IL-13-stimulated human keratinocytes preferentially attract CD4+CCR4+ T cells: possible role in atopic dermatitis

Skin inflammation in atopic dermatitis (AD) is characterized by the predominant infiltration of T-helper (Th)2-cells in lesional skin. However, the mechanism of recruitment of these cells in lesional skin of AD is not yet fully elucidated. In this study, we investigated the role of IL-13-stimulated human primary keratinocytes (HPKs) in the recruitment of lymphocytes and further delineated the mechanism of enrichment of these cells. In the migration assays, we observed preferential enrichment of CD4(+)CCR4(+) T cells towards IL-13-stimulated HPKs. Interestingly, CD4(+)CCR4(+) T cells from AD showed a higher chemotactic response than those from healthy individuals. We observed a significant increase in the expression of CCL22 in IL-13-stimulated HPKs as compared to unstimulated cells. Blocking of CCL22 in IL-13-stimulated HPKs by a neutralizing antibody resulted in 70-90% inhibition in migration of CD4(+)CCR4(+) T cells. Moreover, IL-13 upregulated IFN-gamma-induced chemokines, CCL2 and CCL5, in HPKs. Taken together, our data suggest that IL-13-stimulated HPKs participate in a positive feedback loop by preferentially enriching Th2-cells in lesional skin of acute AD patients. However, in chronic phase, IL-13 may act in synergy with IFN-gamma resulting in lymphocytes recruitment of a mixed phenotype at the site of inflammation, thus contributing to the chronification of eczema.     

天疱疮患者外周血CD4+ T细胞活化状态及其表面共刺激分子的表达

【摘要】 目的 探讨天疱疮患者和健康人外周血CD4+ T细胞的活化状态及其表面共刺激分子的表达情况。 方法 收集天疱疮患者90例,其中初发24例,稳定期51例,复发期15例;健康对照30例。运用流式细胞仪检测天疱疮患者外周血CD4+ T细胞的活化标志CD69及其表面共刺激分子,包括细胞间黏附分子-1（ICAM-1）、诱导性协同刺激分子（ICOS）、CD40配体（CD40L）和OX40的表达情况,并和健康对照组进行比较。用Graphpad 5.0软件进行统计分析,两组间比较采用非参数（Mann-Whitney test）检验。 结果 天疱疮患者外周血CD4+ T细胞表面CD69表达比例为（2.46 ± 0.19）%,显著高于健康对照组［（1.26 ± 0.19）%,P < 0.01］,且初发、稳定期和复发期天疱疮患者CD4+ T细胞表面CD69的表达均显著高于健康对照组（P < 0.01）,但不同病期患者间差异无统计学意义。天疱疮患者CD4+ T细胞表面ICAM-1、CD40L和OX40的表达比例分别为（55.88 ± 1.67）%、（2.23 ± 0.22）%和（2.55 ± 0.29）%,分别显著高于健康对照组［（47.75 ± 2.52）%、（0.73 ± 0.07）%和（0.62 ± 0.17）%,P < 0.05或0.01］,但在初发、稳定期和复发期患者之间差异无统计学意义;ICOS的表达在初发患者中显著高于健康对照组,分别为（3.73 ± 0.60）%和（2.39 ± 0.16）%（P < 0.05）。 结论 天疱疮患者外周血CD4+ T细胞处于相对活化的状态,其表面的共刺激分子也呈高表达,表明CD4+ T细胞可能通过高表达的共刺激分子和B细胞相互作用,参与疾病的发生和发展。     

CD4+辅助性T细胞在特应性皮炎的研究进展

特应性皮炎是慢性复发性炎症性皮肤病,皮肤屏障功能破坏、金黄色葡萄球菌定植造成的持续抗原刺激、免疫细胞调节功能失衡是其发病的关键因素.免疫细胞调控网络结构复杂,其通过细胞因子合成分泌的相互调节、受体表达的相互调控、生物学效应的相互影响发挥重要作用.关于CD4+辅助性T细胞在特应性皮炎发病机制中的作用,近年来基础及临床研究均有进展.     

