Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell-ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell-collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture.

Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells.

Quantitative videomicroscopic analysis of the sociologic behavior of non-invasive and invasive tumor cell lines.

To analyze the spatial distribution of tumor cell lines with different invasive properties, we used time-lapse videomicroscopic recordings associated with software programs we have developed for quantification. We observed that non-invasive tumor cells rapidly formed small clusters which aggregated to form larger clusters, whereas highly invasive tumor cells remained isolated and did not form clusters. An attraction index computed from a cellular automaton model was used to quantify the degree of attraction-repulsion between cells. The results suggest that the cluster formation by noninvasive cells is not related to a global attraction model and that the random (dispersed) distribution of invasive cells is not related to cell repulsion. According to these results, we can conclude that random cell movement combined with the intrinsic properties of cells explains the phenomenon of cluster formation.

Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer.

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue. Moreover, we studied the effects of CFTR gene transfer mediated by a replication-defective adenovirus containing the wild-type CFTR gene (AdCFTR) on CFTR expression and on cAMP-stimulated chloride secretion. Distribution of the CFTR protein was evaluated in regenerating PD airway cells by light fluorescence microscopy and scanning laser confocal microscopy. The cAMP-mediated regulation of cell membrane chloride secretion was investigated using the whole-cell patch clamp and SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) techniques. Compared with the absence of CFTR expression and cAMP-regulated chloride secretion in nontransduced regenerating PD cells of either non-CF or CF origin, transduction with AdCFTR induces a CFTR expression and a cAMP-regulated stimulation of the cell membrane chloride secretion in the regenerating PD cells. These results suggest that, out of the context of CF, remodeled and poorly differentiated airway epithelium may present abnormalities in ion transport. Moreover, our data suggest that, in the context of CF gene therapy, adenoviral vectors can be efficient in correcting, at least partially, the chloride secretion defect in the remodeled CF airway epithelium.

Regenerating cells in human airway surface epithelium represent preferential targets for recombinant adenovirus.

To investigate the efficiency of adenovirus-mediated gene delivery in regenerating human respiratory epithelium, we have performed infections with an E1- and E3-deleted type 5 recombinant adenovirus containing the Escherichia coli LacZ reporter gene on different culture models of regenerating human nasal polyp surface epithelium. These models included: (i) an ex vivo organ culture of nasal polyp tissue, (ii) an explant outgrowth cell culture, and (iii) an in vitro wound repair model, on dissociated cells. In ex vivo nasal polyp tissue, transduced cells were not detected in normal pseudostratified areas, but were found in areas of the surface epithelium with a morphology reminiscent of regenerating airway tissue. In the explant outgrowth cell culture, adenovirus-infected cells were preferentially detected at the periphery of the outgrowth. These transducible epithelial cells, representative of epithelial cells present in vivo during the process of surface airway epithelium regeneration, were shown to be migrating and poorly differentiated cells, which were proliferating or not. In the in vitro wound repair model, the efficiency of cell transduction was much higher in cells present in the wound area than in those far from the wound area. These results indicate that regenerating cells from human airway surface epithelium represent preferential targets for transgene expression, and suggest that efficiency of CFTR gene transfer by recombinant adenovirus vectors may be higher in regenerating CF airway mucosa than in normal tissue. However, since these cells do not show endogenous CFTR expression, the relevance of their preferential transduction for the functional correction of the ion transport defect in cystic fibrosis needs further investigations.

Effect of terbutaline on mucociliary transport and sputum properties in chronic bronchitis.

A double-blind study with placebo control was carried out in 20 patients with chronic bronchitis to analyze the effect of oral terbutaline on mucociliary transport and sputum properties. Terbutaline (2.5 mg tablets) was given 3 times a day for 1 week. The clinical score, forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) significantly improved after treatment with terbutaline and confirmed the bronchodilating action of this drug. The mucociliary clearance measured with a radioactive aerosol tracer did not significantly differ after treatment with terbutaline and placebo although the initial site of aerosol deposition was similar in the two groups before and after treatment. The viscoelastic properties of sputum and their in vitro transport rate on the frog palate were not modified by terbutaline. The decrease in sputum volume and serum albumin concentration suggests a bronchial anti-inflammatory effect of this medication.

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation.

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

Role of phospholipid lining on respiratory mucus clearance by cough.

