HLA-A2 restricted, melanocyte-specific CD8(+) T lymphocytes detected in vitiligo patients are related to disease activity and are predominantly directed against MelanA/MART1.

Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8(+) T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-gamma staining for the detection of specific reactive CD8(+) T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8(+) T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-gamma staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8(+) T cells seems to be closely related to disease activity.     

人真皮间充质干细胞对白癜风患者皮损周围CD8+ T淋巴细胞表达和分泌白介素13的影响

【摘要】 目的 探讨人真皮间充质干细胞（DMSC）对白癜风患者皮损周围CD8+ T淋巴细胞分泌表达白介素（IL）13的影响。 方法 细胞增殖检测法（MTS）检测重组IL-13（rIL-13）对黑素细胞增殖的影响。逆转录聚合酶链反应（RT-PCR）和Western印迹法分别检测6例进展期寻常型白癜风患者皮损周围及外周血中CD8+ T淋巴细胞中IL-13基因和蛋白表达。实时荧光定量反转录聚合酶链反应（qRT-PCR）技术和酶联免疫吸附试验（ELISA）分别检测DMSC与白癜风患者皮损周围CD8+ T淋巴细胞共培养前后细胞IL-13 mRNA水平和上清蛋白水平。 结果 不同浓度（10、50、100、250、500 μg/L）的rIL-13作用黑素细胞24、48、72、96 h后黑素细胞的增殖与对照组比较均未见明显变化（均P > 0.05）。白癜风患者皮损周围及外周血中CD8+ T淋巴细胞均可表达IL-13,但皮损周围CD8+ T淋巴细胞表达IL-13更显著。将皮损周围CD8+ T淋巴细胞和DMSC共培养,CD8+ T淋巴细胞IL-13 mRNA（0.100 0 ± 0.002 4）和蛋白［（1 509.62 ± 48.44） ng/L］的表达水平均显著低于单独培养的CD8+ T淋巴细胞［mRNA：0.383 2 ± 0.018 7,蛋白：（5 507.98 ± 34.11） ng/L,均P < 0.05］。 结论 白癜风患者皮损周围CD8+ T淋巴细胞高表达IL-13,DMSC能够有效地抑制其表达IL-13,或许可作为白癜风的治疗靶点之一。     

白癜风复色区黑素细胞特异性抗原表达的研究

目的：研究白癜风复色区黑素细胞分布及gp100、酪氨酸酶（TYR）等抗原的表达。方法：对白癜风患者复色区皮肤活检的组织标本以免疫组化染色识别gp100、TYR、酪氨酸酶相关蛋白（TRP）-1和TRP-2,以HPIAS-1000彩色病理图文报告系统定量分析阳性表达的强弱。结果：色素脱失斑处未见活性黑素细胞,复色区黑素细胞数目与正常人皮肤比较差异无统计学意义（P〉0．05）。色素脱失斑内岛状复色区皮肤,近毛囊部位黑素细胞数目较多,远离毛囊部位数目较少;色素脱失斑边缘复色区内,黑素细胞在表皮及毛囊的基底层中分布较均匀。复色区黑素细胞树突短粗、数目变少。复色区阳性细胞gp100和TRP-2图像灰度值与正常皮肤比较差异无统计学意义（P〉0．05）,TYR低于正常皮肤（P〈0．05）,而TRP-1明显低于正常皮肤（P〈0．01）。结论：色素脱失斑处有功能黑素细胞缺失。复色区内可见黑素细胞,其结构、功能皆完整,数目与正常人无明显差别。复色区黑素细胞可能来源于毛囊组织．亦可能来源于色素脱失斑边缘正常黑素细胞。TRP-1的高表达可能导致复色区黑素细胞的形态异常。     

Direct evidence to support the role of antigen-specific CD8(+) T cells in melanoma-associated vitiligo

Vitiligo is a cutaneous pigmentary disorder characterized by the loss of melanocytes. An autoimmune mechanism is strongly suspected to be involved in this affection given that it is frequently associated with autoimmune hormonal disorders, and because antibodies directed against melanocytic antigens are found in the serum of patients with vitiligo. We examined the role of cellular immunity in melanoma-associated vitiligo by expanding infiltrating lymphocytes from fresh biopsy specimens of vitiligo patches in melanoma patients. The vitiligo-infiltrating lymphocytes were almost exclusively T lymphocytes, and most were CD8(+). Following in vitro expansion, vitiligo-infiltrating lymphocytes remained predominantly CD8(+) and expressed the cutaneous homing receptor CLA. Furthermore, vitiligo-infiltrating lymphocytes had a clonal or oligoclonal T cell receptor profile, possibly reflecting specific antigenic stimulation. Finally, vitiligo- infiltrating lymphocytes specifically recognized differentiation antigens shared by normal melanocytes and melanoma cells. This direct demonstration of CD8(+) T cell involvement in vitiligo suggests that, in melanoma patients, vitiligo may be a visible effect of a spontaneous antitumoral immune response.     

