蜂膠主成份咖啡酸苯乙酯對於人類樹突細胞及T細胞的免疫調節功能及其機轉之探討

Abstract

氣喘是兒童主要的慢性病，近年來治療的進步，如吸入性類固醇 (目前的主要治療)、茶鹼、長效型beta2-adrenergic agonist、白三烯素拮抗劑和抗IgE抗體等已大幅減少了氣喘的死亡率和併發症，但仍無法完全控制困難治療的氣喘病患，因此我們想探討其他的治療方法及其機轉。 蜂膠是一種古老的傳統草藥，將蜂膠萃取物用於輕度至中度的氣喘病人作為輔助治療已被報告可以改善夜間症狀及肺功能。 雖然蜂膠的成分因不同地區的植物不同而有差異，其中的一種獨特成分－咖啡酸苯乙酯 [ caffeic acid phenethyl ester (CAPE) ] 廣泛存在於大部分的蜂膠中，且被報告有抗腫瘤、抗發炎、抗氧化及抗病毒的作用。 由於樹突細胞及Th2細胞在氣喘的致病機轉中扮演著重要的角色，因此本研究以咖啡酸苯乙酯與來自健康人及過敏病人被刺激活化的樹突細胞及T細胞共同培養，以探討咖啡酸苯乙酯對於健康人及過敏病人的樹突細胞及T細胞的免疫功能之影響。 首先，我們抽取人類週邊血分離出單核細胞中的CD14+ 單核球，再以GM-CSF及IL-4培養出樹突細胞 (monocyte-derived dendritic cells)，以此體外培養系統來研究咖啡酸苯乙酯對於樹突細胞的影響。 我們以咖啡酸苯乙酯與塵蟎萃取物或LPS共同刺激樹突細胞，以ELISA的方式檢測樹突細胞分泌的數種激素； 我們發現咖啡酸苯乙酯可以抑制來自於健康人被LPS刺激的樹突細胞所分泌的 IL-12 p40、IL-12 p70及IL-10，及被塵蟎萃取物刺激所分泌的IL-12 p40、IL-10 及IP-10； 在塵蟎過敏的病人中，咖啡酸苯乙酯可以抑制被塵蟎萃取物刺激的樹突細胞所分泌的IL-10 及IP-10。 接著我們以流式細胞儀分析樹突細胞的表面分子及其吞噬dextran-FITC的量以了解樹突細胞的成熟狀態； 我們發現咖啡酸苯乙脂並不影響MHC分子及costimulatory分子在細胞表面的表現以及樹突細胞的吞噬能力。 我們將刺激處理過的樹突細胞與自體naïve CD4+ T細胞共同培養，結果T細胞產生的細胞激素及複製增生並不受影響，換言之，咖啡酸苯乙酯並不影響樹突細胞呈現抗原給T細胞的抗原呈現能力。 為了進一步了解咖啡酸苯乙酯抑制細胞激素的機轉，我們使用LPS刺激樹突細胞並萃取其細胞蛋白質以西方點墨法來測定其訊息傳導路徑的改變； 我們發現咖啡酸苯乙酯會抑制被LPS刺激的樹突細胞所產生的I kappa B alpha磷酸化及NF-kappa B活化，但不影響MAPK家族的磷酸化。 因此，咖啡酸苯乙酯可以藉由抑制NF-kappa B的訊息傳導路徑來抑制樹突細胞分泌細胞激素的能力，且對於Th2細胞激素 (IL-10) 的抑制比率比Th1細胞激素 (IL-12) 的抑制比率高。 接著，我們抽取人類週邊血分離出單核細胞中的CD4+ T細胞，以研究咖啡酸苯乙酯對於T細胞直接的影響。 首先，我們使用soluble anti-CD3及anti-CD28單株抗體來刺激素T細胞以EILSA測定其所分泌的細胞激素，並使用tritiated thymidine incorporation的方式來測定T細胞的複製增生； 我們發現咖啡酸苯乙酯可以抑制來自於健康人及過敏病人被刺激的T細胞所分泌的IFN-gamma及IL-5，以及淋巴球的複製增生。 接著我們以西方點墨法來測定其息傳導路徑以了解咖啡酸苯乙酯的抑制機轉； 結果咖啡酸苯乙酯會抑制被soluble anti-CD3及anti-CD28單株抗體刺激的T細胞所產生的IKK及I kappa B alpha磷酸化、NF-kappa B活化及Akt磷酸化，但不影響p38 MAPK的磷酸化。 此外我們利用流式細胞儀來分析被咖啡酸苯乙酯處理過的T細胞內所表現的早期凋亡因子active caspase-3； 結果咖啡酸苯乙酯會使T細胞表現早期凋亡因子active caspase-3，而且CCR4+CD4+ T 細胞比 CXCR3+CD4+ T細胞更容易表現active caspase-3。 因此，咖啡酸苯乙酯可以藉由抑制NF-kappa B及Akt的訊息傳導路徑來抑制T細胞分泌細胞激素及細胞複製增生，且咖啡酸苯乙酯也會使T細胞表現早期凋亡因子active caspase-3，尤其是CCR4+CD4+ T 細胞，因此使這種似Th2細胞較容易產生細胞凋亡。 經由上述的研究我們發現了咖啡酸苯乙酯可以藉由抑制NF-kappa B及Akt的訊息傳導路徑來抑制樹突細胞及T細胞分泌細胞激素及淋巴球增生，因而抑制了下游的發炎反應； 咖啡酸苯乙酯也會使T細胞表現早期凋亡因子active caspase-3，尤其是CCR4+CD4+ T 細胞，因此使這種似Th2細胞較容易產生細胞凋亡，進而減少氣喘的後期發炎反應。 本研究建立了咖啡酸苯乙酯在治療氣喘及其他過敏疾病的角色，進而提供蜂膠在治療過敏疾病的學理基礎。