Phospholipid lining, present at the respiratory mucus-mucosa interface, may have an important role in the protective function of the airways by its abhesive properties and may also facilitate mucus transport. To mimic respiratory mucus-mucosa interface, monolayers of three different forms of phosphatidylglycerol (PG) have been deposited on glass slides by the Langmuir-Blodgett technique. Mucus adhesion and clearance by cough of mucus on these PG-coated or noncoated surfaces have been analyzed and compared, using frog respiratory mucus as "normal" mucus. Among the three PG types studied, the phosphatidylglycerol distearoyl, which is the phospholipid with the longest saturated fatty acid chain, was found to significantly improve the mucus cough clearance by decreasing the mucus work of adhesion compared with the noncoated surfaces. On the other hand, phosphatidylglycerol dipalmitoyl did not improve mucus cough clearance although it decreased mucus adhesion, and phosphatidylglycerol dioleyl did not improve either mucus cough clearance or mucus adhesion.

Relationships between the lipid content and the rheological properties of airway secretions in cystic fibrosis.

The lipid composition and rheological properties of expectorated airway secretions have been analyzed in 16 patients with cystic fibrosis (CF), separated into two groups according to whether their secretions were superinfected or not. The total lipid content was higher in the superinfected CF secretions. The content in cholesterol and GL1, GL3 and GL4 glycosphingolipid fractions were significantly higher in the superinfected in comparison to the non-superinfected group. The viscosity was two-fold higher in the CF superinfected group. A significant correlation (r = 0.72, p less than 0.01) was observed between the apparent viscosity and the total lipid content of CF secretions. Cholesterol, glycosphingolipids and sphingomyelin were the lipid components which were the most closely and positively correlated to the viscosity. On the opposite, phosphoglycerol was negatively correlated (r = -0.72, p less than 0.05) to the viscoelastic properties of CF airway secretions.

Carbocisteine improves the mucociliary transport rate in rats with SO2-induced bronchitis.

In order to study the effect of carbocisteine on the mucociliary function of the respiratory tract, we performed a double-blind study on rats with SO2-induced (400 ppm) hypersecretion. During the experimental bronchitis, the treated group of rats received carbocisteine through a stomach tube at a dose level of 500 mg/kg for 15 days, whereas the untreated group of rats received distilled water. After killing the rats, and following lung excision, the respiratory mucus was scraped off and collected by using a glass capillary. The mucus degree of purulence was macroscopically estimated and the mucus transport rate was measured by using the frog palate technique. The mean mucus relative transport rate, measured on the frog palate, was 0.60 +/- 0.17 in the untreated group and was significantly higher (P < 0.01) in the treated group (0.73 +/- 0.14). Carbocisteine also significantly altered (P < 0.01) the mucus macroscopical aspect, leading to a decrease in the number of rats with purulent mucus. These results suggest that carbocisteine maintains an efficient mucus transport rate, leading to a less infected respiratory tract.

Phospholipid composition and surface-active properties of tracheobronchial secretions from patients with cystic fibrosis and chronic obstructive pulmonary diseases.

Among the various components of tracheobronchial secretions, lipids and particularly phospholipids have been shown to influence rheological properties of airway secretions in patients with cystic fibrosis. We studied the phospholipid composition of tracheobronchial secretions, collected from patients suffering from cystic fibrosis (CF) and other chronic obstructive pulmonary diseases (COPD), and we analyzed the possible relationship between the phospholipid profile and the wettability of tracheobronchial secretions evaluated by the measurement of contact angle. Although total phospholipid content and contact angle of tracheobronchial secretions were significantly increased (P less than 0.01) in CF compared to COPD, no significant relationship existed between these two parameters. The concentrations of the different phospholipid subclasses were not homogeneously modified according to the origin of the secretions. Compared to COPD secretions, the CF secretions were characterized by a significant (P less than 0.001) increase in rigidifying fractions such as sphingomyelin and phosphatidylserine/phosphatidylinositol and a significant (P less than 0.001) decrease in surface-active fractions, such as phosphatidylcholine and phosphatidylglycerol (PG) (P less than 0.001). In the two groups, the surface-active phospholipid fraction, PG, was negatively correlated to the contact angle of tracheobronchial secretions. These results suggest that a decrease in PG content in CF secretions may be one factor responsible for an increase in their adhesivity to the respiratory mucosa, and, consequently, for mucus stasis and severity of bronchial obstruction in cystic fibrosis.

Influence of rheological properties of human bronchial secretions on the ciliary beat frequency.

