特应性皮炎患者趋化因子及其受体的研究

目的 探讨几种重要的趋化因子及其受体的表达在特应性皮炎(AD)发病中的作用。方法 采用酶联免疫吸附试验检测39例AD患者及正常人血清中γ干扰素诱导蛋白-10(IP-10)、基质细胞衍生因子-1α(SDF-1α)、嗜酸粒细胞趋化因子、胸腺和活化调节的趋化因子(TARC)及巨噬细胞来源的趋化因子(MDC)等水平;同时用流式细胞仪分析外周血CD4⁺T细胞表面趋化因子受体CXCR3、CXCR4、CCR3、CCR4及CCR5的表达。结果 与正常人对照组相比,AD患者血清SDF-1α、TARC和MDC水平显著升高(P<0.001),IP-10及嗜酸粒细胞趋化因子水平则无明显改变(P>0.05),外周血CXCR3、CCR3、CCR4及CCR5在CD4⁺T细胞表达水平显著增加(P<0.001);血清TARC和MDC水平的变化与疾病严重程度相关(r分别为0.669及0.409,P分别为<0.001及<0.01)。结论 具有生物活性趋化因子及其受体介导的T细胞和嗜酸/嗜碱粒细胞的聚集、激活后释放的炎性介质在AD发病中起着重要作用。     

EB病毒感染与系统性红斑狼疮相关性的研究

目的 探讨EB病毒感染与系统性红斑狼疮发生、发展的关系.方法 用流式细胞仪双色解析法检测SLE患者外周血CD19⁺、CD23⁺B淋巴细胞中EB病毒潜伏膜蛋白LMP1和复制期蛋白ZEBRA的表达.结果 活动期患者CD19⁺细胞和CD23⁺细胞LMP1的表达率均高于稳定期患者,两组患者均高于对照组,且患者CD23⁺细胞LMP1的表达率高于CD19⁺细胞,而对照组两者差异无显着性;ZE-BRA的表达仅见于SLE患者外周血B淋巴细胞中,活动期患者CD19⁺、CD23⁺细胞ZEBRA的表达率均高于稳定期患者,且在活动期患者中,CD23⁺细胞ZEBRA的表达率高于CD19⁺细胞,而在稳定期患者中,CD23⁺细胞和CD19⁺细胞ZEBRA的表达率差异无显着性.结论 EB病毒的感染和复制与B淋巴细胞的活化及SLE的发生和发展有关.     

CCL27和CCR10在寻常性银屑病皮损的表达及与PASI的相关性

目的 探讨皮肤特异性趋化因子CCL27及其受体CCR10在寻常性银屑病皮损中的表达和表达强度与疾病活动性的相关性.方法 SP免疫组化染色检测20例寻常性银屑病皮损区皮损和10例正常人皮肤中CCL27、CCR10的表达,以PASI评分评价银屑病患者疾病活动性.结果 ①CCL27在20例寻常性银屑病皮损和10例正常人皮肤中表达的阳性率分别为95%和20%,两者差异有统计学意义(X²=17.86,P<0.01);CCR10在20例寻常性银屑病皮损和10例正常人皮肤表达的阳性率分别为85%和10%,两者的差异有统计学意义(X²=15.63,P<0.01).②CCL27和CCR10在皮损中的表达水平与PASI评分均呈明显正相关,r值分别为0.82和0.83,P值均<0.01.结论 CCL27和CCR10的过度表达与银屑病患者的疾病活动性相关.     

