An improved method for culture of epidermal keratinocytes from newborn mouse skin

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca²⁺ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin v6. The expression level of v6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.     

The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity

FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.     

Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes

Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data have shown that AKT activation is involved in the cell survival process. Yet, the potential mechanism of niacin in protection against UV-induced skin damage has thus far not fully been eluvidated. We observed that niacin pretreatment enhances UV induced activation of AKT (Ser473 phosphorylation) as well as that of the downstream signal mTOR (S6 and 4E-BP1 phosphorylation). The PI3K/AKT inhibitor, LY294002, and the mTOR inhibitor, rapamycin, largely neutralized the protective effects of niacin, suggesting that AKT and downstream signaling mTOR/S6 activation are necessary for the niacin-induced protective effects against UV-induced cell death and cell apoptosis. Collectively, our data suggest that niacin may be utilized to prevent UV-induced skin damage and provide a novel mechanism of its photoprotective effects against the UV?radiation of sunlight by modulating both AKT and downstream mTOR signaling pathways.     

The selective cyclooxygenase-2 inhibitor rofecoxib suppresses brain inflammation and protects cholinergic neurons from excitotoxic degeneration in vivo

Brain inflammatory processes underlie the pathogenesis of Alzheimer's disease, and non-steroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this work was to study in vivo whether attenuation of brain inflammatory response to excitotoxic insult by the selective cyclooxygenase-2 inhibitor, rofecoxib, may prevent neurodegeneration, as a contribution to a better understanding of the role inflammation plays in the pathology of Alzheimer's disease. We investigated, by immunohistochemical methods, glia reaction, the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway with an antibody selective for the phosphorylated form of the enzyme and the number of choline acetyltransferase-positive neurons and, by in vivo microdialysis, cortical extracellular levels of acetylcholine following the injection of quisqualic acid into the right nucleus basalis of adult rats. Seven days after injection, a marked reduction in the number of choline acetyltransferase-positive neurons was found, along with an intense glia reaction, selective activation of p38MAPK at the injection site and a significant decrease in the extracellular levels of acetylcholine in the cortex ipsilateral to the injection site. The loss of cholinergic neurons persisted for at least up to 28 days. Rofecoxib (3 mg/kg/day, starting 1 h prior to injection of quisqualic acid) treatment for 7 days significantly attenuated glia activation and prevented the loss of choline acetyltransferase-positive cells and a decrease in cortical acetylcholine release. The prevention of cholinergic cell loss by rofecoxib occurred concomitantly with the inhibition of p38MAPK phosphorylation. Our findings suggest an important role of brain inflammatory reaction in cholinergic degeneration and demonstrate a neuroprotective effect of rofecoxib, presumably mediated through the inhibition of p38MAPK phosphorylation.     

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.     

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.     

The tripeptide KdPT protects from intestinal inflammation and maintains intestinal barrier function

Treatment options for inflammatory bowel disease (IBD) are incompletely helpful, and surgery is often needed. One promising class of future therapeutic agents for IBD is melanocortin-related peptides, which exhibit potent immunomodulatory effects. We investigated KdPT, a tripeptide derivative of the C-terminus of α-melanocyte-stimulating hormone, as an anti-inflammatory small molecule in vivo and in vitro. Intestinal inflammation was studied after oral administration of dextran sodium sulfate and in IL-10 gene-deficient mice. The effects of KdPT on key colonic epithelial cell functions were studied in vitro and in vivo by evaluating proliferation, wound healing, transepithelial resistance, and expression of tight junction proteins. Melanin assays were performed to determine the melanotropic effects of KdPT. KdPT-treated animals showed markedly reduced severity of inflammation in both colitis models. In colonic epithelial cells, KdPT increased proliferation, accelerated closure of wounds, and improved transepithelial electrical resistance after stimulation with interferon-γ/tumor necrosis factor-α. Moreover, treatment with KdPT also prevented the loss of tight junction protein expression and improved barrier function in vivo. KdPT acted independently of IL-1 receptor type I in vivo and did not affect melanogenesis in vitro. KdPT is capable of attenuating the course of experimental colitis in different models and maintains epithelial cell function. Furthermore, KdPT does not induce pigmentation, emphasizing the potential of this small molecule for the future treatment of IBD.     

