Airway Epithelial Repair, Regeneration, and Remodeling after Injury in Chronic Obstructive Pulmonary Disease Edith Puchelle, Jean-Marie Zahm, Jean-Marie Tournier, and Christelle Coraux Institut National de la Sante´ et de la Recherche Me´dicale, INSERM UMR-S 514, and Centre Hospitalier Universitaire Maison Blanche, Reims, France
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. Keywords: airway epithelial differentiation; bacterial injury; inflammation; remodeling; wound repair
Because of their diversity, the epithelial cells lining the airways are particularly well adapted to the protection of the airway mucosa from major sources of injury (e.g., tobacco smoke, pollutants, viruses, and bacteria). These cells fulfill a number of critical functions in innate airway defense mechanisms. At the surface of the airway epithelium (bronchi and bronchioles), these specialized cells include columnar ciliated cells, mucous (goblet) cells, Clara cells, and basal cells. At the submucosal level, the glands form tubules that feed into a collecting duct and then into a ciliated duct that is continuous with the airway surface. Tubules are lined with mucous cells (proximal region) and serous cells (distal acini). More than 90% of the airway mucus is provided by these glands. These airway cells can rapidly change
(Received in original form May 16, 2006; accepted in final form July 7, 2006 ) Supported by INSERM, Association Vaincre la Mucoviscidose, and Adult Stem Cells Thematic Concerted Action, with a grant from INSERM and the French Ministry of Research, Cance´ropo ˆ le Grand Est, ARD/Soliance (France), and GlaxoSmithKline (United Kingdom). Correspondence and requests for reprints should be addressed to Edith Puchelle, Ph.D., INSERM UMR-S 514, Centre Hospitalier Universitaire Maison Blanche, 45 rue Cognacq Jay, 51092 Reims Cedex, France. E-mail:
[email protected] Proc Am Thorac Soc Vol 3. pp 726–733, 2006 DOI: 10.1513/pats.200605-126SF Internet address: www.atsjournals.org
their structure and functions, either to adapt to changes in the local environment or to repair the epithelium after injury. In the upper and lower airways, the surface airway epithelium is normally pseudostratified. This implies that all cells are attached to the basement membrane but not all reach the airway lumen. In the proximal bronchioles, the epithelial cells become more cuboidal and, in addition to ciliated cells, contain secretory Clara cells. In the most distal bronchioles, only Clara cells are identified (1).
DEFENSE FUNCTIONS AND ALTERATIONS OF THE AIRWAY EPITHELIAL BARRIER IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE The epithelial lining of the airways provides an efficient barrier against microorganisms and aggressive molecules through interdependent functions including the following: mechanical clearance of the mucus; homeostasis of ion and water transport; biochemical antibacterial, antioxidant, and antiprotease functions; and a cellular barrier function by means of intercellular epithelial junctions. All these functions are fundamental to the protection and maintenance of the integrity of the airway epithelium, which may be rapidly disturbed after any infectious or inflammatory-related injury in diseases such as chronic obstructive pulmonary disease (COPD). In normal airway epithelium, it is widely reported that ciliated cells are terminally differentiated cells that are not able to divide and are very sensitive to injury. Nevertheless, it has been recently reported that ciliated epithelial cells spread and may transdifferentiate into squamous cells within hours after bronchiolar injury to maintain the integrity of the epithelium before redifferentiation (2). Ciliated cells are not only directly involved in the elimination of exogenous particles through the mucociliary clearance; they also possess, at their apical membrane, a number of ion channels. The cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENac) regulate the proper quantity of ions and water on the airway surface via transepithelial transport, and therefore directly participate in the formation of a low viscous periciliary lining fluid (PCL) required for active ciliary beating (3). This PCL also acts as a lubricant to prevent adhesion of the highly viscous mucous gel, which contains the mucins, proteins, and peptides packed in the mucous secretory granules and released after exocytosis to form the protective-gel upper layer mucus. The thickness and rheologic properties of the airway surface liquid (ASL) are closely regulated. The absence or incorrect localization of CFTR, associated with impaired mucociliary clearance, airway mucus plugging, repeated airway infections, and excessive inflammation, is not only a hallmark of CF disease. Mall and coworkers (4) have shown that increased airway epithelial sodium absorption produces CF-like lung diseases in mice. Cantin and colleagues (5) have recently reported that the CFTR expression of epithelial cells may be modulated by oxidant stress that leads to a CFTR deficiency, decreased cAMP-dependent efflux, and increased water absorption due to increased expression
