uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration.

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.

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.

Stimulation of bacterial adherence by neutrophil defensins varies among bacterial species but not among host cell types.

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.

Cell wall-associated protein A as a tool for immunolocalization of Staphylococcus aureus in infected human airway epithelium.

Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated.

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2.

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.

Differentiated and functional human airway epithelium regeneration in tracheal xenografts.

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.

Fibronectin and alpha5beta1 integrin mediate binding of Pseudomonas aeruginosa to repairing airway epithelium.

Initial infection of the airway by Pseudomonas aeruginosa may occur through a variety of bacterial strategies including binding to epithelial receptors present at the surface of the respiratory epithelium. In order to characterize the adherence sites for P. aeruginosa in damaged and repairing bronchial tissue, an ex vivo model of airway epithelial injury and repair was developed using primary cell cultures of nasal cells from 14 subjects with polyposis. P. aeruginosa strongly adhered to flattened dedifferentiated (FD) bronchial and nasal cytokeratin 13-positive epithelial cells in the process of migration for repair. In in vitro experiments, competitive binding inhibition assays demonstrated that alpha5beta1 integrins and cellular fibronectin, in particular the RGD sequence, are receptors involved in P. aeruginosa adherence to FD nasal epithelial cells. Fluorescent cell sorting analysis and immunofluorescence techniques revealed that the alpha5beta1 integrins are overexpressed and apically exposed in FD nasal epithelial cells. One 50 kDa outer membrane protein was identified in piliated and nonpiliated strains of P. aeruginosa that was involved in binding to cellular fibronectin and alpha5beta1 epithelial integrins. These results demonstrate that Pseudomonas aeruginosa adherence is related to the dedifferentiation of airway epithelium during the repair process which unmasks and upregulates the alpha5beta1 integrin expression and induces active synthesis of cellular fibronectin. These epithelial receptors are then used by a Pseudomonas aeruginosa 50 kDa outer membrane protein as sites of bacterial adherence.

Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity.

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts.

The composition of airway surface liquid (ASL) is partly determined by active ion and water transport through the respiratory epithelium. It is usually stated that in cystic fibrosis (CF), CF transmembrane conductance regulator protein abnormality results in imbalanced ion composition and dehydration of ASL, leading to abnormal rheologic and transport properties. To explore the relationship between ion composition, water content, and viscosity of airway liquid (AL), we used a human xenograft model of fetal airways developed in severe combined immunodeficiency (SCID) mice. Six non-CF and six CF portions of fetal tracheas were engrafted subcutaneously in the flanks of SCID mice raised in pathogen-free conditions. AL accumulated in the closed cylindric grafts was harvested 9 to 17 wk after implantation. At the time of AL sampling, all tracheal grafts displayed well-differentiated pseudostratified surface epithelium and submucosal glands. The viscosity of AL was measured using a controlled-stress rheometer. The ion composition of AL was quantified by X-ray microanalysis. No significant difference was observed for AL viscosity between non-CF (0.6 +/- 0.5 Pa. s) and CF (0.2 +/- 0.1 Pa. s) samples. In AL from non-CF and CF samples, the ion concentrations were Na: 63.9 +/- 7.6, 79.7 +/- 11.6; Cl: 64.9 +/- 13.2, 82.6 +/- 15.7; Mg: 1.9 +/- 0.3, 2.2 +/- 0.4; S: 4.9 +/- 1. 3, 4.8 +/- 0.5; K: 2.4 +/- 0.5, 3.2 +/- 1.6; and Ca: 1.2 +/- 0.3, 2.6 +/- 0.8 mmol/liter, respectively. The ion composition of AL from CF versus non-CF xenografts was not significantly different. These results suggest that prior to inflammation and infection, the viscosity and ion composition of the fetal AL do not differ in CF and non-CF.

Association of fibroblastoid features with the invasive phenotype in human bronchial cancer cell lines.

