Inflammation and infection in naive human cystic fibrosis airway grafts

Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease. Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial. We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice. This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways. Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001). CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria. In non-CF grafts, these three sets of events were not observed before 6 h. Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state. After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.     

Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis

Cystic fibrosis (CF) at an advanced stage of the disease is characterized by airway epithelial injury and remodelling. Whether CF remodelling is related to infection and inflammation or due to an abnormal regenerative process is still undecided. We have recently established the expression and secretion profiles of interleukin (IL)-8, matrix metalloproteinase (MMP)-7, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 during non-CF airway epithelial regeneration in a humanized nude mouse xenograft model. To enhance our understanding of CF remodelling, we compared the regeneration process of non-infected human CF and non-CF nasal epithelia. In both CF and non-CF situations, epithelial regeneration was characterized by successive steps of cell adhesion and migration, proliferation, pseudostratification, and terminal differentiation. However, histological examination of the grafts showed a delay in differentiation of the CF airway epithelium. Cell proliferation was higher in the regenerating CF epithelium, and the differentiated CF epithelium exhibited a pronounced height increase and basal cell hyperplasia in comparison with non-CF epithelium. In addition, while the number of goblet cells expressing MUC5AC was similar in CF and non-CF regenerated epithelia, the number of MUC5B-immunopositive goblet cells was lower in CF grafts. The expression of human IL-8, MMP-7, MMP-9, and TIMP-1 was enhanced in CF epithelium, especially early in the regenerative process. Together, our data strongly suggest that the regeneration of human CF airway surface epithelium is characterized by remodelling, delayed differentiation, and altered pro-inflammatory and MMP responses.     

Protection of human respiratory epithelium from Pseudomonas aeruginosa adherence by phosphatidylglycerol liposomes.

The ability of phosphatidylglycerol (DSPG) liposomes to prevent adherence of Pseudomonas aeruginosa to primary cultures of non-cystic fibrosis (CF) and delta F508 homozygous CF human respiratory epithelium was studied. The culture model was characterized by the simultaneous presence of various cellular phenotypes: well-differentiated respiratory epithelial cells, ciliated and nonciliated cells, and migrating cells which can be assimilated into a regenerating epithelium after injury. DSPG liposomes significantly decreased the binding of P. aeruginosa to migrating cells of both non-CF and delta F508 homozygous CF cultures compared with control cultures (35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2 versus 23.9 x 10(-3) +/- 2.5 x 10(-3); P < 0.01 for non-CF cultures and 88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per micron 2 versus 29.1 x 10(-3) +/- 0.6 x 10(-3), P < 0.001 for CF cultures). After treatment with DSPG liposomes, the size of P. aeruginosa aggregates bound to migrating cells in both non-CF cultures and delta F508 homozygous CF cultures was significantly decreased (14.4 +/- 3 bacteria per aggregate versus 11.9 +/- 2.5 bacteria per aggregate [P < 0.05] and 29.9 +/- 8.4 bacteria per aggregate versus 17.3 +/- 2.3 bacteria per aggregate [P < 0.01], respectively). Moreover, the control cultures were characterized by a differential P. aeruginosa adherence according to both the cellular phenotype and the mutation. The migrating cells bound more bacteria than the stationary cells of both non-CF and delta F508 homozygous CF cultures. The CF migrating cells bound significantly more bacteria than the non-CF migrating cells (88.8 x 10(-3) +/- 17.2 x 10(-3) bacteria per microns 2 versus 35.5 x 10(-3) +/- 8.1 x 10(-3) bacteria per micron 2, P < 0.001). These results suggest that DSPG liposomes are able to decrease P. aeruginosa adherence to CF and non-CF respiratory epithelium, particularly to migrating cells, which mimic a regenerating epithelium after injury. DSPG liposomes could also represent a hydrophobic barrier limiting the deleterious action of P. aeruginosa exoproducts.     

Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis

BACKGROUND: Airway inflammation and infection are early events in cystic fibrosis (CF) pathogenesis. The existence of an imbalance in the immune cell population of the CF fetal airway before infection remains completely unknown. OBJECTIVE: The aim of this study was to determine whether early signs of inflammation are observed in CF airways during human fetal development. METHODS: Tracheas and lungs were collected from 21 CF and 16 non-CF fetuses. In tissue sections, the numbers of neutrophils, mast cells, macrophages, and B and T lymphocytes were quantitatively analyzed by means of image cytometry. The presence of IL-4, IL-6, IL-8, IL-10, RANTES, IFN-gamma, TNF-alpha, and NF kappa B and its inhibitor I kappa B-alpha was qualitatively evaluated by immunofluorescent staining. RESULTS: During fetal airway development, epithelial and glandular differentiation, as well as the distribution of inflammatory markers, was similar in CF and non-CF tissues. Significant differences between CF and non-CF fetal airways were observed only in the numbers of mast cells and macrophages. In the CF trachea, the mast cell number increased slowly but continuously, whereas in the non-CF trachea this number rapidly reached a plateau. In the CF lung, the macrophage number increased with time, whereas in the non-CF lung it decreased. CONCLUSION: Although no intrinsic inflammation was demonstrated, we observed a distinct appearance of mast cells and macrophages in CF airways in comparison with non-CF airways during fetal development. These 2 cell populations were greater in CF airways at a late stage of fetal development, suggesting their possible involvement in the early onset of inflammation in CF infants.     

Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na⁺-channel-mediated Na⁺ absorption and stimulation of Cl⁻ secretion through CFTR and the Ca²⁺-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid.

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.     

Measurement of the airway surface liquid volume with simple light refraction microscopy

In the cystic fibrosis (CF) lung, the airway surface liquid (ASL) volume is depleted, impairing mucus clearance from the lung and leading to chronic airway infection and obstruction. Several therapeutics have been developed that aim to restore normal airway surface hydration to the CF airway, yet preclinical evaluation of these agents is hindered by the paucity of methods available to directly measure the ASL. Therefore, we sought to develop a straightforward approach to measure the ASL volume that would serve as the basis for a standardized method to assess mucosal hydration using readily available resources. Primary human bronchial epithelial (HBE) cells cultured at an air-liquid interface develop a liquid meniscus at the edge of the culture. We hypothesized that the size of the fluid meniscus is determined by the ASL volume, and could be measured as an index of the epithelial surface hydration status. A simple method was developed to measure the volume of fluid present in meniscus by imaging the refraction of light at the ASL interface with the culture wall using low-magnification microscopy. Using this method, we found that primary CF HBE cells had a reduced ASL volume compared with non-CF HBE cells, and that known modulators of ASL volume caused the predicted responses. Thus, we have demonstrated that this method can detect physiologically relevant changes in the ASL volume, and propose that this novel approach may be used to rapidly assess the effects of airway hydration therapies in high-throughput screening assays.     

Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.     

Respiratory epithelial gene expression in patients with mild and severe cystic fibrosis lung disease

Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with DeltaF508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between DeltaF508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between DeltaF508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype.     

Ion and liquid transport across the bronchiolar epithelium

The proper homeostasis of the airway surface liquid (ASL) depends on transepithelial ion and fluid transport and is critically important for lung defence, and more specifically for mucociliary transport. In cystic fibrosis (CF), abnormal ion and fluid transport lead to depleted ASL volume resulting in mucus plugs and recurrent lung infections. Like bronchi, human bronchioles exhibit amiloride-sensitive Na(+) absorption and cyclic-AMP and Ca(2+)-activated Cl(-) secretion. However, cyclic-AMP-stimulated Cl(-) and fluid secretion appears to be quantitatively more important in bronchioles than in bronchi. In CF bronchioles, like in CF bronchi, the ASL height is reduced because of an abnormally persistent Na(+) absorption, combined with a lacking CFTR-dependent Cl(-) secretion. The precocity and severity of the bronchiolar disease in CF could be attributed in part to the more important role of CFTR-dependent Cl(-) secretion and fluid secretion, and the lack of compensatory ATP-driven Cl(-) secretion and fluid secretion, in bronchioles compared to bronchi.     

