Cellular processes occurring during migration. The transition from a stationary state to a migratory phenotype involves several distinct mechanisms, Rho-like GTPases regulate the majority of these processes and the individual GTPases most directly linked to each are: (1.) Rac-dependent actin (blue) polymerisation leads to lamellipodium extension at the leading edge of the cell, (2.) new ECM adhesions are formed at the lamellipodium via focal contacts (red), also regulated by Rac, (3.) cell body contraction is dependent on actomyosin contraction (blue arrows) and is dependent on Rho activity, (4.) tail detachment is possibly affected by GTPase activity but the protease calpain is important for degradation of focal adhesions at the rear of migrating cells, (5.) cadherin-mediated cell-cell adhesions (green) are dependent on Rho and Rac activity and are often downregulated during migration. MMP-mediated ECM degradation is necessary for migration, even in cells migrating over a basement membrane.
1.1. Clinical assessment of asthma
Pulmonary function tests (PFTs/spirometry) are routinely used clinically to diagnose asthma. Forced expiratory volume in one second (FEV1) is a measure of airflow and is influenced largely by the resistance in the airways. During an “asthma attack”, the airways narrow owing to mucus hypersecretion and smooth muscle contraction, which results in a sharp increase in airway resistance and reduction in FEV1. In the clinics, this can be reproduced by exposing patients with asthma to non-specific smooth muscle contractile agonists such as methacholine. A fall in FEV1 at a relatively low dose of methacholine indicates airway hyperresponsiveness (AHR). In asthmatic patients this drop in FEV1 can be reversed by inhaled bronchodilators such as β2-agonists (e.g. salbutamol), which relaxes airway smooth muscles. AHR is often used as a diagnostic criterion for asthma.
Asthma is increasingly considered a syndrome, with diverse overlapping pathologies and phenotypes contributing to significant heterogeneity in clinical manifestation, disease progression, and treatment response . Severe uncontrolled asthmatics make up approximately 5-10% of the population of asthmatics, yet they consume around 50% of the treatment resources . Spirometric tests are incapable of exposing the underlying pathologies or phenotypes which combine within an individual to produce the asthmatic disease. In contrast specific biomarkers may enable more accurate sub-phenotyping of disease by indicating the pathology within an individual, and assist clinicians to better tailor the type and/or dose of therapy. In this way biomarkers have the potential to guide more effective personalized treatment regimes in asthma.
1.2. The causes of asthma
Several studies have used lung biopsies taken from non-asthmatic patients and those who died from fatal asthma to show the airways of asthmatics possess characteristic structural differences, shown in Figure 1. These include but are not limited to; increased eosinophil numbers in asthmatics, and areas of focal bronchial epithelial denudation . The contents of the bronchial lumen can also be examined using the bronchoalveolar lavage (BAL) technique, in which warm saline is instilled into a segmental bronchus of a patient and removed. BAL fluid-based studies confirmed the previous research that inflammatory cell influx and epithelial damage occur in the airways of asthmatics  . It is a point of some debate as to whether the epithelial damage is caused by inflammation, is a cause of inflammation, or whether epithelial damage and airway inflammation are concurrent but unrelated processes. We shall discuss the potential role of the epithelium and epithelial repair in asthma in more detail later.
1.3. Inflammation in asthma
The rationale for treating severe asthmatics with steroids is centred on the assumption that the disease is driven by uncontrolled airway inflammation. The classical paradigm of the acute inflammatory response in allergic asthma is briefly; inhaled allergenic antigens are captured by professional antigen-presenting dendritic cells, macrophages, or epithelial cells. Antigens presented by these cells are recognized by T-cells which proliferate and differentiate. Primed Th2 cells bind to B cells and release cytokines including triggering maturation of antigen-specific B cell populations into plasma cells. Plasma B cells release antigen-specific IgE that binds to IgE-receptors on mast cells in the airways, causing release of histamine-containing granules. Extracellular histamine released from mast cells binds to membrane receptors on airway smooth muscle cells triggering a rise in intracellular calcium, muscle contraction and airway narrowing. Although this paradigm has gained acceptance as an important mechanism in asthma, there is significant variation in specific cell types involved between individuals. In the setting of chronic asthma, eosinophils are thought to play a major role in maintaining airway inflammation in the long-term. Neutrophils are occasionally the predominate inflammatory cell present in the airways of chronic asthmatics, suggesting multiple underlying pathologies of disease. Neutrophilic vs. eosinophilic asthma are clinically indistinguishable by pulmonary function testing, but such pathological variation may have important implications regarding treatment as patients with “neutrophilic asthma” tend to be relatively unresponsive to steroid treatment . A rapid and definitive method for determining the nature of airways inflammation in a diagnosed asthmatic is therefore needed to guide the clinician’s selection of therapy.