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.     

In situ localization of IFN-gamma-positive cells in psoriatic lesional epidermis.

Interferon-gamma (IFN-gamma) is believed to be an important mediator in the cytokine cascade of psoriasis. Lesional T cells in the epidermis may play a role in psoriasis. We examined whether IFN-gamma-producing T cells were present in the epidermis of psoriasis in situ by immunohistochemical techniques. Mixtures of CD4+ T cells and CD8+ T cells were found to be present in the papillary dermis and the epidermis of the psoriatic lesions. CD8+ T cells seemed to be dominant in the epidermis. Considerable amounts of IFN-gamma-positive cells were detected in infiltrates of the papillary dermis. IFN-gamma-positive cells were found to be present in the epidermis. The pattern of IFN-gamma staining appeared to be a combination of intracellular staining in mononuclear lymphoid cells and extracellular deposition in the surrounding areas. The staining was considered to be highly specific because it could be completely blocked by preabsorption with recombinant IFN-gamma. Our data suggest that psoriatic epidermal T cells produce and secrete IFN-gamma within the lesion and that these T cells are involved in the pathogenesis of psoriasis.     

Psoriasis confined strictly to vitiligo areas – a Koebner‐like phenomenon?

Vitiligo and psoriasis are both common skin disorders. However, psoriasis strictly confined to pre-existing vitiligo areas is rare and suggests a causal relationship. We report here on two patients with a strict anatomical colocalization of vitiligo and psoriasis. The histopathological examinations showed typical changes for both diseases together with a dense infiltrate of CD4+ and CD8+ T cells. By immunohistochemistry, intracytoplasmatic granzyme B and tumour necrosis factor alpha (TNF-alpha) were detected within the T-cell population, suggesting the functional activity of these cells and the creation of a local T helper 1 (Th1)-cytokine milieu. Additionally, in one patient we could identify anti-melanocytic T cells by tetramer staining and enzyme-linked immunospot (ELISPOT) analysis. These skin-infiltrating lymphocytes might trigger, by the local production of Th-1 cytokines such as TNF-alpha and interferon-gamma (IFN-gamma), the eruption of psoriatic plaques in patients with a genetic predisposition for psoriasis.     

CD8~+T细胞对白癜风患者黑素细胞的杀伤机制

目的:明确CD8~+T细胞对白癜风患者黑素细胞的杀伤机制。方法:分选白癜风患者及正常人外周血CD8~+T细胞,分别与原代黑素细胞及永生化正常黑素细胞系PIG1及永生化白癜风黑素细胞系PIG3V建立共孵育杀伤模型,采用流式细胞术检测靶细胞被杀伤情况,酶联免疫吸附法(ELISA)检测杀伤环境中IFN-γ、TNF-α、颗粒酶B及穿孔素的分泌水平。结果:白癜风患者外周血CD8~+T细胞杀伤原代黑素细胞及PIG3V能力,分泌IFN-γ、颗粒酶B及穿孔素水平均高于健康对照,而杀伤PIG1能力和TNF-α水平并无显著差异;白癜风患者CD8~+T细胞IFN-γ分泌水平与细胞杀伤率呈明显正相关。结论:白癜风患者CD8~+T细胞杀伤黑素细胞是白癜风发病的重要机制,且该杀伤作用可能依赖IFN-γ、颗粒酶B及穿孔素分泌水平的上调。     

Cytotoxic T lymphocyte reactivity to gp100, MelanA/MART-1, and tyrosinase,in HLA-A2-positive vitiligo patients