The acquisition of a metastatic phenotype by epithelial cells implicates a series of changes altering their differentiation, their overall behavior and morphology. In the present study, we have examined the relationships between the cellular morphology, E-cadherin expression, matrix metalloproteinases expression and in vitro invasive properties in two human bronchial immortalized cell lines. The (16HBE14o-) cell line which did not show any invasive abilities in the Boyden chamber assay displayed a typical epithelial morphology in monolayer, expressed high levels of E-cadherin and synthesized neither MMP-2 and MT1-MMP nor vimentin. In contrast, the BZR cell line which was highly invasive displayed a more elongated phenotype in monolayer, did not produce E-cadherin but expressed vimentin, MMP-2 and MT1-MMP. Our data therefore suggest that the metastatic progression of broncho-pulmonary cancer cells results in a cellular dedifferentiation and the gain of some mesenchymal attributes (loss of E-cadherin and expression of vimentin) associated with enhanced degradative properties (expression of metalloproteinases).

Revisited physicochemical and transport properties of respiratory mucus in genotyped cystic fibrosis patients.

We investigated the physicochemical and transport properties of sputum samples collected in physiotherapy from a well-documented group of 27 cystic fibrosis (CF) patients with identified CF genotypes. Sputum samples were characterized ex vivo for their water content, surface properties (surface tension and contact angle), rheologic properties (viscosity and elastic modulus), and transport properties (mucociliary and cough transport). These data were analyzed in relation to the clinical status of the patients (FEV1, FVC, Shwachman score, Brasfield score, nutritional status), their genotype, and the degree of infection of their sputa (leukocyte and Pseudomonas aeruginosa counts). We observed negative and significant correlations between mucociliary transport and elastic modulus of the patients' sputum (r = -0.63, p < 0.01), and between the cough transport and contact angle of the sputum (r = -0.81, p < 0.0001), respectively. The P. aeruginosa count was also significantly correlated with the sputum water content (r = -0.53, p < 0.02) as well as with the cough transport of the sputum (r = -0.62, p < 0.01). In CF patients with a sputum leukocyte count > 2,000/mm3, the sputum water content (p < 0.02), FEV1 (p < 0.05) and FVC (p < 0.02) were significantly lower than those of CF patients with a leukocyte count < or = 2,000/mm3. CF patients with a homozygous delta F 508 genotype had significantly greater values of sputum water content (p < 0.05), and cough-transport capacity (p < 0.05) than did heterozygous patients. No correlation was observed between the sputum properties and any of the clinical data. These results suggest that the control of infection should be emphasized in CF, since it can directly or indirectly modulate the degree of hydration, and therefore the physicochemical and transport properties, of airway secretions.

Receptors in the Pseudomonas aeruginosa adherence to injured and repairing airway epithelium.

In the normal respiratory tract, the airway epithelial surface is protected from pathogenic bacterial colonization by the mucociliary clearance. The mucins present in the gel mucus layer exhibit a high diversity of carbohydrate receptors that allow specific bacterial recognition followed by bacterial and mucus elimination. As soon as the mucociliary clearance mechanism is impaired, the bacterial attachment to mucins in association with mucus stasis represent critical pathways for bacterial colonization of the airway epithelium. Several sources of injury may damage the epithelial integrity and induce partial or complete epithelial shedding, exposing cellular receptors and unmasked extracellular matrix (ECM) components that can be recognized by bacterial adhesins. Laminin and type I and IV collagens represent sites of Pseudomonas aeruginosa attachment to the ECM components. During airway epithelium repair after injury, particularly in cystic fibrosis (CF), the repairing cells exhibit apical receptors such as asialylated gangliosides (asialo GM1) to which P. aeruginosa adheres. The identification of the different receptors for P. aeruginosa, present either on the ECM proteins or on the apical surface of the remodeled airway epithelium, particularly in repairing respiratory CF epithelial cells, is a prerequisite to further therapeutic strategies to prevent airway colonization by P. aeruginosa.

Pseudomonas aeruginosa adherence to remodelling respiratory epithelium.

