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Am J Physiol Lung Cell Mol Physiol 288: L471-L479, 2005. First published October 29, 2004; doi:10.1152/ajplung.00066.2004
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NF-{kappa}B activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

Theresa Joseph,1 Dwight Look,2 and Thomas Ferkol1

1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; and 2Department of Internal Medicine, University of Iowa, Iowa City, Iowa

Submitted 27 February 2004 ; accepted in final form 26 October 2004

The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-{kappa}B (NF-{kappa}B) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-{kappa}B in cells isolated from five CF ({Delta}F508/{Delta}F508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-{kappa}B subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-{kappa}B activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-{kappa}B inhibitor I{kappa}B{alpha} were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-{kappa}B to nuclei of primary CF epithelial cell cultures, but intranuclear I{kappa}B{alpha} may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

interleukin-8; I{kappa}B; nuclear factor-{kappa}B; cystic fibrosis


Address for reprint requests and other correspondence: T. Ferkol, Division of Pediatric Allergy and Pulmonary Medicine, Dept. of Pediatrics, 660 S. Euclid Ave., Campus Box 8208, St. Louis, MO 63110 (E-mail: ferkol_t{at}kids.wustl.edu)




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