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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 5, May, 2001 569-576

Intratracheal Instillation of Lipopolysaccharide in Mice Induces Apoptosis in Bronchial Epithelial Cells
No Role for Tumor Necrosis Factor-alpha and Infiltrating Neutrophils

Juanita H. J. Vernooy, Mieke A. Dentener, Robert Jan van Suylen, Wim A. Buurman, and Emiel F. M. Wouters

Departments of Pulmonology and General Surgery, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University; and Department of Pathology, University Hospital Maastricht, Maastricht, The Netherlands

This study investigated apoptosis in lungs after local exposure to lipopolysaccharide (LPS). Mice were instilled intratracheally with 5 µg LPS, which corresponds to the amount acquired by smoking approximately 25 cigarettes, and killed at different time points after exposure. Our data demonstrate that local LPS exposure resulted in apoptosis in lungs from 2 h and peaked at 24 h, as detected by ligation-mediated polymerase chain reaction. Morphologic examination and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end label staining demonstrated apoptosis in bronchial epithelial cells early after intratracheal (IT) LPS challenge, whereas infiltrating neutrophils displayed positive staining at 24 and 72 h after exposure. Apoptosis in lungs clearly preceded pulmonary neutrophil infiltration, confirming that neutrophils did not contribute to pulmonary apoptosis at early time points. Further, using three experimental approaches---namely, anti-tumor necrosis factor (TNF)-alpha treatment, IT TNF-alpha instillation, and TNF/ lymphotoxin-alpha knockout mice---we demonstrate that TNF-alpha , which was elevated in lungs at both messenger RNA and protein levels after IT LPS challenge, was no primary mediator in LPS-induced apoptosis at early time points. Thus, local LPS exposure in mice resulted in early apoptosis of bronchial epithelial cells independent of infiltrating neutrophils and TNF-alpha , which suggests that apoptosis of bronchial epithelium may be involved in airway injury during exposure to LPS.

Abbreviations: bronchoalveolar lavage, BAL; BAL fluid, BALF; base pair(s), bp; enzyme-linked immunosorbent assay, ELISA; immunoglobulin, Ig; intratracheal, IT; LPS binding protein, LBP; ligation-mediated, LM; lipopolysaccharide, LPS; lymphotoxin, LT; monoclonal antibody, mAb; myeloperoxidase, MPO; messenger RNA, mRNA; polymerase chain reaction, PCR; recombinant, r; reverse transcriptase, RT; standard error of the mean, SEM; Toll-like receptor, TLR; tumor necrosis factor, TNF; terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, TUNEL; ultraviolet, UV.




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