Cystic Fibrosis Respiratory Epithelia Research Articles

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.

173 Upregulated caspase activity in cystic fibrosis respiratory epithelium – a link to increased apoptosis?

173 Upregulated caspase activity in cystic fibrosis respiratory epithelium – a link to increased apoptosis?

Ibuprofen Modulates NF-ĸB Activity but Not IL-8 Production in Cystic Fibrosis Respiratory Epithelial Cells

Background: High-dose ibuprofen is clinically effective in cystic fibrosis (CF); however, its molecular mechanisms are poorly understood. Objective: To test the hypothesis that clinically relevant concentrations of ibuprofen suppress activation of nuclear factor (NF)-ĸB and thus down-regulate stimulated interleukin (IL)-8 production in CF respiratory epithelial cells. Methods: The majority of experiments were conducted in CFTE29o– cells (F508del-mutated CF transmembrane regulator, CFTR). Key experiments were confirmed in CFBE41o– cells (F508del-mutated CFTR) and 1HAEo– cells (wild-type CFTR). NF-ĸB and IL-8 were stimulated with tumour necrosis factor (TNF)-α or IL-1β. NF-ĸB and IL-8 suppression by ibuprofen (480 µM) was compared to dexamethasone (5 nM). Results: Both TNF-α and IL-1β activated NF-ĸB and stimulated IL-8 production. Both ibuprofen and dexamethasone demonstrated comparably modest suppression of NF-ĸB transcriptional activity. However, ibuprofen had no effect on stimulated IL-8 mRNA and protein. By contrast, dexamethasone significantly down-regulated stimulated IL-8 mRNA and protein. Conclusions: The present data do not support the hypothesis that ibuprofen down-regulates IL-8 production in response to TNF-α and IL-1β in CF respiratory epithelium. Suppression of NF-ĸB transcriptional activity does not discriminate between anti-inflammatory drugs with or without effects on IL-8 production. We speculate that NF-ĸB-independent mechanisms may be responsible for anti-IL-8 effects of dexamethasone.

The Trapezoid Waveform High Frequency Chest Compression Therapy Increases Water Secretion by the Cystic Fibrosis Respiratory Epithelium

increase in water in the mucus and sputum produced by patients with cystic fibrosis (CF) who used high fre quency chest compression (HFCC) twice a day. They used pulse generators that were thought to produce a square or a triangle waveform. The increase in water in the sputum was not observed in patients using a sine waveform for HFCC therapy. Part of the problem was resolved when new technology showed that both the square and triangle waveforms were an error in the inventors’ patents. The pulse generators of both of these machines produce trapezoid waveforms, which change to a saw tooth waveform in the vest applied to the patient chest and become a triangle waveform in the airways. In two studies of sputum, half of the CF patients used trapezoid waveform generators for half an hour twice a day. We found the sputum had significantly more water in this case than the other branch of the study. We wonder if there may exist an “aqua pore”—still to be discovered—that may normally react to the triangle waveforms in the body tissues that stimulates exocrine tissues to increase water secretion. We believe there are three ways to explain this increase in secreted water by the exocrine mucus cell and glands, which may work separately or together: The increase water secretion may be a new mechano 3 -aquaphore.

Liquid movement across the surface epithelium of large airways

The cystic fibrosis transmembrane conductance regulator CFTR gene is found on chromosome 7 [Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., Tsui, L.C., 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073-1080; Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., et al., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073] and encodes for a 1480 amino acid protein which is present in the plasma membrane of epithelial cells [Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J., 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263, L1-L14]. This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl(-) channel [Winpenny, J.P., McAlroy, H.L., Gray, M.A., Argent, B.E., 1995. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268, C823-C828; Jia, Y., Mathews, C.J., Hanrahan, J.W., 1997. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978-4984]. In the superficial epithelium of the conducting airways, CFTR is involved in Cl(-) secretion [Boucher, R.C., 2003. Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch. 445, 495-498] and also acts as a regulator of the epithelial Na(+) channel (ENaC) and hence Na(+) absorption [Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., Gatzy, J.T., 1986. Na(+) transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78, 1245-1252; Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C., Boucher, R.C., 1995. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850]. In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium.

