AIRWAY INFLAMMATION AND REMODELLING IN ASTHMA CAUSE AND EFFECT?

Stephen T. Holgate and Donna E. Davies. Respiratory Cell and Molecular Biology Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton, SO16 6YD, UK

Introduction:
Since the seminal description of asthma as a disease of reversible airway obstruction by Salter in 1859, therapy has been targeted to relax smooth muscle including 2 agonists and xanthines. The discovery that oral and then inhaled corticosteroids could prevent episodic bronchoconstriction and reduce bronchial hyperresponsiveness in parallel with resolution of mucosal inflammation provided the basis for considering asthma as a chronic inflammatory disease. Analysis of mucosal biopsies and cells recovered by lavage has revealed polarisation of the CD4+ T cell repertoire to Th-2 like T cells that exhibit co-ordinate secretion of cytokines encoded in a cluster on chromosome 5q31-33, IL-3, IL-4, IL-5, IL-9, IL-13 and GM-CSF [1]. These cytokines have the capacity to support the mast cell, basophil and eosinophil and isotype switching of B cells to IgE. In the respiratory mucosa, mast cells, basophils and eosinophils are themselves sources of Th-2 cytokines and, under certain circumstances, can induce local B cell IgE production. The demonstration that corticosteroids can inhibit production of these cytokines and induces eosinophil and mast cell apoptosis provides an explanation for the therapeutic efficacy of this drug class.

Increasingly it is being recognised that chronic asthma is accompanied by striking changes in the structure and function of the formed elements of the lower airways, including epithelial metaplasia to one enriched in mucus secreting goblet cells, increased matrix deposited in the submucosa, increased smooth muscle and proliferation of airway blood vessels and nerves. Based on these findings, a linear model for the development of chronic asthma has been proposed that sequentially involves aeroallergen sensitisation, Th-2 inflammation that becomes persistent leading to structural and functional airway wall remodelling (Figure 1). A variable

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interaction between mediators released from inflamed airways such as cysteinyl leukotrienes, prostanoids, proteolytic enzymes and kinins and the remodelled airway leads to hyperresponsiveness, variable airflow obstruction and asthma symptoms.

Figure 1: The traditional paradigm for asthma pathogenesis in which airway remodelling follows a sequential path involving atopy and persistent Th-2 inflammation.

Difficulties with the Role of Atopy in a Linear Model for Asthma:

Atopy is one of the strongest risk factors associated with asthma. However one difficulty in trying to explain asthma purely in terms of lower respiratory allergy is the observation that, while up to 50% of a population may exhibit atopy, less than 10% develop persistent asthma, and in those who become sensitised to aeroallergens, the level of early life exposure is not associated with the development of asthma (2). A pivotal role for eosinophils in the inflammatory response of asthma is also being challenged, since studies with an anti-IL-5 blocking monoclonal antibody (3) and rhIL-12 (5) have failed to reveal efficacy despite markedly reducing circulating and airway eosinophil numbers. Thus, while being associated with asthma, atopy and airway eosinophilia would not seem to be critical requirements for disease expression. This proposal is supported by genetic studies have also demonstrated that atopy and bronchial hyperresponsiveness (BHR) have different patterns of inheritance (5). These findings imply that locally operating factors play an important role in predisposing individuals to asthma and provide an explanation for epidemiological evidence that identifies pollutant exposure (6), diet (7) and respiratory virus infection (8) as other important disease risk factors.

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While corticosteroids are considered the mainstay controller therapy for asthma, in a significant proportion of patients the clinical response is incomplete, necessitating the use of supplementary drugs such as inhaled long acting 2 agonists to relieve symptoms of BHR. Morphometry has revealed that thickening of asthmatic airways accounts for a large component of BHR and excessive airway narrowing that is observed in established disease (9). This remodelling response has also been linked to the progressive decline in pulmonary function observed in asthma in population studies (10) and the development of corticosteroid refractory severe chronic asthma with fixed airflow obstruction and gas trapping behind occluded airways and impaired gas exchange. Although remodelling has been considered to be secondary to longstanding inflammation, a recent biopsy study has identified tissue remodelling as an early and consistent component of childhood asthma with fibroblast proliferation and collagen deposition in the subepithelial lamina reticularis being of greater diagnostic significance than tissue eosinophilia (11). Furthermore, biopsy studies in young children have shown tissue restructuring up to four years before the onset of symptoms (12). Early life development of corticosteroid-insensitive airway remodelling would also explain the outcome of the recent Childhood Asthma Management Program Research Group (CAMP) study in 5-11 year old children. This showed that the initial beneficial effect of an inhaled corticosteroid on the postbronchodilator improvement in airway function observed during the first year of treatment was lost over the following 3 years (13). These studies strongly suggest that remodelling processes begin early in the development of asthma and occur in parallel with, or may be obligatory for, the establishment of persistent inflammation.

