Allergen-induced airway remodelling

In vitro cell cultures

Classical in vitro cell-culture systems have been enormously valuable in delineating some of the functions that lung structural cells might play in the development of airway remodelling. These systems have not specifically examined allergen-induced events but rely on the addition of mediators such as T-helper cell (Th) type 2 cytokines to the cells in culture. Cultures of epithelial cells, fibroblasts and smooth muscle cells using immortalised cell lines established that these cells were capable of more than merely maintaining the structural integrity of the lung. Experiments have determined that these cells are able to respond to, and in many cases secrete, a range of inflammatory mediators, including cytokines and chemokines, as well as profibrotic factors such as transforming growth factor (TGF)-β. However, in order to try and obtain information more relevant to the lung, primary cells have also been isolated from the lung. Normal lung fibroblasts, epithelial cells, smooth muscle cells and vascular endothelial cells can be isolated from surgical specimens or post mortem, and are now available commercially. In addition, it is possible to obtain these cells from allergic as well as nonallergic patients 2, enabling comparisons to be made. In vitro cultures of these cells have enabled researchers to examine the effects of inflammatory or profibrotic mediators on a wide range of functions, including proliferation, mediator secretion, extracellular matrix metabolism, protease secretion and, in some cases, migration. The culmination of these experiments is the recognition that lung resident cells are integral to the development and maintenance of lung inflammation and remodelling.

In vivo models

What is the relationship between airway function and airway remodelling?

Perhaps one of the most urgent questions relating to the development of airway remodelling is that of the functional consequences of the remodelling for the patient. The attempts to make correlations between the development of tissue structural changes and changes in lung function parameters have been controversial and remain the subject of intense discussion and investigation. Changes in airway function include hypersensitivity (a leftward shift in curves for bronchoconstrictor dose–response), hyperreactivity (increased slope of these curves) and a greater maximum degree of induced bronchoconstriction. Longitudinal studies, however, imply that AHR should be considered as an independent risk factor for development of asthma rather than as an outcome itself 43. Multiple studies have determined that changes in airway function are accompanied by inflammatory infiltrates composed of eosinophils, T-cells, monocytes and neutrophils and cytokines characteristic of a classical Th2 response. This implies that AHR is at least partially dependent on inflammatory events 44–46, but the exact contribution of inflammation to airway dysfunction remains ill-defined 47. The observation that AHR persists in patients despite prolonged treatment with anti-inflammatory corticosteroids 48–50 implies that other mechanisms account for a major component of airway dysfunction.