Asthma is the leading chronic disease in children. The mortality and morbidity of asthma has been decreased recently due to the advance of management of asthma. The treatment of asthma includes inhaled corticosteroid (the main drug), theophylline, long-acting beta2-agnoist, leukotriene antagonist and anti-IgE, etc. However, some severe difficult to treat patients still not achieve controlled under these treatments. We therefore intend to evaluate other potential treatments and their mechanism. Propolis, an ancient herbal medicine, has been reported the beneficial effect both in asthma patients and murine model of asthma, but the mechanism was not clearly understood. Caffeic acid phenethyl ester (CAPE), a unique active component in propolis, is known to have anti-tumor, anti-inflammatory and antioxidant properties. Since Th2 cells and dendritic cells play important roles in the pathogenesis of asthma, we use CAPE to treat CD4+ T cells and monocyte-derived dendritic cells (MoDCs) from healthy subjects and allergic patients to evaluate the effects of CAPE on the functions of human MoDCs and primary human CD4+ T cells in vitro. First, peripheral blood mononuclear cells (PBMCs) were isolated from non-atopic healthy subjects and mite-sensitized allergic patients. The CD14+ monocytes were isolated by autoMACS and cultured with GM-CSF and IL-4 to become MoDCs. We check the cytokine levels of stimulated MoDCs by ELISA. We found that CAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients’ MoDCs stimulated by crude mite extract. Further, we evaluated the surface markers and dextran-FITC by flow cytometry. We found that the upregulation of costimulatory molecules and phagocytosis alibity in mature MoDCs was not altered by CAPE. Therefore, the antigen presenting ability of MoDCs was not inhibited by CAPE by evaluating the cytokine levels and lymphoproliferation of the co-cultured autologous naïve CD4+ T cells. To study the mechanism of the inhibitory effect of CAPE, we extract the protein from LPS-stimulated MoDCs and check the signaling transduction by Western blot. CAPE inhibited I kappa B alpha phosphorylation and nuclear factor (NF)-kappa B activation but not mitogen-activated protein kinase (MAPK) family phosphorylation in human MoDCs. Together, CAPE inhibited the cytokine production of MoDCS by the inhibition of NF-kappa B activation and the inhibition rate of Th2 cytokine (IL-10) was higher than that of Th1 cytokine (IL-12). Next, we isolated CD4+ T cells from PBMCs of healthy subjects and mite-sensitized asthma patients by autoMACS. We check the cytokine production from stimulated T cells by ELISA and the lymphoproliferation by tritiated thymidine incorporation method. CAPE significantly suppressed IFN-gamma and IL-5 production and proliferation of CD4+ T cells stimulated by soluble anti-CD3 and anti-CD28 monoclonal antibodies both in healthy subjects and asthmatic patients. The signaling pathways were evaluated by Western blot. CAPE inhibited NF-kappa B activation and protein kinase B (Akt) phosphorylation, but not p38 MAPK phosphorylation in T cells. Besides, we evaluated the intracellular active caspase-3 expression by flow cytometry. CAPE could induce active caspase-3 expression in CD4+ T cells; CCR4+CD4+ T cells were more sensitive to CAPE induction than CxCR3+CD4+ T cells. Therefore, CAPE inhibited the cytokine production and lymphoproliferation of CD4+ T cells by the inhibition of NF-kappa B and Akt activation. CCR4+CD4+ T cells were more sensitive to CAPE induced active caspase-3 expression than CxCR3+CD4+ T cells, which indicated that Th2-like cells were more vulnerable to apoptosis and the late phase of allergic inflammation may decreased by CAPE treatment. To summary, our study indicated that CAPE inhibited cytokine and chemokine production by MoDCs and cytokine production and proliferation of T cells which might be related to the NF-kappa B and Akt signaling pathways. CCR4+CD4+ T cells are more sensitive to CAPE inhibition, which would result in decreased downstream inflammatory processes. This study provided a new insight into the mechanism of CAPE in immune response and the rationale for propolis in the treatment of asthma and other allergic disorders.