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of ENac. This suggests that CFTR deficiency may contribute to the physiopathology of cigarette smoke–induced diseases, such as COPD. CFTR is normally present at the apical plasma membrane of the ciliated cells and the serous glandular cells and also has been identified at the membrane of intracellular secretory granules (SGs) where it can regulate the salt and water dynamics required for a proper mucus exocytosis (6). Dynamic videomicroscopy studies in airway glandular cells have shown that CFTR inhibition impairs the swelling of the SGs and markedly delays the mucus expansion due to an impaired hydration of the mucins packed in the SGs (7). This in turn leads to an inadequate clearance of mucus. At the airway surface epithelium, the loss of functional CFTR leads to an increased and isotonic-related liquid absorption from the PCL. This suggests that any “acquired” abnormal CFTR expression may contribute to decreased mucociliary clearance and mucus load. Recent data from Tarran and colleagues (8) have shown that the deposition of excess ASL, as observed in CF and COPD, stimulates sodium absorption, suggesting that ASL volume regulation is very sensitive to changes in the concentration of soluble mediators in ASL. In peripheral airways and in submucosal glands, neutrophil infiltration occurs in smokers with COPD (9, 10), which may also induce a redistribution of ion channels and a lack of electrolyte-driven fluid associated with inadequate mucus exocytosis and abnormal clearance of mucus from glands. The remodeling of the airway epithelium, such as squamous metaplasia and mucous hyperplasia, that occurs during injury and disordered repair, may considerably disturb the innate immune functions of the airway epithelium. We have previously shown that abnormal expression and distribution of CFTR protein is not only caused by mutations of the CF gene but is also observed in non-CF inflamed and/or remodeled airway tissues (11). In nasal polyps, the extensive inflammatory infiltration and remodeling of the epithelium, similar to that observed in COPD, is frequently associated with a diffuse cytoplasmic CFTR distribution or absent expression of CFTR protein in areas of squamous metaplasia. However, apical epithelial residual expression of CFTR could be observed in areas of CF epithelium where pseudostratified and polarized epithelium was still preserved (12). The normal processing and apical targeting of membrane proteins as well as the correct localization of scaffolding proteins, such as ezrin, actin, and tight junctional proteins that stabilize them at the membrane, require polarization of the epithelial cells. In COPD, the airway epithelial remodeling may therefore induce an abnormal localization and expression of ion channel proteins such as CFTR, which may cause abnormal epithelial functions, such as periciliary epithelial dehydration, as observed in CF due to the defective CFTR protein. Presumably, in the submucosal glands, the hyposecretion due to incorrect mucus exocytosis could also be responsible for a decreased release in the airway lumen of glycoproteins, proteins, and peptides secreted by the mucous and serous cells, which normally represent the biochemical barrier against microorganisms. Mucins and proteins such as sIgA, lactoferrin, and glycosaminoglycans actively participate in the airway antibacterial defense. Other molecules, such as defensin peptides, are also involved in the defense of the airway epithelium. It has been recently reported that the human cathelicidin antimicrobial peptide has a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis (13, 14). Surfactant proteins (SP-A, SP-B, SP-D), also called collectins, play an important role in the defense of the epithelium (15). These human lung collectins are synthesized by alveolar epithelial cells, nonciliated bronchiolar cells, and some epithelial cells lining the larger airways and airway glands. At the bronchiolar level, the Clara cell
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secretory protein (CCSP), also called CC10, is able to modulate lung inflammatory and immune response (16–18). At the airway epithelial cellular level, the tight junction (TJ)– associated proteins, such as ZO1, occludin, and claudins, play a central part in the epithelial cytoprotection by maintaining a physical selective barrier between external and internal environments. Apart from their barrier function, the TJ intercellular proteins interact with actin filaments and actively participate in epithelial signaling. The TJ proteins are highly labile structures whose formation and structure may be very rapidly altered after injury. Their function may be disturbed by airway inflammation. Coyne and colleagues (19) have shown in primary cultures of airway epithelial cells that TJ proteins are regulated by proinflammatory cytokines and that the combined exposure to tumor necrosis factor and IFN-␥ induces drastic effects on TJ expression and barrier functions, with significant alterations in the epithelial barrier permeability. TJs not only restrict the permeability of the epithelium but also participate in the polarization by establishing potential differences between the luminal and serosal side. After injury, the junctional barrier becomes “leaky,” facilitating the access of bacteria and the release of soluble virulence factors to basolateral receptors that are never exposed to pathogens in normal conditions. Numerous bacterial toxins released by Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, such as elastase, exotoxin A, and alpha toxin, are able to degrade junctional adhesion molecules and induce severe alterations of the epithelial