Amiloride‐insensitive nasal potential difference varies with the menstrual cycle in cystic fibrosis

There is no adequate explanation for gender-based differences in rates of mortality and of deterioration in pulmonary function in cystic fibrosis (CF) patients. One potential explanation is that gender hormones (sex steroids) may modulate the severity of CF lung disease, the principal cause of mortality in CF, by altering respiratory transepithelial ion transport. To determine whether respiratory epithelial ion transport varied during the menstrual cycle of CF females. The nasal transepithelial electrical potential difference (NPD) was determined as a measure of ion transport across human respiratory epithelium, coincident with measurements of endogenous serum hormone levels in the luteal and follicular phases of the menstrual cycle in CF females aged 16-22 years. The component of the NPD that is insensitive to the Na(+) transport blocker amiloride, but not the amiloride-sensitive component, changed in association with endogenous, menstrual cycle-induced changes in serum levels of progesterone and estrogen (P=0.02, n=7, paired t-test). Measurements using Cl(-) free perfusates suggested that the changes are not a result of Cl(-) conductance. Our results suggest that in CF respiratory epithelium amiloride-insensitive, but not amiloride-sensitive, ion transport is altered by female gender hormones in vivo. We speculate that amiloride-insensitive ion transport may contribute to the regulation of human airway surface fluid.

Dysregulated interleukin-8 secretion and NF-κB activity in human cystic fibrosis nasal epithelial cells

Background It is not clear whether cystic fibrosis (CF) airway inflammation is a consequence of bacterial infection or is intrinsically dysregulated. The aim of this study was to investigate IL-8 secretion and NF-κB activity in primary respiratory epithelial cells cultured from nasal polyps obtained from CF and non-CF subjects. Methods NF-κB activity was studied by electrophoretic mobility-shift and quantitative colorimetric assays in nuclear extracts. Immunoreactive IL-8 levels were assessed by ELISA in cell culture supernatants. Both parameters were studied at baseline and following challenge with Pseudomonas aeruginosa or stimulation with pro-inflammatory cytokines. Results Under basal conditions, CF cells presented a significant higher activity of NF-κB than non-CF cells ( P = 0.0004). P. aeruginosa challenge and IL-1β/H 2O 2 co-stimulation caused four and two fold induction of NF-κB activity in non-CF and CF cells, respectively. IL-8 levels in unstimulated CF cells were significantly higher than in non-CF cells ( P = 0.0025). Upon incubation with P. aeruginosa and IL-1β/H 2O 2, non-CF cells produced 6.3 times more IL-8 than unstimulated cells, whereas IL-8 secretion increased only of 1.4 times in CF cells. Conclusions CF respiratory epithelial cells exhibit a basal dysregulated production of IL-8 that partially correlates to enhanced NF-κB activity. Our data corroborate the hypothesis of a basal exaggerated inflammatory response in the CF respiratory epithelium.

Long-Term Cultures of Polarized Airway Epithelial Cells from Patients with Cystic Fibrosis

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies.

Inhibition of amiloride-sensitive epithelial Na(+) absorption by extracellular nucleotides in human normal and cystic fibrosis airways.

Cystic fibrosis (CF) airway epithelia are characterized by enhanced Na(+) absorption probably due to a lack of downregulation of epithelial Na(+) channels by mutant CF transmembrane conductance regulator. Extracellular nucleotides adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate alternative Ca(2+)-dependent Cl(-) channels in normal and CF respiratory epithelia. Recent studies suggest additional modulation of Na(+) absorption by extracellular nucleotides. In this study we examined the role of mucosal ATP and UTP in regulating Na(+) transport in native human upper airway tissues from patients with 16 patients with CF and 32 non-CF control subjects. To that end, transepithelial voltage and equivalent short-circuit current (I(SC)) were assessed by means of a perfused micro-Ussing chamber. Mucosal ATP and UTP caused an initial increase in lumen-negative I(SC) that was followed by a sustained decrease of I(sc) in both non-CF and CF tissues. The amiloride-sensitive portion of I(SC) was inhibited significantly in normal and CF tissues in the presence of either ATP or UTP. Both basal Na(+) transport and nucleotide-dependent inhibition of amiloride-sensitive I(SC) were significantly enhanced in CF airways compared with non-CF. Nucleotide-mediated inhibition of Na(+) absorption was attenuated by pretreatment with the Ca(2+)-adenosine triphosphatase inhibitor cyclopiazonic acid but not by inhibition of protein kinase C with bisindolylmaleimide. These data demonstrate sustained inhibition of Na(+) transport in non-CF and CF airways by mucosal ATP and UTP and suggest that this effect is mediated by an increase of intracellular Ca(2+). Because ATP and UTP inhibit Na(+) absorption and stimulate Cl(-) secretion simultaneously, extracellular nucleotides could have a dual therapeutic effect, counteracting the ion transport defect in CF lung disease.