Efficient production of bioactive insulin from human epidermal keratinocytes and tissue-engineered skin substitutes: implications for treatment of diabetes

Despite many years of research, daily insulin injections remain the gold standard for diabetes treatment. Gene therapy may provide an alternative strategy by imparting the ability to secrete insulin from an ectopic site. The epidermis is a self-renewing tissue that is easily accessible and can provide large numbers of autologous cells to generate insulin-secreting skin substitutes. Here we used a recombinant retrovirus to modify human epidermal keratinocytes with a gene encoding for human proinsulin containing the furin recognition sequences at the A-C and B-C junctions. Keratinocytes were able to process proinsulin and secrete active insulin that promoted glucose uptake. Primary epidermal cells produced higher amounts of insulin than cell lines, suggesting that insulin secretion may depend on the physiological state of the producer cells. Modified cells maintained the ability to stratify into 3-dimensional skin equivalents that expressed insulin at the basal and suprabasal layers. Modifications at the furin recognition sites did not improve proinsulin processing, but a single amino acid substitution in the proinsulin B chain enhanced C-peptide secretion from cultured cells and bioengineered skin substitutes 10- and 28-fold, respectively. These results suggest that gene-modified bioengineered skin may provide an alternative means of insulin delivery for treatment of diabetes.     

Regulatory mechanisms of galectin-9 and eotaxin-3 synthesis in epidermal keratinocytes: possible involvement of galectin-9 in dermal eosinophilia of Th1-polarized skin inflammation

BACKGROUND: Skin eosinophilia is a common feature of allergic skin diseases, but it is unclear how epidermal and dermal eosinophil infiltration is controlled. To investigate regulation of localization of eosinophils in skin, we examined the regulatory mechanisms of expression of eosinophil-specific chemoattractants in dermal fibroblasts and epidermal keratinocytes. METHODS: We analyzed production of eotaxin, eotaxin-2, eotaxin-3 and galectin-9 by dermal fibroblasts and epidermal keratinocytes in response to several stimuli in vitro. RESULTS: Dermal fibroblasts produced eotaxin and eotaxin-3 in response to stimulation by interleukin (IL)-4 and/or tumor necrosis factor-alpha. Similarly, IL-4 stimulated epidermal keratinocytes to secrete eotaxin-3. However, we did not detect eotaxin mRNA expression or protein secretion by keratinocytes stimulated in vitro. Interferon (IFN)-gamma induced galectin-9 expression on dermal fibroblasts. Conversely, expression of galectin-9 on epidermal keratinocytes was dose-dependently inhibited by IFN-gamma. The immunohistochemical assays revealed that dermal fibroblasts (but not epidermal keratinocytes) in the lesional skin of psoriasis vulgaris (a Th1-polarized disease) express significant levels of galectin-9. CONCLUSION: Eotaxin-3 contributes to dermal and epidermal eosinophil infiltration in Th2-polarized skin inflammation in which IL-4 is produced. In contrast, IFN-gamma-dominated inflammation appears to mediate eosinophil extravasation into the dermis and eosinophil adhesion to dermal fibroblasts via galectin-9 in association with decreased chemoattractant activity of epidermal galectin-9. The present results reveal a novel mechanism of dermal eosinophilia in IFN-gamma-mediated skin inflammation, and reflect concerted chemoattractant production involving dermal and/or epidermal eosinophilia during changes in the local cytokine profile.     

Progranulin is preferentially expressed in patients with psoriasis vulgaris and protects mice from psoriasis‐like skin inflammation

Progranulin (PGRN) is a multi-functional protein known to be involved in inflammation. Recent studies have found that PGRN has dual roles in inflammation and exerts anti-inflammatory and pro-inflammatory function in different diseases. However, the role of PGRN in psoriasis has not been fully elucidated. Here, we detected preferential expression of PGRN in human psoriatic lesions and serum. Moreover, serum PGRN/tumour necrosis factor-α ratio was negatively correlated with disease severity. To investigate the role of PGRN in the pathogenesis of psoriasis, we used wild-type (WT) and PGRN^−/− mice in a model of 12-O-tetradecanoylphorbol 13-acetate (TPA) -induced psoriasis-like inflammation. We demonstrated that PGRN expression was dramatically enhanced in the psoriasis-like lesions of TPA-treated WT mice, in accordance with human psoriatic lesions. Surprisingly, PGRN^−/− mice were more sensitive to the development of TPA-induced psoriasis-like inflammation. The mechanism underlying this increased sensitivity of PGRN^−/− mice to TPA-induced psoriasis-like inflammation was impaired differentiation of regulatory T cells in lymph nodes and decreased recruitment of these cells in the affected skin, which results in more severe inflammation. Hence, in WT mice, PGRN promotes differentiation and recruitment of regulatory T cells at the site of inflammation, which protects the skin from an exaggerated psoriasis-like inflammatory response.     

