immunohistochemistry, enzyme-linked immunosorbent assays, and the thymocyte co-stimulation assay. Using supernatants obtained from epidermal cell cultures, we found a significant but small increase of interleukin 1 alpha protein release from involved CTCL epidermis compared to normal epidermis from healthy individuals. Both keratinocytes and leukocytes could release interleukin-1 alpha, but the majority was derived from the keratinocytes. Interleukin-1 beta protein was not detectable. In the thymocyte assay, interleukin-1 alpha was found to be biologically active. When lymphokines derived from a T-cell clone obtained from involved CTCL skin were co-cultured with epidermal cells, an enhanced release of epidermal interleukin-1 alpha could be demonstrated. Because interleukin 1 alpha was increased, we investigated the presence of interleukin 1-inducible keratinocyte-derived interleukin 8 and found it increased in CTCL epidermis compared to normal epidermis from healthy individuals. This study demonstrated an elevated epidermal IL-1 alpha level and IL-8 immunoreactivity in CTCL epidermis, which suggests that this elevated level is induced by lymphokines released from activated T cells.     

Solar simulated irradiation modulates gene expression and activity of antioxidant enzymes in cultured human dermal fibroblasts

Exposure of skin to solar irradiation generates reactive oxygen species that damage DNA, membranes, mitochondria and proteins. To protect against such damage, skin cells have evolved antioxidant enzymes including glutathione peroxidase (GSH-Px), copper and zinc-dependent superoxide dismutase (SOD1), the mitochondrial manganese-dependent superoxide dismutase (SOD2), and catalase. This report examines the effect of a single low or moderate dose exposure to solar-simulating combined UVB and UVA irradiation on the gene expression and activities of these antioxidant enzymes in cultured normal human fibroblasts. We find that both doses initially decrease GSH-Px, SOD2 and catalase activities, but within 5 days after irradiation the activities of the enzymes return to pre-irradiation level (catalase) or are induced slightly (SOD1, GSH-Px) or substantially (SOD2) above the basal level. For SOD1, SOD2 and catalase, the higher dose also detectably modulates the mRNA level of these enzymes. Our results indicate that the effects of a single physiologic solar simulated irradiation dose persist for at least several days and suggest that skin cells prepare for subsequent exposure to damaging irradiation by upregulating this antioxidant defense system, in particular the mitochondrial SOD2. Our findings are consistent with the existence of a broad-based SOS-like response in irradiated human skin.     

Protective and determining factors for the overall lipid peroxidation in ultraviolet A1-irradiated fibroblasts: in vitro and in vivo investigations

BACKGROUND: Lipid peroxidation (LPO) is one major effector mechanism by which ultraviolet (UV) A contributes to photoageing and the promotion of skin cancer. It is a fingerprint of photo-oxidative stress within the skin, and is initiated by several pathways, with different reactive oxygen species (ROS) and iron ions being involved. OBJECTIVES: To elucidate factors involved in UVA1-induced LPO in human dermal fibroblasts and mouse dermis, and the role of antioxidant enzymes in protecting cells against LPO. METHODS: Using a highly sensitive high-performance liquid chromatography procedure, we measured malondialdehyde (MDA), a specific metabolic tracer molecule for LPO, to determine the overall LPO produced by a given UVA1 dose in vitro and in vivo. By using the iron chelator desferrioxamine (DFO), the hydroxyl radical scavenger dimethylsulphoxide (DMSO) and fibroblasts that specifically overexpress single antioxidant enzymes, we further indirectly assessed the protective effect of manganese superoxide dismutase (MnSOD), catalase and phospholipid hydroperoxide glutathione peroxidase (PHGPx) as well as the relative importance of different ROS and the role of transitional iron for the total amount of LPO induced by a distinct UVA dose. RESULTS: UVA1 irradiation resulted in a time- and dose-dependent increase in MDA levels in vitro, and the in vitro results were shown to have in vivo relevance. Fibroblasts incubated with DFO or DMSO produced lower levels of MDA than controls, as did fibroblasts overexpressing MnSOD, catalase or PHGPx. CONCLUSIONS: The cellular iron pool and hydroxyl radicals were the most important determining factors for the total amount of MDA produced after a given UVA1 dose, and PHGPx overexpression had the greatest protective effect against LPO.     

