Danelishvili Lia, McGarvey Jeffery, Li Yong-Jun, Bermudez Luiz E
Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, CA 94115, USA.
Cell Microbiol. 2003 Sep;5(9):649-60. doi: 10.1046/j.1462-5822.2003.00312.x.
Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.
结核分枝杆菌与肺肺泡空间中的巨噬细胞和上皮细胞相互作用，在这两种细胞类型中它都能够侵入并复制。结核分枝杆菌对这些细胞的细胞毒性已有充分记载，但宿主细胞死亡的机制尚不清楚。我们研究了强毒株（H37Rv）和减毒株（H37Ra）结核分枝杆菌对人巨噬细胞（U937）和II型肺泡上皮细胞（A549）凋亡和坏死的诱导作用。通过酶联免疫吸附测定（ELISA）和TdT介导的dUTP缺口末端标记（TUNEL）测定来确定凋亡，而通过乳酸脱氢酶（LDH）的释放来评估坏死。强毒株和减毒株结核分枝杆菌均可诱导巨噬细胞凋亡；然而，感染5天后，减毒株导致的凋亡明显多于强毒株。相比之下，肺泡细胞的细胞毒性是坏死的结果，而非凋亡。尽管感染结核分枝杆菌菌株导致单层细胞中有14%的细胞凋亡，但感染5天后，在59%的肺泡上皮细胞中观察到与坏死相关的细胞死亡。结核分枝杆菌感染抑制了激酶抑制剂星形孢菌素诱导的肺泡上皮细胞凋亡。由于我们的研究结果表明结核分枝杆菌可以调节巨噬细胞和上皮细胞的凋亡反应，我们对人巨噬细胞和肺泡上皮细胞进行了凋亡途径特异性的cDNA微阵列分析。在感染（48小时）的肺泡上皮细胞中，凋亡抑制因子bcl-2和Rb上调超过2.5倍，而促凋亡基因bad和bax下调。当U937巨噬细胞感染结核分枝杆菌时，观察到相反的情况。用结核分枝杆菌感染肺泡上皮细胞后，由半胱天冬酶-1、半胱天冬酶-3和半胱天冬酶-10的表达所确定的凋亡产生受到抑制。细胞内细菌复制的抑制导致两种细胞类型的凋亡增加。我们的结果表明，巨噬细胞和肺泡上皮细胞之间凋亡诱导的差异代表了结核分枝杆菌在宿主体内存活的特定策略。