1.4. IL-13 and asthma
Interleukin-13 (IL-13) is a pleiotropic Th2 cytokine secreted from a variety of inflammatory cells and structural cells, including the airway epithelium. IL-13 is upregulated in the airways of patients with allergic asthma exposed to allergen; segmental allergen challenge of atopic asthmatics triggered an increase of IL-13 protein and transcript in bronchoalveolar lavage, and the source was identified as mononuclear inflammatory cells . Experiments in small mammals and
1.5. Current treatments for asthma
Asthma is rarely fatal but often leads to significant patient morbidity. For over thirty years the mainstay therapy for asthma has been inhaled β2-agonists (βA), which relax the airway smooth muscles, causing bronchodilation, and inhaled glucocorticosteroids (i.e. steroids), which ameliorate airway inflammation and reduce the risk of asthma exacerbations. Steroids are anti-inflammatory drugs but also have many unwanted side-effects, particularly at high doses, including; weight-gain due to changes in body metabolism, and a reduction in bone mineral density, potentially leading to brittle bones. Therefore the decision to treat a patient with steroids should not be taken lightly. Inhaled β2-agonists (s) are the most effective bronchodilators available and their main mechanism of action is via the relaxation of airway smooth muscle . βAs interact with G-protein-coupled cell surface β2-adrenoceptors (β2AR) on bronchial smooth muscle cells and activate adenyl cyclase, causing a rise in intracellular cAMP, this results in relaxation of the smooth muscle and bronchodilation. Long term βA treatment can result in a loss of response which shows variation between individuals. Desensitisation due to chronic βA treatment is caused by a down-regulation of receptor transcription, secondary to a reduced activity of the CREB (cyclic AMP response element binding protein) transcription factor . The β2-adrenoceptor gene possesses at least three possible GRE binding sites for the activated GCR, and GCs have been shown to increase β2AR transcription two-fold in human lung preparations
Monoclonal antibodies have gained recent credence as a potential means to target therapy against specific receptors and signalling pathways. Omalizumab, a biologic agent that binds to IgE, has shown some promise as a means of controlling some difficult to treat asthma patients who are poorly controlled with standard inhaled steroid and β-agonist combination therapy (reviewed in ). Interleukin-5 (IL-5) is an important cytokine for eosinophil differentiation, maturation, migration into the circulation and survival. Various studies have implicated the eosinophil as being the primary cell responsible for airway hyperresponsiveness in asthma, thus anti-IL-5 antibodies have been developed (such as mepolizumab) with the intention of reducing airways hyperresponsiveness by preventing eosinophil recruitment and survival in the airway. Clinical trials have demonstrated such inhaled anti-IL-5 therapy does indeed reduce airway eosinophilia, but does not have a clinically-beneficial effect on lung function [18, 19]. Considering the highly important role IL-13 is thought to play in asthma, it is unsurprising that therapies are being developed to specifically target IL-13. Lebrikizumab, an anti-IL-13 monoclonal antibody, has just completed phase II clinical trials in which patients with poorly-controlled asthma were given the drug subcutaneously at 4 weekly intervals for 6 months . This study found anti-IL-13 therapy did significantly improve lung function, but only in a sub-group of asthma patients who exhibited high serum periostin levels, a surrogate marker of high airway IL-13 . These studies demonstrate highly-targeted therapies may be useful in asthma, but each individual drug will probably only prove effective in a specific sub-group of asthmatic patients. The potential for such personalised medicine highlights the importance for accurate phenotyping of asthmatic individuals, discussed in the biomarkers section of this chapter (Section 5).
1.6. Carbohydrates and asthma
Carbohydrate decoration of proteins has been associated with asthmatic disease. Two case-control studies have investigated the role of the histoblood group antigens; H(O), A, B, or AB in asthma susceptibility. They found in human subjects the O-secretor mucin glycan (H-antigen) phenotype is associated with an increased susceptibility to recurrent asthma exacerbations .
2. Normal airway structure
From the level of the bronchioles upwards, the stratified epithelium lining the airway lumen rests on a basal lamina or ‘true’ basement membrane, a specialised ‘mat’ of extracellular matrix proteins. Below the basal lamina is a layer of collagenous matrix, termed the reticular basement membrane, in which fibroblasts are embedded in a sporadic arrangement. Farther below the fibroblastic layer are arranged bundles of smooth muscle myocytes, blood vessels and afferent nerve endings.
2.1. Structural changes to the airway in asthma
Although asthma is considered an inflammatory disease, there are many structural changes in the airways. Figure 1. shows cross-sections through the large airways of two patients, one normal and one severe asthmatic. The Movat’s pentachrome stain clearly highlights the various architectural remodeling events occurring in the asthmatic airway. Obstruction of the airways from excessive mucus production is a common finding in severe asthmatics, blue staining in the epithelium and the lumen demonstrates mucous cell hyperplasia, with excessive mucus deposition into the airway. Under the asthmatic epithelium a thicker basement membrane is present which contains several different extracellular matrix (ECM) factors (including tenascin-C) compared to normals . Deeper into the airway, red-stained muscle mass is increased in the asthmatic owing to a combination of smooth muscle hypertrophy and hyperplasia (reviewed in ). The airways of severe asthmatics also demonstrate a fibrotic response with increased connective tissue deposition and fibroblast and myofibroblast proliferation. Opinion is divided as to whether inflammation precedes airway remodeling, or whether the two occur in parallel (reviewed in [15, 24, 25]). Evidence tends to favour the latter because; firstly, remodeling occurs very early on in the disease, and in some cases in the absence of inflammation , secondly, there is only a weak link between airway inflammation and symptoms , and thirdly, epidemiological data demonstrate steroids do not work in all asthmatics . In reality it is likely effective therapies will need to target both airway inflammation and remodeling.
2.2. Airway epithelial structure and function
The bronchial epithelium lines the inner wall of the respiratory tract in a continuous layer; it forms the interface between inspired air and the internal milieu as well as being the primary target for inhaled respiratory drugs. In the larger airways and down the respiratory tree to the level of the bronchioles the epithelium is at least two cells thick (reviewed. in ). Although there are at least eight morphologically and functionally distinct epithelial cell types present in the respiratory tract, they can be classified into three main groups; basal, ciliated columnar, and secretory columnar (reviewed in ).