A New Paradigm for Asthma Pathogenesis:

Even though asthma has a strong genetic basis, the increase in prevalence of this disease over the last 30 years has occurred in too short a time for new genetic changes to be responsible. This leads to the proposal that environmental changes have uncovered a pre-existing susceptibility within the population (1). This is supported by limited investigations in primates that have shown that intermittent exposure to ozone in the presence of allergen sensitisation creates a phenotype resembling chronic asthma (14) and by epidemiological studies identifying environmental risk factors such as diets low in anti-oxidants, intra- and extra-uterine exposure to cigarette smoke, exposure to environmental pollutants (e.g. diesel particles, NOx and ozone) and
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repeated respiratory virus exposure (15, 16, 17). Recognising that environmental agents impact on the bronchial epithelium, the protective layer of cells that line the airways, any difference in the ability of the asthmatic epithelium to withstand environmental insults would provide a plausible, locally acting gene by environment interaction.

In the following sections, we propose a new paradigm for asthma pathogenesis in which exaggerated inflammation and remodelling in the airways are a consequence of abnormal injury and repair responses arising from the susceptibility of the bronchial epithelium to components of the inhaled environment. As illustrated in (Figure 2), this modifies the function of the epithelium and its ability to communicate with the underlying mesenchymal cells to provide the appropriate microenvironment to promote tissue remodelling and to sustain persistent inflammatory responses characteristic of chronic asthma. By placing atopy in parallel with the altered tissue response, this provides an explanation for the failure of a significant proportion of atopic individuals to develop asthma. However, by functionally interacting with the altered tissue responses, the atopic predisposition serves to magnify or prolong events in this sequence.

Figure 2: Parallel model for asthma pathogenesis in which an inherited or acquired epithelial susceptibility to environmental agents leads to induction of stress/injury and repair responses. Growth arrest and prolonged repair enhances cell-cell communication within the EMTU leading to myofibroblast activation and propagation of remodelling responses into the submucosa. At each level Th-2 cytokines are able to interact with the EMTU to enhance or amplify these responses.

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Epithelial Injury and Repair in Asthma:

The normal differentiated bronchial epithelium is a stratified structure consisting of a columnar layer comprising ciliated and secretory cells supported by basal cells. This polarised structure is the physical barrier that protects the internal milieu of the lungs from inhaled pollutants, infectious agents and other particulate matter. The bronchial epithelium is actively engaged in defence of the airways by secreting mucus and cytoprotective molecules that trap and inactivate inhaled components which are then removed through ciliary beat activity. It also responds to environmental stimuli by signalling to, and interacting with, cells of the innate and adaptive immune systems through secretion of cytokines and chemokines and expression of adhesion molecules such as ICAM-1 and CD40 (18). This interaction enables the epithelium to work in conjunction with the immune system to provide a mechanism whereby extra protection can be

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recruited when the natural epithelial barrier is compromised and, once recruited, the immune and inflammatory cells can themselves promote tissue repair by removing cell debris and providing a transient supply of locally acting growth factors.