Nitric oxide has no beneficial effects on ion transport defects in cystic fibrosis human nasal epithelium.

Nitric oxide (NO) has been reported to activate Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) and inhibit epithelial Na+ absorption mediated by amiloride-sensitive epithelial Na+ channels (ENaC). These ion transport systems are defective in cystic fibrosis (CF): Cl- secretion by CFTR is impaired and Na+ absorption by ENaC is dramatically increased. By activating CFTR and depressing ENaC, NO is a potentially beneficial therapeutic agent for ion transport defects in human CF respiratory epithelia. To assess the effects of NO on human respiratory epithelial cells, the NO donors sodium nitroprusside (SNP) and spermine NONOate were applied to primary cultured nasal cells, surgically obtained from non-CF and CF patients. Measurements of transepithelial short-circuit current (ISC) showed that NO has no inhibitory potency against amiloride-sensitive nasal ENaC (nENaC) or amiloride-insensitive Na+-absorbing mechanisms in non-CF and CF epithelia. Furthermore, NO had no stimulatory effect on Cl- secretion by CFTR or any other Cl- conductance pathway in either tissue. Although NO elevated the intracellular Ca2+ concentration, we did not detect any activation of Ca2+-dependent Cl- channels. These results demonstrate that NO has no beneficial effect on CF epithelial cells of the upper airways.

THE EFFECT OF ALGINATE ON THE INVASION OF CYSTIC FIBROSIS RESPIRATORY EPITHELIAL CELLS BY CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA

Chronic infection in the cystic fibrosis (CF) lung is characterized by Pseudomonas aeruginosa strains that overproduce the mucoid exopolysaccharide, alginate. Previous experiments have shown that long-term survival of P. aeruginosa in the CF lung may be facilitated by increased adherence and decreased invasion of respiratory epithelial cells. Therefore, mucoid and nonmucoid clinical isolates of P. aeruginosa were assayed for their ability to associate with and invade the CF respiratory epithelial cell line, CF/T43. Association assays and gentamicin exclusion assays demonstrated that mucoid P. aeruginosa associates with and invades CF/T43 cell monolayers significantly less than nonmucoid P. aeruginosa strains (P =. 004,. 02) . Fluorescence microscopy invasion assays confirmed these results. The differences in association and invasion by the P. aeruginosa strains were not due to differences in lipopolysaccharide phenotype or cytotoxicity for CF/T43 respiratory epithelial cells. Exogenous bacterial alginate had no effect on the invasion of CF respiratory epithelia by a nonmucoid strain. Invasion assays with the wild-type P. aeruginosa strain PAO1 and isogenic algU and mucA mutant strains failed to show differences in invasion (P =. 25). We conclude that (i) mucoid P. aeruginosa isolates associate with and invade CF/T43 respiratory epithelial cells with less efficiency than nonmucoid P. aeruginosa, (ii) these differences are not due to variations in lipopolysaccharide phenotype between strains, (iii) neither exogenous nor endogenous alginate affects the ability of P. aeruginosa to invade CF/T43 respiratory epithelial cells, and (iv) invasion of CF/T43 respiratory epithelial cells by a laboratory reference strain of P. aeruginosa does not appear to be regulated by AlgU.

Interleukin-8 production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-alpha and respiratory syncytial virus stimulation.

High levels of neutrophils and the neutrophil-attracting chemokine interleukin (IL)-8 have been observed in the airways of patients with cystic fibrosis (CF). We hypothesized that CF respiratory epithelium produces excessive amounts of IL-8 either at baseline or after stimulation. To test this hypothesis we compared immunoreactive IL-8 release by primary nasal epithelial cell (NEC) cultures established from young children with or without CF, at several time points after stimulation of cultures with tumor necrosis factor-alpha (TNF-alpha) or infection with respiratory syncytial virus (RSV). Both stimuli induced significantly increased IL-8 release by both CF and control cultures. However, there was no difference between CF and control cells in either the magnitude or duration of the IL-8 response. The effect of transduction of CF cells with Ad5-CBCFTR, an adenovirus vector mediating expression of cystic fibrosis transmembrane regulator (CFTR), on IL-8 production was also determined. TNF-alpha stimulated IL-8 production was not different in Ad5-CBCFTR-transduced, -untransduced, or Ad5-CMVLacZ-transduced control cells. Lastly, immortalized CF tracheal epithelial cell lines, both uncorrected and retrovirally corrected with CFTR, were compared. Again, TNF-alpha-stimulated IL-8 production did not differ significantly between cell lines with and without functioning CFTR. Our data suggest that isolated CF NECs cultured under these conditions do not produce more IL-8 than do non-CF control cultures, either at baseline or after incubation with the nonspecific stimuli TNF-alpha and RSV. We conclude that the absence of functioning CFTR alone is not sufficient to cause excessive production of IL-8.

