TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The I kappa B kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-kappa B activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.     

Variable expression of Ia antigens on the vascular endothelium of mouse skin allografts

Ia antigens are membrane-bound glycoproteins that play a part in antigen recognition and subsequent cell-cell interactions in the immune response. In the mouse they are coded for by the I region of the major histocompatibility complex H-2 and have been demonstrated on B lymphocytes, monocytes, activated T cells, macrophages and dendritic cells, including Langerhans cells. Ia-like antigens have also been detected on the vascular endothelium in man and on epidermal keratinocytes in rats but expression on the latter cells was induced by a graft-versus-host reaction or by contact hypersensitivity. In the mouse, previous studies have suggested that Ia antigens in skin are restricted to epidermal Langerhans cells and it was thought that these were the targets for Ia-dependent rejection of skin allografts. The results presented here show that Ia antigens in mouse allografts are also present on the vascular endothelium but their expression is variable and dependent on the immunological status of the recipient. These findings suggest that vascular endothelial cells can act as targets in Ia-incompatible skin allograft rejection.     

Specific interaction between HPV-16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network.

The human papillomaviruses (HPV) are associated specifically with epithelial lesions, ranging from benign warts to invasive carcinoma. The virus encodes three late proteins, which are produced only in terminally differentiating keratinocytes, two of which are structural components of the virion. The third, E1-E4, is derived primarily from the E4 open reading frame, which represents a region of maximal divergence between different HPV types. E1-E4 does not seem to be a component of the virus particle or to be needed for transformation in vitro, but accumulates in the cytoplasm, where in certain benign lesions it can comprise 20-30% of total cell protein. We show here that expression of the HPV-16 E1-E4 protein in human keratinocytes (the natural host cell for HPV infection) results in the total collapse of the cytokeratin matrix. Tubulin and actin networks are unaffected by E1-E4, as are the nuclear lamins.     

Keratinocytes blocked in phorbol ester-responsive early stage of terminal differentiation by sarcoma viruses

It has been suggested that the initiation step in mouse skin carcinogenesis involves an alteration in epidermal-differentiation, as mouse basal keratinocytes exposed to initiators resist the arrest of cell growth that is normally associated with the induction of terminal differentiation by calcium ions. The growth of epidermal basal cells infected by Kirsten (Ki) or Harvey (Ha) sarcoma viruses is, however, arrested in response to calcium ions, although the cells do not progress through their entire maturation programme when a functioning ras gene of those viruses is expressed. If continuous proliferation in the differentiating cell layers is a requirement for tumour formation in skin, the response of sarcoma virus-infected cells seems inconsistent with the suggestion that an activated ras gene is sufficient to initiate skin carcinogenesis. We now show that sarcoma virus-infected keratinocytes, when induced to differentiate, are blocked at an early, reversible stage of maturation. Furthermore, the cells respond to phorbol ester tumour promoters by undergoing a phenotypic reversion to a less mature stage. These results suggest that activation of a ras gene can produce conditionally initiated cells, in which the full expression of tumorigenicity depends on exposure to tumour promoters.     

Epidermal Langerhans cells are derived from cells originating in bone marrow

Langerhans cells constitute a morphologically well characterised subpopulation (3--8%) of mammalian epidermal cells which, in contrast to the bulk of epidermal cells, bear Fc-IgG and C3 receptors, express immune response-associated (Ia) antigens and function as antigen-presenting cells and allogeneic stimulatory cells to primed T lymphocytes. The ontogeny of Langerhans cells has been a subject of considerable debate since their discovery. Although some studies suggest that Langerhans cells are of mesenchymal as opposed to neural or melanocytic origin, direct evidence for this has not been presented. In this study we demonstrate that, after 3 weeks, most of the Langerhans cells (LC) in parenteral skin which had been transplanted on to F1 hybrids were of recipient origin whereas keratinocytes remained of donor origin; this indicates that the LC are derived from a mobile pool of cells. Furthermore, in studies of skin from radiation-induced bone marrow chimaeric animals we found that, depending on the strain combination, up to 80% of the epidermal LC were derived from the bone marrow of the donor animals.     

白介素24融合蛋白的表达、纯化和促伤口愈合功能的鉴定

本研究通过优化小鼠白介素24(mIL-24)基因序列,构建以Fc-tag为标签的mIL-24-Fc融合蛋白.对CHO进行MTX分级加压筛选,获得了稳定、高表达mIL-24-Fc蛋白的阳性克隆,同时优化了CHO细胞无血清驯化与稳定表达蛋白体系.经protein A亲和层析纯化mIL-24-Fc蛋白并利用AP binding实验检测生物学活性后,小鼠皮内注射mIL-24-Fc蛋白和伤口愈合实验证明:注射mIL-24-Fc蛋白后,小鼠表皮产生2~3层细胞增生,伤口愈合速度加快.     

新型雌激素受体ERα36与窖蛋白-1在皮肤组织细胞中的表达和分布特征

采用蛋白免疫印迹杂交和免疫细胞荧光等方法首次研究了新型雌激素受体ERα36和窖蛋白-1（Caveolin-1）在大鼠皮肤组织中以及角质细胞系中的表达,探讨了二者的表达关系和分布特征.结果发现：（1）大鼠皮肤中有ERα36的表达,其表达的量与Caveolin-1的表达量呈负相关,且存在性别差异和部位差异;（2）表皮角质细胞中有ERα36的表达,主要分布在细胞膜上,且与Caveolin-1共定位.提示新型雌激素受体ERα36存在于皮肤细胞中,且可能参与Caveolin-1介导的雌激素信号转导,在雌激素治疗皮肤疾病过程中发挥一定作用.     