We studied the relations between the mucociliary beat frequency (MF) measured photometrically on the depleted frog palate and the rheological properties of sputum collected in patients with chronic obstructive pulmonary disease. MF was lower (p less than 0.001) with sputa (11.3 +/- 3.3 Hz) than with frog mucus (16.9 +/- 3.3 Hz) used as controls. The relative transport rate (Tr) of sputa was closely correlated (r = 0.81, p less than 0.001) to the relative MF. Significant correlations were observed between MF on the one hand and viscosity (r = -0.68, p less than 0.01), elastic modulus (r = -0.70, p less than 0.01) and spinability (r = +0.49, p less than 0.05) on the other. These results suggest that abnormalities in the rheological properties of bronchial secretions may impair the mucociliary transport rate by first decreasing the ciliary beat frequency.

Spinability of bronchial mucus. Relationship with viscoelasticity and mucous transport properties.

Spinability of normal mucus and of sputum collected in chronic bronchitic patients was measured using an automatic device derived from that developed by CHRETIEN et al (1977) for cervical mucus. Spinability of sputum decreased as the purulence increased. Although significant correlations were found between spinability and apparent viscosity (r = - 0.50, p less than 0.05) or elasticity (r = 0.54, p less than 0.02), large variations in spinability were observed for sputum samples ranging in a zone of low viscosity and elasticity. Sputum with high spinability exhibited normal transport rate on the depleted frog palate although their viscosity and elasticity were abnormally low. The significant correlation obtained between spinability and sputum transport rate (r = 0.61, p less than 0.01) suggests that this rheological factor, along with viscosity and elasticity, plays an important role in the mucociliary transport mechanism.

Rheological properties controlling mucociliary frequency and respiratory mucus transport.

Respiratory mucus and mucosa possess highly hydrophilic structures which are difficult to preserve using standard fixative methods. The close interaction between cilia and mucus can be observed after instantaneously interrupting the ciliary movement using ultra rapid and cryosubstitution fixation methods. Mucus possess several rheological properties such as pseudoplasticity, elastothixotropy, spinability and adhesiveness. Rheological properties of mucus may control, per se, the ciliary beating frequency. By measuring the mucociliary frequency on the excised mucus-depleted frog palate of native mucus and xanthan gum using a simulant of mucus, we observed that beyond an optimal value of viscosity (close to 12 Pa.s) the mucociliary frequency and transport rate decrease in parallel. Other rheological factors such as adhesion and spinability of mucus can also be implicated in the regulation of the mucociliary transport rate.

Effect of air humidity on spinability and transport capacity of canine airway secretions.

The effect of varying the inspired air humidity on a rheological property (spinability) and transport capacity of airway mucus has been analyzed in 10 mongrel dogs. Tracheal mucus was collected in anesthetized dogs inspiring through an endotracheal tube the air of a climate chamber maintained at constant temperature (T degrees:20 degrees C). In one test, the dogs inspired air at an absolute humidity (AH) of 9 g water/m3 air directly through the endotracheal tube. In the other test, the dogs inspired through an artificial nose connected to the endotracheal tube giving a AH of 30 g water/m3 air. Tracheal mucus was collected at the external distal end of the endotracheal tube. The spinability (Sp) or thread-forming properties of mucus was measured. The relative mucociliary transport rate (TR) of mucus was analyzed on a frog palate epithelium preparation. The transport rate was significantly (p less than 0.01) lower (range: 0.59-0.80) when the AH of the inspired air was low in comparison to that obtained with high AH (range: 0.70-1.13). The variations in mucus Sp due to changing AH were positively and significantly correlated (r = 0.80, p less than 0.01) with the corresponding variations in TR. These results suggest that lowering the AH of air induces a decrease in the transport capacity which appears to be dependent on the change of spinability that occurs in the mucus.

Dynamics of cell movement during the wound repair of human surface respiratory epithelium.

Epithelial wound repair represents an important process by which the epithelial barrier integrity recovers after wounding. To evaluate and quantify the dynamics of surface airway cell movement during the wound repair process, we developed an in vitro wounding model of human respiratory cells in culture and we analyzed the wound repair by using videomicroscopic and image analysis techniques. We observed that wound closure occurred within 6 hours, due to the spreading and migration of the cells surrounding the wounded surface. The migration rate of the cells at the leading edge of the wound surface increased progressively up to 26 microns/h during the repair process which was characterized by a uniform centripetal direction of cell movement. The distance travelled by these cells was 2.5 fold longer than the distance travelled by ciliated cells which were located far from the wound area. These results suggest that cell migration after wounding is an important process by which the respiratory epithelial barrier integrity is maintained.

The role of mucus gel viscosity, spinnability, and adhesive properties in clearance by simulated cough.

