The airway epithelium: Structural and functional properties in health and disease

Authors

  • Darryl A. KNIGHT,

    Corresponding author
    1. Asthma and Allergy Research Institute, Sir Charles Gairdner Hospital, Nedlands,
    2. School of Medicine and Pharmacology, University of Western Australia, Crawley, Western Australia, Australia and
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  • Stephen T. HOLGATE

    1. School of Medicine and Pharmacology, University of Western Australia, Crawley, Western Australia, Australia and
    2. The Brooke Laboratory, Division of Infection, Inflammation and Repair, School of Medicine, Southampton General Hospital, Southampton, United Kingdom
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Darryl Knight, Asthma & Allergy Research Institute, Ground Floor, E Block, Sir Charles Gairdner Hospital, Verdun Street, Nedlands, Western Australia 6009, Australia. Email: dknight@cyllene.uwa.edu.au

Abstract

Abstract:  The major function of the respiratory epithelium was once thought to be that of a physical barrier. However, it constitutes the interface between the internal milieu and the external environment as well as being a primary target for inhaled respiratory drugs. It also responds to changes in the external environment by secreting a large number of molecules and mediators that signal to cells of the immune system and underlying mesenchyme. Thus, the epithelium is in a unique position to translate gene–environment interactions. Normally, the epithelium has a tremendous capacity to repair itself following injury. However, evidence is rapidly accumulating to show that the airway epithelium of asthmatics is abnormal and has increased susceptibility to injury compared to normal epithelium. Areas of detachment and fragility are a characteristic feature not observed in other inflammatory diseases such as COPD. In addition to being more susceptible to damage, normal repair processes are also compromised. Failure of appropriate growth and differentiation of airway epithelial cells will cause persistent mucosal injury. The response to traditional therapy such as glucocorticoids may also be compromised. However, whether the differences observed in asthmatic epithelium are a cause of or secondary to the development of the disease remains unanswered. Strategies to address this question include careful examination of the ontogeny of the disease in children and use of gene array technology should provide some important answers, as well as allow a better understanding of the critical role that the epithelium plays under normal conditions and in diseases such as asthma.

INTRODUCTION

Mammalian airways are lined by a variety of specialized cells that fulfil a number of critical functions related to normal homeostasis. These functions include regulation of lung fluid balance, metabolism and/or clearance of inhaled agents, attraction and activation of inflammatory cells in response to injury, and the regulation of airway smooth muscle function via secretion of numerous mediators. However, the epithelium is also the site of first contact for both inflammatory and physical environmental stimuli. Thus, damage to the epithelium may contribute substantially to inflammation, and the bronchoconstriction and oedema seen in asthma and a number of other respiratory diseases. The present review focuses on recent advances in our understanding of the structure of the epithelium and its function(s) in healthy and asthmatic airways.

ANATOMY OF THE EPITHELIUM

At least eight morphologically distinct epithelial cell types are present in human respiratory epithelium, although based on ultrastructural, functional and biochemical criteria these may be classified into three categories: basal, ciliated and secretory.1 In addition, immune cells, inflammatory cells and phagocytic cells migrate to and remain within the epithelium or transit through to the lumen. The terminal processes of sensory and cholinergic nerves are also observed.

Columnar ciliated epithelial cells

Ciliated epithelial cells are the predominant cell type within the airways, accounting for over 50% of all epithelial cells.1 Ciliated epithelial cells arise from either basal or secretory cells and until recently were believed to be terminally differentiated.2 Typically, ciliated epithelial cells possess up to 300 cilia/cell and numerous mitochondria immediately beneath the apical surface, highlighting the primary role of these cells, namely the directional transport of mucus from the lung to the throat.3

Mucous cells (goblet cells)

Mucous cells are characterized by membrane-bound electron-lucent acidic–mucin granules, secreted to trap foreign objects in the airway lumen.4,5 Production of the correct amount of mucus and the viscoelasticity of mucus are important for efficient mucociliary clearance. It is thought the acidity, due to the sialic acid content of the glycoprotein, determines the viscoelastic profile and hence the relative ease of transport across cilia.3 Release of acid mucins from these granules can be increased by acute exposure to stimuli such as inhalation of sulphur dioxide or tobacco smoke. In normal human trachea, it is estimated that there are up to 6800 mucus-secreting cells/mm2 of surface epithelium, although in chronic airway inflammatory diseases such as chronic bronchitis and asthma, mucous cell hyperplasia and metaplasia is a common pathological finding and is thought to contribute to the productive cough associated with these diseases.6 These cells are thought to be capable of self-renewal and may also differentiate into ciliated epithelial cells.7

Serous cells

Serous cells morphologically resemble mucous cells, although ultrastructurally their granule content is electron-dense, rather than electron-lucent.8 Until recently, these cells had only been described in rodent airways. However, two populations of these relatively rare cells have been observed in the small airways of the human lung.8 The chemical composition of the granules has not been extensively characterized, although the same cell-type in rat airways contains neutral mucin and an unidentified non-mucoid substance.

