2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647720摘要：背景及構想:專門表現Foxp3 的調節性T 細胞，具有抑制免疫反應的功能，因此對於免疫反應的調節非常重要。調節性T 細胞之數量或功能的缺乏，會導致過敏性疾病的發生。異位性皮膚炎，是一種慢性反覆發作的皮膚過敏性發炎疾病。皮膚淋巴球相關抗原(cutaneous lymphocyte-associated antigen，CLA)，是引導T細胞自動移至皮膚的T 細胞表面受體。異位性皮膚炎病人的皮膚上非常容易有金黃色葡萄球菌的增生與感染，此菌會分泌許多外毒素，其中以金黃色葡萄球菌腸毒素B (staphylococcal enterotoxin B，SEB)最為常見。這些金黃色葡萄球菌外毒素是超級抗原，它們會刺激帶有特定的T 細胞受體b鏈變異區(variable region of b chain of T cell receptor，TCRVb)的T細胞，造成異位性皮膚炎皮膚過敏性發炎反應的持續與惡化。最近關於異位性皮膚炎病人的調節性T 細胞之數量及功能的一些研究，各自顯示了不同的結果。T細胞活化後引起的細胞凋亡，可以清除被抗原活化的T細胞，使發炎反應得以終止。反覆的T 細胞受體的刺激，也會使調節性T 細胞發生活化後引起的細胞凋亡。本研究的目標，是在異位性皮膚炎病人、無異位性皮膚炎的呼吸道過敏病病人、及健康人之間，比較金黃色葡萄球菌超級抗原對CCR6+亞型與CCR6-亞型的CLA+調節性T細胞之數量比例、功能、及細胞凋亡的影響。特定目的:本研究的第一個目的，是在異位性皮膚炎病人、氣喘或過敏性鼻炎病人、及健康人之間，比較SEB 的刺激對周邊血CLA+ CD4+ Foxp3+ 調節性T細胞之數量的影響。第二個目的，是比較這三組人的CLA+CD4+ Foxp3+ 調節性T細胞，在SEB刺激後，所引起的CCR6+ / CCR6- 數量比例的改變。我們也會比較這三組人的CCR6+亞型與CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T細胞當中，SEB反應型(TCRVb3+and Vb12+ and Vb17+) T細胞所佔的數量比例。第三個目的，是比較這三組人的CCR6+亞型與CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T細胞，在SEB刺激後，所引起的第一型T輔助細胞激素干擾素-g、第二型T 輔助細胞激素介白質-4、介白質-5、介白質-13、抗發炎細胞激素介白質-10、第三型T 輔助細胞激素transforming growth factor-b (TGF-b)、及第十七型T輔助細胞激素介白質-17的製造。第四個目的，是比較這三組人的CCR6+亞型與CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T細胞，在SEB刺激後，所引起的細胞凋亡標記caspase-3的活化、細胞內抗凋亡蛋白Bcl-2、Bcl-XL及促凋亡蛋白Bax含量的變化。方法及實施進度:30位異位性皮膚炎病人、30位氣喘或過敏性鼻炎病人、及30位健康人的周邊血單核細胞，分別給予SEB(20 ng/ml)刺激18小時。之後，以免疫螢光染色法併流式細胞儀分析，來偵測CLA+ CD4+ Foxp3+ 調節性T細胞上CCR6的表現、以及CCR6+亞型與CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T 細胞中的SEB反應型(TCRVb3+ and Vb12+ and Vb17+) T細胞、細胞內的細胞激素干擾素-g、介白質-4、介白質-5、介白質-10、介白質-13、介白質-17、TGF-b、及細胞內的active caspase-3、Bcl-2、Bcl-XL、Bax。初步結果:SEB的刺激對異位性皮膚炎病人、氣喘或過敏性鼻炎病人、及健康人的CLA+ CD4+ Foxp3+ 調節性T細胞之數量並無影響。在異位性皮膚炎病人，SEB減少CLA+ CD4+ Foxp3+ 調節性T細胞中CCR6+ / CCR6-的數量比例;而在氣喘或過敏性鼻炎病人及健康人，SEB增加CCR6+ / CCR6- 的數量比例。在這三組人的CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T 細胞，SEB主要引起第二型T輔助細胞激素介白質-5的製造;而在CCR6+亞型細胞，SEB 主要引起抗發炎細胞激素介白質-10 的製造。和健康人比起來，異位性皮膚炎病人的CLA+ CD4+ Foxp3+ 調節性T細胞在SEB刺激後製造較多的IL-5及較少的IL-10。和另一種亞型比起來，異位性皮膚炎病人的CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T細胞與氣喘或過敏性鼻炎病人及健康人的CCR6+亞型細胞比較不容易發生SEB引起的caspase-3的活化。和其他人比起來，異位性皮膚炎病人的CCR6-亞型之CLA+ CD4+ Foxp3+ 調節性T細胞與健康人的CCR6+亞型細胞比較不容易發生SEB引起的caspase-3的活化。<br> Abstract: Objective, background and rationale:Regulatory T (Treg) cells, which “specifically” express transcription factor Foxp3, play a key role in theregulation of immune responses by their immunosuppressive function. A deficiency in the number or functionof Treg cells can lead to the development of allergic diseases. Atopic dermatitis (AD) is a chronically relapsingTH2-dominanted inflammatory skin disorder. Cutaneous lymphocyte-associated antigen (CLA) is askin-homing receptor involved in the selective migration of T cells to the skin. AD skin exhibits a strikingsusceptibility to colonization and infection with Staphylococcus aureus, which secrete various exotoxinsincluding staphylococcal enterotoxin B (SEB) being the most common. These exotoxins are staphylococcalsuperantigens (SsAgs). They may stimulate T cells bearing specific variable region of b chain of T cell receptor(TCRVb) and contribute to the persistence and exacerbation of allergic skin inflammation in AD. Previousstudies showed controversial results about the number and function of Treg cells in AD patients.Activation-induced apoptosis of T cells provides