Inhibition of Fas-induced apoptosis in cultured cells infected with Trypanosoma cruzi

本实验室以引起恰加斯病的原生动物鞭毛虫--克氏锥虫和感染的HeLa细胞为模型系统，研究宿主细胞的细胞生物学和病理学。我们在此报道，克氏毛滴虫感染抑制了Fas介导的HeLa细胞的凋亡。将指数生长的HeLa细胞接种到24孔板的每孔中，并在37ﾟC下生长2天。用克氏锥虫感染细胞，继续培养4-5天。加入抗Fas单抗诱导对照和感染HeLa细胞发生凋亡。进一步培养，用PBS洗涤，用Giemsa或Hoechst 33342染色，在显微镜下观察。用荧光素标记的磷脂酰乙醇胺探针对Fas诱导的HeLa细胞进行荧光染色和流式细胞术分析，观察磷脂从膜内到膜外的移位情况。加入抗Fas抗体后1d，对照HeLa细胞大多呈圆形，并从孔中脱落。相反，一些感染的细胞显示出明显的正常形态，附着在井上。在Fas介导的细胞凋亡诱导后6h，Hoechst 33342染色显示59.5%的对照细胞发生了凋亡，而感染的细胞中有30.0%发生了凋亡，这表明克氏锥虫感染对宿主细胞的凋亡有明显的抑制作用。对照HeLa细胞比感染细胞含有更多的荧光素阳性细胞。这意味着磷脂酰乙醇胺从质膜内侧向外侧的移位发生在细胞凋亡的早期阶段。抑制Fas介导的细胞凋亡可能构成克氏锥虫一种新的免疫逃逸机制。来自寄生虫和宿主细胞的潜在关键分子现在正在调查中。

In our laboratory, Trypanosoma cruzi, the parasitic protozoan flagellate that causes Chagas' disease, and infected HeLa cells are utilized as a model system in which to investigate the cell biology and pathology of the host cells. We report here that T.cruzi infection inhibits Fas-mediated apoptosis in HeLa cells. Exponentially growing HeLa cells were inoculated into each well of a 24-well plate, and allowed to grow at 37ﾟC for 2 days. The cells were infected with T.cruzi trypomastigotes and further incubated for 4-5 days. Apoptosis was induced in control and infected HeLa cells, by the addition of anti-Fas monoclonal antibody. The cultures were further incubated, washed with PBS, and stained with Giemsa or Hoechst 33342 for observation under a microscope. To examine the translocation of phospholipids from the inside to the outside of the plasma membrane, Fas-induced HeLa cells were stained with a fluorescein-labeled probe specific for phosphatidylethanolamine and analyzed by flow cytometry. One day after the addition of anti-Fas antibody, most of the control HeLa cells appeared roundish and detached from the well. In contrast, some infected cells showed an apparently normal morphology, adhering to the well. Six hours after the induction of Fas-mediated apoptosis, nuclear staining with Hoechst 33342 showed that 59.5 % of control cells were undergoing apoptosis, whereas 30.0 % of the infected cells were apoptotic, indicating that T.cruzi infection markedly inhibits host-cell apoptosis. The control population of HeLa cells contained more fluorescein-positive cells than the infected cell population. This implies that the translocation of phosphatidylethanolamine from the inner side to the outer side of the plasma membrane takes place in an early stage of apoptosis. Inhibition of Fas-mediated apoptosis may constitute a novel immune escape mechanism for T.cruzi . Potentially critical molecules from parasite and host cells are now under investigation.