Basal cells

Basal cells are ubiquitous in the conducting epithelium, although the number of these cells decreases with airway size.7,9 There is a direct correlation between the thickness of the epithelium and the number of basal cells as well as the percentage of columnar cell attachment to the basement membrane via the basal cell. The basal cell has a sparse electron-dense cytoplasm that contains bundles of low molecular weight cytokeratin.10 Within the epithelium, basal cells are the only cell that are firmly attached to the basement membrane3,7,9 and as such, play a role in the attachment of more superficial cells to the basement membrane via hemidesmosomal complexes.11 While interepithelial cell attachment is mediated by desmosomes, only basal cells appear to express hemidesmosomes which have the necessary integrins (α6β4) to attach to the basement membrane.

Similar to the skin, the basal cell is thought to be the primary stem cell, giving rise to the mucous and ciliated epithelial cells. Boers et al. undertook a systematic study of the distribution and contribution of basal and parabasal cells to proliferation in autopsy specimens of normal human lung.12 Basal cells occupied 51% of the proliferation compartment in large airways (> 4 mm ID), and 81% in small (< 2 mm ID) airways, suggesting that these cells are likely to be progenitor cells.12 In smaller airways, where basal cells are sparse or absent, Clara cells perform the primary stem cell role.

In addition to their progenitor and structural roles, basal cells are also thought to secrete a number of bioactive molecules including neutral endopeptidase, 15-lipoxygenase products and cytokines.

Clara cells

In humans, Clara cells are located in large (bronchial) and small (bronchiolar) airways. The cells contain electron-dense granules, thought to produce bronchiolar surfactant and are also characterized by agranular endoplasmic reticulum in the apical cytoplasm and granular endoplasmic reticulum basally. In addition to their secretory role, Clara cells are believed to metabolize xenobiotic compounds by the action of p450 mono-oxygenases and may also produce specific antiproteases such as secretory leukocyte protease inhibitor.13 More recent evidence suggests that these cells play an important stem cell role, serving as a progenitor for both ciliated and mucus-secreting cells.14

PULMONARY NEUROENDOCRINE CELLS

Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells found throughout the bronchial tree as solitary cells or in clusters (neuro-epithelial bodies). PNEC have traditionally been thought of as rare cells, with early reports suggesting numbers ranging from 2.1 PNEC/10 000 epithelial cells in normal human lung, increasing to almost 6/10 000 epithelial cells in chronic bronchitis.15,16 More recently, Boers et al. using human airways from autopsy specimens reported approximately 40 PNEC/10 000 cells.17 We have investigated the distribution of PNEC in human airways using 3-dimensional confocal microsopy and found approximately 45 PNEC/10 000 cells in human airway whole mounts18(Fig. 1) similar to the number reported by Boers and colleagues. These cells secrete a variety of biogenic amines and peptides, which are thought to play an important role in foetal lung growth and airway function. For example, PNEC may play a role as hypoxia sensitive airway chemoreceptors19 as well as being involved in regulating localized epithelial cell growth and regeneration.20

Figure 1.

Confocal microscopy image of a whole mount of human airway epithelium stained for PGP 9.5, imaged from the lumen. Patches of varicosed nerves are present. Pulmonary neuroendocrine cells are abundant and possess a dendritic cell-like morphology when stained with PGP 9.5 (arrows).

Intra-epithelial nerves

The epithelium is richly innervated by nerves derived from subepithelial plexuses. In humans, immunoreactivity for the pan-neuronal marker protein gene product (PGP) 9.5 reveals a large number of subepithelial nerves, which penetrate the basement membrane at focal points where they branch and spread along the basement membrane with terminal ends extending between epithelial cells and terminating at the airway lumen.21 Recently, we have shown that some of these nerves are in direct apposition to pulmonary neuroendocrine cells, suggesting a bi-directional interaction between these two cell types.18 Depending on the species and site, sensory nerves containing tachykinins (substance P (SP) and neurokinin-A) and calcitonin-gene related peptide (CGRP) predominate. Unfortunately, while PGP9.5 immunoreactivity suggests nerves are present, SP immunofluorescence has rarely been detected in human airway epithelium,22,23 although occasionally sensory nerves can be seen ramifying immediately beneath the basement membrane (Fig. 2). Rapid enzymatic degradation and the effects of age or depletion by smoking-related stimuli probably contribute to this effect. Our confocal microscopic analysis of whole mounts of airways from non-smoking, young (< 37 years) subjects show only rare SP+ve nerve fibres. Using a variety of techniques, acetylcholinesterase or catecholamine have not been identified in the terminal fibres, suggesting that these are not motor neurons.

