Furugen Makoto, Higa Futoshi, Hibiya Kenji, Teruya Hiromitsu, Akamine Morikazu, Haranaga Shusaku, Yara Satomi, Koide Michio, Tateyama Masao, Mori Naoki, Fujita Jiro
Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-Town, Okinawa 903-0215, Japan.
Respir Res. 2008 May 1;9(1):39. doi: 10.1186/1465-9921-9-39.
Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells.
Nuclear deoxyribonucleic acid (DNA) fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling method (TUNEL method) and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila.
The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining) and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1) protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 muM) did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells.
Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a major virulence factor of L. pneumophila, is involved in the effects we measured in alveolar epithelial cells. Methyl prednisolone may modulate the interaction of Legionella and these cells.
嗜肺军团菌肺炎常加重急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）。肺泡上皮细胞凋亡被认为在ALI和ARDS的发病机制中起重要作用。在本研究中，我们调查了嗜肺军团菌诱导A549肺泡上皮细胞发生凋亡的精确机制。我们还研究了甲泼尼龙对这些细胞凋亡的影响。
采用末端脱氧核苷酸转移酶介导的三磷酸脱氧尿苷（dUTP）-生物素缺口末端标记法（TUNEL法）和比色法半胱天冬酶活性测定，评估嗜肺军团菌感染的A549肺泡上皮细胞中的核脱氧核糖核酸（DNA）片段化和半胱天冬酶激活情况。本研究使用了有毒力的嗜肺军团菌菌株AA100jm和无毒力的dotO突变体并进行比较。此外，我们研究了甲泼尼龙是否对嗜肺军团菌感染的A549肺泡上皮细胞中的核DNA片段化和半胱天冬酶激活有任何影响。
嗜肺军团菌的有毒力菌株在A549肺泡上皮细胞内生长，并以剂量依赖的方式诱导随后的细胞死亡。无毒力的dotO突变体未显示出这种作用。嗜肺军团菌的有毒力菌株在A549细胞中诱导了DNA片段化（通过TUNEL染色显示）以及半胱天冬酶3、8、9和1的激活，而无毒力菌株则没有。高迁移率族蛋白B1（HMGB1）从感染有毒力军团菌的A549细胞中释放出来。甲泼尼龙（53.4 μM）不影响嗜肺军团菌在肺泡上皮细胞内的细胞内生长，但影响感染的A549细胞的DNA片段化和半胱天冬酶激活。
嗜肺军团菌感染A549肺泡上皮细胞导致程序性细胞死亡、各种半胱天冬酶的激活以及HMGB1的释放。dot/icm系统是嗜肺军团菌的主要毒力因子，参与了我们在肺泡上皮细胞中测量的效应。甲泼尼龙可能调节军团菌与这些细胞的相互作用。
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