Production and auto-induction of transforming growth factor-alpha in human keratinocytes

Transforming growth factor-alpha (TGF-alpha) is a polypeptide which is structurally related to epidermal growth factor (EGF) and binds to the EGF receptor. TGF-alpha synthesis occurs in a variety of neoplastic cells and during early fetal development but has not been reported in normal cells of the adult organisms. TGF-alpha has therefore been regarded as an embryonic growth factor which is inappropriately expressed during neoplasia. Here we report that primary cultures of normal human keratinocytes synthesize TGF-alpha. Furthermore, we show that addition of EGF or TGF-alpha to these cultures induces TGF-alpha gene expression, suggesting that a mechanism of auto-induction exists. Analysis of normal skin biopsies using in situ hybridization and immunohistochemistry demonstrates the in vivo presence of TGF-alpha messenger RNA and protein in the stratified epidermis.     

Pathogenesis and therapy of psoriasis

Psoriasis is one of the most common human skin diseases and is considered to have key genetic underpinnings. It is characterized by excessive growth and aberrant differentiation of keratinocytes, but is fully reversible with appropriate therapy. The trigger of the keratinocyte response is thought to be activation of the cellular immune system, with T cells, dendritic cells and various immune-related cytokines and chemokines implicated in pathogenesis. The newest therapies for psoriasis target its immune components and may predict potential treatments for other inflammatory human diseases.     

IkappaB kinase-alpha acts in the epidermis to control skeletal and craniofacial morphogenesis

IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.     

Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide

Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.     

端粒酶转染防治角膜内皮失代偿的研究

目的评价端粒酶转染对角膜内皮细胞形态和功能的影响。方法将体外培养的猫角膜内皮细胞随机分为3组,兔端粒酶基因转染组、正常对照组和表皮生长因子组。培养30代后观察传代细胞形态和细胞周期各时相细胞比例的变化,检测端粒酶逆转录酶蛋白和Ⅳ型胶原的表达。结果兔端粒酶基因转染组细胞数目较正常对照组和表皮生长因子组增多,伸展贴壁能力增强,表型正常,G2～M期和S期细胞比例显著增加,端粒酶逆转录酶蛋白和Ⅳ型胶原的表达也较正常对照组增多。结论兔端粒酶转染可以促进猫角膜内皮细胞增殖且表型正常,可以增强其伸展粘附能力和分泌功能,有利于防治角膜内皮失代偿。     

HSP27和CFL-1在角质细胞急性刺激反应中相关性的研究

研究急性刺激条件下,角质细胞中热休克蛋白27(heat shock protein 27,HSP27)和Cofilin-1(CFL-1)的表达是否具有相关性,初步探讨皮肤急性刺激反应的作用机理.通过免疫印迹法检测十二烷基硫酸钠(SDS)诱发的急性刺激反应下,角质细胞中HSP27和CFL-1蛋白的表达,并通过RNAi技术...     

Fibronectin inhibits the terminal differentiation of human keratinocytes

In the epidermis proliferation of keratinocytes is restricted to the basal layer, which is in contact with the basement membrane, and cells undergo terminal differentiation as they move upwards through the suprabasal layers. In stratified cultures of human keratinocytes, upward migration is a consequence, not a cause, of terminal differentiation and occurs because keratinocytes become less adhesive to their substratum and to one another. Most keratinocytes can be induced to differentiate to completion by placing them in suspension in methylcellulose: within 12 h DNA synthesis is irreversibly inhibited and by 24 h most cells express involucrin (ref 4; P. A. Hall, J.C.A. and F.M.W., unpublished observations). Here we report that when fibronectin is added to the methylcellulose, keratinocytes still withdraw from the cell cycle, but induction of involucrin expression is largely inhibited. The effect of fibronectin is concentration- and time-dependent and is mediated by a receptor of the integrin family. These results provide an explanation for why overt terminal differentiation is normally restricted to suprabasal cells, whereas cell-cycle withdrawal occurs within the basal layer; they also have important implications for the mechanism of epidermal wound healing. Furthermore, our data show that the binding of an extracellular matrix protein to its receptor can regulate differentiated gene expression in the absence of changes in cell shape.     

Enhancement of polyoma virus middle T antigen tyrosine phosphorylation by epidermal growth factor

Polyoma virus codes for three proteins involved in host cell transformation: the large, middle and small T antigens. Middle T antigen is a major transforming protein which is responsible for the induction of the phenotype of transformed cells and, without it, transformation does not occur (reviewed in refs 1-4). Middle T antigen alone can transform established cell lines, although large, and possibly small, T antigens are also required for the full expression of the phenotype of transformed cells in media with a low concentration of serum. A subfraction of middle T antigen is associated with a protein kinase activity which phosphorylates middle T antigen in vitro on tyrosine. There is a strong correlation between the level of this kinase activity and the degree of expression of the phenotype of transformed cells. We report here that epidermal growth factor (EGF) stimulates tyrosine phosphorylation of middle T antigen, suggesting the possibility that mitogenic growth factor(s) regulates this phosphorylation activity.     

蒙药治疗80例寻常型银屑病临床观察

目的：观察蒙药珍宝丸等对寻常型银屑病的疗效.方法：将80例寻常型银屑病患者随机分为治疗组和对照组各40例;治疗组口服珍宝丸等蒙药进行治疗,对照组口服银屑灵膏治疗,一个疗程21天,共4个疗程.观察皮损,记录PASI评分,评定疗效.结果：治疗组痊愈24例,显效8例,好转5例,无效3例,总有效率80.0%;对照组痊愈10例,显效16例,好转9例,无效5例总有效率65.0%.结论：蒙药治疗优于中药治疗.