Figure 2.

Single sensory nerve stained for substance P (green) and CGRP (red) situated immediately beneath the basement membrane. This nerve travels as a single nerve and then bifurcates into individual nerves staining positive for either substance P or CGRP. Scale bar = 50 mm.

Immune cells

In addition to the resident structural cells, a variety of cells migrate to and reside within the epithelium. These cells include mast cells, intraepithelial lymphocytes, dendritic cells and macrophages. It is beyond the scope of this article to comprehensively review immune cell biology. The reader is referred to several excellent articles dealing with this topic.24–26

Epithelial adhesion molecules

The behaviour of epithelial cells is profoundly influenced by the biochemical composition of the extracellular environment and the health or otherwise of adjacent cells. Similarly, the organization of epithelial cells into a patent 3-dimensional structure is highly dependent on the interaction of cells, both with their neighbours and with the extracellular matrix (ECM).27 The effects of ECM proteins are mediated through specific cell surface receptors or integrins.28

Epithelial integrins

Integrins are heterodimeric transmembrane glycoproteins consisting of α and β chains, with ligand specificity conveyed through the α chain and signal transduction through the β chain.28–30 To date, 18 α and eight β subunits and more than 23 integrin combinations have been identified. Epithelial cells express nine different integrins.29,30 Two of these, α3β1 and α6β4 recognize the components of the basement membrane and function as true adhesion molecules.31 The remaining six integrins recognize ECM proteins that are not normally in contact with these cells. Indeed, many of the ECM ligands for these integrins are part of a provisional matrix produced in response to injury and inflammation.

αv integrins: Receptors for vitronectin as well as tenascin, osteopontin and fibronectin

All five αv integrins (αvβ1, αvβ6, αvβ3, αvβ5, αvβ8) bind vitronectin (VN) and all are expressed on human bronchial epithelial cells. Considerable attention has focused on the αv integrins since αvβ6, αvβ3 and αvβ5 appear to play major roles in several processes relevant to remodelling, such as cell migration,32–37 proliferation38,39 and differentiation10,40–43 as well as binding and activation of matrix metallopro-teinases44–46 and TGFβ (Table 1).47–49 Signalling by αvβ3 integrins also inhibits apoptosis by elevating levels of the anti-apoptotic molecule Bcl-2.50,51 In contrast, the αvβ8 integrin inhibits epithelial cell proliferation as well as the formation of focal contacts and actin cytoskeleton, suggesting that this integrin acts as a negative regulator of epithelial cell function.52

Table 1.   Putative function of av-integrins
IntegrinLigandEffects related to airway remodelling
avb1VN, FN, Osteopontin (OP)Binds latent form of TGFb1
avb3VN, TN-C, fibrinogen, thrombospondin, FN, OPDirectly binds active Matrix Metalloproteinases (MMP’s); co-operative signalling with RTKs; promotes angiogenesis and remodelling.
avb5VNDirect involvement in wound healing; epithelial cell proliferation.
avb6FN, VN, TN-CDirectly binds and activates TGFb1; knockout mice have marked airway inflammation and AHR; direct involvement in wound healing
avb8VN, FNNegative regulator of epithelial cell proliferation

Other epithelial adhesion molecules

CD44 is a widely expressed family of adhesion molecules that represent alternatively spliced and post-translationally modified products of a single gene.53–55 CD44 is the major receptor for the glycosaminoglycan, hyaluronan but also binds fibronectin, collagen and some cytokines.55 Like integrins, CD44 connects the extracellular environment with intracellular machinery and actin cytoskeleton, via the linking proteins ezrin and moesin.55 Under normal conditions, CD44 appears to regulate hyaluronan metabolism, but more recent evidence suggests that it may also play an important role in epithelial repair.56 Increased expression of CD44 in areas of normal epithelium undergoing repair is markedly upregulated in asthma.57 However, increases in CD44 expression in vivo do not occur in areas of cell–matrix interactions, suggesting that this molecule may play a role in cell–cell communication.57

