Role of airway surface liquid and submucosal glands in cystic fibrosis lung disease

Cysticfibrosis (CF) is caused by mutations in the CF transmembraneconductance regulator (CFTR) protein, an epithelial chloride channelexpressed in the airways, pancreas, testis, and other tissues. Acentral question is how defective CFTR function in CF leads to chroniclung infection and deterioration of lung function. Several mechanismshave been proposed to explain lung disease in CF, including abnormalairway surface liquid (ASL) properties, defective airway submucosalgland function, altered inflammatory response, defective organellaracidification, loss of CFTR regulation of plasma membrane iontransporters, and others. This review focuses on the physiology of theASL and submucosal glands with regard to their proposed role in CF lungdisease. Experimental evidence for defective ASL properties and glandfunction in CF is reviewed, and deficiencies in understanding ASL/glandphysiology are identified as areas for further investigation. New modelsystems and measurement technologies are being developed to makeprogress in establishing lung disease mechanisms in CF, which shouldfacilitate mechanism-based design of therapies for CF.

     

Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR?(/)? pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR?(/)? epithelia showed markedly reduced Cl? and HCO?? transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR?(/)? pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl? conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl? and HCO?? in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.     

Airway epithelial cell inflammatory signalling in cystic fibrosis

Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians, estimated to affect one out of 2500-4000 new-borns. In patients with CF, lack of CF transmembrane conductance regulator (CFTR) Cl(-) channel function leads to progressive pulmonary damage and ultimately to death. Severe and persistent polymorphonuclear neutrophil-dominated endobronchial inflammation and chronic bacterial infection are characteristic hallmarks of CF lung disease. Whether CFTR dysfunction results directly in an increased predisposition to infection and whether inflammation arises independent of infection remains to be established. The loss of functional CFTR in airway epithelial cells promotes depletion and increased oxidation of the airway surface liquid. Activated neutrophils present in airways produce large amounts of proteases and reactive oxygen species (ROS). Together these changes are associated with diminished mucociliary clearance of bacteria, activation of epithelial cell signalling through multiple pathways, and subsequent hyperinflammatory responses in CF airways. The NF-kappaB pathway and Ca(2+) mobilization in airway epithelial cells are believed to be of key importance for control of lung inflammation through regulated production of mediators such as interleukin-8 that participate in recruitment and activation of neutrophils, modulation of apoptosis, and control of epithelial barrier integrity. In this review, the current understanding of the molecular mechanisms by which airway epithelial cells contribute to abnormal lung inflammation in CF, as well as the anti-inflammatory strategies that can be proposed are discussed.     

Cystic fibrosis: a disease of vulnerability to airway surface dehydration

Cystic fibrosis (CF) lung disease involves chronic bacterial infection of retained airway secretions (mucus). Recent data suggest that CF lung disease pathogenesis reflects the vulnerability of airway surfaces to dehydration and collapse of mucus clearance. This predisposition is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in (i) the absence of CFTR-mediated Cl- secretion and regulation of epithelial Na+ channel (ENaC) function; and (ii) the sole dependence on extracellular ATP to rebalance these ion transport processes through P2 purinoceptor signaling. Recent clinical studies indicate that inhalation of hypertonic saline osmotically draws sufficient water onto CF airway surfaces to provide clinical benefit.     

Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by chronic inflammation and an imbalance in the concentrations of alveolar and lung oxidants and antioxidants, which result in cell damage. Modifications in lung glutathione concentrations are recognized as a salient feature of inflammatory lung diseases such as cystic fibrosis, and glutathione plays a major role in protection against oxidative stress and is important in modulation of apoptosis. The cystic fibrosis transmembrane conductance regulator (CFTR) is permeable to Cl(-), larger organic ions, and reduced and oxidized forms of glutathione, and the DeltaF508 CFTR mutation found in cystic fibrosis patients has been correlated with impaired glutathione transport in cystic fibrosis airway epithelia. Because intracellular glutathione protects against oxidative stress-induced apoptosis, we studied the susceptibility of epithelial cells (HeLa and IB3-1) expressing normal and mutant CFTR to apoptosis triggered by H(2)O(2). We find that cells with normal CFTR are more sensitive to oxidative stress-induced apoptosis than cells expressing defective CFTR. In addition, sensitivity to apoptosis could be correlated with glutathione levels, because depletion of intracellular glutathione results in higher levels of apoptosis, and glutathione levels decreased faster in cells expressing normal CFTR than in cells with defective CFTR during incubation with H(2)O(2). The pro-apoptotic BCL-2 family member, BAX, is also activated faster in cells expressing normal CFTR than in those with mutant CFTR under these conditions, and artificial glutathione depletion increases the extent of BAX activation. These results suggest that glutathione-dependent BAX activation in cells with normal CFTR represents an early step in oxidative stress-induced apoptosis of these cells.     