系统性硬皮病CD4+T细胞中CD70mRNA及蛋白表达水平的观察

目的:研究系统性硬皮病(SSc)患者外周血CD4+T细胞中CD70的表达水平.方法:应用密度梯度离心法分离17例SSc患者(女性12例,男性5例)和13例对照者(女性8例,男性5例)的外周血单个核细胞,磁珠分选CD4+T细胞.RT-PCR检测CD4+T细胞中CD70 mRNA的表达水平;流式细胞术检测CD70蛋白在CD4+T细胞的表达水平.结果:与对照组相比,SSc患者CD4+T细胞中CD70 mRNA和CD70蛋白表达均明显升高(P=0.001;P=0.007).结论:CD70在SSc的发生发展中可能起着重要作用.     

特应性皮炎患儿血清和CD4+CD25+ T细胞分泌IL-10水平的检测

目的 探讨儿童特应性皮炎血清和CD4+CD25+T分泌细胞因子IL-10表达,分析其与病程及严重程度的相关性。 方法 特应性皮炎患儿按SCORAD指数分3组,轻度10例、中度16例、重度20例。抽取46例特应性皮炎患儿和31例健康对照儿外周血,用免疫磁珠分离获得CD4+CD25+T细胞,用ELISA法分别检测患病组和健康对照组血清及CD4+CD25+T细胞培养液IL-10水平,并分析IL-10水平与SCORAD评分的相关性。结果 轻、中、重度AD各组血清IL-10水平分别为（43.10 ± 25.07）、（68.40 ± 36.65）、（55.55 ± 41.97） pg/ml,与健康对照组（58.27 ± 36.84） pg/ml比较,差异均无统计学意义（P ＞ 0．05）,与患儿SCORAD评分无相关性。轻、中、重度AD组CD4+CD25+T细胞分泌IL-10含量分别为（52.96 ± 11.69）、（49.86 ± 9.18）、（27.25 ± 7.01） pg/ml,重度AD组低于健康对照组（55.15 ± 11.15） pg/ml （P < 0．05）;轻、中度组与健康对照组差异无统计学意义（P ＞ 0．05）。CD4+CD25+T细胞分泌IL-10的水平与患儿疾病严重程度SCORAD评分呈明显负相关（r值分别为-0．757,P < 0．01）。结论 CD4+CD25+T细胞及相关因子IL-10可能参与儿童特应性皮炎的发病。     

Severe atopic dermatitis is associated with a reduced frequency of IL-10 producing allergen-specific CD4+ T cells

BACKGROUND: Several studies have investigated levels of T-cell-derived interleukin (IL)-10 in individuals with atopic dermatitis, with conflicting results. AIMS/HYPOTHESIS: In order to address whether stratification of disease severity may help resolve the different findings, the hypothesis was tested that individuals with severe atopic dermatitis have a lower frequency of circulating IL-10-producing, allergen-specific CD4+ T cells than do individuals with mild disease. METHODS: Using peripheral blood mononuclear cells derived from individuals with severe (n=12) and mild atopic dermatitis (n=10) and from nonatopic controls (n=10), we investigated production by CD4+ T cells of tumour necrosis factor (TNF)-alpha, IL-4, IL-5, IL-13 and IL-10 in response to phorbol myristate acetate/ionomycin and Der p1 allergen. RESULTS: It was observed that there were significantly higher frequencies of allergen-specific circulating CD4+ T cells producing TNF-alpha- IL-4-, IL-5- and IL-13, and lower frequencies of these cells producing IL-10 in individuals with severe atopic dermatitis compared with mildly affected individuals and nonatopic controls (P<0.01 for all comparisons). Furthermore, the Der p1-specific CD4+ T cells were enriched within the subset of cells positive for cutaneous lymphocyte-associated antigen. CONCLUSIONS: Analysis of levels of allergen-specific CD4+ T-cell production of IL-10 in relation to disease severity argues in favour of a role for IL-10 in the control of atopic dermatitis.     

Increased frequency of intracellular interleukin (IL)-13 and IL-10, but not IL-4, expressing CD4+ and CD8+ peripheral T cells of patients with atopic dermatitis