THE BASEMENT MEMBRANE

The epithelial basement membrane serves a number of critical roles within the airway: (i) it acts as an anchor for the epithelium and facilitates adhesion and migration of epithelial cells; (ii) it is essential for regulating the phenotype of epithelial cells as well as establishing and maintaining their correct polarity and (iii) it acts as a barrier between the surface epithelium and the underlying mesenchymal compartments.58,59 Under normal conditions, there is little or no direct contact between epithelial cells and other resident structural cells. However, infiltrating inflammatory and immune cells are able to move freely between epithelial and subepithelial compartments. The upper layer of the basement membrane, the lamina densa, consists predominantly of type IV collagen and type V laminin and is secreted by the epithelial cells themselves. In contrast, the lower, thicker lamina reticularis, consists of types III and V collagen and fibronectin and is synthesized primarily by subepithelial fibroblasts.60

BASEMENT MEMBRANE PORES

It has long been assumed that infiltrating cells found within the epithelium gained access through enzymatic digestion of the ECM within the basement membrane. However, recent studies by Howat and coworkers have demonstrated the presence of pores within the basement membrane, analogous to those seen in the instestine and conjuctiva.61,62 These pores occur at an average density of 737–863/mm2 and have a mean diameter of 1.76 µm. This size is large enough to allow passage of infiltrating cells, and given that the pores traverse the full thickness of the basement membrane, they would provide a conduit for these cells without the obvious need for matrix degradation.61,62 Whether the number, size and distribution are different in asthmatic airways is unknown.

EPITHELIAL FUNCTION

Traditionally, the epithelium was considered to be an inert barrier between the external environment and the inner tissues of the lung. However, it is now generally accepted that the epithelium plays a pivotal role in controlling many airway functions.63–65 In this regard, the repertoire of mediators that epithelial cells can produce, both basally and upon stimulation, indicate its central role in modulating airway function.

Barrier function

The mucociliary layer and the intercellular adhesion complexes provide a physical barrier function.66,67 Tight junctions (zonula occludens) located between the apices of adjacent cells restrict paracellular diffusion of electrolytes and other molecules. Desmosomes, intermediate and gap junctions also maintain the structural integrity of the epithelium.

Secretory function

The epithelium can produce an incredibly diverse array of lipid mediators, growth factors, and bronchoconstricting peptides as well as chemokines and cytokines (Table 2). It is beyond the scope of this article to comprehensively review each of them individually. The reader is referred to several excellent review articles specifically dealing with this topic.68–70

Table 2.  Molecules expressed/produced by bronchial epithelial cells. Owing to space limitations we have omitted references for the information given here. However, a com-plete set of references is available from DAK on request
MoleculeStimulus
Lipid mediators
 PGE2Leukotrienes, bradykinin, allergens, O3
 PGF2x, TXB2PAF
 LtC4, LtD4 LtE4ET-1, histamine
 9-HODE, B-HODE
 Cytochrome P450-derived acosanoidsO3, PAF
Cytokines
 IL-1, TNFa
 IL-6, IL-11, LIFViruses, IL-1b, TGFb
 IL-10
Chemokines
 IL-8, GRO-a, GRO-8TNFa, IL-1b, viruses,
 RantesTNFa, allergen, rhinovirus
 Mip-1a
 MCP-1,4TNFa, IL-1b
 Eotaxin-1, -2Rhinovirus
Growth factors
 TGFbEGF, IL-1b, hypoxia
 EGF (family)
 PDGF
 GM-CSF, CSF-1, G-CSFIL-4, IL-13
 IGF, bFGFTGFb, IL-1b
ECM proteins
 Collagen I, IV
 FN, LN, HyaluronanDamage, EGF
 CD44PDGF
 mmp-1, -2, -3TNFa, PMA
 mmp-9, matrilysin (mmp-7)Bacteria
 TIMP-1, -2TGFb
Peptide mediators
 EndothelinEndotoxin, pro-inflammatory cytokines, IL-6, TGFb
Adhesion molecules
 ICAM-1IL-1b, TNFa
 Ep-CAM
 E, N, C-cadherins
Integrins
 a1,2,3,4,5,6,8,9EGF
 b1,4,6,8
Gases
 NO, COIL-1b, Allergen, TNFa, LPS, IFNg
Others
Oxygen Radicals

Arachidonic acid metabolites

The epithelium is a major source of arachidonic acid metabolites which help regulate airway smooth muscle tone, epithelial mucus secretion, neurotransmitter release and inflammation.

