Bronchial Epithelium as a Key Regulator of Airway Allergen Sensitization and Remodeling in Asthma

STEPHEN T. HOLGATE, PETER LACKIE, SUSAN WILSON, WILLIAM ROCHE, and DONNA DAVIES

Respiratory Cell and Molecular Biology Division, School of Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom

	INTRODUCTION

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

Asthma is a chronic inflammatory disorder of the airways manifesting as intermittent airflow limitation, which over time may become progressive (1). Both in the allergic and nonallergic forms of the disease there is evidence of an altered local T cell response in favor of Th2 cytokine release resulting in B cell isotype switching to IgE; mast cell, eosinophil, and basophil recruitment; and activation and release of a wide range of inflammatory mediators (2). However, it has become clear that, by itself, inflammation is not able to explain many of the features characteristic of chronic asthma and that restructuring of the airway wall is also required (3). This "remodeling" response presumably accounts for the incomplete therapeutic efficacy of corticosteroids, with persistence of bronchial hyperresponsiveness (BHR), and the progressive decline in pulmonary function over time that occurs in those asthmatic individuals with more chronic and severe disease (1). Although atopy, the propensity to generate allergen-specific IgE, is one of the strongest risk factors for asthma, especially in children and young adults, only 10% of atopics develop chronic asthma and, as adults, asthma occurs in the absence of atopy, most notably in those with more severe and chronic disease. It seems plausible that, rather than IgE-mediated inflammation being the initiator of disordered airway function, the epithelium itself is abnormal and it is this that predisposes the individual with asthma toward local allergen sensitization, and the injurious effects of respiratory viruses and air pollutants (including tobacco smoke). A disordered epithelium could also provide a basis for airway remodeling in asthma.

	EPITHELIAL ABNORMALITIES

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

The fully differentiated normal bronchial epithelium is a stratified structure consisting of a columnar layer comprising ciliated and secretory cells supported by basal cells. As originally described by Laitinen and coworkers (4) and confirmed in our own studies (5), the bronchial epithelium in asthma is structurally disturbed, with separation of columnar cells from their basal attachments and the extent of epithelial damage correlating with BHR (6) and epithelial permeability (7). Epithelial shedding is characteristic of asthma (5) and does not occur in other airway pathologies such as chronic obstructive pulmonary disease (COPD), even though both diseases are associated with increased inflammatory cell infiltration. Thus, in asthma it seems that the epithelium is either more susceptible to damage or has an altered response to injury. While ultrastructural analysis of undamged areas of asthmatic epithelium has shown that the epithelial cells are able to form normal cell- cell junctions (8), there is evidence of abnormalities in the responses of asthmatic epithelial cells to environmental stimuli (9). These altered responses are preserved in vitro through several generations (10), consistent with an intrinsic functional abnormality or an acquired repopulation of the epithelium with cells of disordered function. Thus, the sensitivity of the epithelial barrier to the action of different components of the inhaled environment may play a key role in determining the asthmatic phenotype.

	SUBEPITHELIAL AND SUBMUCOSAL FIBROSIS

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

When assessed by high-resolution computed tomography (CT), patients with severe asthma have thicker airways when compared with normal subjects or those with mild disease (11). This involves thickening and increased density of the subbasement membrane (SBM) collagen layer, and an increase in smooth muscle (12) and microvascular networks (13). On the basis of measurements made in human airways (14) and in a guinea pig model of chronic antigen exposure, SBM thickening reflects that of the entire airway wall (15), and has been shown to correlate with disease severity, chronicity, and BHR (16, 17).

We have shown that SBM thickening is due to the deposition of interstitial collagens Types I, III, and V and fibronectin in the lamina reticularis (18), which is accompanied by laminin 2 and 2 chains (19) and tenascin (20). Matrix proteins originate from myofibroblasts, whose numbers and activity are increased in asthma (21). A report by Phunek and coworkers (22) describes deposition of SBM collagen in children up to 3 yr before asthma becomes clinically manifest, suggesting the possibility that epithelial dysfunction may be a primary rather than a secondary abnormality. If this is a primary abnormality, then it could explain why inhaled allergens lead to local sensitization and inflammation in asthma, whereas most individuals who become atopic do not acquire the asthma pheonotype. One important area deserving further attention is the interaction of dendritic cells with the epithelium and the way this could lead to polarization of these cells to a type 2 dendritic cell (DC2) phenotype. The finding of interleukin 4 (IL-4) being expressed on bronchial epithelial cells (23) and the capacity of this receptor to augment epithelial chemokine production (24) provides an example of communication between innate and acquired mucosal immunity in asthma.

Many of the "repair" molecules that are expressed in chronic asthma are the same ones that are involved in fetal lung development and airway branching (e.g., epidermal growth factor [EGF], keratinocyte growth factor [KGF], transforming growth factor  [TGF-], bone morphogenic proteins [BMPs], basic fibroblast growth factor [bFGF]), creating an important link between early life events that influence lung growth and maturation, and subsequent development of airways susceptible to asthma. As a starting point, we have focused our attention on interactions between epithelial damage and the subsequent remodeling response. We have shown that polyarginine, a surrogate for eosinophil basic proteins, or mechanical damage to confluent monolayers of bronchial epithelial cells grown on a collagen gel seeded with human myofibroblasts leads to enhanced prolferation and increased collagen gene expression due to the combined effects of bFGF (FGF-2), insulin-like growth factor I (IGF-I), platelet-derived growth factor BB (PDGF-BB), TGF-, and endothelin 1 (ET-1) (25). Thus, any defect in epithelial repair may result in maintaining the epithelium in a prolonged state of activation associated with the production of profibrogenic cytokines that drive airway remodeling.

	REGULATION OF EPITHELIAL MAINTENANCE AND REPAIR

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

Epithelial Injury

As the barrier between the external environment and the internal milieu, the bronchial epithelium is exposed to viruses, inhaled pollutants and allergens that have the potential to cause tissue injury directly by cytotoxicity or proteolysis and indirectly by involving immune and inflammatory cells. In asthma, the products of eosinophils and mast cells are major causes of epithelial damage. Allergens may also have direct effects on epithelial integrity through their protease activity disrupting intercellular adhesion, increasing paracellular permeability, and initiating cell death (26), while oxidants increase epithelial permeability by damaging tight junctions. In addition to their direct effects on epithelial structure, many of these agents alter cell function through induction of a stress response vial receptor-mediated processes (e.g., protease-activated receptor [PARs]) or by generation of reactive oxygen with activation of proinflammatory transcription factors. Taken together, the widespread proinflammatory activity of the bronchial eptihelium observed in asthma is indicative of an abnormally "stressed" barrier reacting to tissue-damaging agents.

Epithelial Repair

Depending on the extent of injury, qualitatively different repair responses are triggered. In the case of subcytotoxic damage, a stress response occurs, while in the case of minor injury increasing the normal processes of regeneration that replace senescent cells is sufficient. For more extensive injury such as columnar cell loss, as occurs in mild-moderate asthma, either columnar epithelial cells dedifferentiate and migrate to restore the stratified structure or basal cell stratification occurs and is followed by differentiation. These processes are regulated by the activities of inducible genes whose expression is coordinated by intracellular biochemical networks that reflect extracellular signals provided by adhesion molecules and growth factors. These convey essential local spatial and environmental information to direct responses that are appropriate for a given type and phase of repair.

EGFRs as Primary Regulators of Epithelial Repair

The involvement of epidermal growth factor receptors (EGFRs) in responses to acute lung injury is well documented with, for example, EGF enhancing repair of sheep tracheal epithelium after cotton smoke injury (27). The EGFR serves a central role as a primary regulator of epithelial function, transducing extracellular signals from its activating ligands into intracellular signaling cascades through dimerization and transphosphorylation catalyzed by the intrinsic tyrosine kinase (28). EGFR activation can elicit rapid responses such as changes in cell shape through phosphorylation of intracellular substrates, but other phenotypic changes follow from signal transduction leading to induction of new gene expression (29). The best characterized signaling pathway that is activated by the EGFR is the extra cellular regulated kinase/mitogen-associated protein kinase (ERK/MAPK) pathway, which usually leads to induction of DNA synthesis and cell proliferation (Figure 1).

View larger version (27K): [in this window] [in a new window]   Figure 1.   The central role of EGFR signal transduction pathways in bronchial epithelial cells.

Cell Surface Regulation of the EGFR

The activity of the EGFR is controlled on the cell surface by members of the EGF ligand family (EGF, TGF-, heparin-binding EGF-like growth factor [HB-EGF], amphiregulin [AR], betacellulin, and epiregulin) whose expression we have demonstrated in bronchial mucosa (30). These growth factors are all synthesized as transmembrane precursors, with the membrane-associated and soluble forms exhibiting differing potencies. Thus, the sphere of influence and potency of EGFR ligands can be controlled by juxtacrine, autocrine, and paracrine mechanisms. Cleavage of the transmembrane precursor of HB-EGF involves matrix metalloproteinases (MMPs) such as MMP-3, which is overexpressed in asthmatic bronchial epithelium (31). HB-EGF plays a particularly important role in wound repair as it is present in wound fluid (32) and is expressed along the leading edge of wound keratinocytes along with EGFRs (33). The effects of EGFR ligands are not identical, responses being dependent both on properties of the ligand and the surface of the target cell (28). For example, the heparin-binding domains of HB-EFG and AR influence ligand potency through their interaction with cell surface heparan sulfate proteoglycans (HSPGs) including CD44v3 (34), expressed by bronchial epithelial cells. This interaction localizes the growth factor on the cell surface and serves to direct the tyrosine kinase activity of the EGFR toward specific intracellular targets such as ezrin, whose phosphorylation results in linkage of CD44 to the actin cytoskeleton (35). Ligation of the EGFR results in a further level of complexity with formation of activated dimeric receptor complexes comprising either homodimers (EGFR₂) or heterodimers involving other members of the c-erbB family. Heterodimeric combinations modulate receptor function with diverse effects on intracellular signaling and cellular responses. We have shown that normal bronchial epithelial cells coexpress several members of the c-erbB Family (30) and that heterodimerization may control cellular functions within the bronchial epithelial cells.

Coordination of Intracellular Signaling Pathways by the EGFR

The pivotal role that the EGFR exerts in controlling the behavior of the bronchial epithelium is emphasized by the finding that several other regulators of epithelial function exploit the EGFR to achieve a response. Induction of bronchial epithelial cyclooxygenase 2 (COX-2) expression by interferon  (IFN-) occurs by autocrine activation of the EGFR after upregulation of EGFR ligand expression by IFN- (36). EGFRs can also be trans-activated, independently of ligand, by G protein-coupled receptors including those activated by proteases (PARs), ET-1, and bradykinin, as well as by cytokine receptors and by environmental stress via a mechanism involving reactive oxygen (Figure 1). Our studies have shown that the extent of EGFR involvement in bronchial epithelial cell behavior is extended by its ability to be activated by oxidant stress (37) and to be trans-activated by the thrombin receptor (PAR-1). This convergence of extracellular stimuli on the EGFR tyrosine kinase identifies the EGFR as a critical focus and switch point responsible for coordinating many of the intracellular signals elicited by exposure to a variety of external and internal stimuli pertinent to asthma (Figure 1).

EGFR-regulated pathways are also the focus of control and integration with other major regulators of cell behavior (Figure 1). Thus, mucosecretory differentiation of bronchial epithelial cells is controlled by coordinate regulation of signals from the EGFR, the extracellular matrix, and the retinoic acid receptor with phosphorylation of p66^shc by the EGFR (but notably not by insulin) being a key regulatory point (38). Growth inhibition of lung epithelial cells by members of the TGF- family is also due to convergence of signals from EGFRs and TGF--like receptors, in this case at the level of Smad-1 (one of ten proteins derived from the Sma and MAD gene homologues in Caenorhabditis elegens and Drosophilia melanogaster) (Figure 1). Hence, proliferative and antiproliferative signals are coordinated by specific intracellular signaling molecules so that the balance (or imbalance) determines the normal (or abnormal) response of the epithelial cell to injury.

EGFRs in Normal, Injured, and Asthmatic Airways

In our studies of bronchial biopsies from normal airways, there is marked EGFR expression in areas of local epithelial damage, including the apical surface, which is usually devoid of EGFRs (29). This facilitates its exposure to ligands present in airway lining fluid and so enhances repair. We have shown that closure of scrape-wounded monolayers of human bronchial epithelial cells in vitro is enhanced by EGF or HB-EGF, but not by keratinocyte growth factor (KGF) (39). In this system, direct activation of the EGFR occurs even in the absence of exogenous ligand and probably involves release of autocrine ligands as both spontaneous and EGF-stimulated wound closure can be inhibited with an EGFR-selective tyrophostin, AG1478. Consistent with enhanced EGFR expression in repairing bronchial epithelium, we have found that mechanical damage, oxidant stress, TNF-, or enzymatically active allergen extracts enhance surface EGFR levels in bronchial epithelial cells. Unlike the bronchial epithelium in normals, where elevated EGFR expression was observed only in areas of structural damage (29), in asthma we have found a striking disease-related overexpression of the EGFR both in damaged and morphologically intact epithelium (40). Further contrasting with normal epithelium, EGFR immunostaining in asthma occurs throughout the epithelial layer, indicative of widespread functional changes. This confirms a Japanese study using asthmatic bronchial tissue postmortem or from surgical resections (41). As our in vitro findings indicate that EGFR expression occurs as an early response to damage, it is possible that high EGFR expression is a "reporter" of damage and that the extent of epithelial injury in asthma is more widespread than previously appreciated. Alternatively, there may be a failure to downregulate EGFR expression after restitution, with the consequence that the epithelial cells are inappropriately "held" in a repair phenotype. In view of the central role that EGFR-regulated pathways have in coordinating signals that control bronchial epithelial cell behavior, it is likely that the high level of EGFR expression in asthma has widespread consequences on the epithial phenotype, irrespective of its cause(s).

Overexpression of the EGFR in psoriasis and carcinomas is accompanied by hyperplasia driven by an EGFR autocrine loop. However, in asthma, the epithelium is not hyperproliferative. Proliferating cell nuclear antigen (PCNA) is expressed at a low level by asthmatic epithelial cells (42) but is markedly increased on corticosteroid treatment (43), supporting the observation that this "controller" drug increases epithelial repair, which may be expedited by reducing inflammatory damage. As our studies indicate that high EGFR levels persist in intact bronchial epithelium of steroid-treated patients with asthma with persistent symptoms, failure to downregulate EGFR expression after resolution is suggestive of a steroid-insensitive component and a key abnormality. While the phenotype of the asthmatic epithelium is not consistent with excessive mitogenic signaling by EGFRs, other functions may remain. In colonic epithelial cells, we have shown that EGFR activation causes destabilization of adherens junctions and internalization of desmosomes, phenotypic changes associated with cell movement (44). Thus, if the EGFRs are highly active in asthmatic epithelium but not linked to proliverative pathways, they could contribute to epithelial fragility by interfering with junctional integrity.

Modulation of EGFR Signaling in Asthma

Although high EGFR expression is suggestive of increased intracellular signaling, this may not be the case in asthma. A variety of mechanisms could operate in the asthmatic epithelium to prevent effective EGFR signal transduction, explaining the high level of EGFR expression and the extent of epithelial damage as a consequence of impaired EGFR-mediated repair. As impairment of EGFR-mediated epithelial repair is paralleled by enhanced release of profibrogenic growth factors, this defect could account for the associated remodeling responses linked to severe and chronic asthma. There are several ways in which EGFR-mediated repair responses may be handicapped in asthma. These include (1) ligand availability, (2) ligand function, (3) receptor modification, and (4) cosignaling.

	CONCLUSION

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

Taken together, we postulate that conditions that favor collagen biosynthesis by the subepithelial myofibroblasts would also contribute to disease chronicity by retarding epithelial repair (45). In addition, enhanced cytokine and chemokine synthesis by an altered epithelium could augment and prolong airway inflammatory responses while structural changes to the epithelium and modification of signaling between epithelial cells, dendritic cells, and T cells could create the necessary microenvironment for Th2 polarization and subsequent production of IgE. Thus, rather than atopy driving asthma, we suggest that the development of asthma, and its concomitant epithelial abnormalities may predispose the airway to local sensitization that, when established, would have the effect of aggravating the inflammatory and remodeling response.

	DISCUSSION

TOP INTRODUCTION EPITHELIAL ABNORMALITIES SUBEPITHELIAL AND SUBMUCOSAL... REGULATION OF EPITHELIAL... CONCLUSION DISCUSSION REFERENCES

Djukanovi: Are the changes to the epithelium relevant to sensitization? Is there any interaction between dendritic cells and epithelial cells?

Holgate: If the epithelium is altered this might change the response to allergen. Otherwise damaged epithelium could allow easier access of allergens. Moreover, protease activity of allergens could itself activate epithelium and thus function as an adjuvant to sensitization.

Holt: We have recently shown that one of the roles of NO, at least in one particular target cell, which happens to be a T cell, is dephosphorylation of a number of tyrosine kinases. In asthma, there is now increasing evidence that hyperproduction of NO occurs in children a long time before they express the full asthma phenotype, particularly children that are skin test positive to more than one inhalant.

Holgate: Yes, the nitration of proteins within the bronchial epithelium is very impressive. One of the things we are planning to do is to look at the effects of this nitration of the tyrosine residues.

Hiemstra: You describe that EGF receptor expression is increased in the asthmatic epithelium and is not associated with proliferation, suggesting that there is no proliferative response to EGF stimulation in those cells. There are data in the literature suggesting that also in tumor cells that overexpressed EGFRs don't respond to EGF by proliferation and that higher concentrations of EGF may even decrease the proliferation. So, my question is: Is there an analogy between tumor cells and asthmatic epithelial cells in the way they handle the signal or is this merely an effect of overexpression of EGFR, which is a downregulatory signal in proliferation?

Holgate: I am not able to answer this question. Comparison with cigarette smoke-induced lung injury shows that EGFR expression is only apparent in those areas of the epithelium when there is hypertrophy, not throughout the epithelium.

Quanjer: Does your theory apply to other epithelium as well?

Holgate: Yes, I think so. In eczema a retarded response to injury is also observed. Of course, bronchial epithelium is unique in the mediators it is able to express in the airways.

Martinez: Burrows described that the level of IgE in parents has nothing whatsoever to do with the likelihood of asthma developing in the children. Moreover, the likelihood of being sensitized on the part of the parents has absolutely nothing to do with the risk of asthma in the children. But, on the other hand, the likelihood of being sensitized on the part of the parents has a lot to do with the likelihood of the child having allergic rhinitis. In addition, the work by Sears suggests that the most striking risk factor for asthma is the number of things you are sensitized to. These data suggest that rather than just developing asthma because you are sensitized to something, it is the other way around: Because you have something called asthma, you become sensitized to a lot of things in the environment.

Jansen: I want to go back to the first question of Dr. Djukanovi. The consequences of allergen peptides applied to defective epithelial cells may result in the facilitated cross-talk between activated dendritic cells in the epithelial layer and sensitized T cells, but we know from the studies by Holt that such events primarily take place in the regional lymph nodes. On the other hand, preferential homing of allergen-specific Th2 cells is present after allergen challenge. Does that mean that other, target organ-specific factors may be important?

Holgate: Recently, some new T cell homing receptors have been described. To my knowledge no unique homing receptors have been identified in the airway epithelium, but it would be very worthwhile to pursue this line of investigation.

Vercelli: Where is your STAT-6 in the epithelium localized, in the nucleus or in the cytoplasm? This makes a difference. As a cytoplasmic protein, STAT-6 is ubiquitous, but if it is nuclear, then it means that the cell has been activated by IL-4 or IL-13.

Holgate: We did not look in detail. The staining appeared both cytoplasmic and nuclear, but more careful analysis of the sections will be needed.

	Footnotes

Correspondence and requests for reprints should be addressed to Prof. S. T. Holgate, Southampton General Hospital, Univ. Medicine, Level D, Centre Block, Mail Point 810, Tremona Road, Southampton SO16 6YD, UK. E-mail: S.Holgate @

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