银屑病角质形成细胞p14ARF基因甲基化的研究

目的:检测银屑病皮损角质形成细胞p14ARF基因启动子区甲基化,探讨其与银屑病发病的关系。方法:利用甲基化特异性PCR技术检测37例银屑病及35例正常人角质形成细胞p14ARF基因启动子区甲基化状态。结果:37例银屑病皮损角质形成细胞p14ARF基因启动子区甲基化阳性率为43.2%(16/37),显著高于正常对照(χ2=13.5,P<0.05)。结论:p14ARF基因启动子区甲基化与银屑病角质形成细胞过度增殖具有一定的关系。     

Effect of calcipotriol on epidermal cell populations in alefacept-treated psoriatic lesions

BACKGROUND AND OBJECTIVES: The effect of the established antipsoriatic treatment with topical calcipotriol (with a maximum of 100 g per week) in addition to systemic treatment with alefacept, a new biological agent for psoriasis, on epidermal cell populations in the psoriatic lesion was investigated using a combination of the Zenon labelling technique and microscopic image analysis. Epidermal cell populations were measured quantitatively with this sensitive method. PATIENTS/METHODS: Frozen sections of non-treated psoriatic epidermis and psoriatic epidermis treated with either alefacept intramuscular or alefacept intramuscular in combination with topical calcipotriol for 12 weeks were compared immunohistochemically. Antibodies against keratin 6, 10 and 15 were labelled with the Zenon technique, whereas antibodies against the Ki-67 antigen and beta-1 integrin were covalently Fluorescein Isothiocyanate (FITC)-labelled. Using image analysis, these markers were measured in the epidermis in a standardized manner. RESULTS AND CONCLUSIONS: Treatment of psoriasis with alefacept resulted in a good clinical response in several patients and in a normalization of epidermal expression of the immunohistochemical parameters for differentiation and proliferation. The addition of topical calcipotriol resulted in a faster clinical improvement with a similar overall clinical response and a similar response of epidermal cell populations as compared to treatment with alefacept monotherapy after 12 weeks of treatment. This study also suggests that the appearance of keratin 15 has a predictive value for the duration of remission. It can be concluded that the addition of a low-dose calcipotriol treatment does not contribute to the clinical efficacy of alefacept, both at the clinical level and with respect to markers for epidermal proliferation and differentiation.     

CD103+T细胞在银屑病皮损中的表达

目的：明确银屑病患者皮肤CD103+T细胞的表达及其与银屑病严重程度的关系。方法：免疫组化检测29例银屑病患者皮损和非皮损皮肤及6名健康对照皮肤中表皮及真皮CD103+T细胞的表达。计算银屑病患者PASI值。结果：CD103+T细胞主要在真皮表达。银屑病患者皮损和非皮损真皮中每个高倍视野CD103+T细胞百分率分别为（26.06%±11.72）%和（12.82±4.5）%（P<0.05）;健康人对照皮肤真皮内CD103+T细胞百分率为（7.47±1.3）%,明显低于银屑病非皮损区（P<0.05）。银屑病患者皮损中,CD103+T的表达与PASI值正相关（P<0.05）。 结论：真皮中CD103+T细胞可能与银屑病的发病及严重程度有关。     

银屑病患者角质形成细胞对T淋巴细胞CD25、CD69表达的影响

目的 探讨银屑病患者角质形成细胞（KC）对T淋巴细胞CD25、CD69表达的影响。方法 分离10例银屑病患者KC，密度梯度离心法分离单一核细胞（PBMC）；流式细胞仪检测混合培养后T细胞活化标志CD25、CD69的表达。结果 银屑病患者皮损KC作用的自体外周血T细胞CD25、CD69表达水平分别与非皮损KC作用组及自体T细胞自然增殖组相比显著增高，银屑病非皮损KC+自体T细胞共培养组与自体T细胞自然增殖组相比，差异无统计学意义。银屑病皮损、非皮损KC作用的正常人T细胞CD25、CD69表达均显著高于正常人外周血T细胞自然增殖组，银屑病皮损KC+正常人T细胞共培养组与非皮损组相比，差异无统计学意义。结论 银屑病患者局部存在慢性炎症反应，可能是由于皮损KC免疫表型发生改变从而作为自身抗原，启动自身免疫反应。     

Reduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patients

Abstract: Background: T cells have been shown to be highly relevant in psoriasis. CD26 is a novel T‐cell activation marker involved in various T‐cell functions, e.g. (i) co‐stimulation, (ii) migration and (iii) T‐cell memory response. In particular, CD26bright peripheral blood T cells have been shown to be altered in several autoimmune diseases. Objective: To characterize CD26‐expression of T‐cell subsets in psoriatic patients compared to healthy subjects. Methods: Peripheral blood was obtained from 15 untreated patients with severe psoriasis and from nine healthy subjects. The presence of specific CD26‐related T‐cell subsets was assessed by flow cytometry. Results: The CD26bright expression of CD8+ lymphocytes revealed a statistically significant (P<0.05) decrease in psoriatic patients. The majority of CD4+CD26bright cells are CD45RO+, whereas the minority of CD8+CD26bright cells are CD45RO+ in both groups. Conclusions: The present study demonstrates that the CD8CD26bright T‐cell population is markedly decreased in peripheral blood of psoriatic patients. Moreover, CD26 expression did not show a restriction to memory T cells. As CD26 is of relevance for T‐cell functions, future investigations should focus on elucidating these functions in psoriasis. It is attractive to speculate that the reduction in the CD8CD26bright subpopulation may represent a biomarker for recompartimentalization of activated T cells and a reduced CD8CD26bright count may correlate with increased responsiveness to T‐cell targeted treatments.     

Leu-11b (CD16) antigen expression on human keratinocytes

Archives of Dermatological Research -     

银屑病患者骨髓CD34+细胞定向分化的T细胞对角质形成细胞增殖的影响

目的:探讨家族史阳性的银屑病患者骨髓CD34 细胞体外定向分化的T细胞对表皮角质形成细胞(KC)增殖调控蛋白表达的影响.方法:将银屑病患者骨髓CD34 细胞体外定向分化的T细胞经链球菌超抗原活化后与KC共培养,分别采用免疫组化法及ELISA法检测KC C-myc、bcl-xL、p53及Ki67蛋白表达及培养上清白介素(IL)-8、干扰素(IFN)-γ水平.结果:①受银屑病患者CD34 细胞定向分化T细胞作用的KC C-mye及Ki67蛋白表达与自然增殖组及正常人CD34 细胞定向分化的T细胞作用组相比显著增强(P＜0.05),而bcl-xL及p53蛋白表达3组间的差异无统计学意义(P＞0.05);②受正常人CD34 细胞定向分化T细胞作用的Kc C-myc、bcl-xL、p53及Ki67蛋白表达与自然增殖组比较差异亦无统计学意义(P＞0.05);③银屑病CD34 细胞定向分化的T细胞作用组培养上清IL-8及IFN-γ水平显著高于正常对照组(P＜0.01).结论:银屑病患者骨髓造血细胞定向分化的T细胞可影响KC增殖状态,显示类似银屑病外周血T细胞的活性特点.     

Upregulation of cathepsin S in psoriatic keratinocytes

Please cite this paper as: Upregulation of cathepsin S in psoriatic keratinocytes. Experimental Dermatology 2010; 19 : e80–e88. Abstract: Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II‐mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS‐immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T‐ and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS‐immunostaining is also detectable in keratinocytes. We show that cocultivation with T‐cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T‐cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II‐associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.     

角蛋白K6、K16在寻常性银屑病皮损中的表达及其临床意义

目的：探讨角蛋白K6、K16在银屑病发病机制中的地位。方法：用免疫组化法，检测30例银屑病患者皮损处和10例正常对照者皮肤角质形成细胞中角蛋白K6和K16的表达水平。结果：①银屑病组患者皮损处表皮角质形成细胞中K6、K16的表达水平与正常对照组相比，差异具有显著性，其中，病例组棘细胞层K6、K16的表达与对照组相比，差异具有极显著性；②角蛋白K6、K16在银屑病进行期和稳定期的表达差异无显著性，但分别与消退期银屑病相比差异均具有显著性；③角蛋白K6、K16的表达在急性点滴状和慢性斑块状银屑病间差异无显著性。结论：角蛋白K6、K16在银屑病发病机制中可能起重要作用，这两个指标有望作为监测银屑病活动性的指标。     

Abnormal distribution of epidermal protein antigens in psoriatic epidermis.

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.     