Basal epithelial cells are pyramidal-shaped cells with a low cytoplasm to nucleus ratio, although found throughout the airway, their contribution to epithelial volume decreases with airway size (reviewed in ). Basal cells are anchored to the underlying basement membrane and to other cells via specialised adhesion structures discussed below (Figure 2.) [4, 30]. Historically the basal cell has been considered to be the stem cell of the bronchial epithelium (reviewed in ), however animal studies suggest there may be alternative stem cells located in the airway epithelium, including secretory cells, and cells located at specialised niches including the broncho-alveolar junction and the neuroepithelial bodies.
Columnar ciliated cells
Columnar epithelial cells lie above the basal cell layer and line the airway lumen, in direct contact with the inspired air. Ciliated cells are terminally differentiated columnar cells and are the most common cell-type in the bronchial epithelium, accounting for around 50% of all epithelial cells and 80% of terminally-differentiated apical cells in the human trachea , reviewed in . Their main function is the removal of particulate matter by means of the mucociliary pathway. Ciliated cells arise from either a basal or secretory cell pre-cursor and possess around 300 cilia per cell, directly beneath the cilia are observed numerous mitochondria and a dense microtubule system, reflecting the high metabolic demands of particle clearance  .
Secretory cells are also present at the apical surface, comprising 15 to 20% of the normal tracheobronchial epithelium . In the larger airways the mucus-secreting goblet cell represents the predominant secretory cell and is the main source of airway mucus. These cells are characterised by membrane-bound electron-lucent acidic-mucin granules which are released into the airway lumen to trap inhaled particles and pathogens, prior to removal from the respiratory tract by coordinated cilia beating. Serous cells and Clara cells are relatively rare secretory cells in the larger airways present in large airways, but Clara cells are the main secretory epithelial cell type of the bronchioles, particularly the most distal bronchioles (reviewed in ). Several studies in rodents indicate the progenitor cell of the bronchial epithelium is not the basal cell but it is in fact the non-ciliated, secretory columnar cell. Both goblet and clara cells have been observed to undergo de-differentiation and proliferation during
2.3. Airway epithelial carbohydrate expression
The airway epithelial surface is covered by a layer of airway surface liquid (ASL), mainly of epithelial origin. A layer of mucins form a mucous layer that overlies the thin watery periciliary layer (PCL). The predominant mucins in the airways are MUC5AC and MUC5B secreted by goblet cells and submucosal glands respectively. Mucins are highly glycosylated proteins; 70-80% of their molecular weight are carbohydrate, and the structural characteristics of the mucous layer depends on interaction between the carbohydrate side-chains of mucin proteins. Studies of airway mucins in disease, demonstrate mucins are both oversulphated and hyper-sialylated in patients with cystic fibrosis and in chronic bronchitis patients with significant infections . Pseudomonas aeruginosa, the pathogen responsible for the majority of morbidity and mortality in cystic fibrosis patients, uses the sialylated and sulphated Lewis-x determinants as attachment sites. Thus mucin glycosylation patterns may significantly affect infection in the airway. Specific lectin-binding assays have been used to characterise carbohydrate expression patterns in differentiated human airway epithelium . Out of 38 lectin probes tested, seven bound specifically to basal cells, seven to columnar cells, and three specifically labelled secretory cells. The 1HAEo- and 16HBE14o- airway epithelial cell lines were also probed with the same lectin panel, revealing identical carbohydrate expression to the basal epithelial cells in the human tissue . Thus the different cell types in the pseudostratified epithelium of the airways, express specific patterns of carbohydrates, possibly reflecting their different functions.
2.4. Airway epithelial functions
The primary function of the bronchial epithelium is to serve as a continuous physical barrier and as such it functions as part of the non-specific immune system, preventing pollutants, bacteria, viruses, allergens, and other potentially noxious substances transferring from inspired air into the underlying mesenchyme. This defence is mediated by several adhesive mechanisms which have been elucidated through transmission electron microscopy and immunohistochemical studies of bronchial epithelial biopsies   (Figure 2.). Belt-like tight junctions (zonula occludens) seal the lateral apices of columnar cells, regulating para-cellular transport, whilst columnar cells adhere to each other via classical E-cadherin mediated adherens junctions . Desmosomal cadherins mediate strong cell-cell adhesion and desmosomes are present between basal cells and particularly at the junction of basal and columnar cell layers . Basal cells are in turn anchored to the basement membrane via specialised integrin-mediated cell-ECM junctions. Integrin heterodimers function by dynamically linking the contractile machinery of the cell’s cytoskeletal network to the external matrix at specialised sites termed focal adhesions or focal contacts. The airway epithelium expresses several integrin heterodimers, including the α6β4 integrin which binds to laminin-5 in the basement membrane forming a hemidesmosome anchor with the ECM.