In asthma, the epithelium shows evidence of activation linked to structural damage and goblet cell metaplasia. Epithelial stress is seen in the form of widespread activation of the transcription factors nuclear factor-kappa B (NF-B) (19, 20) , activator proteins (e.g. AP-1) (21) and signal transducer and activation of transcription-1 (STAT-1) (22) and by the increased expression of heat shock proteins (23) and the cyclin-dependent kinase inhibitor, p21waf (24). Consistent with an injured phenotype, the asthmatic epithelium is an important source of autacoid mediators, chemokines and growth factors (18). that sustain ongoing inflammation. It has been proposed that epithelial damage is artefactual (25), however, our findings of enhanced expression of the epidermal growth factor receptor (EGFR, HER1, c-erbB1) (26) and the epithelial isoform of CD44 (27) indicate that injury has occurred in vivo. We have found that EGFR expression in asthma increases with disease severity and is evident throughout the epithelium suggesting that stress and damage is widespread (26). Although the extent of injury may reflect damage by immune cell products, the fact that EGFR expression persists in the face of corticosteroid treatment (26) suggests that environmental stimuli make a primary contribution to epithelial stress and injury in asthma.

Although it is difficult to determine experimentally in vivo whether there are any intrinsic differences in the susceptibility of the asthmatic bronchial epithelium to environmental insults, we have recently shown that cultures of asthmatic bronchial epithelial cells grown in vitro are more susceptible than normal to oxidant-induced apoptosis (28), while Bayram et al have demonstrated that bronchial epithelial cells from mild atopic asthmatics release more IL-8 and GM-CSF than non-asthmatic individuals when stimulated with DEPs (29). In being preserved through several generations in vitro, these differences are unlikely to be a secondary effect of airway inflammation. Since epidemiological studies have identified multiple interacting risk factors for asthma, including diets low in anti-oxidants (7) and inhalant pollutants (eg. particulate matter (PM10) and ozone) (16), we propose that the effect of environmental stimuli on such a susceptible epithelium provides a plausible triggering mechanism for the induction of epithelial

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activation and damage in asthma. Once initiated, the resulting inflammatory cell influx will cause secondary damage through production of endogenous reactive oxygen. Thus, it is proposed that, as a result of activation by an environmental trigger(s), the susceptible asthmatic epithelium creates a microenvironment that supports chronic cycles of injury and inflammation.

In addition to being more susceptible to injury, evidence suggests that the repair processes are also compromised in asthma. For example, the disease-related increase in epithelial EGFR in asthma is paradoxical because it is not matched by increased proliferation to replace columnar cells that have been shed (24). The cell cycle is regulated by the cyclin/cyclin dependent kinase (CDK) system that controls passage from G1 into S phase and from G2 into mitosis. The CDK inhibitor, p21waf, is a negative regulator of G1 cyclins and can be induced by stress and injury and the anti-proliferative transforming growth factors, (TGF-_ 1 & TGF-_ 2) whose levels are elevated in asthma (30). Since p21waf is strongly expressed by the bronchial epithelium in severe asthma (24), this may underlie the lack of a proliferative response in asthma and, as a consequence, prolong the period of epithelial repair.

The Epithelial Mesenchymal Trophic Unit and Airway Remodelling:

Recent evidence suggests that epithelial damage and the ensuing repair responses orchestrate airway remodelling in asthma by activating myofibroblasts that lie directly under the epithelial layer in the lamina reticularis (31). This signalling between the epithelium and myofibroblasts involves the provision of growth factors that support the growth and survival of mesenchymal cells that are likely to contribute to the corticosteroid unresponsive component of asthma. This proposal is supported by in vitro studies in which injury to epithelial monolayers results in increased release of fibroproliferative and profibrogenic growth factors including fibroblast growth factor (FGF-2), insulin growth factor (IGF-1), platelet derived growth factor (PDGF), endothelin, (ET-1) and TGF-_ 2 (32). Furthermore, slowing epithelial repair with an EGFR selective inhibitor augments release of TGF-_ 2 (26) that plays a key role in promoting transformation of fibroblasts into myofibroblasts. In vivo, EGFR over expression in asthmatic bronchial epithelium is insensitive to the action of corticosteroids and is positively correlated with the thickness of the lamina reticularis (26) linking epithelial injury to underlying remodelling. In mild-moderate asthma, inhaled corticosteroids reduce airway inflammation and levels of IGF-1, but provide
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minimal improvement in BHR and have no effect on TGF- levels (33). As corticosteroid treatment reduced inflammation, persistently high TGF- in BAL fluid most likely derive from the injured and repairing epithelium and associated matrix turnover rather than from eosinophils. Since both epithelial EGFR expression (26) and TGF- production (33) are refractory to corticosteroids, we propose that the combined effects of these signalling pathways on the EMTU promote remodelling and explain the incomplete resolution of lung function with inhaled corticosteroids observed in chronic asthma.

While myofibroblasts are recognised as key effector cells in tissue fibrosis through their enhanced ability to synthesise interstitial collagens, those from asthmatic bronchial biopsies also release ET-1 and vascular endothelial growth factor (VEGF) (34) which are potent mitogens for smooth muscle and vascular endothelial cells respectively. In this way, remodelling responses initiated at the cell surface are propagated and amplified through the submucosa via the subepithelial myofibroblasts.

Communication between the epithelium and the subepithelial fibroblast sheath is reminiscent of the processes that drive physiological remodelling of the airways during embryogenesis where the epithelium and mesenchyme act as a 'trophic unit' to regulate airway growth and branching (35). Consequently, we propose that the 'epithelial-mesenchymal trophic unit' (EMTU) is reactivated in asthma to drive pathological remodelling of the airways (36). In subjects with asymptomatic BHR, longitudinal studies have shown that those who progress to asthma show parallel changes in inflammation and remodelling (37). Thickening of the lamina reticularis in bronchial biopsies from young children is also present several years before asthma becomes clinically manifest (12). During lung development, epithelial and mesenchymal growth is in part regulated by the balance of EGF and TGF- signalling as we suggest occurs in chronic asthma. In susceptible individuals, we propose that environmental factors interact with the EMTU in early life to initiate structural changes in the airways (Figure 3) which may account for the decrease in lung function observed in young children who are susceptible to early wheezing (38) and for the loss of corticosteroid responsiveness on baseline lung function observed in the CAMP study (13). This is supported by the studies in non-human primates where intermittent exposure to ozone in the presence of allergen creates a phenotype resembling chronic asthma

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(14). Therefore, we hypothesise that bronchial epithelial susceptibility either precedes or occurs in parallel with factors predisposing to Th-2 mediated inflammation and is an absolute requirement to establish the micro-environment for inflammation to become persistent in the airways and for remodelling to occur.

Figure 3: It is proposed that the early life origins of asthma involve developmentally regulated genes that control branching morphogenesis and the structure of the airways and their interactions with the maternal and external environment.

The Interaction between IL-4 and IL-13 and the EMTU:

Th-2 type inflammation of the airways is a characteristic feature of asthma, irrespective of atopy. In transgenic mice, expression of IL-13 or IL-4 transgenes in the bronchial epithelium leads to many of the inflammatory responses characteristic of asthma, but submucosal remodelling is only evident in the case of IL-13 (39, 40) . Using fibroblasts derived from asthmatic mucosal biopsies, we have shown that IL-13 is able to induce myofibroblast transformation but it is two

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orders of magnitude less potent than TGF- and is equipotent with IL-4 in this effect (34). However, since IL-13 causes a corticosteroid-insensitive increase in release of TGF-2 from bronchial epithelial cells, we postulate that IL-13 mediated submucosal remodelling is initiated largely through the bronchial epithelium (34). However, in human epithelial cells, IL-4 is as effective as IL-13 in promoting TGF- release, raising the possibility of an important species difference in epithelial IL-4 and IL-13 function.

While the remodelling effects of IL-4 and IL-13 can be attributed to epithelial activation, these cytokines also have direct proinflammatory effects on both epithelial cells and fibroblasts. Cultures of bronchial epithelial cells respond to IL-4 and IL-13 with increased STAT-6 phosphorylation accompanied by enhanced GM-CSF and IL-8 production which is further augmented by enzymatically active extracts of house dust mite, Dermatophagoides pteronyssinus, (41, 42). We have also found enhanced release of eotaxin from asthmatic fibroblasts (34) which may help explain the accumulation of eosinophils beneath the lamina reticularis in asthma. Thus, by interacting with the EMTU, IL-4 and IL-13 have the potential to augment ongoing inflammation and remodelling responses.