钙泊三醇外用治疗可减少银屑病皮损中ICAM-3阳性浸润细胞的表达

目的 了解钙泊三醇局部外用治疗对银屑病皮损中细胞间粘附分子3(ICAM-3)表达的影响.方法 采用ABC免疫组化法对20例银屑病患者皮损与非皮损部位以及皮损部位治疗前后的ICAM-3、Ki-67和其它免疫分子的表达进行了研究.结果 银屑病皮损部位ICAM-3阳性浸润细胞的表达均明显高于非皮损部位及正常对照组(P均<0.01), 而且ICAM-3的表达和皮损部位Ki-67.CD3等免疫分子的表达呈正相关.经钙泊三醇外用治疗6周后、皮损部位ICAM-3、Ki-67和CD3的表达均明显减少(P均<0.01).结论 外用钙泊三醇治疗银屑病的机制除了有抗角朊细胞增殖的作用外, 对可能参与银屑病发病机制的ICAM-3阳性浸润细胞也有作用.     

趋化性细胞因子受体CCR4和CXCR3在特应性皮炎患者外周血的表达

目的为研究特应性皮炎患者外周血趋化性细胞因子受体CCR4和CXCR3在特应性皮炎的发病过程中的作用。方法采用三色流式细胞仪测定20例特应性皮炎患者和30例健康对照者外周血趋化性细胞因子受体CCR4和CXCR3的表达水平。结果特应性皮炎患者外周血CCR4+CD4+T细胞的水平明显高于对照组(P<0.01);特应性皮炎患者外周血CCR4/CXCR3比率明显高于对照组P<0.01);特应性皮炎患者外周血CXCR3+CD4+T细胞的水平与对照组差异无统计学意义。结论趋化性细胞因子受体CCR4可能促进了Th2细胞从血液进入特应性皮炎患者炎症皮损。     

寻常型银屑病皮肤中皮肤归巢T细胞免疫组化研究

目的 探讨皮肤归巢T细胞在寻常型银屑病(PV)发病中的作用。方法 采用间接免疫荧光双标法研究正常人皮肤和PV患者皮肤中浸润的皮肤归巢T细胞分类及其变化。结果 ①正常人皮肤及PV皮损中浸润的T细胞绝大多数表达皮肤淋巴细胞相关抗原(CLA),CLA⁺细胞高度表达CD45RO,只有个别为CD45RO阴性。②进行期皮损CD4⁺CLA⁺及CD8⁺CLA⁺T细胞数高于静止期皮损(P<0.05),静止期皮损CD4⁺CLA⁺细胞数高于消退期皮损(P<0.05),消退期皮损CD8+CLA+细胞数高于PV外观正常皮肤(P<0.05),进行期皮损周边外观正常皮肤CD4⁺CLA⁺及CD8⁺CLA⁺细胞数高于静止期皮损周边外观正常皮肤(P<0.05).③部分病例皮损的表皮中见大量CLA⁺树突状细胞,正常人皮肤未见此细胞。结论 正常人皮肤及PV皮损中T细胞绝大多数为皮肤归巢T细胞;进行期及静止期PV皮损中浸润的细胞主要为CD3⁺、CD4⁺、CD45RO⁺、CLA⁺T细胞,CD3⁺、CD4⁺、CD45RO⁺、CLA⁺T细胞可能在PV发病中起重要作用。     

银屑病患者中性粒细胞中CXC型趋化因子受体CXCR1、CXCR2 mRNA的表达

目的 了解银屑病患者外周血中性粒细胞中CXC型趋化因子受体CXCR1及CXCR2的表达情况。方法 应用逆转录-聚合酶链反应(RT-PCR)法检测了进行期斑块状银屑病患者及健康人对照各30例(其中治疗后患者14例)外周血中性粒细胞中CXCR1及CXCR2mRNA的表达,并将结果与患者皮损面积及严重程度指数(PASI)进行了相关性分析。结果 斑块状银屑病患者外周血中性粒细胞中CXCR1及CXCR2mRNA表达水平分别为1.30±1.18和1.62±0.97,明显高于正常人对照(分别为0.56±0.36、0.74±0.58,P<0.01),并随治疗后患者PASI评分的下降而降低,治疗后CXCR1、CXCR2分别为0.49±0.34、0.51±0.51,(P<0.01),银屑病患者外周血中性粒细胞中CXCR2mRNA水平高于CXCR1mRNA水平,二者均与PASI呈显著正相关。CXCR1:r=0.60,P<0.001;CXCR2:r=0.84,P<0.001。结论 银屑病患者外周血中性粒细胞中增高的CXCR1与CXCR2可能与白细胞向皮损部位的移行和聚集有关,CXCR1及CXCR2参与了银屑病的发病机制。     

