Airway epithelial integrity may be impaired by bacterial exoproducts, which are able to degrade tight junction–associated proteins such as zonula occludens 1 (ZO-1). We have investigated the protective effect of salmeterol, a long-acting β2-adrenergic agonist, on Pseudomonas aeruginosa–induced alteration of the epithelial junctional barrier. We demonstrate in human airway epithelial cells (HAEC) that salmeterol induces a time-dependent increase in ZO-1 protein, although no significant change in ZO-1 transcripts was observed. When HAEC cultures were exposed to P. aeruginosa (PAO1) supernatants, apical expression of ZO-1 protein was maintained in salmeterol-pretreated HAEC cultures, whereas it disappeared after PAO1 exposure in cultures not pretreated with salmeterol. Western blot experiments showed that the 220-kD ZO-1 protein was decreased after PAO1 incubation but was still present in salmeterol-pretreated HAEC extracts. The functional activity of ZO-1 protein was monitored by measuring transepithelial r...

The regulation of the volume and composition of airway surface liquid is achieved through epithelial ion transport processes. In humans, these processes have been characterized in proximal but not distal airways. Segments of human bronchioles were dissected from surgically removed lung pieces. The transmural potential difference of microperfused bronchioles was inhibited by luminal exposure to amiloride and increased when exposed to the Cl secretagogues forskolin and ATP in the presence of amiloride. Human bronchiolar epithelial cells were cultured on permeable supports and studied in Ussing chambers. They generated a short circuit current (Isc) that decreased in response to amiloride and increased in response to forskolin and to ATP in the presence of amiloride. In low-Cl Kreb's Ringer bicarbonate, the baseline Isc and amiloride-induced decrease in Isc were not different, whereas the forskolin- and ATP-induced increases in Isc were smaller. Fluid transport measurement in excised bronchioles revealed a basal absorptive flow that was reduced by amiloride, whereas forskolin and ATP combined induced a secretory flow in the presence of amiloride. We conclude that human bronchioles actively absorb Na and fluid in unstimulated conditions and are capable of active Cl and fluid secretion when exposed to forskolin and to ATP.

In numerous airway diseases, such as cystic fibrosis, the epithelium is severely damaged and must regenerate to restore its defense functions. Although the human airway epithelial stem cells have not been identified yet, we have suggested recently that epithelial stem/progenitor cells exist among both human fetal basal and suprabasal cell subsets in the tracheal epithelium. In this study, we analyzed the capacity of human adult basal cells isolated from human adult airway tissues to restore a well-differentiated and functional airway epithelium. To this end, we used the human-specific basal cell markers tetraspanin CD151 and tissue factor (TF) to separate positive basal cells from negative columnar cells with a FACSAria cell sorter. Sorted epithelial cells were seeded into epithelium-denuded rat tracheae that were grafted subcutaneously in nude mice and on collagen-coated porous membranes, where they were grown at the air-liquid interface. Sorted basal and columnar populations were also analyzed for their telomerase activity, a specific transit-amplifying cell marker, by the telomeric repeat amplification protocol assay. After cell sorting, the pure and viable CD151/TF-positive basal cell population proliferated on plastic and adhered on epithelium-denuded rat tracheae, as well as on collagen-coated porous membranes, where it was able to restore a fully differentiated mucociliary and functional airway epithelium, whereas viable columnar negative cells did not. Telomerase activity was detected in the CD151/TF-positive basal cell population, but not in CD151/TF-negative columnar cells. These results demonstrate that human adult basal cells are at least airway surface transit-amplifying epithelial cells.

BACKGROUND:For a better understanding of the early stages of cystic fibrosis (CF), it is of major interest to study respiratory epithelial cells obtained as early as possible. Although bronchoalveolar lavage has been proposed for this purpose, nasal brushing, which is a much less invasive technique, has seldom been used in CF infants. The aim of the present study was to examine in a few infants the feasibility of a nasal brushing technique for studies of airway epithelial functions in very young CF infants. METHODS:In 5 CF (median age 12, range 1-18 months) and 10 control infants (median age 5, range 1-17 months), a nasal brushing was performed by means of a soft sterile cytology brush, after premedication with oral paracetamol (15 mg/kg body weight) and rectal midazolam (0.2 mg/kg body weight). Samples were used for microbiological, cytological and functional studies. RESULTS:The procedure was well tolerated. Number of cells collected was similar in CF and non-CF patients (CF: median 230x10(3), range 42x10(3)-900x10(3); non-CF: median 340x10(3), range 140x10(3)-900x10(3)). Median number of viable cells was 67% (range 31-84%). Freshly obtained samples were successfully used for studies of ciliary beating frequency and cAMP-dependent chloride efflux. In 7 out of 17 cell cultures, confluence was obtained (CF: 2 out of 7; non-CF: 5 out of 10). The feasibility of studying protein release and mRNA expression of IL-8, IL-6 and TNF-alpha, under basal conditions and after stimulation by Pseudomonas aeruginosa, was demonstrated. CONCLUSIONS:By means of a simple nasal brushing technique easily performed and well tolerated, it is feasible, in infants, to harvest respiratory cells in sufficient amounts to study the airway epithelium using a broad range of techniques including cell culture.