BACKGROUND: A number of studies exist demonstrating the increased expression of type 2 cytokines and decreased capacity to produce interferon-gamma (IFN-gamma) in peripheral blood mononuclear cells (PBMCs) of patients with atopic dermatitis (AD). OBJECTIVES: To clarify the results of recent studies concerning the role of interleukin (IL)-4 and IL-13 in PBMCs of AD patients, we analysed the activation status of lymphocyte subpopulations. METHODS: We measured the intracellular expression and serum levels of certain type 1 and type 2 cytokines, using cell surface and intracellular cytokine staining, flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: The frequency of IL-10 and IL-13 producing CD4+ and CD8+ T cells was significantly higher in patients with AD, while the frequency of IFN-gamma secreting helper and cytotoxic T cells was significantly lower in patients with AD than in control subjects. The serum levels of IL-10 and IL-13 were also significantly increased. There were no significant differences observed between the experimental groups in the frequency of IL-4 producing CD4+ and CD8+ cells. CONCLUSIONS: This study demonstrates a type 2 cytokine production in the CD4+ and CD8+ T cells of AD patients, which is characterized by an elevated IL-13, but not by IL-4 secretion, and by an increased level of the immunoregulatory IL-10, which can contribute to a decrease in IFN-gamma expression.     

High frequency of IL-4-producing CD4+ allergen-specific T lymphocytes in atopic dermatitis lesional skin.

In atopic dermatitis (AD) hypersensitivity reactions to allergens are commonly observed and are assumed to make a major contribution in the pathomechanism of the disease. It may be expected that allergen-reactive Th cells play a central role in these reactions. In the present study the occurrence and function of allergen-specific T lymphocytes in dermal inflammatory lesions were studied. To this aim panels of randomly cloned CD4+ T cells from lesional skin biopsies of two housedust mite Dermatophagoides pteronyssinus (Dp)-allergic AD patients were screened for reactivity with Dp allergens. The results were compared with similar tests for Dp reactivity of T-lymphocyte clones (TLC) from the peripheral blood of these patients. In the panels of TLC generated from lesional skin (S-TLC), a considerable number of TLC appeared to be Dp-specific, 47% (n = 17) and 10% (n = 29), respectively. In the panels from the peripheral blood, the percentages of Dp-specific TLC were only 0% (n = 22) and 3% (n = 34), suggesting accumulation or expansion of these T cells in lesional skin. The function of these TLC was studied by assaying the secretion of IL-4 and IFN-gamma, which have been shown to be produced in aberrant ratios by Dp-specific TLC from the peripheral blood of AD patients (Wierenga et al: J Immunol 144:4651, 1990). All Dp-specific S-TLC produced IL-4 in combination with no or low levels of IFN-gamma, whereas many of the non-Dp-specific S-TLC and blood-derived TLC (B-TLC) were observed to produce high levels of IFN-gamma without significant amounts of IL-4. A functional consequence of these cytokine profiles was demonstrated by the finding that TLC producing substantial amounts of IL-4 enhanced expression of the low-affinity Fc receptor for IgE (CD23) on antigen-presenting cells to a greater extent than did IFN-gamma-producing TLC.     

Characteristics of peripheral blood CD4+CD25+ regulatory T cells and related cytokines in severe atopic dermatitis

Background

Regulatory T cells (Tregs) have been suggested to play a role in the pathogenesis of atopic dermatitis (AD). However, alterations in the ability of Tregs remain to be determined.

Objectives

To investigate the expression of various surface receptors on CD4⁺CD25^high regulatory T cells and to investigate their capacity for inhibiting the proliferation of CD4⁺ CD25^- effector T cells (Teffs).

Materials and methods

Peripheral blood samples were obtained from 15 patients with severe atopic dermatitis (AD) and 20 control subjects. FACs was then carried out to analyze the expression levels of FoxP3, CD152 (CTLA-4), CD39, CD73, CD223 (LAG-3), CCR4, CCR5, and CCR10 on Tregs. The proliferative responses ofTeffs were assessed in the absence or presence of autologous Tregs and the TGF-β1 and IL-10 levels in the culture supernatant and sera were detected by enzyme-linked immunosorbent assay (ELISA).

Results

The CD152, CD39, CD73, CCR4, and CCR5 expression levels on Tregs were higher in patients with severe AD than in the controls. Tregs showed an attenuated suppressive function of the proliferation of autologous Teffs in severe AD. The concentrations of IL-10 and TGF-β in the culture supernatants of Tregs were lower in the AD group than in the control.

Conclusion

The attenuated ability of Tregs to suppress Teff proliferation may be responsible for the autoimmune reaction of severe AD.