The bronchial epithelium is more than a passive barrier; it performs a variety of roles allowing it to function as a dynamic regulator of the innate immune system . The mucociliary pathway represents the principle mechanism of particle clearance in the airway. Inhaled particles and pathogens (bacteria, viruses) are trapped in mucus prior to removal from the respiratory tract by coordinated cilia beating. In addition to producing mucus, secretory cells are the source of a variety of mediators capable of having direct effects on inhaled noxious agents; including anti-oxidants and a variety of anti-bacterial agents; lactoferrin, lysozyme, β-defensins and also opsonins, components of the complement system that coat bacteria to facilitate phagocytosis by macrophages (reviewed in ). Various immunoglobin isotypes are secreted onto mucosal surfaces where they act in a protective manner by opsonising bacteria and other pathogens, rendering them relatively harmless and activating phagocytosis and digestion by tissue macrophages. Allergen-specific IgA is secreted by activated B lymphocytes (plasma cells) in the sub-mucosa, dimeric IgA (dIgA) selectively binds to the polymeric Ig receptor (pIgR) expressed on the basal surface of basal airway epithelial cells. The pIgR-IgA complex is subsequently internalised and is transported to the apical surface via the endosomal pathway ready for secretion into the airway lumen. Dimeric secretory IgA (sIgA) is released into the airway lumen as a 1:1 combination of dIgA and a portion of the pIgR known as secretory component (SC), which protects the IgA from digestion by proteases on the mucosal surface [40, 41]. SC is a highly glycosylated protein containing 15% N-linked carbohydrate. Glycosylated SC binds IL-8, inhibiting IL-8-mediated neutrophil chemotaxis and transendothelial migration. This interaction is dependent on SC glycosylation state as de-glycosylation with peptide N-glycosidase F abolishes the SC-IL-8 complex .
3. Epithelial injury and repair
All epithelial tissues are regularly damaged as a consequence of exposure to environmental insults. Rapid repair following injury is crucial for restoring adequate barrier function, with subsequent cellular differentiation required to regenerate normal epithelial structure and function. The repair processes in all epithelial types have common elements producing an orderly progression of events including; cell spreading at the margins of the wound, cell migration into the wound, cell proliferation and finally re-differentiation (Figure 3).
Proliferation is also an intrinsic component of epithelial repair. In the aforementioned hamster tracheal injury studies, mitosis rates at 12h post-injury are low (0.4%) but by 24h a wave of proliferation occurs primarily in secretory cells, resulting in a multi-layered epidermoid metaplasia . At 48h post-wounding proliferation rates decrease (although mitosis is still significantly above basal levels), from this time point onwards the upper-most cells of the metaplastic epidermis start to slough off, resulting in a gradual loss of the metaplastic phenotype, with subsequent regeneration of a functional mucociliated phenotype achieved by apical cell re-differentiation. In small wounds normal mucociliary structure is restored by 120h but some persistent metaplasia persists in larger wounds through 168h post-wounding . Although both secretory and basal cells are involved in epithelial repair, secretory cells play a dominant role in these experiments, consistently demonstrating higher mitosis rates than any other epithelial cell type [35-38].
3.1. Airway epithelial wound repair is compromised in asthmatics
The patho-physiological changes observed in the asthmatic airway may be due to reactivation of the epithelial-mesenchymal trophic unit (EMTU), triggered by an abnormal epithelium that is held in a repair phenotype .
3.2. Mechanisms of cellular migration
Many disorders characterized by impaired re-epithelialization, are not a result of inadequate proliferation, but are due to impaired cell migration over the denuded site [47-49]. Cell migration requires a coordinated, highly complex series of events including; changes to the cell cytoskeleton, modification of the surrounding ECM and modulation of adhesions to ECM substrate and to other cells. The majority of these processes are regulated by the Rho-like family of small GTPases including, RhoA, B, C, E, Rac1, 2, 3 and Cdc42 (Figure 3.) (reviewed in [50, 51]) that function by acting as “molecular switches”, cycling between an
inactive GDP-bound state and an active GTP-bound form at the plasma membrane . GTPases are able to regulate the transmission of signals from cell surface receptors, such as integrin-mediated cell-ECM adhesion, or growth factor ligation, to downstream intracellular signalling pathways, influencing the cell cytoskeleton, gene transcription, and the cell cycle (reviewed in ). Rho induces formation of stress fibres, integrin-mediated focal adhesions, and actomyosin-mediated cell body contraction , Rac-dependent actin polymerisation leads to lamellipodium extension and membrane ruffles at the leading edge of the cell , and Cdc42 induces formation of microspikes/filopodia .
3.3. The effect of intracellular protein glycosylation on epithelial repair
The activity of the Rho-GTPase family is highly regulated by glycosylation.
3.4. Role of cell surface glycoconjugates in epithelial repair
Many proteins essential for normal cell physiology including adhesion molecules and cell surface receptors are glycosylated [65,66]. Alteration in the glycosylation pattern of many glycoproteins leads to changes in their function. It has been shown that impaired glycosylation of receptors often leads to abnormal intracellular trafficking, ligand binding and downstream signal transduction ability [67-70]. Tsuda
Lectins are naturally occurring proteins that can be isolated from a variety of plants and animals. Each lectin binds to a specific sugar moiety. As such, lectins are exquisitely selective tools to identify or block specific glycoconjugate motifs and have been extensively employed to study the role of cell-surface sugars and complex carbohydrates in cellular function [77, 78]. A variety of approaches have been employed to unravel the role(s) of carbohydrates in the multiple steps of the repair process. Some studies have investigated the expression pattern of different carbohydrates after injury using lectins as probes. Using three methods for localizing or quantifying lectin-binding sites Gipson
In a previous study, Dorscheid
It has been shown previously by Donaldson
In a study by Trinkaus-Randall
These studies clearly demonstrate the critical role of carbohydrates in the process of epithelial repair. However, carbohydrates must either modify a protein or lipid to regulate its function or participate in binding to a specific receptor to effect the desired action. As such, the biological role of glycans can be broadly divided into two groups. One group depends on the structural and modulatory properties of glycans and the other relies on specific recognition of glycan structures (generally receptor proteins or lectins).