Infection in patients with cystic fibrosis and congenital immune deficiencies

Three models which attempt to account for the interaction between Pseudomonas aeruginosa and cystic fibrosis respiratory epithelium have been developed. These are: (1) malfunction of the defensin system as a result of altered chloride content of the airway fluid; (2) loss of cystic fibrosis transmembrane conductance regulator, which acts as a receptor for Pseudomonas aeruginosa, leading to the failure of internalization of the bacteria; (3) upregulation of asialo GM1 receptors and increased Pseudomonas aeruginosa binding. Other important advances include new uses of antibiotics for resistant strains and the vexed question of infection control strategies.

CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium.

Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.

In Vivo Nasal Potential Difference: Techniques and Protocols for Assessing Efficacy of Gene Transfer in Cystic Fibrosis

Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl- secretion and an accelerated rate of basal Na+ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl- secretion, and the rate of Na+ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients.

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation.

Aldosterone Metabolism and Transepithelial Potential Difference in Normal and Cystic Fibrosis Subjects

The transepithelial potential difference (PD) is raised across cystic fibrosis (CF) respiratory epithelia. This raised voltage reflects active sodium absorption across a relatively chloride impermeable membrane. Because relatively little is known about the regulation of the rate of sodium absorption across mammalian airways, we assessed the possible contribution of aldosterone to the PD in normal and CF respiratory epithelia. Aldosterone excretion in five CF patients was 12.2 +/- 0.9 micrograms/24 h, a mean value not different from normal control subjects (13.6 +/- 1.5 micrograms/24 h, n = 5). Despite similar aldosterone excretion rates, nasal PD was more than 2-fold greater in the CF patients (-53.6 +/- 6.4 mV) than normal subjects (-21.3 +/- 1.4 mV). The effect of an aldosterone antagonist, spironolactone, on aldosterone excretion and nasal and rectal PD was evaluated in four CF patients and five normal subjects. During spironolactone administration, aldosterone excretion increased (2- to 4-fold) and rectal PD decreased in both groups. However, nasal PD was unchanged in each group (CF = -52.1 +/- 4.3 mV pre, -53.6 +/- 1.4 mV during; normal = -21.2 +/- 3.1 mV pre, -21.6 +/- 3.2 mV during). We conclude that neither increased aldosterone secretion rates nor organ sensitivity to aldosterone can account for the abnormally raised PD that characterizes the respiratory epithelium of subjects with CF.

Relative ion permeability of normal and cystic fibrosis nasal epithelium.

The raised transepithelial electric potential difference (PD) across respiratory epithelia in cystic fibrosis (CF) has suggested an abnormality in ion permeation. We characterized this abnormality further by measuring in the nasal epithelia of CF and normal subjects the concentration-PD relationship for amiloride, an inhibitor of cell Na+ permeability, and PD responses to superfusion with solutions of different composition. Amiloride was more efficacious in the CF subjects but the ED50 was not different from that of normals (approximately 2 X 10(-6) M). Na+ replacement by choline induced effects similar to those of amiloride, i.e. a greater depolarization in CF subjects. A 10-fold increase in the K+ concentration of the perfusate induced a small (less than 10 mV) depolarization in both subject populations. When Cl- in the perfusate was replaced by gluconate or SO2-(4) the nasal PD of normal subjects hyperpolarized (lumen became more negative) by approximately 35 mV. A significantly smaller response (less than 17 mV) was induced in CF homozygotes but not in heterozygotes (38 mV). The smaller response of CF subjects appears to reflect an absolute decrease in luminal surface Cl- permeability because pretreatment with amiloride did not increase the response to Cl- free solution (7 mV). Accordingly, three abnormalities (decreased Cl- permeability, raised PD, greater amiloride efficacy) have been identified in CF respiratory epithelia. Whereas "excessive" active Na+ transport can account for these abnormalities and the dessication of airway surface liquid, it is possible that a lower lumenal cell membrane Cl- permeability and inhibition of a potential path of Cl- secretion can also explain the observations.