2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/655010摘要：異位性皮膚炎是一種常見的慢性發炎性皮膚疾病。此病和氣喘、過敏性鼻炎，均為具有異位性體質者，才能引發的免疫疾病。異位性疾病的過敏原皆為環境中普遍存在的蛋白質抗原，其中有許多過敏原具有酵素活性。蛋白質抗原經皮致敏，已被証實為異位性皮膚炎重要的致敏途徑之ㄧ。我們於十幾年前建立小鼠持續性蛋白質貼膚模型(貼膚五天)，來研究”蛋白質抗原經皮致敏的免疫反應及機制”。發現在BALB/c 和B6 小鼠，蛋白質抗原持續經皮致敏引發顯著的第二型T 細胞反應，但所引發的第一型T 細胞反應很弱。最近，本研究室發展出小鼠非持續性蛋白質貼膚模型 (貼膚一天)，發現在BALB/c小鼠中，仍引發顯著的Th2 型反應，但在B6 小鼠中，可引發強烈的Ｔh1 反應，且其Th2 反應較BALB/c 小鼠更高。直接主導 T 細胞亞型分化方向的是樹突狀細胞。樹突狀細胞的性狀 (成熟度、共同刺激因子細胞素)，和其主導Th1/Th2/Th17 的分化，直接相關。對第一型T 細胞的分化而言，樹突狀細胞所產生的IL-12，是最重要的決定因子。對第二型T 細胞的分化而言，因為樹突狀細胞無法產生IL-4，故長久以來，學者們一直在尋找可以快速供給IL-4 的固有性免疫細胞。嗜鹼性淋巴球和自然殺手T 細胞，因可以在被刺激後，快速釋出大量IL-4，而一直被懷疑可能是第二型T 細胞反應的早期IL-4 供應者。近幾年來，關於嗜鹼性淋巴球的了解大有進步，目前已知在某些第二型T 細胞反應的小鼠模型中，尤其是蛋白質抗原具酵素活性者，嗜鹼性淋巴球會被召入淋巴結，釋放出IL-4 和TSLP 來幫助第二型T 細胞的分化。甚至在某些小鼠模型中，嗜鹼性淋巴球可以做為抗原呈獻細胞。在常用的實驗小鼠中，學者早就發現BALB/c 和B6 小鼠，分別易於引發第二型和第一型T 細胞反應。但是嗜鹼性淋巴球和自然殺手T 細胞的質和量，對引發這兩種小鼠的Th2反應的影響，則尚未有報告。本研究將利用非持續性小鼠貼膚模型，來研究嗜鹼性淋巴球和自然殺手 T 細胞，在BALB/c 和B6 小鼠的蛋白質抗原經皮致敏，所引發的Th2 反應中的角色。我們將使用三種抗原: ovalbumin, papain (具酵素活性)和heat-inactivated papain (不具酵素活性)，並測量經皮致敏後淋巴結內的嗜鹼性淋巴球和自然殺手細胞的數量，並分析其和Th2 反應的相關性，我們也將利用生物體內去除或增加嗜鹼性淋巴球或自然殺手細胞等方法，來確立這兩種細胞在BALB/c 和B6 小鼠中，幫助蛋白質抗原經皮致敏引發Th2 反應的角色，同時我們也將測量淋巴結內，以及這兩種細胞產生的細胞激素和細胞素的變化，來釐清其幫助Th2 反應的機制。本計畫將完成三項特定目標: 特定目標一: 確立嗜鹼性淋巴球有助於蛋白質抗原經皮致敏所引發的Th2 免疫反應。特定目標二: 確立自然T 殺手細胞有助於蛋白質抗原經皮致敏所引發的Th2 免疫反應。特定目標三: 釐清嗜鹼性淋巴球和自然殺手T 細胞幫助引發Th2 免疫反應的機制。計劃主持人長期致力於「異位性皮膚炎致病機轉」的研究，尤其著重在「蛋白質抗原經皮致敏的機制」，此計劃為此長期研究的一部分，我們期待這些研究所得到的資料，最終能幫助我們發展出避免過敏原致敏的方法或疫苗，使這些高危險群的兒童，能免受異位性疾病之苦。<br> Abstract: Atopic diseases are clinically common and important disorders. The known allergens ofatopic diseases are all universally existing environmental protein antigens. Many of themposses protease activity. Epicutaneous sensitization has been proven to be one of the mostimportant sensitization routes for atopic dermatitis. Using a murine continuous protein-patchmodel (five-day patch application), which we established over one decade ago, we and otherinvestigators have demonstrated that epicutaneous sensitization with protein antigen inducespredominant Th2, low Th17, and weak Th1 immune responses in BALB/c and B6 mice.Recently, our lab developed a murine non-continuous protein-patch model (one-day patchapplication) and observed that although BALB/c mice showed a predominant Th2 and weakTh1 response, B6 mice showed both strong Th1 and Th2 response.During priming, dendritic cells instruct naïve T cells to differentiate into Th1, Th2 orTh17 cells. Characters of DCs (maturation status, costimulator expression and cytokineproduction) have been proved to be crucial for their Th instructing capabilities. For Th1differentiation, IL-12 produced by dendritic cells is a key determinator. For Th2differentiation, due to the inability of dendritic cells to produce IL-4, it has been a long-termpursuit to search for early IL-4 provider in immune system. Basophils and iNKT cells bothare considered to be candidates due to their capability to release large amount of IL-4 rapidlyafter activation. Recently, the role of basophils in initiating Th2 responses has beenemphasized. Studies showed that basophils could be recruited into draining lymph nodes andsecrete IL-4 and TSLP to initiate Th2 differentiation in some experimental systems especiallywhen using protease as an antigen. Moreover, in some model systems, basophils function asantigen presenting cells to initiate Th2 differentiation. In murine experimental system,BALB/c and B6 mice have long been demonstrated to be Th2- and Th1-prone micerespectively. However, the contributions of basophils and/or iNKT cells to the development ofTh2 response in these two kinds of mice have never been explored.In this project, we will use murine non-continuous protein-patch model (one-day patchapplication) to investigative the roles of basophil and iNKT cell in development of Th2response by epicutaneous sensitization with protein antigen in BALB/c and B6 mice. We willuse three kinds of antigen: ovalbumin, papain (possessing enzyme activity), heat –inactivatedpapain (no enzyme activity). We will measure the amount of basophils and iNKT T cellsrecruited into regional lymph nodes after sensitization and search for correlation of numbersof basophils and iNKT cells with potency of Th2 responses. In addition, in vivo depletion orpromotion of basophil or iNKT cells will be performed to establish the roles of these cells indeveloping Th2 response. Finally, the cytokine and chemokine expressions in regional lymphnodes and the production of cytokines by basophil and iNKT cell after sensitization will bemeasured to elucidate the mechanisms. This project has the following specific aims: 1. Toestablish the role of basophil in epicutaneously-induced Th2 response. 2. To establish the roleof iNKT cell in epicutaneously-induced Th2 response. 3. To elucidate the mechanisms ofinitiating Th2 response with the keep of basophil and iNKT cell.We have focused on the research of “pathogenesis of atopic dermatitis “especially on”mechanisms of epicutaneous sensitization with protein antigens” for many years. This projectis a part of our long-term pursuit. We hope that the results from this project will help us tounderstand the mechanisms of allergen sensitization and finally we could develop usefulstrategy or vaccines to prevent atopic children from developing atopic diseases.蛋白質抗原經皮致敏嗜鹼性淋巴球自然殺手T 細胞第二型T 細胞反應protein antigenepicutaneous sensitizationbasophiliNKT cellTh2 response