Cyclooxygenase, lipoxygenase (LO) and mono-oxygenase are the three primary enzymes respon-sible for eicosanoid synthesis in human airway epithelium.71

The prostaglandins PGE2, PGI2 and small amounts of PGF and TxA2 are all produced in airway epithelium following activation of cyclooxygenase enzymes, with PGE2 being produced in the greatest amounts. PGE2 is generally described as a bronchoprotective mediator due to its inhibitory effects on airway smooth muscle, mucus secretion and nerve activity.72–75 However, PGE2 may also promote a Th-2 bias for intraepithelial dendritic cells, suggesting that it has complex effects within the airways.76

Originally, it was thought that bone-marrow derived cells were the main cells expressing lipoxygenase enzymes. However, 5-, 12- and 15-LO have been localized to basal and ciliated cells of human airway epithelium.77,78 Indeed, the epithelium is an abundant source of 15-HETE and can also produce significant amounts of other lipoxygenase products such as 5-HETE, 12-HETE and di-HETEs.79–82 The function of 15-HETE in the airways is uncertain, but increased amounts have been found in the epithelium of asthmatics and chronic bronchitics.83 Early studies had shown 15-HETE to indirectly cause contraction of airway smooth muscle by stimulating the release of leukotrienes.84

The mono-oxygenase pathway of arachidonic acid metabolism, mediated by NADPH cytochrome p450 reductase, is also present in human airway epithelium. It produces a variety of epoxyeicosatrienoic acids. The functions of these metabolites are unknown, although they can induce smooth muscle relaxation in vitro.85

Nitric oxide

Epithelial-derived nitric oxide (NO) plays a prominent role in cell signalling within the respiratory tract and has been implicated in the pathogenesis of a number of airway diseases.86–88 A variety of lung cells may produce NO from l-arginine through the action of the enzyme nitric oxide synthase (NOS).87 Constitutive isoforms of NOS appear to be involved in the regulation of endothelial and nerve cell functions.88 Expression of the inducible isoform of NOS (iNOS) in the airway epithelium is markedly upregulated after exposure to pro-inflammatory cytokines and oxidants, and for this reason, has been implicated in the pathogenesis of airway inflammation.86,89 Moreover, patients with asthma have a marked increase in exhaled NO,90,91 and also show an increased immunolabelling for iNOS in epithelial biopsies when compared with control subjects. Altered levels of NO production may be important in asthma due to its bronchodilatory effects.92 However, whether NO production is related to the presence of asthma per se or related to an underlying predisposition such as atopy remains unknown.

GROWTH FACTORS

Epidermal growth factor

Among the signals that regulate epithelial restitution, the EGF family of growth factors are particularly important, serving as primary regulators of migration, proliferation and differentiation and enhancing several phases of epithelial repair.93,94 Expression of epidermal growth factor receptor (EGFR) appears to be markedly upregulated in the airways of asthmatics, and appears to be disease related and in morphologically intact cells.95,96 Paradoxically, expression of EGFR does not appear to correlate with proliferation of damaged cells in vivo.96,97 EGF interacts with a specific receptor that is one of a family of four different receptor tyrosine kinases (RTK).98,99 Activation of the intrinsic receptor tyrosine kinase generates an intracellular signal that can proceed down two pathways: (i) phosphorylation and activation of transcription factors leading to expression of new genes and (ii) activation of mitogen activated protein kinase (MAPK) pathways, leading to proliferation and cell migration.100 However, EGFR has also been detected in the nucleus in many tissues, suggesting that EGF may act directly as a transcription factor under some conditions.101 Recent data have shown that transacti-vation  of EGFR by  other  receptors including the IL-4Rα occurs, demonstrating that other cytokines/growth factors including IL-13, exploit the EGFR for optimal signalling.102

The function of EGFR can be positively regulated by integrin-mediated adhesion to the ECM. These interactions are thought to lead to clustering of the receptors, thereby enhancing their signalling efficiency. Binding of RTKs to integrins may also protect them against dephosphorylation and/or prevent their internalization following binding of growth factors.103 The maintenance of active growth factor receptors on the cell surface would lead to sustained signalling. In addition, the ability of integrins to organize the cytoskeleton may control the spatial organization of signalling molecules, facilitating convergence of separate signalling pathways. Thus it appears that cooperative, rather than individual signalling by integrins and RTKs is a critical process responsible for regulating cellular functions such as adhesion, cell-cycle progression and apoptosis.104,105 Using primary cultures of human bronchial epithelial cells obtained by bronchial brushing, we have commenced experiments examining localization (Fig. 3) and cooperative signalling by αvβ5 integrin/EGFR complexes.

Figure 3.

Colocalization of (a) avb5 integrins and (b) epidermal growth factor receptor in primary cultures of human bronchial epithelium.

