Denis Pierrot

Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory Epithelium

The respiratory epithelium is frequently injured by inhaled toxic agents or by micro-organisms. The epithelial wound repair represents a crucial process by which surface respiratory cells maintain the epithelial barrier integrity. The repair process involves both cell migration and proliferation, but as yet, the kinetic of these two mechanisms has not been extensively studied. Using an in vitro model of human respiratory epithelium wound repair, proliferative cell immunofluorescent staining and a computer-assisted technique allowing the tracking of living cells, we studied the cell proliferation and migration during the wound repair process. Respiratory epithelial cells were dissociated from human nasal polyps and cultured on a collagen I matrix. At confluency, a chemical wound was made on the culture. We observed that the cell mitotic activity peaked at 48 h after wounding (23% of the cells) and mainly concerned the cells located 160 to 400 microns from the wound edge. The migration speed was highest (35 to 45 microns/h) for the spreading cells at the wound edge and progressively decreased for the cells more and more distant from the wound edge. The temporal analysis of the cell migration speed during the wound repair showed that it was almost constant during the first 3 days of the repair mechanism and thereafter dropped down until the wound closure was completed (after 4 days). We also observed that over a 1-hour period, the intra-individual and interindividual variation of the cell migration speed was 43% and 37%, respectively. These results demonstrate that cell proliferation and cell migration during respiratory epithelial wound repair are differently expressed with regard to the cell location within the repairing area.

Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium.

Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti‐cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6‐6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose‐dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium.

ATP Depletion Induces a Loss of Respiratory Epithelium Functional Integrity and Down-regulates CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Expression

To mimic the effect of ischemia on the integrity of airway epithelium and expression of cystic fibrosis transmembrane conductance regulator (CFTR), we induced an ATP depletion of the respiratory epithelium from upper airway cells (nasal tissue) and human bronchial epithelial 16HBE14o− cell line. Histological analysis showed that 2 h of ATP depletion led to a loss of the epithelium integrity at the interface between basal cells and columnar cells. The expression of connexin 43 (Cx43, subunit of the gap junctions) and desmoplakins 1 and 2 (DPs 1 and 2, major components of the desmosomes) proteins was inhibited. After 90 min of ATP depletion, a significant decrease of the transepithelial resistance (25%) was observed but was reversible. Similar results were obtained with the 16HBE14o− human bronchial epithelial cell line. ATP depletion led to actin filaments depolymerization. The expression of the mature CFTR (170 kDa) and fodrin proteins at the apical domain of the ciliated cells was down-regulated. The steady-state levels of CFTR, Cx43, DPs 1 and 2 mRNAs, semiquantified by RT-polymerase chain reaction kinetics, remained constant throughout ATP depletion in nasal tissue as in the homogeneous cell population of 16HBE14o− human bronchial epithelial cell line. This suggests that the down-regulation of these proteins might be posttranscriptional. The intercellular diffusion through gap junctions of Lucifer dye was completely inhibited after 90 min of ATP depletion but was reversible. The volume-dependent and the cAMP-dependent chloride secretion were inhibited in a nonreversible way. Taken together, these results suggest that an ATP depletion in human airway epithelium, mimicking ischemia, may induce a marked alteration in the junctional complexes and cytoskeleton structure concomitantly with a loss of apical CFTR expression and chloride secretion function.

Nasal Epithelial Cell Culture as a Tool in Evaluating Ciliary Dysfunction

Cultures of respiratory epithelial cells were obtained from nasal polyps collected in patients with and without primary ciliary defect. The ciliary beating frequency and the ciliary beating heterogeneity were determined on native and cultured tissues. We observed a significantly higher (p < 0.01) ciliary beating frequency of cultured ciliated cells, when compared with ciliated cells from the native tissue. The ciliary beating frequency of the cultured ciliated cells from the patient with primary defect (7.9 +/- 2.1 Hz) was significantly lower when compared with the beating frequency of the ciliated cells from the control subject (12.4 +/- 2.0 Hz). In addition, the percentage of ciliated cells characterized by a beating frequency lower than 8 Hz was 90.7% in the native tissue and 47.5% in the cultured tissue from the patient with ciliary primary defect. In the patient without ciliary primary defect, 90% of the cultured ciliated cells had a homogeneous ciliary beating, whereas in the patient with primary ciliary defect, only 47% of the ciliated cells had a homogeneous ciliary beating. These results suggest that the culture of respiratory cells associated with the functional activity measurement of the ciliated cells represent another way of precisely determining the extent of the primary ciliary dyskinesia defect.