The clinical consequences of airway remodelling remain uncertain but contributions to AHR and fixed airflow obstruction have been suggested. Asthmatic subjects show an accelerated decline in lung function compared with nonasthmatics, and this is more marked in those asthmatics that smoke 51, 52. Research has been hampered by the fact that there are no noninvasive methods of assessing airway remodelling at present, and human airway research is limited to mainly morphological and in vitro experimental work that is constrained further by ethical limitations. Nevertheless, recent bronchoscopic studies have enabled hypotheses to be formed regarding the role of particular mediators and cells in the development of structural changes following allergen challenge 36, 43, 53. In addition, high-resolution computed tomography (HRCT) shows promise in evaluating structure–function relationships in asthma 54. HRCT reveals that an increase in the ratio of bronchial wall thickness to bronchial diameter exists in the large airways in asthma 55. Interestingly, wall thickness as assessed by HRCT correlates with subepithelial fibrosis, as assessed by biopsy 56. In addition, HCRT wall thickness is associated with increased airflow limitation and correlates with asthma duration, as well as traditional indices of asthma severity and control 43. In these computational models the increase in reactivity and maximal response caused by airway thickening is related to one or a combination of mechanisms. Increase in the airway wall thickness internal to the smooth muscle layer can amplify the airway narrowing for a given degree of airway smooth muscle shortening. Increase in the thickness of the adventitial layer has the potential to uncouple the airway smooth muscle layer from the surrounding parenchyma, allowing the smooth muscle to shorten more before being balanced by parenchymal tethering. Increase in the smooth muscle layer could result in greater force development and therefore shortening against the loads that normally attenuate airway muscle shortening 57. Paradoxically, a recent study determined that airway reactivity negatively correlated with airway wall thickness. Airway sensitivity was related to the degree of airway inflammation as determined by sputum eosinophilia, but not to any index of airway wall thickness. It was concluded that airway wall thickening was perhaps protective against excessive airway narrowing in asthma 58. This apparent contradiction of accepted dogma might be explained by the fact that the computational studies undertaken are primarily based on altered geometry and do not fully take into account the potential effect of airway wall thickening on the mechanical properties of the airway. It has been argued that there are several potential explanations to this apparent paradox, all relating to the stiffness of the airways. Deposition of ECM could separate the smooth muscle layer from the parenchyma, thus limiting the ability of the smooth muscle to narrow the airway lumen. Similarly, thickening of the airway wall between the smooth muscle layer and the epithelium might stiffen the airway. In addition, although the smooth muscle layer is increased during chronic asthma, it has been argued that it changes from a mainly contractile phenotype to a secretory phenotype 59. Therefore, the apparent paradoxical relationship between airway geometry and AHR described by Niimi et al. 58 may simply be a function of the mechanical properties of the material deposited within the layers of the airway wall. However, these results suggest that the present author's ideas regarding the relationship between airway remodelling and airway function are simplistic; the mechanical properties of the material thickening the airway wall influence the ultimate change in physiological response, not just the amount 57. It is clear that some of the changes characteristic of airway remodelling occur in other chronic lung diseases and are not specific to asthma.

Investigating the relationship between airway remodelling and changes in airway function in humans is difficult, since endobronchial biopsies do not always show the true extent of changes to the airway wall and indeed serial biopsies from the same patient are largely impractical and unethical. At present, there is a lack of reliable noninvasive predictive markers and insufficient data from longitudinal studies to define the natural history of remodelling from childhood wheezing into adulthood. The development of such noninvasive systems is urgently needed in order to further the analysis of the relationship between airway remodelling and changes in lung function. In the absence of suitable human studies, animal models can attempt to dissect the relationship between lung structural changes and lung function after allergen challenge. The majority of mouse models report some change in lung function after prolonged challenge 25, 60–63, which correlates with airway inflammation and airway remodelling. However, the relationship between airway inflammation, airway remodelling and changes in airway function is still ambiguous. Several of these studies argue that airway structural changes can be uncoupled from AHR. One elegant study compared brief or chronic exposure of mice to allergen and determined that airway dysfunction and remodelling persisted beyond the resolution of immune-mediated inflammatory events 62. Airway responses to methacholine were measured and increases in the maximal inducible bronchoconstriction persisted for ≥8 weeks after cessation of allergen challenge. These functional changes were seen in conjunction with increases in contractile tissue in the airway wall. In contrast, observed increases in airway reactivity (rate of increase in respiratory resistance for a given dose of methacholine) only persisted beyond resolution of allergen-induced inflammation in chronically challenged mice. The authors concluded that sustained airway hyperreactivity was not associated with ongoing Th2 inflammatory markers, such as eosinophilia or IL-13, but that sustained dysfunction occurred as a consequence of airway remodelling rather than these immune-mediated events. One can speculate that the initial development of airway dysfunction is dependent on acute inflammatory responses to allergen challenge, such as recruitment of eosinophils and Th2 cells and production of Th2 cytokines, but that sustained dysfunction is dependent on structural changes to the airways, such as changes in the smooth muscle layer and increased deposition of ECM. Subsequent studies using knockout mice revealed that IL-4 and -13, but not IL-5, are critical for the development of sustained airway remodelling and AHR 64. However, it could be argued that since these mice are completely devoid of the individual cytokines for the duration of the entire protocol, the observed effect may reflect differences in the initial development of remodelling or AHR rather than in the persistence. Studies with neutralising antibodies during different phases of the protocol would determine this.