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.     

Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co‐cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o‐ line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F‐actin content was increased in co‐cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co‐cultures had a tendency of increased expression of occludin and ZO‐1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC‐CFBE co‐cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC‐CFBE co‐cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co‐culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.     

Metabolomic Profiling Reveals Biochemical Pathways and Biomarkers Associated with Pathogenesis in Cystic Fibrosis Cells

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.     

Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant DeltaF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-kappaB. G551D mutants also lack Cl- channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-kappaB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked DeltaF508 allele caused a sevenfold activation of NF-kappaB compared with wild-type CFTR or the G551D mutant (P < 0.001). NF-kappaB was also activated in 9/HTEo-/pCep-R cells and in 16HBE/pcftr antisense cell lines, which lack CFTR Cl- channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-kappaB was associated with elevated interleukin-8 expression. Impaired CFTR Cl- channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-kappaB in cells with the CFTR mutation.     

Airway epithelial cells—Hyperabsorption in CF?

Airway epithelial cells transport electrolytes and are central to the disease cystic fibrosis (CF), which is an inherited transport defect affecting smaller airways and a number of other epithelial organs. Clinically, CF is dominated by a chronic lung disease, the main cause of morbidity and mortality. Airway obstruction by thick mucus and chronic infection by Pseudomonas aeruginosa eventually lead to loss of pulmonary function. Loss of function of CFTR Cl^? channels was found to be the cause for CF. However, intensive research on the detailed mechanism of CF lung disease for more than 25 years produced a bewildering number of hypotheses and an endless discussion whether reduced Cl^? secretion, primarily located in airway submucosal glands, or dehydration of the airways, driven by a hyperabsorption of Na⁺ ions, is the primary cause of the disease. Recent results suggest a fine-tuned regulation of the airway fluid layer, but how significant really are Cl^? and Na⁺ transport?     

Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.     

Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions

A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN(-)) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN(-)). Airway epithelial cultures have been shown to secrete SCN(-) in a CFTR-dependent manner. Thus, reduced SCN(-) availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN(-) concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN(-) concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN(-) concentration was somewhat reduced. Among human subjects, SCN(-) concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN(-) levels had better lung function than those with low SCN(-) levels. Thus, although CFTR can contribute to SCN(-) transport, it is not indispensable for the high SCN(-) concentration in ASL. The correlation between lung function and SCN(-) concentration in CF patients may reflect a beneficial role for SCN(-).     

Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) lung disease is characterized by persistent airway inflammation and airway infection that ultimately leads to respiratory failure. Aspergillus sp. are present in the airways of 20-40% of CF patients and are of unclear clinical significance. In this study, we demonstrate that CF transmembrane conductance regulator (CFTR)-deficient (CFTR knockout, Cftr(tm1Unc-)TgN(fatty acid-binding protein)CFTR) and mutant (DeltaF508) mice develop profound lung inflammation in response to Aspergillus fumigatus hyphal Ag exposure. CFTR-deficient mice also develop an enhanced Th2 inflammatory response to A. fumigatus, characterized by elevated IL-4 in the lung and IgE and IgG1 in serum. In contrast, CFTR deficiency does not promote a Th1 immune response. Furthermore, we demonstrate that CD4+ T cells from naive CFTR-deficient mice produce higher levels of IL-4 in response to TCR ligation than wild-type CD4+ T cells. The Th2 bias of CD4+ T cells in the absence of functional CFTR correlates with elevated nuclear levels of NFAT. Thus, CFTR is important to maintain the Th1/Th2 balance in CD4+ T cells.     

Effects of cystic fibrosis transmembrane conductance regulator and DeltaF508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.     