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.     

Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.     

New models of the tracheal airway define the glandular contribution to airway surface fluid and electrolyte composition

Antibacterial defenses in the airway are dependent on multifactorial influences that determine the composition of both fluid and/or electrolytes at the surface of the airway and the secretory products that aid in bacterial killing and clearance. In cystic fibrosis (CF), these mechanisms of airway protection may be defective, leading to increased colonization with Pseudomonas aeruginosa. Submucosal glands, a predominant site of cystic fibrosis transmembrane conductance regulator (CFTR) protein expression in the airway, have been hypothesized to play an important role in protection of the airway. Furthermore, recent studies have suggested that the salt concentration at the airway surface may be a key factor in regulating the activity of antibacterial substances in the airway. To explore these issues, we have used a new model of the ferret tracheal airway to evaluate the contribution of submucosal glands in regulating airway surface fluid and electrolyte composition. Using tracheal xenograft models with and without submucosal glands, we have characterized several aspects of airway physiology that may be important in defining antibacterial properties. These endpoints included the contribution of submucosal glands in defining bioelectric properties of the surface airway epithelium, airway surface fluid (ASF) chloride composition, ASF volumes, and secretion of the antibacterial factor lysozyme. Findings from these studies demonstrate a significantly elevated secreted fluid volume (Vs) and chloride concentration ([Cl](s)) in ASF from airways with submucosal glands (Vs = 47 +/- 4 microl; [Cl](s) = 128 +/- 5 mM), as compared with xenograft airways without glands (Vs = 36 +/- 2 microl; [Cl](s) = 103 +/- 6 mM). Furthermore, a temperature labile factor secreted by submucosal glands appears to alter the baseline activation of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and/or diphenylamine-2-carboxylic acid-sensitive chloride channels in the surface airway epithelium. Lastly, the lysozyme content of tracheal airways with submucosal glands was 8.5-fold higher than were airways without glands. These studies demonstrate that submucosal glands affect both the ionic composition and bioelectric properties of the airway and suggest that models evaluating antibacterial properties of the airway in CF should take into account the contribution of glands in airway physiology.     

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.     

Selective Up-Regulation of Chemokine IL-8 Expression in Cystic Fibrosis Bronchial Gland Cells in Vivo and in Vitro

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the ΔF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1β and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured ΔF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl⁻ concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl⁻ concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.     

Nitric oxide deficiency contributes to impairment of airway relaxation in cystic fibrosis mice

The pulmonary disease of cystic fibrosis (CF) is characterized by persistent airway obstruction, which has been attributed to chronic endobronchial infection and inflammation. The levels of exhaled nitric oxide (NO) are reduced in CF patients, which could contribute to bronchial obstruction through dysregulated constriction of airway smooth muscle. Because airway epithelium from CF mice has been shown to have reduced expression of inducible NO synthase, we examined airway responsiveness and relaxation in isolated tracheas of CF mice. Airway relaxation as measured by percent relaxation of precontracted tracheal segments to electrical field stimulation (EFS) and substance P, a nonadrenergic, noncholinergic substance, was significantly impaired in CF mice. The airway relaxation in response to prostaglandin E2 was similar in CF and non-CF animals. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methylester reduced tracheal relaxation induced by EFS in wild-type animals but had virtually no effect in the CF mice. Conversely, exogenous NO and L-arginine, a NO substrate, reversed the relaxation defect in CF airway. We conclude that the relative absence of NO compromises airways relaxation in CF, and may contribute to the bronchial obstruction seen in the disease.     

Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium.

Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.     

Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous DeltaF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0.001). The number of T lymphocytes was higher at the CF distal level (P = 0.035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0.033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.     

Abnormalities in the pulmonary innate immune system in cystic fibrosis

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.     

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.