Intervention studies in mouse models of chronic allergen challenge have revealed some interesting correlations between airway inflammation, development of airway dysfunction and airway remodelling. Several of these models have shown that it is possible to uncouple airway function from either inflammation or airway remodelling. Mice genetically deficient in eosinophils are protected from development of remodelling but, surprisingly, not from changes in airway function 26. These mice develop robust AHR to acute and chronic allergen challenge but fail to develop airway fibrosis or airway smooth muscle hypertrophy after chronic challenge. Another study has shown that airway inflammation and AHR is preserved in mice that lack mast cells, but that allergen-induced subepithelial fibrosis was partially attenuated 65. Therapeutic administration of anti-TGF-β antibody after the onset of eosinophilic inflammation and AHR had no effect on these parameters but did prevent the development of airway remodelling 66. These studies show that it is possible to affect one parameter of chronic allergen challenge without affecting others, and will permit the testing of potential therapeutic treatment regimens on outcomes of the various aspects of chronic allergen challenge. In the future it will be important to consider the success of these therapeutic regimes on facets of chronic allergen challenge, such as airway remodelling rather than just eosinophil recruitment. In addition, it will be important to test potential therapeutic treatments using therapeutic rather than prophylactic regimens.

These developments in animal models will increasingly allow dissections of potential components contributing to airway remodelling and their impact in airway physiology. However, relating these findings to chronic asthma in humans remains a challenge. The use of monoclonal antibody therapy in asthma and allergen challenge models may give some insights. For example, Flood-Page et al. 53 reported reduced subepithelial tenascin staining after anti-IL-5 treatment of asthmatics, but this was not associated with any change in AHR, lung function or asthma symptoms. Chronic intervention and observational studies are required but are difficult to both fund and execute. Of importance here is the question of which parameters of lung function relate to asthma outcomes and can these be related to remodelling? For example, tailoring treatment to AHR 49 or sputum eosinophilia 67 reduced asthma exacerbation rates, suggesting that these are important determinants of morbidity, but it will also be important to determine whether remodelling is related to the development of fixed airflow obstruction, the rate of change of airway calibre or the response to acute challenge (allergen or infection).

Is inflammation necessary for the development of airway remodelling?

Although it is generally held that airway remodelling occurs as a consequence of chronic inflammation induced by repeated allergen challenge, emerging theories challenge this concept. The idea that reactivation of the EMTU is a key feature of induction of airway remodelling has led to the proposal that inflammation and remodelling are parallel rather than sequential events (fig. 3⇓). Communication between the epithelium and the underlying epithelial fibroblast sheet is reminiscent of the processes that drive branching morphogenesis in the foetus, where the epithelium and the mesenchyme function as a trophic unit. Holgate et al. 68 have proposed that the EMTU becomes reactivated during chronic asthma to drive pathological remodelling. This theory may explain the potentially conflicting findings linking inflammatory cells with markers of remodelling, some of which may be consequent upon inflammation while others are not. In addition, the hypothesis might answer some of the controversies regarding inflammation during asthma, such as why the prolonged use of corticosteroids has little or no effect on the natural history of asthma, even if treatment is instigated in early childhood.

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Fig. 3—

Summary of current concepts in the pathogenesis of airway remodelling in allergen-induced asthma. Exposure to environmental insults can lead to airway damage, and individuals with a genetic predisposition to atopy and/or dysregulated or impaired airway repair may go on to develop sustained inflammation and tissue remodelling. Increased activation of the epithelium leads to signalling to inflammatory cells and also activation of the underlying mesenchymal cells (activated epithelial-mesenchymal trophic unit (EMTU)). Both structural and inflammatory cell sources of growth factors, such as those of the transforming growth factor (TGF)-superfamily as well as vascular endothelial growth factor (VEGF) and interleukin (IL)-13, are thought to be important. Progressive structural changes include increased numbers and size of fibroblasts and airway smooth muscle and vascular remodelling together with excessive and dysregulated extracellular matrix (ECM) deposition, the balance of which may lead to a phenotype that is characterised by increases in airway hyperresponsiveness (AHR) or airway obstruction. In turn the pro-inflammatory environment generated by chronic structural cell activation will sustain and propagate the inflammatory response to ongoing environmental insults. RBM: reticular basement membrane; APC: antigen-presenting cell; ADAM: disintegrin and metalloproteinase; Th2: T-helper 2; Ig: immunoglobulin; FGF: fibroblast growth factor; ASM: airway smooth muscle.