3.5. Galectins, annexins and epithelial injury – repair
Galectins and annexins are two of possibly many families of proteins with carbohydrate binding capabilities. Although their role in epithelial repair remains unclear, recent studies highlight their importance in these processes. Galectins are a specific family of lectins with an affinity for β-galactose-containing oligosaccharides that have no enzymatic activity. To date, as many as 14 galectins have been identified, and they have been found in non-mammalian species such as birds, amphibians, fish, worms, sponges and fungi. The binding affinities of galectins are often greater for the oligosaccharides than the monosaccharide galactose. The expression of galectins is conserved however; their expression is often tissue specific and developmentally regulated. Different cells express a unique complement of galectins. Of the 14 galectins, eight have been identified in the nucleus and cytoplasm and participate in specific cellular functions. Nine galectins have been found extracellularly. Their presentation on the cell surface remains a mystery since they lack a signal sequence for secretion via the classical secretory pathway for membrane proteins.
Studies have shown that galectin-1 and galectin-3 have the potential to mediate cell-matrix interactions [92-95]. Their ubiquitous expression makes it difficult to understand the mechanism of their involvement in this process. In corneal epithelial cells, galectin-3 and galectin-7 have been characterized as mediators of epithelial cell migration, an essential component of epithelial wound repair [96, 97]. Galectin immunostaining in healing wounds is more intense relative to normal epithelium, primarily at the leading edge and at areas of cell-matrix interactions . Furthermore, wound repair was impaired in gal-3-/- mice in the wound model systems of either alkali burn wounds that leave the corneal stroma intact or laser ablation wounds that result in damage to the underlying basement membrane. This impaired repair was not seen in gal-1-/- mice. The authors propose that this effect is mediated by the lectin domain binding complementary glycoconjugates in the ECM and cell surface molecules resulting in enhanced cell-cell and cell-matrix interactions . Similar work has demonstrated that galectin-7 also plays a role in corneal epithelial repair . Although obvious differences exist between tissues, galectins may play a similar role in other epithelial organs and epithelial repair such as the lung and intestinal tract.
Annexins are a family of proteins that bind acidic phospholipids in a calcium dependant manner. Similar to galectins, annexins are largely cytoplasmic, however; several annexins have been detected on the cell surface of a variety of cells. Like galectins, annexins also lack a signal sequence for the cell surface presentation . Annexins are proteins that have been associated with many cellular functions however their role remains poorly understood. Recent work has suggested that annexins possess carbohydrate binding abilities. This was first observed with annexin A4 when it was shown that it binds a variety of glycans . Recent studies have suggested that annexin II (AII) is also capable of binding carbohydrates however this work is still in preliminary stages. Previously, the binding of tissue-type plasminogen activator (t-PA) was shown to bind HepG2 cells, however, following enzymatic removal of α-fucose residues on t-PA, this binding was dramatically decreased . Subsequently, AII has been shown to be a cell surface receptor for t-PA . The importance of α-fucose on t-PA may be a result of the carbohydrate binding of AII. Patchell
As a cell surface molecule, it has been suggested that AII is the human cytomegalovirus receptor . Following the initial interaction with heparan sulfate proteoglycan, the virus particle is primed for membrane fusion and infection [106, 107]. As a membrane fusion protein, AII could potentially act as a receptor to other enveloped viruses such as respiratory syncytial virus (RSV). Recently, AII has been shown to bind RSV and was characterized as a potential cell surface protein that can promote RSV infection . These findings along with data that has identified AII on the surface of airway epithelial cells provide strong evidence AII may be a cell fusion receptor allowing RSV infection of the respiratory tract. Viruses are a common source of lung injury, specifically the epithelium. Epithelial cells, as a protective mechanism, become highly apoptotic following virus infection to prevent further infection and viral persistence . Viral infectivity of several enveloped viruses is dependent upon carbohydrate interactions between the viral proteins and their receptors. The end result is injury to the epithelium that requires repair. Altered glycosylation then may either increase the resulting injury from RSV infection or impair the AII coordinated repair.
3.6. MMPs in asthma, glycosylation and repair
Matrix-metalloproteinases (MMPs) are a family of zinc-dependant endopeptidases that digest all components of the extracellular matrix (ECM) and many non-ECM substrates including growth factors, cytokines, and their receptors [110, 111]. Several MMPs are increased in airway tissue and broncho-alveolar lavage (BAL) fluid of asthmatics including MMP-1, -2, -3, -7, -8 and -9 [112, 113], suggesting a link between increased airway proteolytic activity and the asthma phenotype.