5—羟色胺在银屑病患者皮损中的表达

目的：探讨5—羟色胺在银屑病发病机制中的作用。方法：用免疫组化SP法检测银屑病患者皮损中5—羟色胺(5—HT)的表达。结果：进行期银屑病患者皮损处棘细胞，汗腺腺细胞，皮脂腺细胞，毛囊壁细胞浆中5—HT表达呈中等以上阳性，静止期减弱。寻常型和脓疱型无明显差异，正常人皮肤为阴性。结论：5—HT可能有促进角质形成细胞过度增殖的作用，从而参与银屑病的发病。     

Altered expression of matrix remodelling associated 7 (MXRA7) in psoriatic epidermis: Evidence for a protective role in the psoriasis imiquimod mouse model

Preliminary data mining performed with Gene Expression Omnibus data sets implied that psoriasis may involve the matrix remodelling associated 7 (MXRA7), a gene with little function information yet. To test that hypothesis, studies were performed in human samples and murine models. Immunohistochemistry in normal human skin showed that MXRA7 proteins were present across the full epidermal layer, with highest expression level detected in the basal layer. In psoriatic samples, MXRA7 proteins were absent in the basal stem cells layer, while suprabasal keratinocytes were stained at a higher level than in normal tissues. In an imiquimod‐induced psoriasis‐like disease model in mice, diseased skins manifested similar MXRA7 expression pattern and change as in human samples, and MXRA7‐deficient mice developed severer psoriasis‐like diseases than wild‐type mice did. While levels of propsoriatic genes (eg IL17, IL22, IL23) in imiquimod‐stimulated MXRA7‐deficient mice were higher than in wild‐type mice, keratinocytes isolated from MXRA7‐deficient mice showed increased proliferation upon differentiation induction in culture. These data demonstrated that MXRA7 gene might function as a negative modulator in psoriasis development when propsoriatic factors attack, presumably via expression alteration or redistribution of MXRA7 proteins in keratinocytes.     

寻常型银屑病皮损肥大细胞密度及IL-8表达的研究

目的：探讨肥大细胞和IL-8在寻常型银屑病中的作用。方法：采用免疫组化技术(SABC法)观察寻常型银屑病皮损处MC分布和IL-8在表皮中的表达情况。结果：寻常型银屑病皮损处MC密度明显高于皮肤血管炎组和健康对照组；寻常型银屑病皮损处KC中IL-8的表达明显高于两对照组。结论：结果提示MC和IL-8参与寻常型银屑病的致病过程，并且两者之间存在相关性。     

银屑病患者皮损局部朗格汉斯细胞数量异常机制的研究

目的：探讨银屑病患者皮损局部朗格汉斯细胞(LCs)数量异常的原因。方法：培养银屑病患者皮损处角质形成细胞，通过微孔小室实验检测其上清液对单核细胞的趋化功能；通过酶联免疫吸附法(ELISA)检测上清液中单核细胞趋化蛋白—1(MCP-1)的表达。结果：银屑病患者皮损处角质形成细胞分泌上清液对单核细胞的趋化能力明显强于正常对照组；其分泌的MCP-1水平也高于正常人。结论：银屑病角质形成细胞表达的趋化因子趋化了更多数量的单核细胞至皮损局部，因此，银屑病患者皮损局部LCs的数量理应是增多的。但由于银屑病角质形成细胞表达的其它一些因子也促使了单核细胞衍生的LCs的活化，活化的LCs会迁移至淋巴结或活化后凋亡，又导致其数量减少。因此，银屑病患者皮损局部朗格汉斯细胞数量是一动态变化过程。     

Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis

BACKGROUND: Tumour necrosis factor (TNF)-alpha blockade using infliximab, a chimeric anti-TNF-alpha antibody, is an effective treatment for plaque-type psoriasis, inducing remission in about 80% of patients. OBJECTIVES: To examine infliximab-induced programmed cell death (PCD) of keratinocytes in psoriatic plaques on serial skin biopsy samples. METHODS: Five patients with moderate to severe plaque-type psoriasis received infliximab infusions intravenously (5 mg kg(-1)) at weeks 0, 2 and 6. Biopsies of nonlesional and lesional skin (days 0, 5, 14 and 21) were obtained. Conventional microscopy was used to examine the morphology of the psoriatic keratinocytes. In situ detection of apoptosis was performed by electron microscopy and by immunohistochemical staining with anti-p53 and anti-caspase-3 antibodies. Results Infusion of infliximab induced a clinical response in all five patients with psoriasis, with a mean Psoriasis Area and Severity Index improvement of 24.8% already at day 5. This was accompanied by significant histopathological changes in the skin biopsy samples after infliximab treatment. Light and electron microscopic evaluation revealed apoptosis-like morphological changes in lesional keratinocytes, i.e. nuclear condensation, chromatin fragmentation and cytoplasmic vesiculation, visible already after the first infusion. These damaged keratinocytes stained positively for p53, but not for active caspase-3. CONCLUSIONS: The effects of infliximab in psoriasis extend beyond merely anti-inflammatory actions, and may include caspase-independent PCD of lesional keratinocytes. The PCD of keratinocytes may be an important mechanism that could explain at least in part the rapid and sustained therapeutic effect of infliximab in psoriasis.     