The question as to whether remodelling of the airway wall might precede asthma has been investigated by studies of childhood asthma. Data from longitudinal studies indicate that wheezing in early childhood is a marker for subsequent reduced pulmonary function and asthma. The question is whether or not these children have smaller airway diameters that were genetically or pre-natally determined; whether this would predispose them to wheeze in association with insults such as viral infections; and whether atopy subsequently manifests as wheezing and bronchial asthma. Alternatively, pre-clinical bronchial asthma may cause abnormal airway development or remodelling, which leads to reduced airway diameter and then renders pre-school children more prone to wheezing with viral infections as well as after allergen exposure 69. An early paper compared biopsies from 12-yr-old females with bronchial asthma with post mortem samples from lungs taken from similar aged children who died of severe acute asthma. In the living patients there was evidence of airway remodelling, including goblet cell metaplasia and mucus plugging, together with thickening of the bronchial epithelial basement membrane 70. Furthermore, smooth muscle hypertrophy and inflammatory cell infiltration were also apparent. The children who had died of asthma showed more pronounced peribronchiolar eosinophil infiltration in conjunction with focal loss of the bronchial epithelium. Therefore, it seems that there is evidence of lung structural changes in children; this is reinforced by a retrospective study of bronchial biopsies from children with respiratory symptoms that subsequently developed bronchial asthma 71. The histological features of asthma, including eosinophilia and thickening of the lamina reticularis underneath the bronchial basement membrane, were present even in pre-school children prior to the diagnosis of asthma. Even though the precise relationship between airway inflammation and airway remodelling is unclear, there is evidence that airway remodelling occurs in early childhood and that in some cases it pre-dates onset of symptoms. Other studies report that loss of lung function is determined mainly in childhood, but these physiological findings have not been correlated with histological evidence of airway remodelling 72, 73. Perhaps more worryingly, biopsy studies of schoolchildren who remained relatively asymptomatic due to high doses of corticosteroids showed thickening of the lamina reticularis of the basement membrane compared with nonasthmatic children 74. There is also evidence for early bronchial smooth muscle hypertrophy in biopsies from schoolchildren 75, 76. In contrast, a recent study has looked for even earlier evidence of remodelling in pre-school children 77. Examination of the bronchial mucosa of infants aged <2 yrs demonstrates that the eosinophilic inflammation and RBM thickening that are characteristic of asthma in older children and adults are not present in infants with recurrent wheeze and/or cough or in the presence of reversible airflow obstruction. Moreover, there was no correlation with atopy. Further study is required to determine the relationship between airway inflammation and remodelling in children in order to identify whether early intervention will impact on lung function in later life.

This relationship has not been investigated at all in animal models. All of the studies described thus far have been performed on adult mice and the question of influence of early life events on development of airway remodelling with or without inflammation has not been addressed. Furthermore, the question as to whether remodelling precedes airway inflammation or AHR has also not been addressed, probably due to the lack of suitable models. Current models of allergic inflammation and remodelling, including those described previously, require systemic sensitisation with allergen to induce changes to airway pathology 20. Transgenic models in which various Th2 cytokines have been ectopically expressed specifically in the lung have determined that airway remodelling does occur without immune sensitisation and allergen challenge, but the remodelling occurred in conjunction with airway inflammation, most obviously eosinophilia 27, 28. Therefore, it is difficult to determine which came first, the inflammation or the structural changes. Whether similar results would be obtained if the mice were developed in a strain lacking CD4 T-cells remains to be seen. Models which address these specific questions are urgently required, particularly due to the ethical limitations of performing allergen challenge studies or even biopsy studies in children and infants.

Is airway remodelling reversible?

One of the most critical questions regarding chronic changes to the lung is that of reversibility. It is not clear how effective current asthma therapies are in reducing remodelling. This is critical since it has become obvious that remodelling events occur early in disease development and it is likely that by the time patients present to their physician and are diagnosed they already display markers of airway remodelling processes that are similar to those observed in patients with long-standing disease 78. Thus, the true extent of reversibility of changes in airway wall remodelling is unclear, as is the relationship between these airway wall changes and ongoing exposure to allergen. Although studies have reported an improvement in parameters of inflammation and AHR in children undergoing allergen avoidance at high altitude 79, airway remodelling was not included in this study, probably for ethical reasons. One study has reported that airway remodelling is reversible after cessation of exposure to isothiocyanates in sensitised individuals 80. Reversibility is relatively easy to examine in animal models and investigators have approached this by inducing airway inflammation, AHR and remodelling by chronic allergen challenge and then examining the same parameters at different time points after cessation of allergen exposure. Leigh et al. 62 looked at lungs up to 8 weeks after final allergen exposure and determined that chronic challenge elicited aspects of sustained airway dysfunction and remodelling that persisted beyond the resolution of acute inflammatory events. Similarly, Kumar et al. 81 observed that persistence of airway wall eosinophilia and mucous cell hyperplasia was dependent on continued allergen exposure, whereas subepithelial fibrosis and epithelial hypertrophy were not. The current authors determined that peribronchiolar fibrosis and airway smooth muscle changes persisted for ≥4 weeks following chronic allergen challenge, but inflammation and AHR resolved 82. A recent report has described a different model in which allergic inflammation and remodelling are elicited after chronic intranasal instillation of house dust mite extract without prior sensitisation 21. This protocol resulted in classical Th2-type eosinophilic inflammation concomitant with AHR to methacholine and lung structural changes assessed by goblet cell hyperplasia, collagen deposition and peribronchiolar accumulation of contractile tissue. Interestingly, cessation of house dust mite exposure led to resolution of eosinophilic inflammation within 2 weeks but no resolution of remodelling changes and only partial resolution of AHR 21. The question of reversibility has not been investigated as yet in larger animal models.

These mouse models all allude to the fact that although the inflammatory aspects of allergen challenge are reversible, the lung structural changes are not. However, all of these studies have relied on cessation of allergen challenge to determine reversibility but complete allergen avoidance is not a viable option for many asthmatics. Therefore, it is important to consider whether airway remodelling might be reversible with therapy. Such studies have been undertaken in patients but, unfortunately, the majority of the available evidence regarding regression of airway remodelling in response to classical asthma treatment is contradictory. Reversal of subepithelial fibrosis in allergic asthmatics after prolonged therapy with high-dose corticosteroids has been reported 49, 83, 84 but this is not a universal finding 48, 85, 86. Taken together, these studies indicate that airway inflammation, basement membrane thickening and AHR are interrelated and can be improved by corticosteroids but that this occurs over different time scales and with a different dose–response relationship 87. Unfortunately, it does not seem that corticosteroids are totally protective against long-term airway damage; proof of their effectiveness needs to be collected after further investigation and other therapeutic strategies explored. A recent study with a nonbactericidal dose of a macrolide antibiotic promoted reversal of airflow limitation in chronic lung allograft rejection 88 indicating that, ultimately, it might be possible to reverse airway remodelling.

Again, mouse models have been used to investigate the effect of common asthma therapies on the development of airway remodelling. Corticosteroids have been used at the onset of allergen challenge and fluticasone was shown to be effective in preventing increases in airway laminin but, interestingly, had no effect on AHR 89. In contrast, the current authors have shown that administration of budesonide is effective in resolving established airway eosinophilia and AHR and prevents development of airway remodelling 90. Therapy directed at leukotrienes has also shown that airway remodelling can be prevented 91. However, none of these studies has used a treatment regimen designed to reverse established airway remodelling. In all of the studies, therapeutic agents were administered at the start of allergen challenge 89, 91 or at a time of established inflammation, but before remodelling was observed 90. In the future it will be important to use these models to determine the efficacy of standard asthma treatments in reducing established airway remodelling.

Is airway remodelling genetically determined?

Asthma is a complex inflammatory disorder in which development is thought to be affected by both genetic and environmental considerations. It is commonly held that asthma is caused by multiple interacting genes, some having protective effects and others contributing to disease pathogenesis, but with each gene having its own tendency to work with, and be influenced by, the environment. Therefore, the complex nature of the disease coupled with the substantial locus heterogeneity and environmental influence has made it difficult to uncover the genetic factors that underlie the disorder 92. Linkage analysis uses polymorphic markers throughout the genome to determine whether certain markers segregate in the affected family members, indicating that they might be located near one or more genes that cause the disease under study. Numerous studies have examined the association between asthma or its related traits and genetic variations in candidate genes 92. Most of these studies have utilised common clinical manifestations of the disease, such as AHR, elevated IgE and atopy, in order to look for evidence of genetic linkage. However, there is accumulating evidence that airway remodelling is also genetically linked. Genetic mapping studies have revealed that chromosome 5q31-q33 is of particular interest since it contains the cytokine gene cluster including IL-4, -5, -9 and -13, as well as other growth factors and receptors implicated in asthma pathology 93. It is of interest that transgenic models developed using tissue-specific promoters to drive lung expression of IL-9 and -13 in particular result in airway remodelling as well as the other common features of the allergic syndrome 27, 28. Linkage disequilibrium studies identified multiple polymorphisms in the ADAM33 gene that are associated with asthma 94. Haplotypes comprised of polymorphisms within the ADAM33 gene were significantly associated with asthma in the case–control samples, as well as in family based association tests. ADAM33 is a member of the ADAM subgroup of the zinc-dependent metalloproteinase superfamily and is a very plausible candidate gene for tissue remodelling in asthma. The gene has been found to be expressed on fibroblasts and smooth muscle cells. In addition, polymorphic variations in ADAM33 predict impaired early life functions 95 and single nucleotide polymorphisms in ADAM33 have been linked with accelerated decline in lung function in the general population 96. Although its precise function is not fully understood, it has similarities to other ADAM genes that possess proteolytic activity and this suggests that ADAM 33 may play a role in airway remodelling. The development of a mutant mouse either overexpressing ADAM33 or genetically deficient in ADAM33 is eagerly awaited and might yield some clues as to the role of this molecule in airway remodelling.

The contribution of genes and environment to the development of airway remodelling highlights a major drawback of almost all animal models. Most strains of mice are inbred and are housed in clean environments that may be beneficial when considering the role of a particular pathway, cell or molecule in isolation. However, it is important to consider that innate tissue responses, as well as primary immune responses, are strain-dependent. Differences in the strain of mouse used for particular experiments have resulted in some contradictory results in the past 97 and are an important factor when comparing similar studies. One study has investigated the effect of mouse strain on the development of airway remodelling by assessing the responses of commonly used strains of mice to airway allergen challenge in the absence of systemic sensitisation 98. Using this protocol, persistent AHR, eosinophilic inflammation, collagen deposition and airway wall thickening were all found to be strain-dependent. Changes were minimal in the commonly used Balb/c strain and absent in C57BL/6 and C3H/HeJ mice. Interestingly, the strain that gave consistent remodelling responses to airway intranasal challenge (rather than inhalational) was the A/J mouse, which has been shown to exhibit marked methacholine-induced AHR that is independent of allergen sensitisation and challenge 99. Quantitative locus analysis of the AHR revealed that this naïve AHR is conferred by a major locus on A/J chromosome 2 and is an interacting locus on chromosome 6 100. Interestingly, chromosome 2 also contains the mouse orthologue of ADAM33.