Immunohistochemical localization of ornithine decarboxylase in human skin

The localization of ornithine decarboxylase (ODC) in human skin and cultured keratinocytes was studied with an immuno-histochemical method. ODC was found in the epidermis, hair follicles, sweat glands and errector muscles. Irritation induced by stripping or UV-B irradiation did not change the staining pattern in the epidermis. In psoriasis, the staining was most marked at the tip of the epidermal rete ridges. In basal cell carcinoma, there was a homogeneous labelling of the tumor cells and, in squamous cell carcinoma, the labelling was strong but less homogeneous. Melanoma and dermal naevus also positively stained for ODC. Cultured human keratinocytes also showed ODC positive immunofluorescence. This technique detects the ODC antigen present rather than levels of ODC activity.     

Immunohistochemical localization of alpha 2-macroglobulin receptors in human skin.

The localization of receptors for the proteinase inhibitor alpha 2-macroglobulin was studied in human skin by immunohistochemistry using a monoclonal mouse antibody. No epidermal staining was identified. alpha 2-Macroglobulin receptors were identified on dermal fibroblasts and dermal dendritic cells. Endothelial cells did not stain with the antibody, but positive staining cells were concentrated around dermal vessels. The myoepithelial layer of eccrine glands exhibited receptors; however, there was no staining of the eccrine epithelial layer. The distribution of alpha 2-macroglobulin receptors correlates with the reported distribution of alpha 2-macroglobulin: both are present in the dermal connective tissue and absent in epithelium and endothelium. The distribution of alpha 2-macroglobulin and its receptor in the dermis is consistent with a possible role in regulation of dermal proteolytic activity.     

Production of a monoclonal antibody,DF-5, that identifies cells at the epithelial-mesenchymal interface in normal human skin. APN/CD13 is an epithelial-mesenchymal marker in skin

Epithelial-mesenchymal interactions play a critical role in skin development and differentiation, and similar interactions may also regulate the day-to-day proliferation and differentiation events of the epidermis that occur in normal adult skin. This study was directed at identifying molecules that are selectively located at the dermal-epidermal junction in normal adult skin as they may be involved in regulating these homeostatic events. To this end, monoclonal antibodies were raised against the crude cell membrane fraction of cultured human dermal fibroblasts. Screening of antibodies that recognized cell surface antigen on cultured human dermal fibroblasts was followed by determining which of these antibodies selectively localized cells at sites of epithelial-mesenchymal interactions. Antibody DF-5 fit these criteria and was further characterized. This antibody was found to recognize the cell surface ectopeptidase aminopeptidase N (APN), a molecule homologous to the cluster differentiation antigen CD13. Antibody DF-5 and anti-CD13 antibodies both identified cells at sites of epithelial-mesenchymal interactions in fetal, neonatal, and adult human skin, and the APN/CD13 enzyme activity was also identified at these sites. A second ectopeptidase, dipeptidyl peptidase IV (DPPIV) or CD26, presented a significantly different immunohistochemical and histochemical pattern in skin samples, confirming the specificity of the APN/CD13 studies. The function of APN/CD13 in skin has yet to be determined. Its invariant localization at sites of epithelial-mesenchymal interactions argues for a role particular to this region. It may play a role in regulating the activity of neuropeptides or other signaling peptides that are released in this region of skin or it may have an as yet undefined role in mediating communication between dermal and epidermal cells.     