a mechanism for the deletion of antigen-activated T cells,thereby leading to the resolution of inflammation. Repetitive anti-CD3/anti-CD28 stimulation of Treg cells mayalso increase their sensitivity to activation-induced apoptosis. The objective of this study is to compare theeffects of SsAgs on the ratios, functions, and apoptosis of CCR6+ subtype and CCR6- subtype of skin-homingTreg cells among AD patients, patients with respiratory allergy without AD, and healthy subjects.Specific aims:The first specific aim is to compare the effect of SEB stimulation on the number of peripheral bloodskin-homing CLA+ CD4+ Foxp3+ Treg cells among AD patients, asthma/allergic rhinitis (AR) patients, andhealthy subjects. The second aim is to compare SEB-induced change of CCR6+ / CCR6- ratios in CLA+ CD4+Foxp3+ Treg cells among the three groups of subjects. We also compare the percentages of SEB-reactive(TCRVb3+ and Vb12+ and Vb17+) T cells between CCR6+ subtype and CCR6- subtype of CLA+ CD4+ Foxp3+Treg cells among the three groups of subjects. Third, we aim to compare SEB-induced production of TH1cytokine interferon-g (IFN-g), TH2 cytokines interleukin-4 (IL-4), IL-5, IL-13, anti-inflammatory cytokineIL-10, TH3 cytokine transforming growth factor-b (TGF-b), and TH17 cytokine IL-17 between CCR6+ subtypeand CCR6- subtype of CLA+ CD4+ Foxp3+ Treg cells among the three groups of subjects. Finally, we wouldlike to compare SEB-induced caspase-3 activation (apoptosis marker) and SEB-induced change of intracellularanti-apoptotic proteins Bcl-2, Bcl-XL, and pro-apoptotic protein Bax levels between CCR6+ subtype andCCR6- subtype of CLA+ CD4+ Foxp3+ Treg cells among the three groups of subjects.Methods and experimental plan:Peripheral blood mononuclear cells (PBMCs) isolated from 30 AD patients, 30 asthma/AR patients, and 30healthy subjects are cultured with or without SEB (20 ng/ml) stimulation for 18 hours. Immunofluorescencestaining followed by flow cytometric analysis is used to detect CCR6 expression on CLA+ CD4+ Foxp3+ Tregcells, SEB-reactive (TCRVb3+ and Vb12+ and Vb17+) T cells, intracellular cytokines IFN-g, IL-4, IL-5, IL-10,IL-13, IL-17, TGF-b, and intracellular active caspase-3, Bcl-2, Bcl-XL, Bax in CCR6+ subtype and CCR6-subtype of CLA+ CD4+ Foxp3+ Treg cells.Preliminary results:SEB stimulation had no effect on the number of CLA+ CD4+ Foxp3+ Treg cells in AD patients, asthma/ARpatient, and healthy subjects. SEB decreased the CCR6+ / CCR6- ratios in CLA+ CD4+ Foxp3+ Treg cells fromAD patients and increased the CCR6+ / CCR6- ratios in those from asthma/AR patients and healthy subjects.SEB preferentially induced production of TH2 cytokine IL-5 in CCR6- subtype and anti-inflammatory cytokineIL-10 in CCR6+ subtype of CLA+ CD4+ Foxp3+ Treg cells from AD patients, asthma/AR patient, and healthysubjects. CLA+ CD4+ Foxp3+ Treg cells from AD patients produced more IL-5 and less IL-10 after SEBstimulation than those from healthy subjects. CCR6- subtype of CLA+ CD4+ Foxp3+ Treg cells from ADpatients and CCR6+ subtype of those from asthma/AR patients and healthy subjects were more resistant toSEB-induced caspase-3 activation than the other subtype. CCR6- subtype of CLA+ CD4+ Foxp3+ Treg cellsfrom AD patients and CCR6+ subtype of those cells from healthy subjects were more resistant to SEB-inducedcaspase-3 activation than those from other subjects.