integrity. This likely explains the fragility of surface epithelium and its shedding during acute infections and inflammation. After injury, the barrier function will re-establish after several days, but relative leakiness will correspond to a “low protective” barrier against successive aggressive agents. We have shown that, even after complete wound closure, the integrity of the epithelium is not completely recovered. During several days, the epithelium remains leaky and therefore particularly exposed to the noxious effects of the environment or to bacterial virulence factors (20). There is evidence that bacterial infection is a frequent cause of acute exacerbations of COPD. Most of the bacteria identified in COPD may release virulence factors that impede or delay the migration of the epithelial cells and induce a disordered repair with a relative leakiness of the epithelial barrier function. Although large areas of epithelial detachment are a characteristic feature of asthma not observed in COPD, it is possible that an incomplete restoration of the cellular barrier function after bacterial injury may favor the recurrence of infections in COPD. After injury, the airway epithelial cells modify their structure and functions, either to adapt to changes in the environment of injured areas or to repair the epithelium. The plasticity of the airway epithelium, generally described as abnormal remodeling, involves basal cell and mucous cell hyperplasia, mucous cell proliferation, and squamous cell metaplasia. These changes may represent intermediate steps of repair and regeneration, without a complete restitution of a normal, well-differentiated airway epithelium.
CELLULAR EVENTS DURING AIRWAY EPITHELIAL REPAIR AND REGENERATION Whatever the source of acute injury, by either inhaled toxic agents or microorganisms such as bacteria or viruses, common sequences of injury and wound repair have been described in vivo (21). After a transient mucus release, a rapid shedding of the columnar ciliated cells is observed, whereas large areas of basal cells are still attached to the basal lamina. A variety of animal models developed to analyze the repair process highlight a common process in epithelial repair and regeneration, including spreading and migration of the basal cells neighboring the
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wound, proliferation and active mitosis, and squamous metaplasia, followed by progressive redifferentiation with the emergence of preciliated cells (mixed phenotype of ciliated cells with mucous SGs), and a final step of ciliogenesis and complete regeneration of a pseudostratified mucociliary epithelium (22). The sequential and dynamic changes of the epithelial cell phenotypes during the repair and regeneration steps are representative of the high plasticity of the airway epithelial cells. A squamous metaplasia observed during airway epithelial regeneration in animal models appears to be a “normal” step of regeneration and, in fact, represents a “high protection” cellular response in the dynamic process of injury and repair. However, this step represents a transient step of the regeneration process. The epithelial cells can rapidly change their phenotype and can redifferentiate into secretory or ciliated cells. Nevertheless, any delay in the repair process, or interruption at a given step of dedifferentiation or redifferentiation due to an abnormal response of the other cells interacting with the epithelium (e.g., fibroblasts and effector molecules from inflammatory cells), may affect the dynamic process of repair and regeneration.
IN VITRO AND IN VIVO MODELS OF AIRWAY EPITHELIAL REPAIR AND REGENERATION Numerous cellular and molecular factors are involved in the repair and regeneration of the airway epithelium. These factors are modulated by the MMPs, cytokines, and growth factors released by the epithelial and mesenchymal cells (Figure 1). In vitro and in vivo models are useful to analyze the main steps of the wound repair process and the contribution of various cytokines and extracellular matrix (ECM) molecules that trigger behavioral changes in the epithelial cells involved in wound repair. In Vitro Models of Airway Epithelial Repair and Regeneration
The re-epithelialization pattern of the airway cells in twodimensional (2-D) cell culture has been well described by several authors (23, 24) who have shown that one of the most important and first events occurring during the re-epithelialization of the denuded airway epithelium is cell migration and not proliferation. Interestingly, Erjefalt and colleagues (24, 25) have shown in patients with asthma that gentle removal of airway epithelium produces venular gaps, infiltration of neutrophils, and migration