Pseudomonas aeruginosa is an opportunistic organism, which frequently colonizes the respiratory tract of patients with impaired host defence. In cystic fibrosis (CF) patients, this pathogen causes a progressive destructive bronchitis and bronchiolitis and is responsible for high mortality. Normal respiratory epithelium is protected against bacteria via mucus and mucociliary clearance. Alteration of mucociliary clearance and of glycosylation of mucins in CF facilitates the access of bacteria to the underlying airway epithelial cells. Intact respiratory epithelium does not bind P. aeruginosa, whereas injured respiratory epithelium is highly susceptible to P. aeruginosa adherence. We found that the high affinity of respiratory epithelium, from CF and non-CF sources, for P. aeruginosa, during the wound repair process is related to the apical expression of asialo ganglioside M1 (aGM1). The affinity of repairing respiratory epithelium for P. aeruginosa is time-dependent, and is related to transient apical expression of aGM1 at the surface of repairing respiratory epithelial cells. CF respiratory epithelial cells apically express more aGM1 residues with relation to an increased affinity for P. aeruginosa than non CF cells. High epithelial damage followed by repair represents a major cause of P. aeruginosa adherence to airway epithelium in cystic fibrosis. However, P. aerurignosa adherence and colonization are not restricted to cystic fibrosis disease and P. aeruginosa pneumonia may also occur in severely immunocompromised patients, suggesting that epithelial injury and decreased host-response favour the colonization of the airways by P. aeruginosa.

Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells.

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.

Effects of rhDNase on purulent airway secretions in chronic bronchitis.

Recombinant human deoxyribonuclease (rhDNase) has been demonstrated to reduce in vitro the viscosity and to improve the transport capacity of purulent respiratory mucus in cystic fibrosis. During episodes of exacerbation of chronic bronchitis, the patients generally expectorate purulent mucus. Purulence of mucus is associated with an increased deoxyriboneucleic acid (DNA) concentration. We analyzed in vitro the potential effect of rhDNase on chronic bronchitis mucus transport by the ciliary activity (frog palate model) and by simulated cough (cough machine model), as well as the effect on mucus viscosity (controlled stress rheometer) and surface properties (contact angle). Purulent sputa collected from patients with chronic bronchitis (n = 15) during an episode of exacerbation were incubated for 30 min at 37 degrees C with either rhDNase at two different concentrations (final concentration 2 or 4 micrograms.mL-1) or placebo. The median mucociliary transport rate was significantly improved by rhDNase from 0.68 with placebo to 0.79 and 0.83 with 2 and 4 micrograms.mL-1 of rhDNase, respectively. A significant improvement in mucus cough transport was also induced by rhDNase from 25.5 mm with placebo to 27.0 mm with either 2 or 4 micrograms.mL-1 rhDNase. These improvements in mucus transport capacity were associated with alterations in the physical properties of the mucus. The mucus median control viscosity (511.4 Pa.s) and median contact angle (0.85 rd) significantly decreased to 112.5 Pa.s and 0.74 rd, respectively, in the presence of 4 micrograms.mL-1 of rhDNase. These findings demonstrate that recombinant deoxyribonuclease may exert a beneficial effect on mucus clearance in vitro by altering the viscosity and surface properties of the purulent chronic bronchitic sputum samples.

Dose-dependent in vitro effect of recombinant human DNase on rheological and transport properties of cystic fibrosis respiratory mucus.

Recombinant human deoxyribonuclease (rhDNase) has been demonstrated to reduce the viscosity of purulent cystic fibrosis (CF) respiratory mucus, to improve pulmonary function and to reduce the risk of respiratory tract infectious exacerbations, but its effect on mucus transportability has not so far been investigated. The dose-dependent effect of rhDNase was analysed in vitro on mucus transport rate (tr) by ciliary activity and by simulated cough (cough transport (ct)), as well as on mucus viscosity and surface properties. Purulent CF sputa (n = 15) were incubated for 30 min at 37 degrees C with either rhDNase at three different concentrations (final concentrations 0.2, 2 or 20 micrograms.ml-1 of mucus) or placebo. No significant dose-dependent effect of rhDNase on the mucociliary transport rate was observed when the samples wer statistically analysed together. However, in the larger group of mucus samples (n = 11) with a low initial mucociliary transport rate, the latter was improved at each rhDNase concentration (tr0.2 = 0.69, tr2 = 0.88 and tr20 = 0.87) as compared to placebo (trp = 0.58). In the smaller group of mucus samples (n = 4) with high initial transport rate, a decrease in mucociliary transport rate was observed, particularly at the highest concentration rhDNase assayed, i.e. 20 micrograms.ml-1 of mucus (tr20 = 0.58) as compared to placebo (trp = 0.86). The mucus cough transport was increased by rhDNase (ct0.2 = 25 mm, ct2 = 27.5 mm and ct20 = 31 mm) as compared to placebo (ctp = 23.5 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

Physical and functional properties of airway secretions in cystic fibrosis--therapeutic approaches.