Epidermal growth factor promotes wound repair of human respiratory epithelium

Reepithelialization of the airway mucosa is an essential step toward restoring a normal functional protective barrier during the repair of airway epithelial wounds. We investigated the role of epidermal growth factor in the wound healing of human surface epithelial cells cultured from nasal polyp explants on a type I collagen gel in serum‐free defined medium. By using image analysis techniques, we measured the outgrowth area, the ciliated surface, the ciliary beating frequency, and the in vitro wound repair rate in the presence of different epidermal growth factor concentrations. We observed a significant dose‐dependent increase in the outgrowth area (10‐fold increase with epidermal growth factor doses of 0 to 20 ng/ml), in the percentage of the outgrowth surface covered by ciliated cells (30% without epidermal growth factor and 43% with epidermal growth factor 20 ng/ml) and in the ciliary beating frequency (12.6 to 14.5 Hz). The wound repair rate was improved by 29% in the presence of epidermal growth factor 10 ng/ml. These results suggest that epidermal growth factor could be involved in the wound repair process of the airway epithelium.

The elastase-induced expression of secretory leukocyte protease inhibitor is decreased in remodelled airway epithelium

During airway inflammation, proteinases such as human leukocyte elastase are actively secreted. Secretory leukocyte protease inhibitor is a major serine proteinase inhibitor, secreted by bronchial, bronchiolar and lung epithelial cells. We recently identified secretory leukocyte protease inhibitor in human nasal epithelium, exclusively in remodelled areas of the surface epithelium. We now investigated the influence of remodelling and inflammation of the nasal tissue on the in vitro capacity of these cells to respond to human leukocyte elastase. Primary cultures of surface epithelial cells were established from various nasal polyp samples. At confluency, cell cultures were exposed to different human leukocyte elastase concentrations. The secretory leukocyte protease inhibitor immunocytolocalisation, expression and secretion were then investigated. Immunocytochemistry, showed a human leukocyte elastase dose-dependent increase of secretory leukocyte protease inhibitor containing cells and a basal extracellular localization of secretory leukocyte protease inhibitor after incubation with 100 microg/ml human leukocyte elastase. The relative amount of secretory leukocyte protease inhibitor mRNA transcripts increased with respect to the human leukocyte elastase concentration. Nevertheless, the potential stimulation of secretory leukocyte protease inhibitor secretion by human leukocyte elastase was lower in the more remodelled and inflamed tissue. Our results suggest that the contribution of the surface epithelial cells of poorly remodelled tissues to the protection against the deleterious effect of neutrophil proteinases is severely decreased in highly remodelled and inflamed tissues.

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

Summary— 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.

Quantitation of in vitro ciliated cell growth through image analysis

SummaryCiliated cell cultures can be produced in outgrowths from explants of human respiratory epithelium. An image analysis technique was develope to quantify the percentage of active ciliated cells present in these cultures. The subtraction 2 by 2 of five successive video images of the cultures, followed by the addition of the resulting images, allowed the determinaton of the culture surface covered by ciliated cells. The percentage of this surface varied according to the regions of the explant (27.7% in the outgrowth near the explant and 4.1% at the periphery of the outgrowth). High variations were observed within the same region of an outgrowth, as well as from one outgrowth to another. However, maximal differentiation was observed after 4 d of culture. The quantitation techniques described in the present work might be useful for studying in vitro the respiratory epithelial injury and the subsequent repair processes.

The involvement of metalloproteinases in the wound repair of human respiratory epithelium

During the wound healing of injured epithelia, the extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases and stromelysins, in wound healing, by using an in vifro wound repair model of human respiratory epithehum (ZAHM J-M., CHEWLLARD M. and PUCHELLE E.(1991). Am.J. Kzsptr. Cell Mol. Btol.. 5, 242-248). Surface epithelial cells were isolated from human nasal polyps and cultured on a type 1 collagen gel. Circular wounds were made with NaOH in confluent cell cultures. Over the next-3 to 4 days, the repair kinetics of the wounds were monitored as the cells were proliferating and migrating onto the injured area to cover the denuded surface. Gelatinolytic and caseinolytic matrix metalloproteinases were analvsed bv using substrate zvmographv analysis in the culture supemateiand in-the celiextracts fro& rep%&g~and st&onary cells. Only small amounts of the 72 kDa gelatin% A were expressed by repairing and stationary cells, and the enzyme concentration hid not v-&y‘cluring the wound repair process. In contrast, the 92 kDa gelatinase B and stromelysins were upregulated in the repairing migratory cells with a maximum peak observed at wound closure. Furthermore, a marked gelatinase B activation occured during the wound repair process. By using an immunofluorescence technique, gelatinase B was localized in certain migratory basal cells, recognized by an anti-cytokeratin 14 antibody. The addition to the culture medium, of a monoclonal antibody known to inhibit gelatinase B activity, resulted in a dose-dependent decrease of the cell migration speed. These results suggest that gelatinase B and stromelysins contribute -to the wound repair procesS, and that these matrix metalloproteinases are involved in human respiratory epithelium remodeling.