Vitiligo is a common depigmentation disorder thought to result from autoimmune destruction of melanocytes. Recent studies suggest a role for cell-mediated immune responses to melanocyte differentiation antigens, including gp100, MelanA/MART-1, and tyrosinase, in vitiligo pathogenesis. This study investigated T cell reactivity to MelanA/MART-1, tyrosinase, and gp100, in HLA-A2-positive patients with vitiligo. Melanocyte-specific T cell responses were measured ex vivo via enzyme-linked immunospot assay following stimulation with MelanA/MART-1, tyrosinase, and modified gp100 epitopes. Antigen-specific T lymphocyte reactivity to gp100 peptides was seen in 15 of 17 (88%) patients, with many demonstrating very high reactivity at levels comparable with those observed with common recall antigens. Reactivity to gp100 was noted to be associated with disease activity. Antigen-specific T lymphocyte reactivity to MelanA/MART-1 and tyrosinase peptides was not observed ex vivo in our patients, and only one patient demonstrated responses to MelanA/MART-1 and tyrosinase peptides following in vitro re-stimulation. Our findings implicate T cell reactivity to gp100 in patients with active disease and support the concept of an immunopathologic mechanism in vitiligo, in which cell-mediated responses to normal melanocyte antigens play a crucial part.     

Vitiligo with inflammatory raised borders, associated with atopic dermatitis

A 31-year-old man had had atopic dermatitis since childhood and developed vitiligo with inflammatory raised borders 5 years prior to presentation. Immunohistochemically, CD4+ T cells infiltrated predominantly in the raised border of vitiligo, while CD8+ T cells were present just outside of the borders, suggesting that CD8+ cells were an antecedent to the CD4+ cells. Despite the presence of atopic dermatitis, the percentage of CXCR3+ CD4+ Th1 cells increased in the patient's peripheral blood, compared with a representative atopic patient showing a high percentage of CCR4+CD4+ Th2 cells. This case suggests that vitiligo with inflammatory raised borders can occur even in patients with atopic dermatitis when Th1 cells are activated and overcome the Th2-dominant state.     

Influence of psoriatic peripheral blood CD4+ T and CD8+ T lymphocytes on C-myc, Bcl-xL and Ki67 gene expression in keratinocytes

T cells play a role in the hyperproliferation of keratinocytes, however, direct evidence on what is the main mechanism and which T-cell subset is central is still lacking. Our aim was to elucidate the role and mechanism of CD4+T and CD8+ T lymphocytes in the immunopathogenesis of lesion hyper-proliferation in psoriasis. T cell and CD4+T and CD8+ T cell subpopulations were isolated by immunomagnetic methods from peripheral blood of psoriatic patients and healthy volunteers. Keratinocytes from foreskins were incubated in the absence or presence of T cells and T cell subpopulations for 72 h. The expression of c-Myc, Bcl-xL and Ki67 proteins in keratinocytes were detected by immunohistochemical staining, and then IL-4, IL-8 and IFNgamma level in the supernatant was determined by respective ELISA. T cells of psoriatic origin induced keratinocytes to overexpress C-myc and Ki67 proteins with high amounts of IL-8 and IFN-gamma in the supernatant, while the T cells of normal origin did not. Furthermore, there was a difference in keratinocyte response to CD4+ T cells and CD8+ T cells from psoriatic patients. CD4+ T cells can secrete much higher levels of IL-8 and IFN-gamma and induced much stronger C-myc and Ki67 expression in keratinocytes, compared with CD8+ T cells. We concluded that psoriatic peripheral blood T cells and the T cell subpopulation could induce keratinocytes to over-express pro-proliferation proteins probably by secreting Th1 cytokines. T cells, especially the CD4+ T cell subpopulation, play an important role in the immunopathogenesis of lesion hyper-proliferation in psoriasis.     

Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.     

Candida albicans-specific lymphoproliferative and cytokine (IL-4 and IFN-gamma) responses in atopic eczema dermatitis syndrome. Evidence of CD4/CD8 and CD3/CD16+CD56 ratio elevations in vitro