     

系统性硬皮病CD4~+ T细胞中CD70 mRNA及蛋白表达水平的观察

目的:研究系统性硬皮病(SSc)患者外周血CD4+ T细胞中CD70的表达水平。方法:应用密度梯度离心法分离17例SSc患者(女性12例,男性5例)和13例对照者(女性8例,男性5例)的外周血单个核细胞,磁珠分选CD4+ T细胞。RT-PCR检测CD4+ T细胞中CD70 mRNA的表达水平;流式细胞术检测CD70蛋白在CD4+ T细胞的表达水平。结果:与对照组相比,SSc患者CD4+ T细胞中CD70mRNA和CD70蛋白表达均明显升高(P=0.001;P=0.007)。结论:CD70在SSc的发生发展中可能起着重要作用。     

Expansion of IL-10-producing CD4+ and CD8+ T cells in fixed drug eruption

BACKGROUND: A severe form of fixed drug eruption (FDE) clinically and histologically mimics toxic epidermal necrolysis (TEN) but, unlike TEN, resolves spontaneously upon withdrawal of the causative drug. OBJECTIVE AND METHODS: We reported a case of a severe FDE caused by mefenamic acid that spontaneously resolved without use of systemic corticosteroids. To clarify the phenotype of the T cells responsible for clinical resolution of FDE, we kinetically examined gamma-interferon (IFN-gamma), interleukin (IL)-2, IL-4 and IL-10 production by peripheral blood T cells of the patient before and after oral challenge with the causative drug using flow cytometry. RESULTS: We found that the proportions of CD4+ and CD8+ T cells capable of producing IFN-gamma and IL-4 remained unchanged after challenge, while those of CD4+ and CD8+ T cells capable of producing IL-10 dramatically increased after challenge. The frequency of CD8+ T cells capable of producing IL-2 decreased after challenge. CONCLUSION: These results suggest that expansion of IL-10-producing CD4+ and CD8+ T cells may be responsible for spontaneous resolution of a severe form of FDE.     

特应性皮炎患儿外周血Th17细胞与CD4+CD25+ T细胞失衡的研究

【摘要】 目的 探讨Th17细胞和Treg细胞失衡在特应性皮炎（AD）发病机制中的作用。方法 流式细胞仪检测52例AD患者外周血Th17细胞和Treg细胞的频率、酶联免疫吸附方法（ELISA） 检测外周血中细胞因子IL-6、TGF-β1的表达水平。同时以30例性别、年龄匹配的健康体检者作为对照。结果 AD组外周血Th17细胞（CD3+CD8-IL17+）占CD3+T细胞的百分比为（1.20 ± 0.41）%,高于对照组的（0.54 ± 0.28）% （t = 2.58,P < 0.05）;Treg（CD4+ CD25+）细胞的百分比为（2.29 ± 0.67）%,低于对照组（5.95 ± 0.45）%,（t = 15.23,P < 0.01）。关键调控因子测定结果：IL-6水平,AD组（5.12 ± 0.45）ng/L高于对照组（3.89 ± 0.38） ng/L,差异具有统计学意义（t = 2.59,P < 0.05）;而TGF-β1的表达水平AD组（57.65 ± 10.78） ng/L低于对照组的（81.18 ± 7.78） ng/L,（t = 5.41,P < 0.01）。 结论 特应性皮炎患儿外周血Th17、Treg细胞水平及其关键的调控平衡因子IL-6、TGF-β1发生变化,其比例的失平衡可能参与特应性皮炎的发病。     

特应性皮炎患者外周血CD4+CD25+调节性T细胞的检测

目的 探讨CD4+CD25+调节性T细胞（CD4+CD25+ Treg）在特应性皮炎（AD）发病中的作用机制及临床意义。方法 流式细胞仪分析AD患者外周血中CD4+CD25+ Treg细胞数量,实时荧光定量PCR检测外周血单核细胞（PBMC）中Foxp3 mRNA水平,ELISA检测血清中IL-2、IL-4、IL-10、IFN-γ水平。结果 AD患者外周血中CD4+CD25+ Treg细胞占CD3+ T细胞及CD4+ T细胞的比例均明显低于正常人对照组（t′ = 3.775、4.533,P值均 ＜ 0.01）;外周血中CD4+CD25+ Treg细胞占CD3+ T细胞比例在AD患者急性期明显低于慢性期（t = 2.217,P < 0.05）,而在急性期与亚急性期、亚急性期与慢性期之间差异均无统计学意义（t = 1.558、0.49,P值均 ＞ 0.05）。AD患者PBMC中Foxp3 mRNA的水平低于正常人对照组（z = -2.368,P ＜ 0.05）;其外周血中CD4+CD25+ Treg细胞与血清中IL-2和IL-10成正相关（r = 0.512、0.494,P值均 ＜ 0.05）,与IL-4和IFN-γ的相关性无统计学意义（r = -0.110、-0.237,P值均 ＞ 0.05）。结论 在AD患者中,外周血中CD4+CD25+ Treg细胞数量及Foxp3 mRNA水平均下降,从而可能减少对Th2细胞增生及其细胞因子分泌的抑制,使Th2占优势,参与AD的发病。     