Upregulation of collagen type 1 in aged murine dermis after transplantation of dermal multipotent cells

Background. Multipotent cells can be isolated from dermis, and have wound‐healing and antioxidant effects on human and murine skin. Aim. To investigate the changes in aged dermis after injection of dermal multipotent cells and adult fibroblasts. Methods. Dermal multipotent cells were isolated and cultured from 3‐day‐old BALB/c mice by a regular dermal fibroblast culture system. We used various inducers to confirm the multilineage differentiation of these cells, then the dermal multipotent cells were transplanted into the dermis of aged (12‐week‐old) BALB/c mice. At 2 and 4 weeks after transplantation, we examined dermal thickness, and determined the amounts of collagen type I present, using PCR and western blotting. Result. The dermal multipotent cells exhibited adipogenic and osteogenic phenotypes when cultured in the presence of certain inducers. Dermal thickness and collagen content in the dermis also increased in the mice injected with dermal multipotent cells, which also had increased RNA and protein expression of collagen type I. Conclusions. Collectively, these results indicate that dermal multipotent cells are more effective than fibroblasts in increasing collagen type I and dermal thickness, which may be of importance in preventing skin ageing.     

The effect of UV-A and UV-B irradiation on the skin barrier. Skin physiologic, electron microscopy and lipid biochemistry studies]

In order to gain insight into the effects of UV-irradiation on the skin barrier, functional (skin reactivity), electron microscopic and lipid-biochemical studies were performed. In three different irritation models, both UV-A-irradiated and UV-B-irradiated areas proved to be more resistant to damage than normal skin, providing evidence for improvement of barrier function after UV irradiation. Electron microscopic evaluation showed that UV-B induced a significant increase in horny cell layers, whereas after UV-A no change was detected. However, both UV-B and UV-A exposure resulted in an increase in the amount of all stratum corneum lipids. This was also observed in all major ceramide subfractions, which are believed to be the essential lipid constituents for the epidermal barrier function. These findings may explain the known beneficial effects of phototherapy in dermatoses with impaired barrier function, i.e., atopic dermatitis.     

Effect of nickel on the activation state of normal human keratinocytes through interleukin 1 and intercellular adhesion molecule 1 expression

Patch tests with nickel on sensitive subjects induce a characteristic allergic reaction involving epidermal and dermal cells, as well as modulation of cytokines and adhesion molecule production. In order to gain further insight into the role of keratinocytes in this phenomenon, we assessed their activation state induced by Ni²⁺ by studying interleukin 1 (IL-1) production and intercellular adhesion molecule 1 (ICAM-1) expression, using normal human keratinocytes cultured in defined medium. In comparison with controls, the addition of subtoxic NiSO₄ concentrations (0.1–20 μg/ml) to keratinocyte cultures induced a significant, but low release of IL-1α and IL-1β at 24 and 48 h, detectable by enzyme-linked immunosorbent assay of the supernatants of treated cells, Moreover, IL-1 receptor antagonist was significantly increased in the supernatants and the cell extracts. Using fluorescence-activated cell sorter analysis, ICAM-1 expression at 24h was found to be induced in a dose-dependent manner, reaching a level comparable with that obtained upon interferon-γ (10 IU/ml) stimulation. Overall, these data confirm the existence of direct interactions between Ni²⁺ and keratinocytes, which generate immunological signals of major importance in the pathophysiology of allergic contact dermatitis.     

UVB-induced conversion of 7-dehydrocholesterol to 1alpha,25-dihydroxyvitamin D3 in an in vitro human skin equivalent model.

We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol (1alpha,25-dihydroxyvitamin D3). This study was initiated to test whether the ultraviolet-B-induced photolysis of provitamin D3 (7-dehydrocholesterol), which results in the formation of vitamin D3, can generate calcitriol in an in vivo-like human skin equivalent model made of fibroblasts in a collagen matrix as the dermal component and keratinocytes as the epidermal component. Cultures were preincubated with increasing concentrations of 7-dehydrocholesterol (0.53-5.94 nmol per cm2 human skin equivalent) at 37 degrees C and irradiated with monochromatic ultraviolet B at wavelengths ranging from 285 to 315 nm (effective ultraviolet doses 7.5-45 mJ per cm2). In our in vitro model irradiation with ultraviolet B resulted in a sequential metabolic process with generation of previtamin D3 followed by the time-dependent formation of vitamin D3, 25-hydroxyvitamin D3, and ultimately calcitriol in the femtomolar range. Unirradiated cultures and irradiated cultures without keratinocytes generated no calcitriol. Irradiation of skin equivalents at wavelengths > 315 nm generated no or only trace amounts of calcitriol. The ultraviolet-B-triggered conversion of 7-dehydrocholesterol to calcitriol was strongly inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases. The amount of calcitriol generated was dependent on the 7-dehydrocholesterol concentration, on wavelength, and on ultraviolet B dose. Hence, keratinocytes in the presence of physiologic concentrations of 7-dehydrocholesterol and irradiated with therapeutic doses of ultraviolet B may be a potential source of biologically active calcitriol within the epidermis.     

Tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts by the inhibition of superoxide anion production and glutathione depletion

Background/Objectives: Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may induce an irreversible growth arrest similar to senescence. Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, is a widely used antioxidant to rescue ROS-evoked cell death. The aim of the article was to explore the effects of tiron on skin photoaging and associated mechanisms. Methods: The effects of tiron on cell proliferation were determined using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide. Senescent cells were determined by morphology and senescence-associated β-galactosidase activity analysis. Intracellular hydrogen peroxide, superoxide anion and glutathione concentration were analysed by a fluorescent probe. The concomitant changes of protein expression were analysed with Western blot. Results: Human dermal fibroblasts were induced to premature senescence by sub-cytotoxic doses of irradiated UVB. Strong senescence-associated β-galactosidase activity and increased intracellular superoxide anion were observed in human dermal fibroblasts irradiated by UVB. Tiron blocks UVB-induced glutathione depletion and increase of superoxide anion and protects against UVB-induced senescence-like characteristics in human dermal fibroblasts. Compared with normal fibroblasts, UVB-irradiated human dermal fibroblasts showed a higher ratio of active (hypophosphorylated) to inactive (phosphorylated) forms of Rb and p38, upregulation of p53 or p16 and c-Myc and insulin-like growth factor 1 (IGF-1) downregulation. After treatment with tiron, p53, p16 c-Myc and IGF-1 as well as phosphorylation Rb and p38 could partially recover. Conclusion: These results indicate that tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts via the inhibition of production of superoxide anion and glutathione depletion, and modulation of related senescence proteins.     

小鼠真皮间充质干细胞与成纤维细胞共培养对胶原分泌和TGF-β1表达的影响

目的 探讨真皮间充质干细胞在皮肤组织修复中的作用.方法 采用低血清培养基,消化-贴壁-传代法体外培养、鉴定小鼠真皮间充质干细胞(mdMSC),并与体外分离培养的正常人皮肤成纤维细胞于transwell小室培养体系中共培养,样本碱水解法和ELISA法分别检测第4、8天培养上清液中羟脯氨酸和TGF-β1的变化.结果 共培养第8天,经mdMSC 2.5×104和mdMSC 1×104处理的正常人皮肤成纤维细胞培养上清液中羟脯氨酸含量较单独培养时明显增高(P<0.05).经mdMSC处理的各组正常人皮肤成纤维细胞培养上清液中TGF-β1含量于共培养第8天时均高于单独培养(P<0.01);经mdMSC 1×104处理的正常人皮肤成纤维细胞培养上清液中TGF-β1含量在第4天亦高于单独培养,差异有统计学意义(P<0.05).各不同细胞密度的MSC处理组的羟脯氨酸含量与TGF-β1水平无相关关系(r=0.108,P＞0.05).结论 mdMSC与正常人皮肤成纤维细胞共培养可增加羟脯氨酸和TGF-β1的分泌,可能是mdMSC促进皮肤组织修复的机制之一.     

Human neonatal keratinocytes have very high levels of cellular vitamin A-binding proteins

Since cellular retinol- and retinoic acid-binding proteins (CRBP and CRABP) mediate the effects of vitamin A on epidermal differentiation, the levels of these binding proteins were measured in the epidermal and dermal layers of newborn, human foreskin as well as in primary cultures of keratinocytes and fibroblasts from these layers. Ligand binding assays with saturating concentrations of all trans-[3H]retinol or of all trans-[11-3H]retinoic acid were used to quantitate amounts of binding proteins in cytosols prepared from these skin layers or cultured cells. The epidermal levels of CRABP and CRBP (60.9 +/- 14.4 and 7.3 +/- 1.7 pmol per mg cytosol protein, respectively) were markedly higher than that reported for adult epidermis but were comparable to levels in keratinocytes cultured from neonatal foreskin epidermis (61.8 +/- 7.8 and 10.7 +/- 2.5, respectively). The levels of CRABP were much lower in the foreskin dermis than in the epidermis and the levels measured in the fibroblasts cultured from this dermis were consistent with the dermal levels. However, CRBP levels in cultured dermal fibroblasts were very low and could not account for the dermal CRBP levels, suggesting that another dermal cell type has high levels of CRBP.     