蕈样肉芽肿中恶性T细胞亲表皮性机制研究进展

皮肤T细胞淋巴瘤中最常见的蕈样肉芽肿,早期可见不同程度的亲表皮现象,即恶性T细胞不同程度侵入表皮,对诊断有较大提示意义,当病情进展到肿瘤期时可逐渐消失.目前对此现象产生和维持的机制研究主要集中在以下两个方面,一为角质形成细胞表达的趋化因子及淋巴细胞表面的趋化因子受体相互作用,如IP-10/CXCR3、SDF-1/CXCR4、TARC/CCR4等,二为表皮内的朗格汉斯细胞及调节性T细胞的相互作用.

Abstract：

Mycosis fungoides is the most common cutaneous T cell lymphoma.Epidermotropism,manifested as the infiltration of epidermis with atypical CD4+ T lymphocytes, may be observed at the early stage of mycosis fungoides, and disappear at the tumor stage.Current studies concerning epidermotropism in mycosis fungoides are mainly focused on the following two aspects, i.e., the interaction of chemokines expressed by keratinocytes with their receptors on the surface of lymphocytes, such as IP-10/CXCR3, SDF-1/CXCR4, TARC/CCR4, as well as the interaction between intraepithelial Langerhans cells and regulatory T cells.     

维生素D3抑制角质形成细胞CXCR2的表达

目的 观察维生素D3对角质形成细胞表面表达CXCR2的影响,探讨其在治疗银屑病中的作用机制。方法 用不同浓度的维生素D3处理人角质形成细胞株HaCaT,48h后通过四甲基偶氮唑盐(MTT)法检测了维生素D3抑制HaCaT增殖的有效剂量;通过流式细胞仪(FACS)分析维生素D3处理的HaCaT表达趋化因子受体CXCR2的情况。结果 HaCaT经维生素D3在8.0×10^-8mol/L～5.12×10^-6mol/L浓度范围内处理48h后的A值较未处理组明显降低(P<0.05).HaCaT在5.12×10^-6mol/L维生素D3处理48h后CXCR2的表达明显低于正常对照组。结论 维生素D3治疗银屑病的作用机制部分是通过抑制角质形成细胞表达CXCR2,从而达到抑制角质形成细胞增殖的目的。     

神经性皮炎皮损处神经纤维的数量及其与朗格汉斯细胞关系的研究

目的探讨神经性皮炎患者皮损处神经纤维的数量变化及其与朗格汉斯细胞接触的关系.方法用辣根过氧化物酶结合的链霉亲和素-生物素技术观察24例神经性皮炎患者皮损处神经纤维的表达及数量变化.用免疫荧光双标记及共聚焦激光扫描显微镜技术观察神经性皮炎患者皮损处神经纤维与朗格汉斯细胞接触数量关系.用实时定量PCR方法检测皮损处神经生长因子mRNA的表达情况.结果 神经性皮炎患者皮损处神经纤维长度显著增加,与皮损周边对照(t=6.90,P<0.001)及正常人对照(t=5.71,P<0.001)比较差异有统计学意义.皮损表皮内有神经纤维接触的朗格汉斯细胞占朗格汉斯细胞总数的百分数明显增多,与皮损周边对照(X²=43.91,P<0.001)及正常人对照(X²=46.11,P<0.001)比较差异有统计学意义.皮损处神经生长因子mRNA高表达,与皮损周边对照(t=3.25,P<0.01)及正常人对照(t=3.67,P<0.01)比较差异有统计学意义.结论 神经性皮炎患者皮损处存在高水平活性的NGF导致神经纤维增生.     