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and activation of eosinophils. This suggests that epithelial shedding restitution processes may cause part of the microvascular and leukocyte changes that occur in inflammatory airway diseases. Cell migration involves the anchorage onto type IV collagen via focal contacts, and metalloproteinases control the migration of the repair cells by remodeling the provisional ECM. The 2-D epithelial cell culture has the advantage of allowing the study of the dynamic migration of cells in real time by videomicroscopy but does not allow the complete re-epithelialization of the wound with well-differentiated cells. The air–liquid culture (26) has the advantage of modeling the epithelial steps of differentiation and permits the examination of migration, proliferation, and final mucous and ciliated cell differentiation. This model was particularly useful in analyzing the genesis of mucous cell metaplasia driven by interleukin (IL)-13, that induces airway epithelial cell proliferation and disordered mucous metaplasia observed in COPD (26). Interestingly, it was recently shown that airway mucous metaplasia may be blocked by inhibiting epidermal growth factor receptor (EGFR) antiapoptosis and IL13 transdifferentiation signals (27). The three-dimensional (3-D) model of airway epithelial cells cultured as spheroids describes the kinetics of dedifferentiation, polarization, redifferentiation, and complete mucociliary regeneration (28). The major interest of these cultured cells is related to their capacity to maintain a differentiated state for several months in liquid culture suspension. The use of human airway epithelial cells, collected by brushings after bronchoscopy in COPD, and cultured as native 3-D spheroids, would be of particular interest to study the role of pharmacologic molecules that may stimulate the regeneration of a functional airway epithelium after loss of the cellular epithelial integrity. In Vivo Models of Airway Epithelial Regeneration
The human airway xenograft in nude or severe combined immunodeficient (SCID) mice is one of the most appropriate models to study the role of cellular and molecular mechanisms of airway epithelial regeneration after injury. Initially developed by Zhang and colleagues (29), we adapted this model using dissociated adult airway epithelial cells seeded into the lumen of a rat trachea that was denuded of its epithelium and tied to sterile tubing, allowing the collection of the airway liquid secreted during the different steps of airway epithelial regeneration (30). This assembly
Figure 1. Cellular and molecular factors involved in the repair and regeneration of the airway epithelium. These factors, which closely interact during the different steps of airway epithelial regeneration after injury, are modulated by the components of the extracellular matrix; the matrix metalloproteinases (MMPs), cytokines, and growth factors released by the epithelial cells; and by the mesenchymal cells (fibroblasts, inflammatory cells, and chondrocytes).
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was then subcutaneously implanted in the flank of a recipient nude mouse. The sequence of events analyzed during the regeneration shows that 3 d after implantation in nude mice, tracheas were partly repopulated with flattened, nonciliated, poorly differentiated cells, generated by airway epithelial cells that adhered rapidly to the denuded host basal lamina (regeneration step 1). These cells, very similar to migrating epithelial cells involved in wound repair, were characterized by cytoplasmic expansions. No adhesion molecules were observed and cell contacts were not present. After 2 wk (regeneration step 2), cell proliferation produced, on the entire surface of the rat trachea, an epithelium that was stratified with a morphology similar to squamous epithelium. The pseudostratification of the epithelium was only observed after 25 d (regeneration step 3); the epithelium then became well differentiated after 35 d (regeneration step 4). At this last step of regeneration, basal cells and columnar cells composed of ciliated and secretory cells could be identified by cytokeratins 14 and 18, respectively. Interestingly, at the last stage of complete regeneration, ZO-1 and desmoplakins 1 and 2 were observed, and CFTR was present at the apical domain of ciliated cells. This chimeric model has the main advantage of reconstituting a human adult airway epithelium that is exposed to the air environment in the same way as in adult human airways. A very similar model has been developed in humanized SCID mice as an assay for potential airway progenitor cells. Human fetal tracheal grafts were first repopulated with allogeneic epithelial cells: total epithelial cells dissociated from fetal airways were seeded in host grafts from which native epithelium had been eliminated. A fully differentiated mucociliary epithelium was recovered in all cases, whereas autonomous regeneration did not occur in unseeded control grafts. The donor origin of newly formed epithelia was confirmed in sex-mismatched combinations of hosts and donor tissues. In other experiments, the endodermal pouch dissociated from 5- to 7-wk embryonic lung rudiment, and which forms a homogenous population of respiratory epithelial “stem cells,” was used as donor tissue. Such early anlagen also replenished the denuded host grafts with the full spectrum of surface epithelial cells and glands. These experimental results demonstrate that candidate airway epithelial stem cells can be assayed functionally in this model (31, 32). Using an array of candidate markers, among them the aquaporin-3 (AQP3) water channel, specifically expressed on the surface of human fetal tracheal basal cells, we could separate by flow cytometry AQP3⫹ fetal basal cells and AQP3⫺ fetal ciliated and secretory cells. Functional evaluation of sorted cells in the humanized SCID mice xenograft model showed that AQP3⫹ cells restored a normal pseudostratified mucociliary epithelium, as well as submucosal glands. AQP3⫺ cells also exhibited the same potential, but with faster engraftment, suggesting that they included more committed progenitor cells. These results showed that epithelial progenitors exist among both basal and suprabasal cell subsets within human fetal airways. Together, the results validate the data reported in animals and suggest that basal cells should be considered important airway epithelial cells, which could be pharmacologically stimulated to rapidly restore a fully differentiated airway epithelium after injury. The open-air xenograft model system in nude mice may be very useful in studying the role of bacteria and inflammatory mediators during the epithelial regeneration of cells collected from patients with COPD. Using a murine model of tracheal epithelial regeneration in which male tracheas were transplanted into female mice, Gomperts and colleagues (33) have shown that a population of progenitor epithelial cells exists in the bone marrow and circulation of mice that are positive for the early epithelial marker cytokeratin 5 and the chemokine receptors CXCR4 and CXCL12. Interest-
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ingly, they showed that neutralizing antibodies to CXL12 resulted in a phenotype of squamous metaplasia, frequently seen in COPD, suggesting that the circulating progenitor epithelial cells are necessary for the re-establishment of a normal pseudostratified airway epithelium after injury. The human airway xenograft is also particularly relevant in analyzing the early phase of inflammation in CF airways before infection with P. aeruginosa or S. aureus, either at the bronchial or bronchiolar level. Using non-CF and CF-naive human airway xenografts, an inflammatory imbalance, characterized by a higher intraluminal content of IL-8 and significant accumulation of leukocytes in the subepithelial region of the CF airway grafts, was observed before any infection (34). No histologic abnormality was observed until these CF tissue grafts were challenged with P. aeruginosa. After intraluminal infection, rapid and massive transepithelial migration of leukocytes occurred in CF airway grafts, associated with epithelial exfoliation facilitating the access of bacteria to adherence sites on exposed basal cells and basal lamina. To investigate whether inflammatory pathways are dysregulated in CF bronchiolar airways independently of infection, Tirouvanziam and colleagues (35) used an in vivo model based on the maturation of human fetal CF and non-CF small airways in SCID mice. They showed that uninfected mature CF small airway grafts underwent time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clearcut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection. Evidence for bacteria and viruses as a primary cause of exacerbations in COPD is still discussed, but it has been reported that the association between neutrophilic airway inflammation and presence of bacteria and viruses on sputum and viral culture, respectively, supports a bacterial and/or viral cause of exacerbations (36, 37). Moreover, the increased concentration of neutrophil elastase and the release of inflammatory chemokines and mediators may contribute to the lung injury and to the abnormal sequence of repair associated with a delayed recruitment and proliferation of mesenchymal cells in the subepithelial airway tissue (24).
REMODELING OF THE EPITHELIUM DURING INJURY AND REPAIR FAVORS BACTERIAL INFECTIONS In COPD, the association of inflammation after viral and/or bacterial infections by S. aureus, H. influenzae, and/or S. pneumoniae may pave the way to later-appearing bacteria such as P. aeruginosa. Apical cell surface carbohydrates are altered during cellular differentiation and differ in normal and repairing epithelial cells, and the host–pathogen interactions between bacterial adhesins and host receptors are completely modified. An important contribution to the understanding of the persistent infections in patients with chronic airway diseases was first given by Plotkowski and colleagues (38), who showed that the pneumococcal adherence to respiratory epithelium was maximal in the repairing migratory cells after viral infections. The role of viral/ bacterial coinfection in the exacerbations of COPD has been more recently emphasized (39, 40). Similarly, maximal adherence of S. aureus and P. aeruginosa has been reported in wounded cells that exhibit apical receptors, such as asialylated gangliosides (asialo GM1) and ␣51 integrins normally never exposed to bacteria. In areas of incomplete repair, ECM components (laminin, collagen I and IV, and fibronectin) can also be recognized by bacterial adhesins (41, 42). It has been shown that numerous pathogens interact with the carbohydrate sequence GalNac1-4 Gal (43, 44) found in the
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asialylated ganglioside asialo-GM1, a transmembrane glycolipid receptor present and overexpressed at the surface of poorly differentiated respiratory cells in remodeled epithelia. Using the adult human airway xenograft model, it was shown that S. aureus does not adhere to ciliated cells and that the major sites for bacterial adherence are the undifferentiated airway cells observed during the first migratory step of regeneration. Fibronectin-binding bacterial proteins belonging to the family of staphylococcal surface adhesins called MSCRAMM (microbial surface components recognizing adhesive matrix molecules) are involved in the bacterial adherence to the dedifferentiated cells (45). A high affinity of P. aeruginosa for spreading and migrating human epithelial respiratory cells has been reported (41). Aggregated bacteria surrounded by fibronectin matrix are frequently identified in these dedifferentiated cells during the repair process. It has been speculated that, during the wound repair and inflammation process, soluble cellular fibronectin released by the epithelial cells may interact with P. aeruginosa and mediate bacterial adherence by establishing a bridge between bacteria and cell surface receptors, further favoring P. aeruginosa infection of injured tissue. A different glycosylation pattern of carbohydrates has been reported to be characteristic of epithelial cells during repair: cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration (46). Nevertheless, the specific reactivity of repairing epithelial cells for specific lectins that recognize the terminal disaccharide sequence Gal13GalNAc of asialoGM1 may partly explain the unique affinity of P. aeruginosa for migrating airway epithelial cells. P. aeruginosa binding and internalization occur frequently in airway epithelial cells that have lost their polarity (42) or in airway epithelial cells that do not express CFTR and are dedifferentiated. The transmembrane integrin ␣5 1, a specific ECM ligand for fibronectin, and identified in repairing cells, is largely responsible for P. aeruginosa adherence to remodeled airway epithelium. Common bacteria identified in COPD, such as H. influenzae and S. pneumoniae, bind to ECM matrix type I and IV collagen and laminin as well as to fibronectin. These data demonstrate that different types of bacteria share a great homology in the nature of epithelial receptors to which they can bind and they also share another important common attribute, which is that they bind especially to damaged and repairing cells.
MOLECULAR MECHANISMS RESPONSIBLE FOR NORMAL AND DYSREGULATED AIRWAY EPITHELIAL REPAIR AND THE REGENERATION PROCESS The cellular and molecular factors involved in epithelial wound repair, regeneration, and complete redifferentiation are numerous and closely interacting. Cell migration involves protrusion of the plasma membrane (lamellipodium extension) at the leading edge of the cell, which implies cytoskeleton reorganization. Cell movement also implies the formation of new sites of adhesion to the ECM at the front of the cells but also the release of adhesion sites at the back of the cells. This necessarily implies a coordinated sequence of events involving the following: contraction of the actin and actomyosin cytoskeleton and interaction with ECM proteins and MMPs, with regulation between MMPs and their inhibitors. Furthermore, production of ECM by the airway epithelial cells during the migration process requires a signaling pathway through specific receptors on the airway cell surface. Role of Cytoskeleton Proteins in Epithelial Repair
In spreading and migrating airway epithelial cells, the polymerization of G actin to F filamentous actin leads to an accumulation of F actin in the lamellipodia of the dedifferentiated and flattened basal cells, which form adhesive contacts with the ECM. Cyto-
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chalasin B, which blocks actin polymerization, is known to inhibit cell migration and repair (47, 48).Virulence factors from P. aeruginosa and from S. aureus induce actin skeleton disorganization and overactivation of MMP-2, responsible for delayed epithelial wound closure. In some cases, the epithelial wound healing is incomplete and the wound closure and epithelial junctional integrity are never achieved. Azghani (49) has also shown that elastase and exotoxin A are virulence factors that damage the TJs, disabling the restoration of alveolar epithelial impermeability. Role of MMPs in Epithelial Repair and Regeneration
The MMPs are a family of zinc-dependent endopeptidases that have proteolytic activity against a wide range of extracellular proteins (50). MMP expression is generally described as characteristic of tissue remodeling associated with normal and abnormal biological processes, such as development, inflammation, tumor invasion, and repair. MMPs are involved in epithelial wound repair, especially in the remodeling of the provisional matrix onto which the cells migrate, by degrading components of the ECM. During the re-epithelialization of the injured airway epithelium, MMP-9 (gelatinase B) is overexpressed in the migratory cells. MMP-9 has been shown to play a key role in the migration of epithelial bronchial cells to repair a wound (51, 52). Indeed, cell migration is a step-by-step process in which MMP-9 expression is highly and rapidly regulated: MMP-9 has a specific role in the degradation of type IV collagen present in primordial contacts, the first cell–extracellular contacts present in lamellipodia, which have to be rapidly remodeled to allow migrating cells to form new contacts on which cells can exert traction through actin filament bundles to move further on. MMP-9 inactivation results in a decrease of the rate of wound repair. Stromelysin 1 and 3 (MMP-3 and MMP-11, respectively) are detected in these migratory cells that acquire a typical epithelial– mesenchymal phenotype (53). Interestingly, the epithelial cells spreading at the edge of the wound, and expressing stromelysin, were positive to vimentin and cytokeratin 14, markers of mesenchymal and basal cells, respectively. These data demonstrate that stromelysins are involved in epithelial cell migration and ECM remodeling during wound repair and that the migrating repair cells shift from an epithelial to a mesenchymal phenotype. This epithelial– mesenchymal transition is therefore a necessary step for the airway re-epithelialization. Failure to transition, due to release of bacterial virulence factors able to block actin polymerization in the repairing cells, will inhibit the first step of epithelial repair and therefore prevent a normal airway epithelial regeneration. Unlike most MMPs, matrilysin (MMP-7) is produced by intact, noninjured airway epithelial cells where it functions in host defense by activating the latent form of antimicrobial peptides, such as defensins (54). In models of airway injury, MMP-7 expression is up-regulated in migrating epithelial cells. Matrilysin mediates the shedding of the ectodomain of E-cadherin required for epithelial repair (55). The marked inhibition of tracheal reepithelialization in MMP-7–null mice and in wounded epithelial culture, after addition of a peptide hydroxamate inhibitor of MMP catalytic activity, demonstrates the unique role of MMP-7 in epithelial repair. As reported by Parks and coworkers (54), MMP-7 may serve different complementary functions. It acts on matrix proteins during repair when it is secreted basally (cell migration), but it also has bactericidal properties (defensin activation) when it is secreted apically. We can therefore hypothesize that any remodeling of airway epithelium associated with a loss of polarization may markedly impair the important cytoprotective and pro-regenerative functions of MMP-7. The “air opened” humanized xenograft model, which mimics the regeneration dynamics after severe injury, was used to characterize the cellular and molecular mechanisms involved during
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the different steps of airway epithelial reconstitution (56). The expression and potential involvement of matrilysin (MMP-7) and gelatinase B (MMP-9), and of their inhibitor, the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), as well as of the proinflammatory cytokine IL-8, were analyzed. It was found that during the cell migration and proliferation steps, airway epithelial cells expressed IL-8 at a high level, whereas airway epithelial pseudostratification and surface airway epithelium differentiation were associated with an increased expression of MMPs and a progressive decrease of IL-8. Interestingly, the immunohistochemical detection revealed an exclusive expression of MMPs at the apical part of the well-differentiated regenerated airway epithelium, and incubation of the regenerating epithelial cells with MMP inhibitors led to an abnormal epithelial differentiation demonstrated by immature and mature squamous metaplasia with areas of basal cell hyperplasia. These data provide evidence that epithelial MMP-7 and MMP-9 play a key role, not only in the early steps of, but throughout re-epithelialization and differentiation process. The close association between IL-8 and MMP expression suggests that expression and activation of MMPs can be regulated by an increased level of the IL-8 proinflammatory cytokine. Whether imbalance of MMPs and their inhibitors during epithelial regeneration is responsible for the frequent areas of epithelial remodeling histologically described in COPD is not known. Abnormal repair resulting from repeated episodes of infection will create a dysregulated and prolonged activation of MMPs and an imbalance between MMPs and their inhibitors, which will create an ongoing epithelial inflammatory response associated with a remodeling of the airways. Role of Cytokines and Growth Factors in Airway Epithelial Repair
The activation of chemokines, interleukins, growth factors, and colony-stimulating factors has been frequently described during the early inflammatory and chemotactic response of the airway epithelium. All these factors are secreted by mesenchymal cells, endothelial cells, and macrophages, but are also secreted by epithelial cells during injury and repair. Transforming growth factor (TGF)-1 modulates the composition of the provisional matrix over which the epithelial cells migrate and has been shown to increase in vitro airway wound repair via MMP-2 upregulation (57): an increase of MMP-2 in response to TGF1 would promote airway repair in conditions of homeostasis, whereas MMP-9 could be critical in an inflammatory context characterized by a prolonged remodeling process. In a normal, well-differentiated airway epithelium, epidermal growth factor (EGF) is expressed at the apical domain and is separated by TJs from its receptor, EGFR (also called Erb B), which is localized at the basolateral domain (58, 59). Although the differentiation of human epithelial cells has been recently reported to be dependent on ErB2 (60), the activation of EGFR and of the IL-13 receptor (IL-13R) is known to lead to mucous cell metaplasia and mucin synthesis (60, 61). IL-13 can directly drive mucin gene expression in physiologic conditions of airway epithelial cell culture, and IL-4 is often overexpressed in the setting of mucous cell metaplasia in asthma and COPD (62, 63). IL-9 induces goblet cell hyperplasia during repair of human airway epithelia, whereas its inhibition decreases the repair. IL-9 has therefore been proposed to modulate the repair process and to decrease airway remodeling (64). Airway mucous cell metaplasia may be blocked by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals. During wound repair, trefoiled peptides, such as TFF2, exhibit a synergistic effect with EGF and enhance epithelial migration by activation of the protein kinase-C and extracellular signal-regulated kinase (ERK)
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signaling pathways (65). The inhibition of EGFR tyrosine kinase completely inhibits the re-epithelialization process. Airway epithelial migration is also induced by other growth factors and motogen peptides, including insulin, insulin-like growth factors, hepatocyte growth factor (HGF), calcitonin gene–related peptides, and the cathelicidin LL-37 peptide (66–68, 70, 71). The stimulation of wound closure by this latter antimicrobial peptide is mediated through EGFR, a G protein–coupled receptor, and mitogen-activated protein (MAP)/extracellular regulated kinase (68). It has been also recently shown that the bombesin receptor subtype 3, increased in ozone-stressed animals, activates the wound repair and proliferation of bronchial epithelial cells through the protein kinase-A (PKA) signaling pathway (69). These results confirm previous data that reported that the activation of PKA accelerates bronchial epithelial cell migration (70). Keratinocyte growth factor (KGF) and HGF may enhance wound repair by acting as a chemotactic or a growth-stimulating factor that in turn may stimulate the synthesis of ECM and facilitate the interaction with MMPs through specific cell receptors (72, 73). It is now established that acetylcholine may function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. Acetylcholine is synthesized and secreted by airway epithelial cells and regulates their adhesion to the ECM though specific nicotinic acetylcholine receptors (nAChRs), activated either by endogenous acetylcholine or exogenous inhaled nicotine. Tournier and colleagues (74) reported that the ␣3␣52 nAChR is involved in human bronchial epithelial cell migration and wound repair and that nicotine increased the intracellular Ca2⫹ in migrating cells. Whether a prolonged exposure to nicotine may reverse this effect remains to be determined inasmuch as acetylcholine and nicotine are chemotactic for pulmonary neutrophils. Furthermore, different nAChRs may exert opposing effects in the nicotinergic control of cell migration. Glycosaminoglycans, such as hyaluronan, participate in a variety of biological processes, including cell–matrix interactions and activation of chemokines, enzymes, and growth factors. Epithelial cell surface hyaluronan is protective against apoptosis through Toll-like receptor–dependent basal activation of nuclear factor-B (75, 76), and accelerated repair of epithelial cells has been reported in the presence of hyaluronan (76, 77).
FUTURE THERAPY TO PROMOTE REPAIR AND REGENERATION IN COPD Repair of the airway epithelium after injury is critical for the maintenance of the barrier function and the limitation of airway hyperreactivity. Future therapy to promote repair and regeneration should aim to improve the cytoprotection of the airway epithelium against injury. Protection of the airway epithelium against the enhanced susceptibility to infection and excessive inflammatory response requires molecules that may prevent bacterial adherence and limit the cellular damages due to virulence factors. These therapies should also be aimed at maintaining a vectorial transport of chloride and water transport, which is crucial for normal functioning (mucociliary clearance, biochemical protection of the epithelium, trafficking and polarization of the membrane receptors, protection of the tight junctional proteins). There is already evidence of enhanced respiratory cytoprotection against viral and bacterial infection when a corticosteroid is combined with a long-acting 2-adrenergic receptor (2AR) agonist (78). The increased CFTR expression associated with 2AR stimulation may induce other beneficial effects on ion and water transport, protein expression, and differentiation (79). Furthermore, the combination of a long-acting 2AR with a glucocorticoid has been shown to attenuate the S. aureus– induced airway epithelial inflammation (80). In the case of severe
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injury, these therapies could enhance wound repair and epithelial regeneration function. Improving our understanding of epithelial function in normal and pathologic conditions, through in vitro and in vivo models that mimic human airway epithelial injury repair and regeneration, may help to develop novel regenerative therapeutics allowing the rapid repair and regeneration of a functional airway epithelium in COPD. Several peptides, such as defensins, trefoiled peptides, and KGF, could represent future important and effective molecules in protection and regeneration of the airway epithelium after injury. Conflict of Interest Statement: E.P. has received research grants to support work carried out in her laboratory from GlaxoSmithKline (United Kingdom) and by Agro Industrie Recherches et De´veloppements (ARD)/Soliance (France). J.-M.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.-M.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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