Plasticity of hair follicle dermal cells in wound healing and induction

The capacity of adult hair follicle dermal cells to participate in new follicle induction and regeneration, and to elicit responses from diverse epithelial partners, demonstrates a level of developmental promiscuity and influence far exceeding that of interfollicular fibroblasts. We have recently suggested that adult follicle dermal cells have extensive stem or progenitor cell activities, including an important role in skin dermal wound healing. Given that up to now tissue engineered skin equivalents have several deficiencies, including the absence of hair follicles, we investigated the capacity of follicle dermal cells to be incorporated into skin wounds; to form hair follicles in wound environments; and to create a hair follicle-derived skin equivalent. In our study, we implanted rat follicle dermal cells labelled with a vital dye into ear and body skin wounds. We found that they were incorporated into the new dermis in a manner similar to skin fibroblasts, but that lower follicle dermal sheath also assimilated into hair follicles. Using different combinations of follicle dermal cells and outer root sheath epithelial cells in punch biopsy wounds, we showed that new hair follicles were formed only with the inclusion of intact dermal papillae. Finally by combining follicle dermal sheath and outer root sheath cells in organotypic chambers, we created a skin equivalent with characteristic dermal and epidermal architecture and a normal basement membrane - the first skin to be produced entirely from hair follicle cells. These data support the hypothesis that follicle dermal cells may be important in wound healing and demonstrate their potential usefulness in human skin equivalents and skin substitutes. While we have made progress towards producing skin equivalents that contain follicles, we suggest that the failure of cultured dermal papilla cells to induce follicle formation in wounds illustrates the complex role the follicle dermis may play in skin. We believe that it demonstrates a genuine dichotomy of activity for follicle cells within skin.     

Mast cell-fibroblast interactions: human mast cells as source and inducers of fibroblast and epithelial growth factors

As mast cells have been implicated in cutaneous repair processes, we have examined the ability of human mast cells to produce important epithelial and fibroblast growth factors or to stimulate the production of such factors in dermal fibroblasts. Isolated, highly purified human dermal mast cells and human leukemic mast cells were examined for mRNA and partly also for protein expression of these molecules as such or after preincubation with interleukin-4, stem cell factor, or with phorbol myristate acetate. In addition, mast cells were studied for their ability to induce fibroblast growth factor 2 and fibroblast growth factor 7 secretion from dermal fibroblasts. Both dermal and leukemic mast cells expressed fibroblast growth factor 2, fibroblast growth factor 7, and heparin-binding epidermal growth factor, but not hepatocyte growth factor at mRNA level, and dermal mast cells expressed fibroblast growth factor 10 in addition. At protein level, spontaneous fibroblast growth factor 2 secretion was noted that was markedly enhanced by phorbol myristate acetate, whereas no fibroblast growth factor 7 protein was detected under these conditions. Instead, human mast cell-1 supernatants induced enhanced fibroblast growth factor 7 secretion from dermal fibroblasts, with phorbol-myristate-acetate-stimulated supernatants being more effective. This effect could be reproduced with histamine and was H1-receptor mediated. Tryptase was ineffective but stimulated instead fibroblast growth factor 2 secretion from fibroblasts. These data demonstrate for the first time the ability of mast cells to express and/or secrete several growth factors of the fibroblast growth factor family as well as heparin-binding epidermal growth factor directly or indirectly via stimulation of fibroblasts, underlining the potentially pivotal role of these cells during human tissue repair and homeostasis.     

Interleukin 6 indirectly induces keratinocyte migration

IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation.