Expression of the T-helper 2-specific chemokine receptor CCR4 on CCR10-positive lymphocytes in atopic dermatitis skin but not in psoriasis skin

BACKGROUND: Atopic dermatitis (AD) and psoriasis are inflammatory skin diseases. AD is generally perceived as a T-helper (Th) 2-dominated disease whereas psoriasis is a Th1-dominated disease. The chemokine cutaneous T-cell attracting chemokine (CTACK) and its receptor CCR10 attract skin-homing lymphocytes to inflamed skin, suggesting that CCR10+ cells in AD and psoriasis should be of Th2 and Th1 type, respectively. The chemokine receptor CCR4 is expressed selectively on Th2 lymphocytes and its ligand thymus and activation-regulated chemokine (TARC) is upregulated in AD lesions, suggesting that the CCR10+ cells in AD lesions should also express CCR4. OBJECTIVES: To examine the coexpression of CCR10 and CCR4 on skin-invading lymphocytes in AD and psoriasis lesions as well as the Th1/Th2 cytokine expression of the CCR10+ lymphocytes. METHODS: Skin biopsies from AD and psoriasis patients were double stained with antibodies against CCR10-CCR4, CCR10-CCR5, CCR10-interleukin (IL)-2 and CCR10-IL-4. RESULTS: The CCR10+ cells in AD showed a mixed IL-2/IL-4 expression pattern, and a minor proportion expressed CCR4, whereas a large proportion of the CCR4+ cells did not express CCR10. In psoriasis the CCR10+ cells only expressed IL-2, and no CCR4 expression was detected. CONCLUSIONS: The CCR10+ lymphocytes invading the skin in AD and psoriasis have different Th1/Th2 profiles, as measured by both their cytokine and chemokine receptor expression. This suggests that the CCR10+ subpopulation of lymphocytes is made up of different Th1/Th2 subsets. However, the Th1/Th2 lymphocytes of AD and psoriasis were either CCR10+ or CCR10-, suggesting that both the Th1 and Th2 subpopulation can be subdivided further. CCR4 was found only in AD skin and on both CCR10+ and CCR10- cells, supporting the hypothesis of TARC and CTACK as being independent lymphocyte-attracting chemokines in AD.     

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.     

CCR4 is expressed on infiltrating cells in lesional skin of early mycosis fungoides and atopic dermatitis

CCR4 is expressed on tumor cells of mycosis fungoides (MF) and Sézary syndrome (SS). In MF, most infiltrating cells in patches and plaques express CXCR3, while tumor cells express CCR4 in advanced stages. Poteligeo Test IHC (CCR4 staining kit) is a newly developed staining kit that can examine the presence of CCR4 expressed on tumor cells of adult T‐cell leukemia/lymphoma, peripheral T‐cell lymphoma and cutaneous T‐cell lymphoma before treatment of anti‐CCR4 antibody using paraffin‐embedded samples. In this study, we analyzed CCR4 expression in lesional skin of MF, SS, atopic dermatitis (AD) and psoriasis with this new kit. CCR4 was expressed on infiltrating cells in lesional skin of patch, plaque, tumor MF and SS, and the number of positive cells increased as the disease progressed. Immunohistochemistry with frozen sections also showed some positive cells scattered in the dermis, although the quality was not high enough to quantify positive cells. There were significant positive correlations between CCR4⁺ cells and serum lactate dehydrogenase levels. Interestingly, CCR4⁺ cells were also detected in AD skin, whose number was larger than that in psoriatic skin. Previous studies showed only scattered CCR4⁺ cells in skin samples by standard immunohistochemical staining. The new, sensitive CCR4 staining kit has revealed that CCR4 is expressed on infiltrating cells in lesional skin of early MF and AD as well as advanced MF and SS. These cells can be therapeutic targets for patients who are resistant to standard treatments.     

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.     

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.     

Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-gamma- and interleukin-4-induced activation of human keratinocytes

Interleukin-17 is a T-cell-derived cytokine, detected in skin affected by allergic contact dermatitis and psoriasis, which regulates keratinocyte expression of adhesion molecules and chemokines. In this study, we have analyzed whether interleukin-17 production segregates with a particular T helper (Th) cell subset, and have examined the capacity of interleukin-17 to modulate the activation of keratinocytes induced by Th1 and Th2 cytokines. A panel of 80 nickel-specific CD4+ T cell clones (36 Th0, 30 Th1, and 14 Th2) was isolated from peripheral blood or lesional skin of allergic contact dermatitis patients. Significant amounts (> 50 pg per ml) of interleukin-17 were released by about 50% of activated Th0, Th1, and Th2 cells. Interleukin-17 alone and in cooperation with interleukin-4, or to a lesser extent with interferon-gamma, decreased the interleukin-1 receptor antagonist to interleukin-1alpha ratio in the supernatants as well as in cell lysates from keratinocytes. In addition, interleukin-17 stimulated the release of growth-regulated oncogene-alpha, granulocyte-macrophage colony stimulating factor, and interleukin-6, with synergistic or additive effects when used together with interferon-gamma or interleukin-4. Interleukin-17 and interleukin-4 also increased stem cell factor release, a function that was inhibited by interferon-gamma. Moreover, interleukin-17 and interleukin-4 enhanced interferon-gamma-induced expression of intercellular adhesion molecule 1, but not CD40, on keratinocytes. The constitutive expression of interleukin-17 and interferon-gamma receptors on keratinocytes was not modulated by interleukin-17, interferon-gamma, or interleukin-4, whereas the interleukin-4 receptor was significantly downregulated by interferon-gamma. As a whole, the results indicate that interleukin-17 can participate relevantly in T-cell-mediated skin immune responses by amplifying both interferon-gamma- and interleukin-4-induced activation of keratinocytes.     

Increased circulating skin-homing cutaneous lymphocyte-associated antigen (CLA)+ type 2 cytokine-producing cells, and decreased CLA+ type 1 cytokine-producing cells in atopic dermatitis

BACKGROUND: Skin-homing T cells are characterized by expression of cutaneous lymphocyte-associated antigen (CLA). Few data are available on the frequency of circulating CLA+ cytokine-producing T cells in atopic dermatitis (AD) patients. OBJECTIVES: We aimed to investigate cytokine synthesis capability vs. CLA expression in phorbol myristate acetate and ionomycin-stimulated, secretion-inhibited peripheral blood T cells of AD patients compared with healthy subjects and psoriatic patients. METHODS: Multiparameter flow cytometry was used. RESULTS: The expression of CLA among CD4+ T cells was significantly elevated in AD patients compared with healthy subjects and psoriatic patients, whereas there was no significant difference between each group in CLA expression among CD8+ T cells. The frequency of interleukin (IL)-4- and IL-13-producing cells in AD patients was significantly higher than in healthy subjects (in both CD4+ and CD8+ T-cell subsets) and psoriatic patients (in CD4+ T cells). In contrast, the frequency of interferon (IFN)-gamma-producing cells was significantly reduced in AD patients, among both CD4+ and CD8+ T cells, compared with healthy subjects and psoriatic patients. Moreover, in AD patients, the frequency of IL-4- and IL-13-producing cells was remarkably increased among the CLA+ subset (in both CD4+ and CD8+ T cells), whereas the frequency of IFN-gamma-producing cells was decreased in the CLA+ subset (in CD4+, but not in CD8+ T cells). CONCLUSIONS: These results provide evidence for the expansion of skin-homing type 2 cytokine-secreting T cells, associated with a reduction in skin-homing type 1 cytokine-producing T cells, in peripheral blood of AD patients.     

CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease expressed early in life. Disease development is primarily determined by as yet unknown genetic factors, leading to the accumulation of activated T lymphocytes in the skin. OBJECTIVES: To investigate the nature of these T cells. METHODS: T-cell lines could be established from AD skin biopsies, but not from normal skin or AD peripheral blood, when placed in RPMI 1640 medium with 10% human AB serum, antibiotics, and the T-lymphocyte growth factors interleukins 2 and 4. The cell lines were subjected to phenotypic analysis using a fluorescence-activated cell sorter and compared with lymphocytes from AD and normal control peripheral blood. RESULTS: T-cell lines from 22 of 24 consecutive skin biopsies taken from 24 adult patients with AD were established. All cells were T lymphocytes expressing several activation markers. A significant proportion of the lymphocytes had stable expression of a CD4+ CD8+ phenotype (26% +/- 6%; mean +/- SEM). Such double-positive T lymphocytes are normally only seen in the thymus and not in the peripheral immune system. CD4+ CD8+ cells in peripheral blood of the patients (12.5% +/- 3.3%) were also detected. CONCLUSIONS: We suggest that a basic pathophysiological change in AD may be a faulty maturation of the T-lymphocyte system, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells. This is likely to lead to skewing of many immune reactions in the patients.     

A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy

BACKGROUND: The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD. OBJECTIVES: To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns. METHODS: Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry. RESULTS: Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells. CONCLUSIONS: These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.     

Differential expression of genes involved in skin homing, proliferation, and apoptosis in CD4+ T cells of patients with atopic dermatitis

CD4+ T cells play a critical role in allergic diseases, both in the affected tissue as well as systemically. Our objective was to investigate the in vivo activation state of peripheral blood CD4+ T cells of atopic dermatitis (AD) patients by analyzing gene expression profiles of unstimulated CD4+ T cells. mRNA samples from blood CD4+ T cells, isolated from five AD patients and seven healthy controls (HC), were analyzed using oligonucleotide arrays. Differentially regulated genes were validated by quantitative PCR (Q-PCR) in a larger group of patients with AD, in a group of patients with allergic asthma (AA), and HC subjects. In addition, "typical" T helper type 1 (Th1)- and Th2-related genes were analyzed by Q-PCR. Microarray analysis revealed differential expression of 52 genes in AD patients. Q-PCR confirmed several differentially regulated genes in AD, including CCR10, CRTH2, C-JUN, and NR4A2. Two groups of genes with highly correlating gene expression levels involved in tissue homing and proliferation or apoptosis, respectively, were identified. No marked differences were found in gene expression levels of typical Th1 or Th2 genes in AD or in AA patients. This study demonstrates that peripheral blood, unstimulated CD4+ T cells in AD patients show differentially expressed genes involved in tissue homing, proliferation, and apoptosis. No marked expression differences of "typical" atopy genes were found.     

The Th2 cytokine,interleukin‐4, abrogates the cohesion of normal stratum corneum in mice: implications for pathogenesis of atopic dermatitis

There is mounting evidence that Th2 cytokines adversely affect skin barrier functions and contribute to the pathogenesis of atopic dermatitis (AD). AD is also characterized by abnormal cohesion in the stratum corneum (SC). However, the contribution of Th2 cytokines to this abnormality remains unknown. This study examined the effects of IL‐4, a prototypic Th2 cytokine, on the cohesion of the SC. Structural and physiological assessments revealed that repeated intradermal injections of IL‐4 compromised the cohesion of the SC of normal hairless mice. Two potential mechanisms were explored to account for the altered cohesion. First, IL‐4 decreased the amount of corneodesmosomes and down‐regulated the expression of desmoglein 1, but not of corneodesmosin (CDSN) or loricrin expression, in murine skin and in cultured human keratinocytes (KC). IL‐4 did not affect the skin surface pH, and in situ zymography revealed no net change in total serine protease activity in the IL‐4‐treated SC. Yet, IL‐4 enhanced expression of kallikrein (KLK)7, while simultaneously down‐regulating KLK5 and KLK14. Finally, IL‐4 did not alter the expression of the lympho‐epithelial Kazal‐type inhibitor (LEKTI) in KC. This study suggests that IL‐4 abrogates the cohesion of SC primarily by reducing epidermal differentiation.     

Production of IL-4, IL-2, IFN-gamma, and TNF-alpha by peripheral blood mononuclear cells of patients with atopic dermatitis.

Recently, T cell-derived cytokines have been postulated to be involved in the pathogenesis of atopic dermatitis (AD) since the synthesis of IgE is profoundly regulated by cytokines such as IL-4, IFN-gamma and IL-2. IL-4 enhances the production of IgE and in contrast, IFN-gamma inhibits this IL-4-mediated IgE production. IL-2 also prevents IL-4-induced production of IgE by a different mechanism from IFN-gamma, suggesting that the level of IgE is regulated by the quantitative balance of these antagonizing cytokines. We examined the production of IL-4, IL-2, IFN-gamma and TNF-alpha by PBMC of AD patients and non-AD controls. Although the production of IL-2 and TNF-alpha by AD patients was significantly lower than that of non-AD controls, the production of IL-4 and IFN-gamma did not show significant differences between AD and non-AD individuals. There was no significant correlation between cytokine production and clinical symptoms in AD patients. There was significant positive correlation between IL-4 and IL-2 production, and between IFN-gamma and TNF-alpha production. Serum IL-4 levels showed no significant difference between AD group and non-AD group.     

Caspase抑制剂对特应性皮炎患者外周血CD4+T细胞亚群分泌细胞因子的影响

目的：明确广谱半胱氨酸天冬氨酸蛋白酶(caspase)抑制剂对特应性皮炎(AD)患者外周血CD4+T细胞亚群分泌细胞因子的影响。方法：广谱caspase抑制剂Z-VAD-FMK与30例AD患者外周血单一核细胞(PBMCs)体外共培养后,PBMCs分为3组,分别加入Z-VAD-FMK溶液、地塞米松溶液和PBS溶液。流式细胞仪检测CD4+T细胞亚群;ELISA方法检测上清液中IFN-γ、IL-4、IL-17的浓度。结果：IFN-γ、IL-4、IL-17水平在PBS溶液组高于Z-VAD-FMK组,差异均有统计学意义(均P<0.01),在Z-VAD-FMK组与地塞米松组间比较,均无统计学意义(均P>0.05)。结论：广谱caspase抑制剂Z-VAD-FMK可抑制AD患者PBMC中CD4+T细胞Th1、Th2、Th17分泌相关细胞因子。     

Distribution of interleukin-2 receptor bearing lymphocytes in the skin. A comparative study of allergic and irritant contact dermatitis, tuberculin reaction and cutaneous T cell lymphoma

The tissue distribution of T cells and interleukin-2 receptor bearing (Tac+) lymphocytes was studied immunohistochemically in frozen sections of biopsies from inflamed skin. In skin lesions caused by irritant or immunologic stimuli, relatively few (less than 5%) of the total dermal infiltrating T cells were Tac+, but in both types of conditions the Tac+ cells showed a predeliction for the epidermis. In skin lesions from patients with cutaneous T cell lymphoma a relatively high proportion of dermal T lymphocytes were Tac+. Tac+ as well as Tac- T lymphocytes may thus migrate to a tissue secondary to several different stimuli. The demonstration of a non-random distribution of Tac+ lymphocytes in the non-malignant inflammatory conditions in the present study indicate that the epidermis may have a special role in the activation of T lymphocytes.