Transforming growth factor β

The TGFβ family of growth factors are produced by many cells that are activated in the asthmatic response and also appear to control multiple processes involved in wound repair.106 TGFβ signals through a heteromeric complex of Type I (TGFR-I) and Type II (TGFR-II) transmembrane serine/threonine receptor kinases. After ligand binding, TαR-I becomes activated and phosphorylated by the constitutively active TαR-II kinase and propagates the signal downstream to Smad proteins, which play pivotal roles in the intracellular signalling of TGFβ.107–110 Signalling downstream of TGF-αR also involves other signal-transducing pathways including MAPK cascades.109,111 TGFβ is thought essential for epithelial homeostasis by virtue of its antiproliferative and pro-apoptotic activity and through its ability to negatively regulate the actions of EGF.106,112 More recently, Howat and colleagues showed that repairing epithelial cell monolayers produce and activate latent TGFβ1 and TGFβ2, but only TGFβ1 produced at the wound edge increases the speed of epithelial repair.113

Degradative enzymes

In addition to being a source of mediators, the epithelium also produces degradative enzymes. Perhaps the best described is neutral endopeptidase (NEP), responsible for the breakdown of kinins (tachykinins and bradykinin), endothelin, vasoactive intestinal peptide and angiotensins I and II.114,115 Enzymes such as acetylcholinesterase and histamine N-methyltransferase have also been identified in human and animal airway epithelium.116,117

Epithelial damage and repair

Tissue repair is dependent on a structured progression of events that re-establishes integrity of the damaged tissue. The precise mechanisms involved in regeneration of the airway epithelium are controversial.118 Much of what is known about epithelium repair comes either from studies in which the epithelium is injured and the repair processes are followed histologically over time, or from cell culture models. However, it is clear that the airway epithelium has a tremendous capacity to repair itself after injury. In a recent in vivo study, Erjefalt et al. showed that in as little as 15 min after an 800-µm wide wound was made in the tracheal epithelium of guinea pigs, epithelial cells on the damaged margin (including secretory and ciliated cells) began to dedifferentiate, flatten and migrate over the denuded area.119 In this model, proliferation of epithelial cells was not observed until some 30 h after the initial stimulus, and after the denuded area was completely covered by a layer of tight, flattened undifferentiated epithelium. Within 5 days a fully differentiated epithelium was present. At approximately the same time as cells begin to differentiate, plasma exudate is also observed.120–122 This exudate contains fibrin and fibronectin as well as other ECM proteins such as vitronectin. Increased expression of specific integrins, notably αvβ1 and αvβ6 occurs on the basal surface of regenerating epithelial cells after the deposition of fibronectin, suggesting that fibronectin in the plasma exudate may induce integrin expression.123 Indeed, fibronectin in addition to two other matrix proteins, collagen IV and laminin, promote directional migration of bovine tracheal epithelial cells in culture.124–126 Following a lag period, subepithelial fibroblasts appear to become activated and begin proliferating (Fig. 3).120

Recent studies have attempted to develop in vivo models using xenografts, in which human bronchi are wounded and then transplanted subcutaneously into SCID mice.127 Alternatively, human dispersed epithelial cells have been cotransplanted with denuded rat tracheae, also into SCID mice.128 These models have demonstrated (i) differences between cystic fibrosis (CF) and non-CF secretions,129,130 (ii) the profile of integrins expressed during wound repair127 and (iii) that repairing or regenerating epithelium is more susceptible to transfection by viral gene vectors than fully differentiated epithelium.131,132 Importantly, when the epithelium of the xenografts was fully differentiated the proportion of proliferating cells, the integrin profile and the ratio of ciliated columnar cells to goblet cells was virtually identical to that seen in normal airways taken at biopsy or transplant.127

The epithelial–mesenchymal trophic unit

Restitution of damaged epithelium is associated with alterations in cell phenotype as well as disruption of the normal patterns of proliferation and apoptosis, reflecting inflammation and repair processes.63,65 These processes are spatially and temporally controlled by local signals generated by a plethora of growth factors and integrins. Repairing epithelial cells also produce a variety of fibrogenic growth factors and peptide mediators such as TGFβ1 and 2,113 insulin-like growth factor,133 basic fibroblast growth factor, platelet derived growth factor and endothelin, which are all capable of inducing proliferation of subepithelial fibroblasts as well as differentiation into activated myofibroblasts.134 These cells, characterized by expression of contractile proteins such as α-smooth muscle actin (α-SMA), are key cells involved in wound healing, and remodelling, through the release of growth factors that drive autocrine proliferation and deposition of collagen and other ECM proteins as well as paracrine effects on the epithelium, airway smooth muscle and blood vessels.43,135–137

This bi-directional communication between epithelial cells and underlying (myo)fibroblasts closely resembles the epithelial–mesenchymal trophic unit (EMTU) that controls physiological remodelling of the airway during branching morphogenesis.138–140 The possibility that overexpression or inhibition of key elements of this unit would result in dysfunctional repair, has led Holgate and coworkers to propose that the remodelling of the airways in asthma is a result of the EMTU remaining active after birth or becoming reactivated in susceptible asthmatics.141–143

