OPTIMIZATION OF CATIONIC LIPOSOME-MEDIATED GENE TRANSFER TO HUMAN BRONCHIAL EPITHELIAL CELLS EXPRESSING WILD-TYPE OR ABNORMAL CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR)

ABSTRACT Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

Download Full-text

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00094.2011 ◽

2011 ◽

Vol 301 (4) ◽

pp. L557-L567 ◽

Cited By ~ 37

Author(s):

Ahmed Lazrak ◽

Asta Jurkuvenaite ◽

Lan Chen ◽

Kim M. Keeling ◽

James F. Collawn ◽

...

Keyword(s):

Cystic Fibrosis ◽

Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Alveolar Type ◽

Transmembrane Conductance Regulator ◽

Wild Type ◽

Transmembrane Conductance ◽

Alveolar Epithelial ◽

Cystic Fibrosis Transmembrane

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous ( Cftr +/−), knockout ( Cftr −/−), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (Po): wild-type=0.21 ± 0.015: Cftr +/−=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr −/−=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the Po and number of channels in ATII cells of wild-type, Cftr +/−, and ΔF508, but not in Cftr −/− mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr +/− and Cftr −/− mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.

Download Full-text