Preferential expression of alphaEbeta7 integrin (CD103) on CD8+ T cells in the psoriatic epidermis: regulation by interleukins 4 and 12 and transforming growth factor-beta

BACKGROUND: Intraepidermal T lymphocytes are a critical element for sustaining the lesional pathology of psoriasis. Integrin alphaEbeta7 (CD103), a ligand for E-cadherin, may play a role in the localization of pathogenic T cells within the epidermis of psoriatic lesions. However, little information is available regarding alphaEbeta7 expression on intraepidermal T cells in psoriasis. OBJECTIVES: To examine alphaEbeta7 expression on intraepidermal T cells in psoriatic lesions and the regulation of alphaEbeta7 expression on T cells in response to cytokines. METHODS: T-cell expression of alphaEbeta7 was examined by immunohistochemistry and flow cytometry. In vitro regulation of alphaEbeta7 expression on CD4+ or CD8+ T cells purified from peripheral blood of healthy donors was also examined. RESULTS: Immunohistochemical staining revealed expression of alphaEbeta7 on a greater proportion of epidermal T cells than dermal T cells. Nearly 30% of intraepidermal CD4+ T cells were found to express alphaEbeta7 on flow cytometry, whereas more than 80% of intraepidermal CD8+ T cells expressed this integrin. In contrast, few T cells expressed alphaEbeta7 in the peripheral blood of psoriatic patients. The in vitro culture experiment confirmed that alphaEbeta7 was preferentially expressed on CD8+ T cells after stimulation with anti-CD3 monoclonal antibodies. Addition of transforming growth factor-beta and interleukin-4 upregulated alphaEbeta7 expression on T cells, whereas interleukin 12 downregulated this. Furthermore, alphaEbeta7 expression on established memory CD8+ T cells was not so reversible as that on CD4+ T cells. CONCLUSIONS: Preferential and stable expression of alphaEbeta7 on CD8+ T cells may be involved in the lesional pathology of psoriasis.     

Isolation of plasma membranes from keratinocytes: A newly developed method allows the sensitive detection of the membrane antigen pattern in normal and psoriatic skin

Distinct differences of proteins in the plasma membranes have been described in psoriatic keratinocytes as compared with normal epidermis. These changes are presumably involved in the pathogenesis of psoriasis. To identify and distinguish glycosylated proteins of the plasma membranes of keratinocytes, a method for mild preparation was developed that avoids the use of degrading or digestive enzymes. After cell lysis, three steps of centrifugation were performed, including the use of a sucrose step gradient. A fine-vesicular membrane fraction was obtained. Using marker enzymes for cell compartments, no contamination of cell nuclei or mitochondria and only 0.4% of endoplasmic reticulum was detectable in the final membrane fraction. Based on this preparation, disc-polyacrylamide-gel electrophoresis, followed by Western blotting, were performed. Staining with five different lectins to visualize glycosylated proteins allowed 75 different membrane glycoproteins to be distinguished. The patterns of normal, transformed (HaCaT), foreskin and psoriatic keratinocytes after cell culture with one passage were compared. Up to six proteins per lectin staining were expressed differently in psoriatic as compared to normal keratinocytes. Psoriatic cells shared similarities with highly proliferative foreskin cells, but not with transformed HaCaT cells. Main alterations of glycosylation were detected in the fucose content. In conclusion, the method developed for isolation of plasma membranes allows selective and sensitive examination of plasma membranes of normal and pathological keratinocytes. The glycosylation patterns observed suggest that distinct membrane proteins may be involved in the pathogenesis of psoriasis.     

Perforin expression is upregulated in the epidermis of psoriatic lesions

BACKGROUND: There are currently very few data regarding the role of cell-mediated cytotoxicity in psoriasis. Both cytotoxic T lymphocytes and natural killer (NK) cells mediate cytotoxicity reactions, mainly by two distinct pathways, the perforin/granzyme and the Fas/Fas ligand pathway. OBJECTIVES: To study the expression and distribution of perforin, T- and NK-cell subsets in psoriatic lesional and nonlesional skin. METHODS: Skin biopsy specimens from both lesional and nonlesional skin of 11 patients with chronic plaque psoriasis and eight healthy controls were analysed by immunohistochemistry. RESULTS: We found a significant increase in CD4+ and CD8+ cells in psoriatic lesions compared with nonlesional and healthy skin. The expression of CD16+ NK cells was significantly lower in lesions compared with healthy skin. Perforin expression was significantly enhanced in the epidermis of psoriatic lesions. CONCLUSIONS: Perforin expression is upregulated in the epidermis of psoriatic lesions, suggesting a potential role for perforin in the creation of the psoriatic plaque.