Epithelial cell–mesenchymal transformation

Remodelling in the asthmatic airway is associated with accumulation and activation of myofibroblasts immediately beneath the lamina reticularis of the basement membrane.144 However, the origin of these α-smooth muscle actin positive cells is unknown.145 One hypothesis argues that bone marrow stromal cells are precursor cells for tissue resident fibroblasts.146 A second hypothesis suggests that fibroblasts are derived from a pool of airway smooth muscle cells. Alternatively, epithelial–mesenchymal transition (EMT) may serve as a local source of interstitial fibroblasts.145,147 EMT occurs during early development where there is a seamless plasticity between epithelial and mesenchymal cells and may also occur in some adult tissues during carcinoma cell invasion and metastasis or following wound repair or organ remodelling in response to injury.148,149 EMT has also been observed in cell culture systems and in this environment cells lose polarity, adherence to adjacent cells and the ECM, and gain mesenchymal cell properties such as motility and the expression of α-smooth muscle actin.150 Growth factors, in particular TGFβ, as well as oncogenes and adhesion molecules such as E-cadherin have all been suggested as modulators of EMT.150,151 Whether or not EMT occurs in human airways in the setting of epithelial damage and repair is unknown.

THE EPITHELIUM IN ASTHMA

The paradigm of epithelial damage and desquamation in asthma has been based on findings of exfoliated cells in BAL samples or induced sputum or on histological assessment of tissue sections. Gross areas of denuded airways provided a plausible explanation for the observed hyperreactivity to non-specific agents. However, whether marked desquamation is a pathological feature of asthma is still far from settled.152,153 Indeed, recent studies have provided morphological evidence that epithelial loss observed on biopsy is an artefact of tissue sampling and that the extent of epithelial loss does not correlate with either the magnitude of airway narrowing or the severity of bronchoconstriction.152,153In vivo models of epithelial regeneration following allergen challenges of sensitized animals suggest that epithelial damage is patchy with focal localization of inflammation to the sites of injury.122

Is asthmatic epithelium abnormal?

Despite the controversy regarding epithelial damage and asthma pathophysiology, there can be little doubt that the airway epithelium of asthmatics is abnormal and has increased susceptibility to injury compared to normal epithelium.65,154 In asthma, the epithelium shows evidence of structural damage and mucus-cell hyperplasia and metaplasia. Increased ‘stress’ is often observed in the guise of increased expression of pro-inflammatory transcription factors (NFκB, AP-1) and heat shock proteins. Sampath and colleagues recently described constitutive activation of signal transducer and activator of transcription (STAT)-1 in asthmatic epithelium compared to epithelial cells from healthy controls or chronic bronchitics.155 Similar findings have been recently reported for STAT-6, the signal transducer used by IL-4 and IL-13 receptors.156 In addition to being more suseptible to damage, it is becoming increasingly apparent that normal repair processes are also compromised. Failure of appropriate growth and differentation of airway epithelial cells will cause persistent mucosal injury. If the basement membrane is damaged, epithelial cells and fibroblasts may directly interact with each other.

Expression of EGFR is markedly upregulated in the epithelium of adult asthmatics, especially where basal cells have lost their columnar cell attachments.95,96 This is evidence that injury has occurred in vivo and is not an artefact of the biopsy procedure. Expression of EGFR is also related to disease severity and is not modified by corticosteroid treatment. However, expression of EGFR does not correlate with the proliferative status of repairing epithelial cells in vivo.96 This paradoxical finding may be explained by recent studies that have shown an abnormally high expression of the cyclin dependent kinase (cdk) inhibitor p21waf1 in asthmatic epithelium.157 P21waf1 plays an important role in regulating cell proliferation and apoptosis and is induced by a variety of growth-inhibitory signals such as DNA damage and cell differentiation as well as secreted molecules such as TGFβ. Given that expression of p21waf1 is related to disease severity and is not influenced by corticosteroid therapy, this further suggests that epithelial cell proliferation is abnormal in asthma.

