NF-κB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated withPseudomonas aeruginosa

Abstract

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-kappaB (NF-kappaB) 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-kappaB in cells isolated from five CF (DeltaF508/DeltaF508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-kappaB 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-kappaB activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-kappaB inhibitor IkappaBalpha 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-kappaB to nuclei of primary CF epithelial cell cultures, but intranuclear IkappaBalpha 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.

IL-8 Dictates Glycosaminoglycan Binding and Stability of IL-18 in Cystic Fibrosis

“…The latter observation can be explained at least in part by the ability of Pseudomonas-derived elastase to cleave IL-18 and also by greater numbers of neutrophils and, by corollary, greater levels of NE entering the microbe-infected CF lung. In addition, it has previously been shown that IL-8 expression is prolonged post P. aeruginosa stimulation (40), thus contributing to excessive neutrophil recruitment and NE burden within the CF airways. In support of our findings, NE inhibitors (A1AT, SLPI, elafin, and CMK) were able to inhibit the cleavage of IL-18, and purified NE was shown to rapidly cleave IL-18 in vitro.…”

Tumor Necrosis Factor-α in Airway Secretions from Cystic Fibrosis Patients Upregulate Endothelial Adhesion Molecules and Induce Airway Epithelial Cell Apoptosis: Implications for Cystic Fibrosis Lung Disease

“…Recent studies have reported higher constitutive expression and increased activation of NF-KB in unstimulated CF airway epithelial cells compared with non-CF cells (25)(26)(27)(28). NF-KB has been shown to exert an anti-apoptotic effect in many cell lines including respiratory epithelial cells (29)(30).…”

Lung and Systemic Inflammation in COPD

“…Joseph and colleagues demonstrated that in vitro activation of NF-κB in human airway epithelial cells isolated from CF (DeltaF508/DeltaF508) and non-CF (NCF) patients when infected by Pseudomonas aeruginosa elevated nuclear levels of IkB in CF cells, although this increase was transient. They also showed increased baseline translocation of NF-κB to nuclei in primary CF epithelial cell cultures; following Pseudomonas aeruginosa infection, activation of IκB might suppress that of NF-κB [84].…”