Abstract
In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRDeltaF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRDeltaF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRDeltaF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRDeltaF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.
Highlights
JULY 16, 2010 VOLUME 285 NUMBER 29 membrane conductance regulator (CFTR).4 Defective CFTR function in the airway epithelium is responsible for cystic fibrosis (CF) lung disease, the most life-threatening complication of CF, characterized by mucus hypersecretion and neutrophil-dominated inflammation
We investigated the role of p38␣ MAPK in mediating proinflammatory cytokine production in airway epithelial cells expressing the most common mutation found in CF, CFTR⌬F508 [20]
KAP-K2 was seen on a biopsy of a healthy lung (Fig. 1). Medium from these cells to stimulate neutrophil migration. These results indicate that the p38 MAPK pathway is more When fresh human neutrophils isolated from peripheral blood activated in the airway epithelium of CF patients
Summary
Materials—SB203580 was obtained from InvivoGen (San Diego, CA). PD184352 was bought from USBiological (Swampscott, MA). One milliliter of cell supernatant was incubated with each membrane according to the manufacturer’s protocol. The CuFi airway epithelial cell line was derived from lung of a 14-year-old female patient with cystic fibrosis by the same method and is homozygous for the CFTR⌬F508 mutation. These cells were purchased from the ATCC or, alternatively, generously provided by Dr Emmanuelle Brochiero (Centre de Recherche, Hotel-Dieu du CHUM, Universitede Montreal, Canada). The same experiment was performed by adding recombinant human IL-6 (200 pg/well) in Dulbecco’s modified Eagle’s medium to the lower chamber instead of conditioned medium. Statistical Analysis—Analyses of variance followed by a multiple comparison test (Bonferroni) were used to test differences in mean between groups. p values of Ͻ0.05 were considered significant
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