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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 5, May, 2001 583-590

Nitrogen Dioxide Induces Death in Lung Epithelial Cells in a Density-Dependent Manner

Rebecca L. Persinger, Wendy M. Blay, Nicholas H. Heintz, David R. Hemenway, and Yvonne M. W. Janssen-Heininger

Departments of Pathology and Civil and Environmental Engineering, University of Vermont College of Medicine, Burlington, Vermont

Nitrogen dioxide (·NO2) is commonly known as an indoor and outdoor air pollutant. Inhalation of ·NO2 is associated with epithelial cell injury, inflammation, and the aggravation of asthma. ·NO2 can also be formed during inflammation, by the metabolism of nitric oxide. We describe a gas-phase exposure system for in vitro exposure of lung epithelial cells to ·NO2. Immunofluorescence revealed 3-nitrotyrosine immunoreactivity of rat alveolar type II epithelial cells exposed to 5 parts per million of ·NO2 for 4 h. Comparative analysis of log-phase and confluent cultures demonstrated that cell death occurred extensively in log-phase cells, whereas minimal death was observed in confluent cultures. Peroxynitrite (ONOO-) or the ONOO- generator 3-morpholinosydnonimine (SIN-1) caused similar amounts of death. Further, exposure of wounded cell cultures to ·NO2 or SIN-1 revealed that death was restricted to cells repopulating a wounded area. Cycloheximide or actinomycin D, inhibitors or protein and messenger RNA synthesis, respectively, significantly reduced terminal transferase reactivity, suggesting that a new protein(s) may be required for cell death. These results suggest that during restitution after pulmonary injury, epithelium may be sensitive to cell death by reactive nitrogen species.

Abbreviations: actinomycin D, Act D; analysis of variance, ANOVA; cycloheximide, CHX; dichlorofluorescein diacetate, DCF; hydrogen peroxide, H2O2; lactate dehydrogenase, LDH; messenger RNA, mRNA; nitric oxide, ·NO; nitrogen dioxide, ·NO2; peroxynitrite, ONOO-; propidium iodide, PI; parts per million, ppm; rat lung alveolar type II epithelial cells, RLE cells; reactive nitrogen species, RNS; standard error of the mean, SEM; 3-morpholinosydnonimine, SIN-1; superoxide dismutase, SOD; single-stranded DNA, ssDNA; labeling with digoxygenin-uridine triphosphate using terminal transferase, TUNEL.




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