成年小鼠背部皮肤角质形成细胞的原代培养及其与乳鼠角质形成细胞的对比性研究

目的:探索成年小鼠背部皮肤角质形成细胞有效且高生长率的原代培养方法;对比乳鼠和成年小鼠背部皮肤角质形成细胞的增殖、集落形成效率与凋亡能力。方法:取正常成年小鼠背部皮肤,分别利用4种方法分离其表皮与真皮,取表皮分离培养角质形成细胞;免疫荧光进行细胞鉴定;调整种板密度、种板方式观察细胞生长状况;利用CCK-8、EdU、结晶紫染色法和TUNEL染色检测乳鼠与成年小鼠背部皮肤角质形成细胞的增殖、集落形成效率与凋亡情况。结果:4种成年小鼠表皮-真皮分离方式中,二步消化法获得的细胞量最大、细胞增殖能力最强、集落形成效率高;水滴状种板法较普通平铺种板法集落形成率高;乳鼠角质形成细胞最适种板密度约为3. 2×10⁴/cm²,成年小鼠背部皮肤角质形成细胞最适种板密度约为1. 6×10⁴/cm²;与成年小鼠背部皮肤角质形成细胞相比,乳鼠角质形成细胞增殖能力较强、集落形成效率较高。结论:成功建立了较为完善的成年小鼠背部皮肤角质形成细胞原代培养体系。     

Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress

Background  

Recent studies have demonstrated a link between the inflammatory response, increased cytokine formation, and neurodegeneration in the brain. The beneficial effects of anti-inflammatory drugs in neurodegenerative diseases, such as Alzheimer's disease (AD), have been documented. Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. The objectives of this study are to determine the effects of acetaminophen on cultured brain neuronal survival and inflammatory factor expression when exposed to oxidative stress.     

Signaling via the RIP2 adaptor protein in central nervous system-infiltrating dendritic cells promotes inflammation and autoimmunity

Peripheral peptidolgycan (PGN) is present within antigen-presenting cells in the central nervous system (CNS) of multiple sclerosis (MS) patients, possibly playing a role in neuroinflammation. Accordingly, PGN is linked with disease progression in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), but the role of specific PGN-sensing proteins is unknown. Here we report that the progression of EAE was dependent on the intracellular PGN sensors NOD1 and NOD2 and their common downstream adaptor molecule, receptor interacting protein 2 (RIP2; also known as RIPK2 and RICK). We found that RIP2, but not toll-like receptor 2 (TLR2), played a critical role in the activation of CNS-infiltrating dendritic cells. Our results suggest that PGN in the CNS is involved in the pathogenesis of EAE through the activation of infiltrating dendritic cells via NOD1-, NOD2-, and RIP2-mediated pathways.     

Innate immunity mediated by epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin

Skin is an immunological organ consisting of epidermal cells, i.e. keratinocytes and Langerhans cells (LCs, antigen-presenting dendritic cells), and both innate and acquired immune systems operate upon exposure of the skin to various external microbes or their elements. To explore the relationship between innate and acquired immunities in the skin, we investigated whether Toll-like receptor (TLR) ligation of epidermal cells enhances the ability of LCs to present a specific antigen to T cells in mice. LC-containing epidermal cells were incubated with CpG oligonucleotide (TLR9 ligand) modified with trinitrophenyl hapten, and cultured with hapten-primed CD4(+) T cells. TLR9 ligand was capable of enhancing the hapten-presenting ability of LCs when LC-enriched epidermal cells, but not purified LCs, were used as the LC source, suggesting that bystander keratinocytes play a role in the enhancement of LC function. Cultivation of freshly isolated epidermal cells with CpG promoted the expression of major histocompatibility complex (MHC) class II and CD86 molecules on LCs. CpG enhanced the production of interleukin (IL)-1alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha by primarily cultured keratinocytes. The addition of a cocktail of neutralizing antibodies against these cytokines abrogated the CpG-promoted, antigen-presenting ability of LC-enriched epidermal cells. Moreover, the addition of culture supernatants from CpG-stimulated keratinocytes restored the ability of purified LCs. Our study demonstrated that although the direct effect of CpG on LCs is minimal, LC function can be up-regulated indirectly by cytokines released by CpG-stimulated keratinocytes. This also implies that innate immunity evoked by TLR ligation of keratinocytes enhances acquired immunity comprising LCs and T cells.     

Pharmacological modification of 12-O-tetradecanoylphorbol-13-acetate induced inflammation and epidermal cell proliferation in mouse skin

Topical application of TPA to mouse skin causes oedema (2–6 h) neutrophil influx (3–24 h) and epidermal cell proliferation (24–48 h). Topical application of a cyclooxygenase inhibitor (indomethacin) dual cyclooxygenase and lipoxygenase inhibitors (phenidone and BW 755C) a selective lipoxygenase inhibitor (AA 861), protein synthesis inhibitors (cycloheximide and actinomycin D) or a glucocorticosteroid (prednisolone) inhibited oedema and neutrophil influx. Systemic administration of an inhibitor of microtubule assembly (colchicine) also prevented neutrophil influx and oedema. These results suggest that the inflammatory response to TPA depends on an interaction between a protein and products of arachidonic acid metabolism to produce a neutrophil dependent oedema. Epidermal cell proliferation was inhibited by topical administration of prednisolone, indomethacin, BW 755C and cycloheximide but not systemically administered methotrexate. This suggests that inhibition of the early inflammatory response to TPA prevents the subsequent epidermal proliferation.     

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.