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. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In chronic obstructive pulmonary disease (COPD), exacerbations are generally associated with several causes, including pollutants, viruses, bacteria that are responsible for an excess of inflammatory mediators, and proinflammatory cytokines released by activated epithelial and inflammatory cells. The normal response of the airway surface epithelium to injury includes a succession of cellular events, varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even complete denudation of the basement membrane. The epithelium then has to repair and regenerate to restore its functions, through several mechanisms, including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. In COPD, the remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia that occur during injury, may considerably disturb the innate immune functions of the airway epithelium. In vitro and in vivo models of airway epithelial wound repair and regeneration allow the study of the spatiotemporal modulation of cellular and molecular interaction factors-namely, the proinflammatory cytokines, the matrix metalloproteinases and their inhibitors, and the intercellular adhesion molecules. These factors may be markedly altered during exacerbation periods of COPD and their dysregulation may induce remodeling of the airway mucosa and a leakiness of the airway surface epithelium. More knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to cytoprotective and regenerative therapeutics, allowing the reconstitution of a functional, well-differentiated airway epithelium in COPD.

Cystic Fibrosis (CF) is the most common genetic autosomal recessive disease caused by mutations in a single gene that encodes a transmembrane glycoprotein of 1,480 amino acids called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). About 70 % of patients with CF are homozygous for the ΔF 508 mutation. Mutations vary in the severity of the disease but even in the ΔF 508 homozygous CF patients, there is a wide variability in the clinical manifestations of the lung disease that is the major cause of the morbidity and mortality. At an advanced stage, CF lung disease is characterized by Pseudomonas aeruginosa lung infection associated with an excessive airway inflammation response, bronchiectasis, severe epithelial lesions with progressive suppurative pulmonary disease which ultimately leads to the death of CF patients (1). 1. Abnormal ion and water transport in CF. The discovery of the CF gene in 1989 demonstrated the basic defect to be in a cAMP regulated chloride channel due to an absent or deficient maturation of the CFTR protein normally localized at the apical domain of the apical airway epithelial cell membrane. The impaired transepithelial chloride secretion is associated to impaired bicarbonate secretion and sodium hyperabsorption resulting in excessive water reabsorption and decreased mucociliary transport (2,3). These constitutive abnormalities, in particular the depletion of the airway surface liquid, contribute to an abnormal mucociliary transport, detected very early in the absence of any infection (4). At the airway level, in healthy subjects, CFTR is localized at the luminal surface of ciliated cells of well-differentiated surface epithelium and in the serous cells of submucosal glands. The finding that the remodelling of non-CF airway epithelium (such as mucous hyperplasia or epithelial metaplasia) was associated with an intracellular localization or absence of CFTR protein and on the opposite, the observation that in CF airways, CFTR immunoreactivity could be observed in areas of well-differentiated surface epithelium, led to the concept that maintaining the integrity, polarity and airway epithelial differentiation could favour a residual functional activity of ΔF508 CFTR (5,6). This concept led to the development of pharmacologic activators and potentiators of the defective protein. Moreover, we have shown that in the CF airway submucosal glandular secretory cells, where CFTR is predominantly localized at the membrane epithelial surface but also at the membrane surface of the secretory granules, the granules that contain mucins and proteins could not correctly expand and disperse their mucus content into the airway lumen (7). This abnormal mucus expansion and exocytosis was due to an abnormal water content inside the granules. Interestingly, in the CF glandular serous cells, we could recover normal water content after lentiviral CFTR cDNA transfection. Changes in the phospholipid profile of airway mucus related to defective intra- cellular acidification and alteration of phospholipids synthesis and trafficking could be responsible for an early stasis of CF airway mucus. A decrease in the concentration of surface active phospholipids such as phosphatidylglycerol and phosphatidylcholine results in less lubricant properties of the airway surface liquid and increased adhesivity of the mucus (8). The inadequate water hydration of mucus and inadequate phospholipids profile favor the retention of mucus, resulting in a decreased muco-ciliary clearance, increased bacterial growth and release of soluble virulence factors that may injure the airway epithelium.