Estrogen and the cystic fibrosis gender gap

Cystic fibrosis (CF) is the most frequent inherited disease in Caucasian populations and is due to a defect in the expression or activity of a chloride channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in this gene affect organs with exocrine functions and the main cause of morbidity and mortality for CF patients is the lung pathology in which the defect in CFTR decreases chloride secretion, lowering the airway surface liquid height and increasing mucus viscosity. The compromised ASL dynamics leads to a favorable environment for bacterial proliferation and sustained inflammation resulting in epithelial lung tissue injury, fibrosis and remodeling. In CF, there exist a difference in lung pathology between men and women that is termed the “CF gender gap”. Recent studies have shown the prominent role of the most potent form of estrogen, 17β-estradiol in exacerbating lung function in CF females and here, we review the role of this hormone in the CF gender dichotomy.     

Myeloperoxidase promoter polymorphism -463G is associated with more severe clinical expression of cystic fibrosis pulmonary disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.     

Sequential analysis of surfactant,lung function and inflammation in cystic fibrosis patients

Background

In a cross-sectional analysis of cystic fibrosis (CF) patients with mild lung disease, reduced surfactant activity was correlated to increased neutrophilic airway inflammation, but not to lung function. So far, longitudinal measurements of surfactant function in CF patients are lacking and it remains unclear how these alterations relate to the progression of airway inflammation as well as decline in pulmonary function over time.

Methods

As part of the BEAT trial, a longitudinal study to assess the course of airway inflammation in CF, we studied lung function, surfactant function and endobronchial inflammation using bronchoalveolar lavage fluid from 20 CF patients with normal pulmonary function (median FEV₁ 94% of predicted) at three times over a three year period.

Results

There was a progressive loss of surfactant function, assessed as minimal surface tension. The decline in surfactant function was negatively correlated to an increase in neutrophilic inflammation and a decrease in lung function, assessed by FEV₁, MEF_75/25%VC, and MEF_25%VC. The concentrations of the surfactant specific proteins A, C and D did not change, whereas SP-B increased during this time period.

Conclusion

Our findings suggest a link between loss of surfactant function driven by progressive airway inflammation and loss of small airway function in CF patients with limited lung disease.     

Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl- channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be 'corrected' through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be 'potentiated', pharmacologically. Importantly, two such small molecules, a 'corrector' (VX-809) and a 'potentiator' (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.     

Innate immune response in CF airway epithelia: hyperinflammatory?

The lack of functional cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in the apical membranes of CF airway epithelial cells abolishes cAMP-stimulated anion transport, and bacteria, eventually including Pseudomonas aeruginosa, bind to and accumulate in the mucus. Flagellin released from P. aeruginosa triggers airway epithelial Toll-like receptor 5 and subsequent NF-B signaling and production and release of proinflammatory cytokines that recruit neutrophils to the infected region. This response has been termed hyperinflammatory because so many neutrophils accumulate; a response that damages CF lung tissue. We first review the contradictory data both for and against the idea that epithelial cells exhibit larger-than-normal proinflammatory signaling in CF compared with non-CF cells and then review proposals that might explain how reduced CFTR function could activate such proinflammatory signaling. It is concluded that apparent exaggerated innate immune response of CF airway epithelial cells may have resulted not from direct effects of CFTR on cellular signaling or inflammatory mediator production but from indirect effects resulting from the absence of CFTRs apical membrane channel function. Thus, loss of Cl^–, HCO₃^–, and glutathione secretion may lead to reduced volume and increased acidification and oxidation of the airway surface liquid. These changes concentrate proinflammatory mediators, reduce mucociliary clearance of bacteria and subsequently activate cellular signaling. Loss of apical CFTR will also hyperpolarize basolateral membrane potentials, potentially leading to increases in cytosolic [Ca²⁺], intracellular Ca²⁺, and NF-B signaling. This hyperinflammatory effect of CF on intracellular Ca²⁺ and NF-B signaling would be most prominently expressed during exposure to both P. aeruginosa and also endocrine, paracrine, or nervous agonists that activate Ca²⁺ signaling in the airway epithelia. Pseudomonas aeruginosa; Toll-like receptor; NF-B; oxidative stress; acidic airway surface liquid; calcium