The airway secretions which line the respiratory tract form a biphasic layer composed of an aqueous 'sol' layer and a more superficial 'gel' layer. In the sol layer, also described as the 'periciliary' layer or 'airway surface fluid', the cilia beat and relax. The lubricant sol layer enables the gel mucus present at the tips of the cilia to be transported by the ciliary beating of the ciliated cells. Due to difficulties with sampling, little is known about the physical and biochemical properties of the sol layer. The gel layer is composed of high molecular weight glycoproteins (mucins) linked with proteins and lipids. They form a gel network with a high water content (95%) and rheologic and physical properties (viscoelasticity, adhesivity) adapted in normal conditions to protect the airway mucosa, particularly through mucociliary transport. The adhesive properties of mucus, which are influenced by its lipid composition and degree of hydration, are very important in controlling the efficacy of mucus transport through ciliary activity and coughing. An intermediate viscosity and elasticity is required for optimal mucociliary transport. In obstructive airway diseases, either of genetic origin, such as cystic fibrosis (CF), or acquired (acute or chronic bronchitis), and particularly during inflammatory and infectious episodes, mucus dehydration is associated with an increase in secreted or transudated molecules and with marked augmentation of DNA content. These abnormalities contribute to the increased viscosity and adhesivity of the airway secretions and are responsible for their abnormally low transport rate by ciliary activity and for inefficient cough clearance. In view of these alterations in the physical and functional properties of CF airway secretions, pharmacologic approaches should aim to rehydrate the mucus and to restore normal mucociliary or cough transport by stimulating chloride ion secretion (i.e. using UTP or ATP associated with amiloride in order to block sodium ion and water reabsorption). During acute episodes of infection, recombinant human DNase (rhDNase) may rapidly prevent mucus stasis by improving its rheologic properties. Lubrication of the mucus at the sol phase interface by 'surfactant' therapy may also represent a very promising therapeutic perspective to reduce the hyperviscosity and hyperadhesivity of airway secretions.

Cytotoxicity of Pseudomonas aeruginosa internal lectin PA-I to respiratory epithelial cells in primary culture.

Pseudomonas aeruginosa is the most important bacterial pathogen associated with chronic airway infection, especially in cystic fibrosis. We addressed the question of whether the galactophilic internal lectin of P. aeruginosa (PA-I) could represent a virulence factor for the respiratory epithelium. PA-I lectin was localized in all the bacteria of P. aeruginosa ATCC 33347 as determined by immunofluorescence staining. We investigated the dose-dependent effect of P. aeruginosa PA-I lectin on the growth, the ciliary beating frequency, and the morphology of human respiratory cells in primary culture of nasal polyps collected from non-cystic fibrosis patients. PA-I lectin significantly (P < 0.01) inhibited the growth of respiratory cells at a concentration of > or = 10 micrograms/ml. The percentage of active ciliated cell surface of the cultures decreased significantly (P < 0.05) at a PA-I lectin concentration of 50 micrograms/ml. Exposed to a low concentration of PA-I lectin (10 micrograms/ml), respiratory epithelial cells showed intracytoplasmic vacuoles when examined by light and transmission electron microscopy. At a higher concentration of PA-I lectin (100 micrograms/ml), major cell damage and severe epithelial shedding occurred. These results demonstrate that the P. aeruginosa internal PA-I lectin has a dose-dependent cytotoxic effect on respiratory epithelial cells in vitro. The P. aeruginosa PA-I lectin may represent a virulence factor by contributing to the respiratory epithelial damage during P. aeruginosa respiratory infections.