Elucidation of developing mechanisms for infectious endocarditis using a proteomic technique

使用蛋白质组学技术阐明感染性心内膜炎的发展机制

• 批准号: 14571953
• 负责人: YAMAGUCHI Noboru
• 金额: $ 2.18万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571953/
• 关键词: periodontopathic bacteria; human endothelial cell; proteomics; inflammatory cytokine; leukotoxin; apoptosis; integrin; caspase

First of all, to elucidate the developing mechanisms for infectious endocarditis, we analyzed some proteins which are induced and produced from human endothelial cells (EA.hy926) infected with Acanobacillus acdnornycetemcomians (Aa) strain SUNY465 using a proteomic technique. This result suggested that several inflammatory cytokines might be induced after the bacterial invasion to the cell. We demonstrated previously that Aa leukotoxin (Ltx) is strongly able to induce apoptotic signal of cells that are positive for lymphocyte function-associated antigen-1 (LFA-1), a cell receptor of Ltx. So we next investigated whether inflammatory cytokines regulate apoptosis of human leukemic HL-60 cells induced by the Ltx. Of these cytokines tested, TNF-α, IL-1α and IL-1β significantly enhanced the Ltx-induced cell apoptosis. Northern and Western blotting analyses showed that TNF-a enhanced the expression of CD11a in the cells at mRNA and protein level, but did not for CD18. TNF-α also enhanced the binding of Ltx to the cells. We also observed by measuring mitochondrial transmembrane potential and the generation of superoxide. anion that the cytokine enhanced Ltx-induced HL-60 cell apoptosis. The enhancing action was strongly neutralized by anti-TNF-receptor 1 antibody, though anti-TNF-receptor 2 antibody did not so. In addition, IL-1α and β enhanced the cell apoptosis as the same mode as TNF-α. Furthermore, caspase-3 intracellular activity assay (PhiPhiLux^<TM>G_1D_2) showed that the Ltx-induced caspase-3 activation was completely neutralized by CD18 antibody treatment, though the significant neutralization was also observed by CD11a antibody one. Taken together, the present study indicates that TNF-α acts as a potent stimulator of the Ltx-induced HL-60 apoptosis via TNF-receptor 1-mediated upregulation for LEA-1 expression.

首先，为了阐明感染性心内膜炎的发育机制，我们分析了一些蛋白质，这些蛋白质是由人类内皮细胞（EA.HHY926）诱导和产生的，这些蛋白质使用蛋白质组学技术感染了Acanobacillus acdnornycetemcomians（aa）菌株Suny465。该结果表明，细菌侵入细胞后可能会诱导几种炎性细胞因子。我们先前证明了AA白细胞毒素（LTX）非常能够诱导细胞的凋亡信号，这些信号对淋巴细胞功能相关的抗原-1（LFA-1），LTX的细胞受体呈阳性。因此，我们接下来研究了炎性细胞因子是否调节LTX诱导的人白血病HL-60细胞的凋亡。在这些测试的细胞因子中，TNF-α，IL-1α和IL-1β显着增强了LTX诱导的细胞凋亡。 Northern和Western印迹分析表明，TNF-A在mRNA和蛋白质水平下增强了CD11a在细胞中的表达，但不适合CD18。 TNF-α还增强了LTX与细胞的结合。我们还通过测量线粒体跨膜电位和产生超氧化物来观察。细胞因子增强了LTX诱导的HL-60细胞凋亡的阴离子。抗TNF受体1抗体将增强作用强烈中和，尽管抗TNF受体2抗体并非如此。另外，IL-1α和β增强了与TNF-α相同模式的细胞凋亡。此外，caspase-3细胞内活性测定（phiphilux^<tm> g_1d_2）表明，尽管通过CD18抗体处理，LTX诱导的caspase-3激活被CD18抗体处理完全中和，但CD11a抗体也观察到了显着的中和。综上所述，本研究表明TNF-α充当LTX诱导的HL-60凋亡的有效刺激剂，通过TNF受体1介导的LEA-1表达上调。

项目成果

Yamaguchi, N., et al.: "TNF-alpha enhances A.actinomycetemcomitans leukotoxin-induced HL-60 cell apoptosis by stimulating LFA-1 expression"Infection and Immunity. 72(1). 269-276 (2004)

Yamaguchi, N. 等人：“TNF-α 通过刺激 LFA-1 表达增强 A.actinomycetemcomitans 白毒素诱导的 HL-60 细胞凋亡”感染和免疫。

Yamaguchi, N., et al.: "TNF-alpha enhances A.actinomycetemcomitans leukotoxin-induced HL-60 cell apoptosis by stimulating LFA-1 expression"Infection and Immunity. 72. 269-276 (2004)

Yamaguchi, N. 等人：“TNF-α 通过刺激 LFA-1 表达增强 A.actinomycetemcomitans 白毒素诱导的 HL-60 细胞凋亡”感染和免疫。

Yamaguchi, N., et al.: "TNF-alpha enhances Actinobacillus actinomycetemcomitans leukotoxin-induced HL-60 cell apoptosis by stimulating LFA-1 expression"Infection and Immunity. 72・1. 269-276 (2004)

Yamaguchi, N., et al.：“TNF-α 通过刺激 LFA-1 表达增强放线放线杆菌白细胞毒素诱导的 HL-60 细胞凋亡”，感染与免疫 72·1 (2004)。