BACKGROUND: Hypersensitivity to cross-reactive mannan polysaccharide allergens of saprophytic yeasts is likely to be involved in the pathogenesis of atopic eczema dermatitis syndrome (AEDS). Mannans induce elevated specific immunoglobulin E and lymphoproliferative responses in peripheral blood mononuclear cells (PBMCs). To gain more detailed data of the involvement of different subpopulations of PBMCs in AEDS after mannan stimulation, changes in the cell-surface marker distribution were analysed. METHODS: The Ficoll-isolated PBMCs of eight yeast hypersensitive AEDS patients and seven non-AEDS controls were stimulated in vitro by mannan (CAM) or whole extract antigen [In-House Reference (IHR)] of Candida albicans or tuberculin [purified protein derivative (PPD)] and after immunofluorescence staining analysed by flow cytometry. The expression of cytokine mRNA was measured by kinetic real-time polymerase chain reaction (TaqMan). RESULTS: After 7-day antigen stimulation, there were significant increases in the CD3/CD16(+)CD56 ratio (P = 0.028 with mannan and P = 0.006 with IHR), CD4/CD8 ratio (P = 0.049 with mannan) and interleukin-4/interferon-gamma (IL-4/IFN-gamma) mRNA ratio (P = 0.028 with IHR) and a decrease in the CD3/CD19 ratio (P = 0.035 with mannan) of AEDS patients' PBMCs as compared with healthy controls' cells. These changes were not seen in cultures with PPD. CONCLUSIONS: The observed CAM and IHR-induced elevations in T cell/natural killer cell, CD4/CD8 and IL-4/IFN-gamma ratios suggest that C. albicans-induced TH(2)-type responses can also play a role in AEDS.     

Reduction of skin-homing cytotoxic T cells (CD8+ -CLA+) in patients with vitiligo

Background: Vitiligo is a frequently acquired, hereditary disease, characterized by achromic macules due to the absence of melanocytes. In contrast with earlier studies, in which the main pathogenic role was attributed to anti‐melanocyte antibodies, recent papers have emphasized a role for CD8⁺ cytotoxic T lymphocytes in melanocyte destruction. Fifteen percent of peripheral T cell express cutaneous lymphocyte‐associated antigen (CLA), responsible for skin‐homing T cell. Phototherapy is used to treat patients with generalized vitiligo and it has been shown to interfere with CLA⁺ T cells in other skin diseases. Objective: To describe peripheral blood T cell subpopulations' frequency and ability to express the skin‐homing molecule (CLA) in patients with non‐segmental vitiligo, before and after photochemotherapy (PUVA). Patients and Methods: Twenty‐two patients with generalized and active spreading vitiligo were submitted to 30 PUVA‐8MOP sessions. Lymphocyte immunophenotyping was performed by flow cytometry using anti‐CD3, anti‐CD8 and anti‐CLA monoclonal antibodies. Fifteen healthy volunteers, sex‐ and age‐matched, were included as a control group. Results: CD8⁺–CLA⁺ T cells were significantly reduced in number in untreated vitiligo patients (P=0.008) when compared with control individuals, albeit with a more intense CLA expression (P=0.028). These findings were not altered after PUVA. No significant difference was noticed in CD4/CD8 ratios nor in CD4–CLA⁺ T cell numbers between vitiligo patients and controls, both before and after PUVA. Conclusions: CD8–CLA⁺ T cells are reduced in peripheral blood of patients with non‐segmental vitiligo. This finding may be related to the previously reported increase of CD8⁺ cells in both lesions and perilesional skin of these 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.     

墨旱莲乙醇提取物动物致色素作用和对小鼠B16黑素瘤细胞黑素生成的影响

目的选择离体实验中对黑色素合成的关键酶—酪氨酸酶(tyrosinase,TYR)有激活作用的墨旱莲(旱莲草),研究其动物致色素作用和对小鼠B16黑素瘤细胞黑素生成及相关基因犤TYR、TYR相关蛋白(TRP-1/2)mRNA犦表达的影响。方法以棕色豚鼠为动物模型,用Schmorl法染色,计数含黑素的细胞;用多巴(DOPA)-氧化酶染色,计算每100个基底细胞中DOPA阳性细胞数。培养的小鼠B16黑素瘤细胞以四甲基偶氮唑盐比色(MTT)法、氢氧化钠(NaOH)法和体外氧化DOPA反应方法分别测定细胞的增殖活性、黑素生成量及TYR活性;逆转录-聚合酶链反应(RT-PCR)测定TYR及其TRP-1/2基因的表达。结果墨旱莲乙醇提取物可使豚鼠表皮基底层中含黑素颗粒细胞增多,使豚鼠表皮内DOPA阳性细胞增多(P<0.05);具有促进小鼠B16黑素瘤细胞黑素合成及TYR活性作用(P<0.05),对细胞TYR基因有上调作用,而对TRP-1/2mRNA的表达无明显作用。结论墨旱莲乙醇提取物具有促进黑素合成及上调酪氨酸酶基因表达的作用,对白癜风色素恢复有较好应用和开发的前景。