Imbalance of CD4+CD29+ and CD4+CD45RA+ T-helper cell subsets in peripheral blood of patients with severe atopic dermatitis

To determine the proportion of T-helper cell subsets in the peripheral blood we studied 16 patients with mild, moderate and severe atopic dermatitis. Lymphocytes were isolated from heparinized peripheral blood and analysed by two-colour flow cytometry. Patients with severe atopic dermatitis had a decreased CD4+CD29+CD4+CD45RA+ ratio (p<0.01). We found a decreased absolute number of CD4+CD29+ cells (p<0.05) and an increased absolute number of CD4+CD45RA+ cells (p<0.05) in the peripheral blood. No significant changes in the CD4+CD29+CD4+CD45RA+ ratio were found in the peripheral blood of patients with clinically mild or moderate atopic dermatitis.     

The role of CD4+ and CD8+ T cells in contact hypersensitivity and allergic contact dermatitis

Allergic contact dermatitis (ACD) and contact hypersensitivity (CHS) are delayed-type hypersensitivity reactions which are mediated by hapten specific T cells. During the sensitisation phases, both CD4+ and CD8+ T cell precursors are activated in the draining lymph nodes by presentation of haptenated peptides by skin dendritic cells. Subsequent hapten skin painting induces the recruitment of T cells at the site of challenge which induces inflammatory signals and apoptosis of epidermal cells, leading to the development of a skin inflammatory infiltrate and of clinical symptoms. There have been major controversies on the respective roles of CD4+ and CD8+ T cells in the development of the CHS inflammatory reaction. Experimental studies from the last 10 years have demonstrated that, in normal CHS responses to strong haptens, CD8+ type 1 T cells are effector cells of CHS while CD4+ T cells are endowed with down-regulatory functions. The latter may correspond to the recently described CD4+ CD25+ regulatory T cell population. However, in some instances, especially those where there is a deficient CD8 T cell pool, CD4+ T cells can be effector cells of CHS. Ongoing studies will have to confirm that the pathophysiology of human ACD is similar to the mouse CHS and that the CHS response to weak haptens, the most frequently involved in human ACD, is similar to that reported for strong haptens.     

Relative impact of CD4+CD25+ regulatory T cells and tacrolimus on inhibition of T-cell proliferation in patients with atopic dermatitis

BACKGROUND: It has been established recently that CD4+CD25+ regulatory T cells (Tregs) play an important role in controlling various immune responses. Immunosuppressive drugs are often used to treat immune dysregulation but are frequently associated with undesirable side-effects. OBJECTIVES: We examined the suppressive capacity of circulating Tregs in patients with atopic dermatitis (AD). Combined effects of Tregs and tacrolimus on the inhibition of T-cell proliferation in vitro were also assessed. METHODS: CD4+CD25+ and CD4+CD25- T cells were isolated from peripheral blood mononuclear cells using immunomagnetic beads. CD4+CD25- T cells were stimulated with purified protein derivative (PPD) or house dust mite allergen (Der p1) for 6 or 7 days, respectively. A dose range of tacrolimus and CD4+CD25+ T cells were added separately, or together. Proliferation was measured by (3)H-thymidine incorporation. RESULTS: CD4+CD25+ T cells from normal controls and patients with AD are anergic and inhibit the proliferation of CD4+CD25- T cells in response to PPD and Der p1 in vitro in a dose-dependent manner. Addition of tacrolimus and Tregs together showed significantly stronger inhibition of proliferation than either on their own. This was true for both antigens and both in normal controls and in patients with AD. CONCLUSIONS: CD4+CD25+ T cells in patients with AD have normal suppressive activity compared with healthy controls. Tregs and tacrolimus have additive effects on the inhibition of proliferation in response to PPD and Der p1.