title: Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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. k068737@eve.u-ryukyu.ac.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Furugen
authors.forename: Futoshi
authors.initials: F
authors.lastname: Higa
authors.forename: Kenji
authors.initials: K
authors.lastname: Hibiya
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Teruya
authors.forename: Morikazu
authors.initials: M
authors.lastname: Akamine
authors.forename: Shusaku
authors.initials: S
authors.lastname: Haranaga
authors.forename: Satomi
authors.initials: S
authors.lastname: Yara
authors.forename: Michio
authors.initials: M
authors.lastname: Koide
authors.forename: Masao
authors.initials: M
authors.lastname: Tateyama
authors.forename: Naoki
authors.initials: N
authors.lastname: Mori
authors.forename: Jiro
authors.initials: J
authors.lastname: Fujita
mesh_terms: D000893:Anti-Inflammatory Agents / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology; D024243:HMGB1 Protein / Q000378:metabolism*; D006801:Humans; D016952:Legionella pneumophila / Q000502:physiology*; D011239:Prednisolone / Q000008:administration & dosage*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology*
chemical_list: D000893:Anti-Inflammatory Agents; D024243:HMGB1 Protein; D011239:Prednisolone; D020169:Caspases