Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H ؉ ,K ؉ -ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent HCO 3 ؊ secretory pathway that was defective in CF: (i) ASL [HCO 3 ؊ ] was higher in NL than CF ASL; (ii) activating CFTR with forskolin͞3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pH ASL is abnormally regulated under basal conditions because of absent CFTRdependent HCO 3 ؊ secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.Among the most serious manifestations of CF are chronic pulmonary infections with the bacterium P. aeruginosa. The basis for hypersusceptibility of CF patients to this bacterium is not well understood, and the role of mutant CFTR, if any, is not clear. Binding and internalization of respiratory pathogens by epithelial cells followed by desquamation could be an important mechanism for clearing bacteria from the lung. This mechanism has been shown to be important in protecting against bladder infections (1).To investigate whether the most common and severe CFTR mutation (ΔF508) affected uptake of P. aeruginosa, we performed bacterial invasion assays (2) with four cell lines: CFT1, an airway epithelial cell line derived from a CF patient homozygous for ΔF508 CFTR and that is transformed with human papilloma virus 18 E6/E7; CFT1-ΔF508, which expresses a third copy of ΔF508 CFTR introduced by a retrovirus; CFT1-LC3, which expresses a control gene (β-galactosidase) introduced by the same retrovirus; and CFT1-LCFSN, which expresses a functional wild-type human CFTR gene (3). We tested a standard laboratory strain of P. aeruginosa, designated PAOI, and two nonmucoid, LPSsmooth clinical isolates from CF patients (4). Compared with CFT1-LCFSN cells, the three lines expressing ΔF508 CFTR internalized significantly fewer bacterial cells (Fig. 1A). The ΔF508 mutation causes inefficient processing of CFTR, a defect that is partially corrected if the cells are grown at 26°C (5). When epithelial cells were cultured for 72 hours at 26°C there were no longer significant differences in uptake of the P. aeruginosa strains by the cells expressing wild-type or mutant CFTR (Fig. 1B). Because the overall uptake of bacteria at 26°C was low, we performed additional experiments with cells grown for 72 hours at 26°C in which the invasion assay was performed at 37°C for 3 hours, conditions under which surface expression of mutant ΔF508 CFTR is maintained (5). No significant difference in bacterial cell uptake was measured (Fig. 1C), and overall amounts of internalization approached those of the CFT1-LCFSN cells at 37°C. Returning cells expressing ΔF508 CFTR to 37°C for 24 hours after growth for 72 hours at 26°C removes CFTR from the cell surface (5); under these conditions internalization of the bacterial strains ...

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.

In patients with cystic fibrosis, adenoviral-vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer.

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor ␣ (TNF-␣), interferon ␥ (IFN-␥), and IL-1␤ in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1␤ treatment led to alterations in TJ ion selectivity, combined treatment of TNF-␣ and IFN-␥ induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-␣ and IFN-␥. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.