CC chemokine receptor 4 expression on peripheral blood CD4+ T cells reflects disease activity of atopic dermatitis

Recent studies indicate that Th1 and Th2 cells differ in their chemokine receptor expression and their responsiveness to various chemokines. Therefore, selective Th2 cell recruitment in Th2-predominant inflammatory diseases such as atopic dermatitis may be under the influence of some chemokines. It is reported that CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells whereas CXC chemokine receptor (CXCR) 3 is selectively expressed on Th1 cells. In this study we examined CCR4 and CXCR3 expression on peripheral blood CD4+ and CD8+ T cells obtained from adult atopic dermatitis subjects, and compared the results with those from patients with psoriasis vulgaris and healthy controls. CCR4 was preferentially expressed on CD4+ T cells from atopic dermatitis subjects and CXCR3 was preferentially expressed on CD4+ T cells from psoriasis vulgaris subjects. This CCR4 expression was prominent especially in severe atopic dermatitis subjects. CCR4 expression on CD4+ T cells in severe atopic dermatitis subjects decreased on improvement of disease activity. CD25 was preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. Cutaneous lymphocyte-associated antigen was also preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. CD4+ T cells in atopic dermatitis skin lesions were predominantly CCR4+ cells. Taken together, this study strongly indicates that CCR4+CD4+ T cells reflect disease activity and suggests that CCR4 expression is important for T cell infiltration into atopic dermatitis lesions. Thus, CCR4 may be a possible target for therapy of atopic dermatitis in the future.     

The role of chemokines in allergic contact dermatitis

Chemokines are important mediators of immune-mediated skin diseases. Allergic contact dermatitis (ACD) is the most thoroughly investigated T cell-mediated disorder because of the ability to easily reproduce the lesions in humans and the availability of an excellent mouse model. Migration of dendritic cells from the skin to lymph nodes is absolutely required for induction of hapten sensitization, and depends upon expression of CCR7 by mature dendritic cells and SLC in the lymph nodes. During expression of ACD, recruitment of T lymphocytes is driven by chemokines exposed on the surface of endothelial cells or released by activated resident skin cells such as mast cells, fibroblasts and keratinocytes. Chemokines are produced in a coordinated and sequential manner, with IL-8 and RANTES induced by TNF-alpha during early stages, and MCP-1, IP-10, Mig, I-TAC, I-309 and MDC induced by IFN-gamma during later stages. Infiltrating monocytes, dendritic cells and T cells are additional sources of chemokines for further leukocyte accumulation. Distinct T cell subsets express different chemokine receptors, with type 2 cells mostly attracted by eotaxin, MDC, TARC and I-309, and type 1 cells sensitive to IP-10, Mig, I-TAC, RANTES and MIP-1beta. MCP-1 is effective on both subsets. T regulatory cells, which inhibit dendritic cell function and are probably involved in the termination of ACD, are sensitive to MCP-1, MIPs and TARC, but express high levels of CCR8 and are more specifically attracted by I-309. Targeting chemokines and chemokine receptors may offer new opportunities for therapeutic interventions in ACD and other chronic inflammatory skin diseases.     

The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions.