Concluding Remarks:
The existence of parallel pathways leading to inflammation and remodelling can account for the variable nature of this chronic and relapsing disease. It would also provide a plausible explanation for the inability of conventional anti-inflammatory therapies to adequately treat chronic persistent disease, which is characterised by BHR and airway wall remodelling and for the failure of corticosteroids to prevent disease progression when given in childhood. As such, our new paradigm of asthma pathogenesis offers a new perspective on the development of new treatment modalities targeting those corticosteroid-unresponsive pathways that operate locally in the airway microenvironment.

References:
1. Holgate, S.T. J Allergy Clin Immunol 1999:: 104: 1139-1146.

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2. Lau, S., Illi, S., Sommerfeld, C., Niggemann, B., Bergmann, R., von Mutius, E.,Wahn, U. Lancet 2000; 356:: 1392-1397. 3. Leckie, M.J., Brinke, A.T., Khan, J., Diamant, Z., O'Connor, B., Walls, C.M., Mathur, A.K., Cowley, H.C., Chung, K.F., Djukanovic, R., Hansel, T.T., Holgate, S.T., Sterk, P.J., Barnes, P.J. Lancet 2000; 356:: 2144-48. 4. Bryan, S.A., Kanabar, V., Matti, S., Leckie, M.J., Khan, J., Rolfe, L., Renzetti, L., Rames, A., Bock, J.A., Boyce, M.J., Hansel, T.T., Holgate, S.T., O'Connor, B.J.,Barnes, P.J. The Lancet 2000; 356:: 2149-53. 5. Skadhuage, L.R., Christensen, K., Kyvik, K.O., Sigsgaard, T. Eur Respir J 1999; 13:: 8-14. 6. Rahman, I., MacNee, W. Eur Respir J 2000; 16:: 534-554. 7. Seaton,A., Devereux,G. Pediatr.Allergy Immunol 2000; 11 Suppl 13:: 37-40. 8. Johnston,S.L. Pediatr.Pulmonol.Suppl 1999; 18:: 141-143. 9. Sont, J.K., Willems, L.N., Bel, E.H., van Krieken, J.H., Vandenbroucke, J.P., Sterk, P.J. Am J Respir Crit Care Med 1999; 159:: 1043-1051. 10. Lange, P., Parner, J., Vestbo, J., Schnohr, P., Jensen, G. N.Engl.J Med 1998; 339:: 11941200. 11. Cokugras, H., Akcakaya, N., Seckin, Camcioglu,Y., Sarimurat, N.,Aksoy, F. Thorax 2001; 56:: 25-29. 12. Pohunek, P., Roche, W.R., Tarzikova, J., Kurdman, J.,Warner, J.O. Eur Resp J 1997; 10 (Suppl 25):: 160s. 13. The Childhood Asthma Management Program Research Group: N.Engl.J Med 2000; 343:: 1054-1063. 14. Plopper, C.G., as cited in New Scientist 2001; 169: p7. 15. Martinez, F.D., Holt, P.G. Lancet 1999; 354 Suppl 2:: SII12-SII15. 16. Nicolai, T. Monaldi Arch.Chest Dis. 1999; 54: 475-478. 17. Johnston, S.L., Pattemore, P.K., Sanderson, G., Smith, S., Campbell, M.J., Josephs, L.K., Cunningham, A., Robinson, B.S., Myint, S.H., Ward, M.E., Tyrrell, D.A., Holgate, S.T. Am J Respir Crit Care Med 1996; 154:: 654-660. 18. Chung, K.F., Barnes, P.J. Thorax 1999; 54:: 825-857. 19. Hart, L.A., Krishnan,V.L., Adcock, I.M., Barnes, P.J.,Chung, K.F. Am.J.Respir.Crit Care Med. 1998; 158:: 1585-1592.