Expression of protease activated receptor (PAR)-2 is also markedly upregulated in the epithelium of asthmatics, and like EGFR, expression of PAR-2 appears refractory to corticosteroid therapy.158 Whether in-creased expression of PAR-2 represents an attempt to modify local inflammatory responses or contributes to asthma pathology is not clear. It has been argued that one of the major functions of epithelium-expressed PAR-2 is bronchoprotection.159 However, PAR-2-deficient mice exhibit delayed inflammatory responses.160 PAR-2 is also expressed on neurons and may mediate neurogenic inflammation, either directly or via the release of tachykinins.161 Activation of PAR-2 on epithelial cells is also associated with IL-6 and IL-8 release.162

Evidence is accumulating that the epithelium of asthmatics is structurally and biochemically abnormal in vivo. For example, asthmatic epithelium releases greater amounts of pro-inflammatory cytokines into BAL, and express elevated levels of activation markers. Importantly, these abnormalities are also seen in vitro.163 Furthermore, asthmatic epithelial cells, but not those from normal subjects, can induce the production of collagen III from cocultured fibroblasts.164 Bayram and colleagues have shown that asthmatic epithelial cells develop increased permeability to O3 and NO2 compared with cells isolated from normal subjects, and also release greater amounts of IL-8 and GM-CSF when stimulated with diesel exhaust particles.163,165,166 Bucchieri and coworkers have also shown that asthmatic epithelial cells are more susceptible than normal cells to oxidant induced apoptosis.167 These differences are unlikely to be due to downstream effects of airway inflammation since they are maintained on a background of serial passaging.

The effect of glucocorticoids on the epithelium in asthma

It is well accepted that the epithelium is an important therapeutic target for the treatment of many airway diseases, including asthma.168,169

Glucocorticoids (GCS) are currently the first line prophylactic therapy for asthma and associated disorders. These drugs are extremely effective anti-inflammatory agents, suppressing the expression of pro-inflammatory cytokines, chemokines and peptide mediators both in vivo170,171 and in vitro.172–174 However, there is conflicting data regarding the effectiveness of these drugs in modulating the structural changes in the remodelled airway wall. For example, these drugs fail to regulate EGF-induced TGFα release or EGFR expression in asthmatic epithelium.175 This may lead to an inappropriate repair response. Indeed, asthmatics taking regular GCS still have substantial epithelial cell damage and loss.

Dorscheid and colleagues showed that GCS induced a time and concentration dependent cell death of primary cultures of normal human epithelial cells as well as in cell lines and there is also in vivo evidence that GCS are associated with epithelial cell apoptosis in asthma.176 This incomplete repair response coupled with enhanced apoptosis leads to a continued production of growth factors and cytokines, perpetuating the cycle of chronic inflammation and remodelling. However, Carayol and coworkers have shown that in epithelial cells derived from GCS-dependent asthmatics, markers of cell survival and proliferation are coexpressed with markers of activation, suggesting that epithelial repair in asthma is associated with persistent activation.177 In contrast to the findings of Dorscheid et al. very few apoptotic cells were identified.

SUMMARY AND CONCLUDING REMARKS

The major function of the respiratory epithelium was once thought to be primarily that of a physical barrier, but recent studies clearly indicate that it is metabolically very active with the capacity to modulate a variety of inflammatory processes through the agency of an array of receptor-mediated events. On activation, it has the capacity to produce a number of pro-inflammatory cytokines, pro-inflammatory or regulatory mediators including arachidonic acid products, nitric oxide, endothelin-1, TGF-β, TNFα, and cytokines such as IL-1, IL-6 and IL-8. As well as upregulating the inflammatory response some of these factors also play roles in fibroblast proliferation, myofibroblast differentiation and collagen production. It has been demonstrated in asthma that increased collagen deposition in the airway walls perpetuates the characteristic airway hyperresponsiveness of asthma. An increase in subepithelial collagen deposition changes the airway mechanics, narrowing the airway lumen and exaggerating the effect of any smooth muscle shortening.

In addition to producing a range of pharmacologically and immunologically active agents, the respiratory epithelium also plays a major role in the recruitment of inflammatory cells through the expression of adhesion molecules, which are upregulated in response to stimuli such as cytokines.

There is mounting evidence to show that asthmatic epithelium is structurally and functionally abnormal. However, the question as to whether the differences observed in asthmatic epithelium are a cause of, or secondary to, the development of the disease remains unanswered. One way to address this question is to carefully examine the ontogeny of the disease in children. Indeed, thickening of the lamina reticularis has been identified in susceptible children several years before the onset of the disease.178 It has been hypothesized that during early life, environmental stimuli interact with the EMTU to initiate the remodelling process. This may also explain the loss of corticosteroid responsiveness of baseline lung function in some children taking part in the CAMP study.179 Molecular genetic studies employing gene array technology may also provide some important answers as well as allow a better understanding of the critical role that the epithelium plays under normal conditions and in diseases such as asthma.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the National Health and Medical Research Council of Australia and the Medical Research Council UK for support of parts of the research presented in this article. We also thank Dr Markus Weichselbaum for provision of the confocal microscopic images.

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