Am. J. Respir. Cell Mol. Biol., Volume 26, Number 5, May, 2002 617-626

Early Mitochondrial Dysfunction, Superoxide Anion Production, and DNA Degradation Are Associated with Non-Apoptotic Death of Human Airway Epithelial Cells Induced by Pseudomonas aeruginosa Exotoxin A

Maria-Cristina Plotkowski, Helvécio C. C. Póvoa, Jean-Marie Zahm, Gérard Lizard, Geraldo M. B. Pereira, Jean-Marie Tournier, and Edith Puchelle

Department of Microbiology and Immunology and Laboratory of Electron Microscopy, UERJ, Rio de Janeiro, Brazil; INSERM UMRS U514, Reims, France; INSERM U498, Dijon, France; and Laboratory of Immunopathology, UERJ, Rio de Janeiro, Brazil

It has been shown that bacterial exoproducts may induce airway epithelium injury. During the epithelial repair process, the respiratory epithelial cells no more establish tight junctional intercellular complexes and may be particularly susceptible to bacterial virulence factors. In this study, we analyzed the effect of Pseudomonas aeruginosa exotoxin A (ETA) at different periods of time and concentrations on 16 HBE 14o human bronchial epithelial cells in culture conditions inducing a phenotype of repairing cells. ETA treatment for 24 and 48 h led to the killing of 40.0 ± 5.7% and 79.0 ± 1.4% of the cells, respectively, as determined by the dimethylthiazole 2,5 diphenyl tetrazolium bromide assay. At 1,000 ng/ml, ETA led to the killing of 25.2 ± 6.6, 59.4 ± 5.9, and 82.3 ± 3.7% of the cells, after treatment periods of 7, 24, and 48 h, respectively. Cell death could not be inhibited by z-VAD-fmk, a broad spectrum caspase inhibitor. By transmission electron microscopy, ultrastructural characteristics described in apoptosis were not detected in ETA-treated cells. Instead, the mitochondria of cells treated for 24 and 48 h with ETA at 100 and 1,000 ng/ml were highly condensed. Human nasal polyp epithelial cells in primary culture exposed to ETA at 1,000 ng/ml did not exhibit characteristic features of apoptotic cells either. Cytofluorometric analysis of ETA-treated 16 HBE 14o cells labeled with DiOC₆(3) and hydroethidine showed a time- and dose-dependent reduction of the mitochondrial transmembrane potential, detected 7 h after ETA treatment, and an increase in superoxide production, detected at 24 h, respectively. By a photometric assay, DNA degradation was also detected 7 h after cell treatment with ETA at 100 and 1,000 ng/ml. Taken together, our results show that ETA-induced death of epithelial respiratory cells was preceded by early mitochondrial dysfunction and superoxide anion production, but was not followed by the classically described apoptotic pathways.

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