3.7. IgCAMs in asthma, repair and the effects of IL-13
Immunoglobulin-cell adhesion molecules (Ig-CAMs) are a diverse family of cell adhesion molecules characterised by the presence of one or more copies of a structure known as the Ig fold . These receptors are expressed on a variety of cell types and are vital for many different biological processes, including neural development and immune system function . In the adult, Ig-CAMs regulate the recruitment and subsequent activation of circulating lymphocytes, studies show they are also expressed in the epithelium and vasculature of the bronchial mucosa and levels of two Ig-CAMs, ICAM-1 and CD44 (hyaluronic acid receptor), are increased in the epithelium of asthmatics [130, 131]. Recent data also suggests a third Ig-CAM, CD147 (EMMPRIN – Extracellular matrix Metalloprotease Inducer) is increased in the epithelium of patients with chronic obstructive pulmonary disease (COPD) . CD147 is over-expressed by metastatic cells of the skin, bladder, breast, and lung . In cancer CD147 stimulates synthesis of various MMPs via homophilic interaction of CD147 oligomers on adjacent cells . CD147 is potentially involved in various signaling pathways relevant to remodeling in airways disease, for an in depth review see . Studies have shown CD147 is endogenously expressed at low levels in the basal cells of various stratified epithelia including the airway. Airway epithelial CD147 expression is increased in murine models of lung injury  and in several inflammatory airway diseases including pulmonary fibrosis  and interstitial pneumonias, where increased CD147 is associated with MMP-2, -7 (matrilysin) and -9 levels in the lungs of patients . Recent studies have demonstrated CD147 is increased in the airway epithelium and BAL of patients with COPD, and signaling via CD147 regulates MMP-9 expression in cultured bronchial epithelial cells . CD147 is potentially important in asthma; in a mouse model of allergic asthma, inhalation of a CD147-neutralizing monoclonal antibody reduced ovalbumin (OVA)-induced airway mucin production and airway hyperreactivity, whilst partially inhibiting the recruitment of eosinophils and effector CD4+ T cells into lung tissue . The authors attributed the reduced airways inflammation to an inhibition of the interaction between CD147 on inflammatory cells with cyclophilin chemokines. The reduction in OVA-induced mucous production suggests CD147 inhibition directly affects the epithelium.
MMP activity is dependent upon the close spatial juxtaposition of enzyme and substrate, therefore targeted MMP localization is a further mechanism of regulation. Specific MMP-substrate interactions can be facilitated and regulated at the cell surface via association of MMPs with chaperone molecules. CD44 is strongly upregulated in injured asthmatic airways and at the leading edge of repairing bronchial epithelial cells [43, 140], yet its function during airway epithelial repair is unknown. Studies suggest CD44 may co-ordinate MMP-substrate interactions at the plasma membrane by functioning as a docking molecule. In repairing reproductive epithelium, the v3 isoform of CD44 (CD44v3) forms a signaling complex with MMP-7 which is presented at the cell surface in close proximity to a potential substrate, pro-Heparin-Binding Epidermal-like Growth Factor (pro-HB-EGF). MMP-7 cleaves pro-HB-EGF into an active soluble form which triggers recruitment and activation of the Erb-B4 receptor (a member of the EGFR family) . The EGF pathway is essential for efficient repair of the bronchial epithelium , and a similar mechanism of targeted MMP-7 presentation may occur during lung epithelial repair. Carbohydrate modification of proteins is essential for the creation of such micro-environments at the cell surface; in this case the interaction of MMP-7 (an non-glycosylated protease) with CD44 is dependent on the presence of branched glycosylated side-chains on the extracellular portion of the CD44 molecule. It is logical to extrapolate that a deficiency in the post-translational modification of CD44 could lead to a lack of MMP-7 binding, a reduction in HB-EGF activation, and a consequent inhibition of epithelial repair.
3.8. How carbohydrates mediate cell-cell interaction and migration
The glycans attached to matrix molecules such as collagens and proteoglycans are important for the maintenance of tissue structure, porosity, and integrity. Such molecules also contain binding sites for specific types of glycans, which in turn help with the overall organization of the matrix. Glycans are also involved in the proper folding of newly synthesized polypeptides in the endoplasmic recticulum and /or in the subsequent maintenance of protein solubility and conformation . In this manner, altered glycosylation of matrix proteins may change adhesion properties and the potential of cell to migrate. This is due to either an altered conformation of the extracellular matrix of a change in the carbohydrate ligands available to bind.
Glycosylation of growth factor receptors has tremendous effects on the receptor function. Several studies have focused on the role of EGF and its family of receptors on epithelial repair [24, 142]. EGF, a well known mitogen for epithelial cells, has been used in to stimulate epithelial wound healing in guinea pigs as well as human airway cell monolayers and differentiated cells
The specificity of carbohydrate interactions allows for the high degree of selectivity found within the cell. Whether it is in regulating receptor activation, cell-cell or cell-matrix interactions or cell migration, defects in these processes may result in abnormalities and altered phenotypes. Their understanding will provide new avenues for research and therapies.
4. IL-13 structure
Interleukin-13 (IL-13) is a type I cytokine, comprised of four short α-helical hydrophobic bundles joined by two disulfide bonds . The cDNA of IL-13 has a single open reading frame that encodes 132 amino acids, including a 20-amino acid signal sequence that is cleaved from the mature secreted protein . IL-13 has four predicted N-linked glycosylation sites and can either exist as a 17kDa glycoprotein or 10-12kDa unglycosylated protein . IL-13 has been demonstrated to be secreted predominantly as the 10-12kDa unglycosylated protein . In a study conducted by Liang
The glycosylation state of IL-13 can also affect its activity and potency to induce certain effector functions such as the up-regulation of low affinity IgE receptor, CD23 on the surface of monocytes. A functional study conducted by Vladich
4.1. IL-13 function
IL-13 is a pleiotropic cytokine that exerts its effect on a wide variety of cell types. IL-13 has a variety of functions which are critical to immune homeostasis and repair, but when uncontrolled contribute to the asthma phenotype. IL-13 is predominantly produced by T helper type 2 (Th2) cells  and to a lesser extent by mast cells, basophils, and eosinophils [117, 118]. IL-13 is also produced by a variety of non-hematopoietic cells including airway epithelial cells  and bronchial smooth muscle cells . As a Th2 cytokine, IL-13 shares numerous overlapping characteristics with IL-4, where both cytokines promote B cell proliferation, IgE class switching and synthesis in B cells, and induce surface expression of antigens such as CD23 and major histocompatibility complex (MHC) class II . These similarities between IL-13 and IL-4 function can be explained by the fact that the cytokines share a common receptor subunit, IL-4 receptor α subunit (IL-4Rα). However unlike IL-4, IL-13 does not exert direct effects on T cells and cannot elicit Th2 differentiation in naïve T cells; IL-13 is instead involved in mediating downstream Th2-effector functions . In monocytes and macrophages, IL-13 can inhibit the production of various pro-inflammatory mediators such as prostaglandins, reactive oxygen species, IL-1, and TNF-α [113, 122]. IL-13 can also enhance the expression of several members of the integrin family that play important roles in adhesion, including CD11b, CD11c, CD18, and CD29 as well as induce surface expression of CD23 and MHC class II in monocytes and macrophages . Furthermore, IL-13 has been reported to have direct effects on eosinophils, including promoting eosinophil survival, activation, and recruitment [123, 124].