L′épithélium respiratoire de surface est en contact permanent avec l’air ambiant, il est donc fréquemment lésé par des agents bactériens ou viraux. En réponse à une lésion, l’épithélium respiratoire de surface est capable de réparer par un certain nombre de mécanismes incluant l’étalement et la migration des cellules basales, puis leur prolifération et leur différenciation. Les facteurs cellulaires et moléculaires impliqués dans le processus de réparation et régénération de l’épithélium respiratoire sont multiples et interagissent étroitement. Parmi ces facteurs, on peut citer les protéines du cytosquelette, les composants de la matrice extra-cellulaire et les intégrines correspondantes, les métalloprotéinases matricielles (MMPs) et leurs inhibiteurs ainsi que les cytokines et facteurs de croissance sécrétés par les cellules épithéliales.

Although Staphylococcus aureus is a major cause of pulmonary infection, the role played by this bacterium in the induction of inflammation of human airway epithelial cells ( HAEC) is poorly understood. In this study, we investigated the inflammatory response of HAEC to S. aureus soluble virulence factors and demonstrate that the combination of a long-acting beta(2)-adrenergic receptor agonist ( salmeterol) with a glucocorticoid ( fluticasone propionate) has an anti-inflammatory effect on HAEC. First, we demonstrate increased expression at both the mRNA and protein levels of interleukin ( IL)-8, IL-6, and tumor necrosis factor ( TNF)-alpha following incubation of HAEC in the presence of S. aureus soluble virulence factors and the increase of 1) the free nuclear factor-kappa B ( NF-kappa B) and activator protein-1 ( AP-1) activities and 2) the phosphorylated ( P-) extracellular signal-regulated kinases 1 and 2 ( ERK1/ERK2), the P-c-Jun NH2-terminal kinase ( JNK), and the P-isoform-alpha of the NF-kappa B inhibitor ( I kappa B alpha). Next, when HAEC were preincubated with the combination of salmeterol and fluticasone propionate, the inflammatory response of HAEC was markedly attenuated in that levels of IL-8, IL-6, TNF-alpha, NF-kappa B, AP-1, P-ERK1/ERK2, P-JNK, and P-I kappa B alpha decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a beta(2)-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation.

Human airway surface epithelium is frequently damaged by inhaled factors (viruses, bacteria, xenobiotic substances) as well as by inflammatory mediators that contribute to the shedding of surface epithelial cells. To regain its protective function, the epithelium must rapidly repair and redifferentiate. The Trefoil Factor Family (TFF) peptides are secretory products of many mucous cells. TFF3, the major TFF in the airways, is able to enhance airway epithelial cell migration, but the role of this protein in differentiation has not been defined. To identify the specific role of TFF3 in the differentiation of the human airway surface epithelium, we analyzed the temporal expression pattern of TFF3, MUC5AC, and MUC5B mucins (goblet cells) and ciliated cell markers beta-tubulin (cilia) and FOXJ1 (ciliogenesis) during human airway epithelial regeneration using in vivo humanized airway xenograft and in vitro air-liquid interface (ALI) culture models. We observed that TFF3, MUC5AC, MUC5B, and ciliated cell markers were expressed in well-differentiated airway epithelium. The addition of exogenous recombinant human TFF3 to epithelial cell cultures before the initiation of differentiation resulted in no change in MUC5AC or cytokeratin 13 (CK13, basal cell marker)-positive cells, but induced an increase in the number of FOXJ1-positive cells and in the number of beta-tubulin-positive ciliated cells (P < 0.05). Furthermore, this effect on ciliated cell differentiation could be reversed by specific epidermal growth factor (EGF) receptor (EGF-R) inhibition. These results indicate that TFF3 is able to induce ciliogenesis and to promote airway epithelial ciliated cell differentiation, in part through an EGF-R-dependent pathway.

In response to bacterial infection, airway epithelium releases inflammatory mediators including cytokines and chemokines that lead to immune cell efflux and could stimulate the adaptive T cell immune response. The aim of our study was to analyze, in a double chamber culture, the chemokine changes in response to Staphylococcus aureus and their consequences for T cells. Our data show that S. aureus stimulates basolateral and apical release of IL-8 and eotaxin by airway epithelial cells. We also observed increased chemokine receptor expression on CD8+ and CD4+ T cells and enhanced chemotaxis of CD4+ T cells toward apical supernatant. Our data strongly suggest that S. aureus interaction with airway epithelium contributes to specific migration of T cells to inflamed sites.