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20. Wilson, S.J., Leone, B.A., Anderson, D., Manning, A., Holgate, S.T. J.Pathol. 1999; 189:: 265-272. 21. Demoly,P., Basset-Seguin, N., Chanez, P., Campbell, A.M., Gauthier-Rouviere, C., Godard, P., Michel, F.B., Bousquet, J. Am.J.Respir.Cell Mol.Biol. 1992; 7:: 128-133. 22. Sampath, D., Castro, M., Look, D.C., Holtzman, M.J. J.Clin.Invest 1999; 103:: 1353-1361. 23. Bertorelli, G., Bocchino, V., Zhuo, X., Chetta, A., Del Donno, M., Foresi, A., Testi, R., Olivieri, D. Clin.Exp.Allergy 1998; 28:: 551-560. 24. Torres-Lozano, C., Puddicombe, S.M., Howarth, P.H., Djukanovic, R., Vrugt, R., Aalbers, R., Holgate, S., Davies, D.E., Wilson, S.J. Am J Respir Crit Care Med 2000; 161:: A260. 25. Ordonez, C., Ferrando, R., Hyde, D.M., Wong, H.H., Fahy, J.V. Am J Respir Crit Care Med 2000; 162:: 2324-2329. 26. Puddicombe, S.M., Polosa, R., Richter, A., Krishna, M.T., Howarth, P.H., Holgate, S.T., Davies, D.E. FASEB J. 2000; 14:: 1362-1374. 27. Lackie, P.M., Baker, J.E., Gunthert, U.,Holgate, S.T. Am.J.Respir.Cell Mol.Biol. 1997; 16:: 14-22. 28. Bucchieri, F., Lordan, J., Richter, D., Buchanan, D., Djukanovic, R., Holgate, S.T., Davies, D.E. Am J Respir Crit Care Med 2000; 161(3):: A153. 29. Bayram, H., Devalia, J.L., Khair, O.A., Abdelaziz, M.M., Sapsford, R.J., Sagai, M., Davies, R.J. J Allergy Clin.Immunol. 1998; 102:: 771-782. 30. Redington, A.E., Madden, J., Frew, A.J., Djukanovic, R., Roche, W.R., Holgate, S.T., Howarth, P.H. Am.J.Respir.Crit Care Med. 1997; 156:: 642-647. 31. Brewster, C.E., Howarth, P.H., Djukanovic, R., Wilson, J., Holgate, S.T., Roche, W.R. Am.J.Respir.Cell Mol.Biol. 1990; 3:: 507-511. 32. Zhang, S., Smartt, H., Holgate, S.T., Roche, W.R. Lab Invest 1999; 79:: 395-405. 33. Hoshino, M., Nakamura, Y., Sim, J.J., Yamashiro, Y., Uchida, K., Hosaka, K., Isogai, S. Clin Exp Allergy 1998; 28:: 568-77. 34. Richter, A., Puddicombe, S.M., Lordan, J., Bucchieri F., Mullings, R.E., Wilson, S.J., Djukanovic, R., Holgate, S.T., Davies, D.E. Am J Respir Cell Mol Biol 2000; submitted. 35. Warburton, D., Schwarz,M., Tefft, D., Flores-Delgado, G., Anderson, K.D., Cardoso, W.V. Mech.Dev. 2000; 92:: 55-81.

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36. Holgate, S.T., Davies, D.E., Lackie, P.M., Wilson, S.J., Puddicombe, S.M., Lordan, J.L. J Allergy Clin.Immunol. 2000; 105:: 193-204. 37. Laprise, C., Laviolette, M., Boutet, M., Boulet, L.P. Eur Respir J 1999; 14:: 63-73. 38. Stick, S. Thorax 2000; 55:: 587-594. 39. Zhu, Z., Homer, R.J., Wang, Z., Chen, Q., Geba, G.P., Wang, J., Zhang, Y., Elias, J.A. J Clin.Invest 1999; 103:: 779-788. 40. Rankin, J.A., Picarella, D.E., Geba, G.P., Temann, U.A., Prasad, B., DiCosmo, B., Tarallo, A., Stripp, B., Whitsett, J., Flavell, R.A. Proc.Natl.Acad.Sci.U.S.A 1996; 93:: 7821-7825. 41. Mullings, R.E., Wilson, S.J., Djukanovic, R., Howarth, P.H., Harper, S., Holgate, S.T.,Davies, D.E. J Allergy Clin Immunol 2000; submitted. 42. Lordan,J.L., Bucchieri F., Richter,A., Holloway,J., Konstantinidis,A., Buchanan,D., Beghe,B., Djukanovic, R., Holgate, S.T., Davies, D.E. Am J Respir Cell Mol Biol 2000; submitted.

This manuscript is reproduced in the IVIS website with the permission of the World Equine Airway Society

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