IL-13 has important functions on non-hematopoietic cells, including endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells. In endothelial cells IL-13 is a potent inducer of vascular cell adhesion molecule 1 (VCAM-1), which plays a role in eosinophil recruitment . IL-13 has been shown to enhance proliferation and cholinergic-induced contractions of smooth muscle cells
4.2. IL-13Rα1 structure and function
IL-13 signals via a receptor system that consists of IL-13 receptor α1 (IL-13Rα1)/IL-4 receptor α subunit (IL-4Rα) and IL-13 receptor α2 (IL-13Rα2). Both IL-13Rα1 and IL-13Rα2 belong to the type I cytokine receptor family, which possess several definitive features, including a W-S-X-W-S motif, four conserved cysteine residues, fibronectin type II modules in the extracellular domain and proline-rich box regions in the intracellular domain . The two receptors share 33% homology and 21% identity at the amino acid level  and their respective genes have both been mapped to the X chromosome . Kinetic analysis have revealed that IL-13Rα1 and IL-13Rα2 both bind specifically only to IL-13 and not IL-4, while IL-13 alone has no measurable affinity for IL-4Rα .
IL-13Rα1 is widely expressed on both hematopoietic and nonhematopoietic cells except human and mouse T cells and mouse B cells . It is a 65-70kDa glycosylated trans-membrane receptor protein with 10 predicted N-glycosylation sites in the extracellular domain . The cDNA for human IL-13Rα1 encodes a 427-amino acid sequence, including a 60-amino acid intracellular domain and 21-amino acid signal sequence . A mutational analysis demonstrated that Leu319 and Tyr321 in the cytokine receptor homology module (CRH) of human IL-13Rα1 are critical residues for binding to IL-13 . Interestingly, these residues are in close proximity to the predicted glycosylation sites of IL-13Rα1, suggesting that the glycosylation state of IL-13Rα1 could potentially influence receptor-ligand interactions.
IL-13Rα1 binds IL-13 with low affinity by itself but binds IL-13 with high affinity when heterodimerized with IL-4Rα to form a functional signalling receptor . This signalling complex is known as the type II IL-4/IL-13 receptor and it also serves as an alternate receptor for IL-4. IL-13 is known to primarily signal through IL-13Rα1/IL-4Rα by first binding to IL-13Rα1 chain with low affinity and then heterodimerizing with IL-4Rα to become a stable, high affinity signalling complex . The formation of IL-13Rα1/IL-4Rα results in the activation of the Jak kinases, Jak1 and Tyk2, followed by the recruitment of the transcription factor, signal transducer and activator of transcription 6 (STAT6) to the receptor. STAT6 is then phosphorylated and forms functional dimers that translocate to the nucleus to bind specific canonic DNA elements and initiate transcription of downstream genes . Some examples of downstream targets of IL-13Rα1 activation include eotaxin , MUC5AC , and, arginase I which is an enzyme important in the development of airway hyperreactivity .
4.3. IL-13Rα2 structure and function
IL-13Rα2 is a 56kDa glycoprotein that is expressed by a variety of cell types including monocytes, airway epithelial cells, fibroblasts, and keratinocytes [144, 145]. The cDNA of human IL-13Rα2 encodes a 380-amino acid protein with a 26-amino acid signal sequence and a 17-amino acid intracellular domain . IL-13Rα2 contains four predicted glycosylation sites  and the glycosylation state of IL-13Rα2 has been demonstrated to be important in its interactions with IL-13 . IL-13Rα2 exists in three cellular compartments: on the cell surface as a single trans-membrane receptor, in the cytosol within large intracellular pools, and in the extracellular space as a soluble form . In fibroblasts and airway epithelial cells, IL-13Rα2 has been demonstrated to be predominantly localized to intracellular pools that can be rapidly mobilized to the membrane upon stimulation by IL-13 and IL-4 .
IL-13Rα2 has been considered for a long time as a decoy receptor which does not directly contribute to IL-13 signaling, but serves as a negative regulator to terminate IL-13 responses by directly binding to IL-13. The notion that this receptor had no signaling function arose from the fact that it has a short cytoplasmic tail that is missing two known signalling motifs  and does not bind JAKs or STATs . IL-13Rα2 has also been shown to be internalized quickly upon IL-13 binding . Unlike IL-13Rα1, IL-13Rα2 alone has very high affinity for IL-13  and this is further increased when IL-13Rα2 exists as a soluble form . N-linked glycosylation in the extracellular domain of IL-13Rα2 has shown to be necessary for optimal IL-13 binding, demonstrating the importance of glycosylation in IL-13 signalling .