We investigated the role of the viscoelastic and adhesive properties of mucus gel simulants on the clearance of mucus by simulated cough. Mucus-like gels with widely varying viscoelastic properties were prepared from polysaccharides crosslinked with sodium borate. Cough was simulated by opening a solenoid valve connecting a model trachea to a pressurized tank. The clearance of gels lining the model trachea was quantified by observing marker particle transport. Viscosity elastic modulus, relaxation time and yield stress were measured with a steady-shear viscoelastometer. Spinnability (thread formation) was determined with a filancemeter. Adhesivity (surface tension) was measured by the platinum ring technique. The viscoelastic and adhesive properties of the mucus gel simulants spanned the ranges observed for bronchial secretions from patients with COPD. The relationship between simulated cough clearance and the viscoelastic and adhesive properties of the gels was analyzed by stepwise linear regression of the non-zero data matrix. The major independent variable relating to clearance was viscosity. Secondary, but highly significant dependences, were also found for spinnability and adhesivity. Elastic modulus, relaxation time and yield stress had no independent effect on cough clearance over the investigated range. The results indicate that, in the absence of airway surface liquid, cough-type clearance relates primarily with mucus gel viscosity. For a given viscosity, clearance is also impaired by spinnability, i.e. the capacity of the mucus to form threads. At constant viscosity and spinnability, clearance is further impaired by an increase in the adhesivity of the mucus. The negative dependence of each of these physical factors can be rationalized in terms of their inhibitory effect on wave formation in the mucus lining layer during high velocity airflow interaction.

The role of mucus sol phase in clearance by simulated cough.

Using a simulated cough machine, we analyzed the effect of adding tensio-active liquids as sol phase simulant on the clearance of gel mucus simulant by cough. Polysaccharides crosslinked with sodium tetraborate were used at different concentration as gel mucus simulant. A drop of gel mucus simulant was deposited either directly on the model trachea or on a sol phase layer simulant (2% sodium dodecyl sulfate in water). The clearance of the mucus simulants was quantified by observing the movement of marker particles in the gel layer. The viscoelastic properties of gel mucus simulants were determined by using a viscoelastometer (SEFAM). The adhesive properties were analyzed by means of the platinum ring technique. The wettability of the mucus simulants was quantified by the automatic measurement of the contact angle of the drop of gel on the model trachea. We found that the addition of a sol phase significantly decreased by about 50% the adhesivity and wettability of the gel mucus simulants. This decrease was associated with a marked enhancement of cough clearance, whatever the viscoelastic properties of the gel mucus simulants. These results suggest that the sol phase is essential in bronchial respiratory mucus clearance by the cough mechanism.

Comparative rheological profile of rat gastric and duodenal gel mucus.

Methods for measuring the adhesiveness, plasticity, viscoelasticity and spinnability of mucus microsamples have been developed. The rheological properties of the rat gastric and duodenal gel mucus have been analyzed and compared. Using a controlled stress rheometer (Carri-Med), flow and creep experiments showed that gastroduodenal mucus exhibits a typically non-newtonian, pseudoplastic and viscoelastic behaviour. The apparent viscosity (7,800 +/- 11,000 Pa.s) and yield stress (24.9 +/- 8.5 Pa) of gastric mucus were significantly higher than the duodenal mucus viscosity (39 +/- 160 Pa.s) and yield stress (12.9 +/- 2 Pa). Spinnability of gastric mucus, measured with a Filancemeter (SEFAM), was significantly lower (4.9 +/- 2.5 mm) in comparison to duodenal mucus (6.9 +/- 1.5 mm). Adhesive properties of gastric mucosa (analyzed with the platinum ring method) were not significantly different in comparison to duodenal mucus (99.9 +/- 31.5 mN/m and 92.8 +/- 11.2 mN/m, respectively).

Elasto-thixotropic properties of bronchial mucus and polymer analogs. I. Experimental results.

The non-newtonian viscous and elasto-thixotropic properties of native and lyophilized pathological bronchial mucus and of polymer solutions (3% and 6% PIB in decalin) used as mucus analogs were analyzed using a cone-plate Carri-Med rheometer and a Couette viscoelastometer that we have specifically developed for measuring the rheological properties of bronchial mucus in clinical practice. The master curves obtained for apparent viscosity under steady conditions as a function of shear rates (gamma: 2.6 X 10(-3) to 6.9 X 10(1) sec-1) were fairly similar, whatever the apparatus used. Under transient conditions, at low shear rate (gamma less than 1.4 sec-1), PIB and mucus exhibited a typical viscoelastic behavior: the shear stress increased slightly up to a steady-state value. At higher gamma, a transitory overshoot of sigma characteristic of the elastothixotropic systems appeared. Such a behavior can be interpreted as resulting from structural changes such as formation and rupture of the three-dimensional network present in bronchial mucus as in polymer solutions.