| Summary: | 博士 === 臺灣大學 === 臨床醫學研究所 === 98 === 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.
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