Recent investigations have suggested that IL-13Rα2 acts as a signaling receptor not merely as a decoy receptor. Dienger
4.4. IL-13 genetic and clinical linkage to asthma
The gene encoding IL-13 consists of four exons and three introns and is located on chromosome 5q31 . The chromosomal region 5q31 also contains genes for other molecules associated with asthma such as IL-4, IL-3, IL-9 and GM-CSF . Previous studies have demonstrated strong associations between asthma and several single nucleotide polymorphisms (SNP) in the IL-13 gene, including +2043G>A and -1111C>T [156, 157]. +2043G>A is within the IL-13 coding sequence and causes a positively charged arginine (R) to be substituted by a neutral glutamine (Q) at position 130, forming the IL-13 R130Q variant . Position 130 is in the α-D segment of the IL-13 molecule, where it has been demonstrated to interact with IL-13Rα1 . Compared to wildtype IL-13, the R130Q variant has previously been reported to have lower affinity for soluble IL-13Rα2 due to slower association rates with the receptor . IL-13R130Q is also more active compared to wildtype IL-13 in inducing STAT-6 activation and CD23 expression in monocytes . The enhanced activity of IL-13R130Q may be explained by its decreased affinity to IL-13Rα2.
4.5. Roles of IL-13 in airway epithelial repair
IL-13 has been demonstrated to be critical in mediating normal airway epithelial wound repair. In an
5. The use of biomarkers in asthma diagnosis and therapeutic intervention
Asthma is increasingly recognised as a heterogeneous syndrome with multiple patient phenotypes reflecting the varied underlying pathologies present in each individual. Pulmonary function testing has severe limitations; spirometry can identify a broad spectrum of asthmatics, but it is incapable of discerning sub-types of disease and therefore which individuals will respond to specific treatment regimes. Tissue biopsies and inflammatory cell counting in induced sputum, are accepted measures of determining airway inflammation, however both techniques are invasive, expensive and difficult to standardize, making them unsuitable for routine clinical use. Biomarkers hold the promise of being able to rapidly and specifically diagnose and monitor various sub-types of asthma in a non-invasive manner. The advantages and disadvantages of current and potential future biomarkers of airway inflammation for the diagnosis and monitoring of asthma in the clinic are reviewed in .
5.1. “Invasive” airway biomarkers
5.2. Exhaled biomarkers
Less invasive means of obtaining airway biomarkers are desirable, particularly as diagnostic or disease monitoring tools in pediatric patients. The composition of exhaled breath is correlated to various disease states. This has been exploited by cancer researchers who have pioneered the use of gas sensor arrays, or “electronic noses”, to aid the diagnosis of lung cancer . As asthma is also a disease of the airways, it follows that the composition of exhaled breath will also be altered in the disease. The advent of so-called “breathomics” promises to revolutionize the way clinicians diagnose and treat asthmatics.
5.3. Non-exhaled biomarkers
One recent study used nuclear magnetic resonance spectroscopy (1H-NMR) to measure levels of 70 metabolites in the urine of children with and without asthma. NMR is an attractive method for urinary biomarker examination as it is able to provide qualitative and quantitative data on multiple compounds in a complex biofluid, without requiring significant pre-treatment of the sample. Urine was collected from control children without asthma (C), with stable asthma in the outpatient department (AO), and in children with unstable disease hospitalized for an asthma exacerbation (AED). NMR examination of urinary metabolites showed a 94% success rate in identifying AO children versus C, and a similar success rate was seen when diagnosing AED versus AO asthma . This study suggests the measurement of urinary metabolites is a potentially valuable technique to help clinicians diagnose and monitor asthma in children.
6. Closing remarks
In addition to forming a barrier against inhaled toxins, pathogens and allergens, the airway epithelium performs numerous innate immune and transport functions necessary for airway health and homeostasis. Damage to the epithelial layer is a common occurrence, thus complex repair mechanisms have evolved to rapidly restore the epithelial barrier, with subsequent regeneration of the fully functional differentiated tissue. In asthmatic airways, these epithelial repair mechanisms are compromised.
The post-translational modification of proteins by glycosylation is capable of drastically affecting function via changes to; sub-cellular localisation and secretion, enzyme substrate binding, receptor-ligand binding, protein stability and degradation amongst others. Many of the proteins involved in the complex repair mechanisms defined in the airway epithelium are dependent on correct glycosylation in order to function correctly. There is accumulating evidence to suggest glycosylation is altered in airway diseases such as asthma. In addition, an important cytokine in asthma, IL-13, has a significant effect on the glycosylation state of the epithelial lining of the airway, and IL-13 signalling itself is affected by glycosylation of its receptors. Thus altered glycosylation of the airway epithelium, will have significant effects on the repair and regeneration of a functional airway epithelial barrier.
Asthma is a complex syndrome, individuals with asthma can be classified into a number of sub-phenotypes who will respond to different types of therapies. In order to facilitate the accurate diagnosis of asthmatics the development of specific bio-markers is required. Considerable research is being conducted into the use of non-invasive exhaled, urinary, or blood-borne markers of asthmatic disease to aid diagnosis in adults and children. To date the majority of biomarker research has concentrated on the identification of proteins linked to disease, rather than identifying differences in post-translational modifications. As glycosylation plays a fundamental role in regulating protein function, it is highly likely that future biomarkers for asthma will need focus on identifying alterations to the carbohydrate structures of proteins, particularly those involved in airway epithelial repair.