Differentiation between allergic and irritant contact dermatitis reactions is difficult, as both inflammatory diseases are clinically, histologically, and immunohistologically very similar. Previous studies in mice revealed that the chemokine IP-10 is exclusively expressed in allergic contact dermatitis reactions. In the present study, we investigated whether the mRNA expression of IP-10 and the related CXCR3 activating chemokines, Mig and IP-9 are also differentially expressed in human allergic contact dermatitis and irritant contact dermatitis reactions. Skin biopsies from allergic (13 cases) and sodium lauryl sulfate-induced irritant patch test reactions (13 cases), obtained 1-72 h after patch testing, were studied by means of an in situ hybridization technique. Results of chemokine mRNA expression were correlated with clinical scoring, histology, and immunohistochemical data including the proportion of inflammatory cells expressing CXCR3, the receptor for IP-10, Mig, and IP-9, and ICAM-1 and HLA-DR expression on keratinocytes. IP-10, Mig, and IP-9 mRNA were detected in seven of nine allergic contact dermatitis reactions after 24-72 h, but not in sodium lauryl sulfate-induced irritant contact dermatitis reactions. ICAM-1 expression by keratinocytes was only found in allergic contact dermatitis reactions and correlated with chemokine expression. Moreover, up to 50% of the infiltrating cells in allergic contact dermatitis expressed CXCR3, in contrast to only 20% in irritant contact dermatitis reactions. In conclusion, we have demonstrated differences in chemokine expression between allergic contact dermatitis and irritant contact dermatitis reactions, which might reflect different regulatory mechanisms operating in these diseases and may be an important clue for differentiation between allergic contact dermatitis and irritant contact dermatitis reactions.     

皮肤淀粉样变性皮损中T/B细胞的检测

目的 探讨原发性皮肤淀粉样变性皮损中T/B细胞变化的差异及其意义.方法 60例患者局部病变皮损标本、29例正常人皮肤标本,分别进行CD3、CD4、CD8、CD20免疫组化染色.结果 与正常人对照组比较,皮肤淀粉样变性皮损中,淋巴细胞总数、CD3⁺T细胞、CD8⁺T细胞的均数增高(P<0.05),CD4⁺T细胞均数无明显增高(P>0.05),CD4⁺/CD8⁺T细胞比值下降(P<0.05),CD20⁺B细胞均数增高(P<0.05).T淋巴细胞、B淋巴细胞在原发性皮肤淀粉样变性苔藓样、斑疹型或双相型三种分型病变间相互比较,差异无统计学意义(P>0.05).结论 原发性皮肤淀粉样变性局部皮损存在有T/B淋巴细胞的异常表达,但三种病变类型间病变机制的免疫学异常无区别.     

Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3)

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.     

Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10

BACKGROUND: Whereas T lymphocytes are widely accepted as effector cells determining the pathogenesis of allergic contact dermatitis, contradictory results have been found regarding the roles of different T-cell subsets. The use of various experimental models, involving long-term cultured T-cell lines or clones, may explain these contradictory results. OBJECTIVE: To investigate the involvement of distinct T-cell subsets in patients with nickel contact allergy. METHODS: Different T-cell subsets were directly isolated from peripheral blood mononuclear cells (PBMCs) of nickel-allergic patients, and their proliferative capacity, type-1 or type-2 cytokine secretion [measured by interferon (IFN)-gamma or interleukin (IL)-5 release] and phenotypical marker expression were analysed after stimulation with nickel. RESULTS: Only CD4+ CLA+ CD45RO+ and not CD8+ T cells proliferate and produce both type-1 (IFN-gamma) and type-2 (IL-5) cytokines in response to nickel. Moreover, cells expressing the marker CLA in combination with CD4, CD45RO or CD69 are increased after nickel-specific stimulation. Interestingly, in addition, CD45RA+ CLA+ cells showed an increased frequency after allergen-specific stimulation. Analysis of nickel-reactive T cells for expression of distinct chemokine receptors showed that both proliferative capacity and cytokine production are restricted to subsets expressing CXCR3, CCR4 but not CCR6. Fluorescence-activated cell sorting analysis of chemokine receptors expressed on nickel-stimulated T cells confirmed these results; a subset of T cells expressing CLA and CXCR3, CCR4 and, most importantly, CCR10 increased in response to allergen, while these CLA+ nickel-reactive T cells were all negative for CCR6. CONCLUSIONS: These findings demonstrate that freshly isolated nickel-reactive T cells can be characterized as CD4+ CLA+ memory T cells which express the chemokine receptors CXCR3, CCR4 and CCR10, but not CCR6.