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Animal models of chronic experimental asthma — strategies for the identification of new therapeutic targets
Journal of Occupational Medicine and Toxicologyvolume 3, Article number: S4 (2008)
Over the last decade mouse models of experimental asthma proved to be a valuable tool for the investigation of mechanisms that underlie acute allergic airway inflammation and development of airway hyperresponsiveness, two of the hallmarks of human asthma. Nevertheless, these acute models fail to reflect the aspects of this chronic disease because they do not represent any signs of chronicity and airway remodelling as it is defined by subepithelial fibrosis, goblet cell hyperplasia and airway smooth muscle cell hypertrophy. Recent mouse models were successful in overcoming these limitations by using chronic allergen-challenges. These new models of chronic experimental asthma now proved as a novel tool to examine the complex interaction of infiltrating inflammatory cells and structural cells such as fibroblasts and smooth muscle cells that ultimately leads to airway remodelling and stable airflow limitation. Recent studies clearly demonstrated that T helper 2 (TH2) cells and their typical cytokines play a critical role not only in airway inflammation but also in the development of airway remodelling. Since the transcription factor GATA-3 is essential for TH2 cell development and the production of several TH2 type cytokines this intracellular molecule represents a new promising target for therapeutic intervention in asthma that might even effect airway remodelling.
Allergic bronchial asthma is a chronic inflammatory disease of the airways that is characterized by allergic airway inflammation and development of airway hyperresponsiveness (AHR). Another hallmark of asthma involves various structural changes of the airway wall such as airway fibrosis and smooth muscle cell hyperplasia summarized as airway remodelling . Over the last 15 years animal models, especially mouse models, of experimental asthma have been intensively used to investigate the immuno-pathological mechanisms that ultimately lead to the formation of this disease. Mostly, these models utilized systemic sensitization to foreign proteins such as ovalbumin (OVA) adsorbed to the adjuvant aluminium hydroxide (alum) to induce a T-helper-2 (TH2) cell triggered immune-response together with marked production of OVA-specific IgE. Once sensitized, these animals developed airway eosinophilia and AHR after inhalation or intra-nasal application of OVA aerosols or OVA-solutions . Such models provided insight into the immunological mechanisms of acute allergic inflammation, helped to identify key cytokines such as interleukin 4 (IL-4), IL-5 and IL-13 that play critical roles in the regulation of the local inflammatory process  and proofed valuable to investigate the neuro-immunological dysregulation underlying the development of AHR .
Mouse models of chronic experimental asthma
However, since allergic airway inflammation in these models is induced by short term exposure to the aerosolized allergen and, therefore, does not persist for longer than 10 days, these models are limited regarding the modelling of the chronic aspects of asthma, more precisely airway remodelling and its patho-physiological consequences that might result in stable airflow limitation. Furthermore, pathological aspects of experimental asthma in these acute models are reduced to the central airways. Since asthma, allergic inflammation and airway remodelling is observed in peripheral airways of asthmatic patients as well [5, 6] and structural changes especially of distal airways play a critical role in the development of irreversible airflow limitation  these aspects represent important hallmarks for disease modelling in animals.
Over the last few years airway remodelling became a field of special interest in asthma research since it is largely resistant to medication  and is an important factor to develop irreversible airflow limitation, resulting in progress of asthma severity . To overcome these limitations recently many advanced models have been established. Most of these models use systemic sensitization to ovalbumin (OVA) adsorbed to aluminium hydroxide (alum) followed by repeated exposure to aerosolized OVA for 4-8 weeks [10–13]. Within these models it is interesting to note that similar results are achieved with a huge variety of OVA-aerosol concentrations (0.1 – 2.5%) as well as the frequency of allergen challenges (2–7 times a week).
However, the modelling of these chronic aspects of asthma in animals requires clear definition of airway remodelling. The pathological analysis on bronchial specimens of asthmatic patients revealed goblet cell hyperplasia of the airway epithelium, hyperplasia of the submucosal glands, thickening of the epithelial basement membrane, extracellular matrix deposition in the subepithelial layer and hypertrophy and hyperplasia of the bronchial smooth muscle cells . To reflect these hallmarks of airway remodelling, we sensitized BALB/c mice to OVA by three intra-peritoneal (i.p.) injection of OVA adsorbed to alum followed by a twelve week long OVA challenge period with two challenges a week on consecutive days . These animals developed chronic allergic inflammation that in contrast to other models affected the entire bronchial tree and even persisted after six weeks of OVA challenge discontinuation. This chronic airway inflammation was further associated with persistent AHR and stable airflow limitation as indicated by reduction of baseline midexpiratory airflow (EF50). In contrast to other models of chronic experimental asthma, proximal as well as distal airways revealed marked deposition of collagen fibres in the airway wall especially in the lamina propria. The airway wall was additionally thickened by appearance of myofibrocytes and enlargement of the airway smooth muscle layer indicating airway smooth muscle hypertrophy. Goblet cell hyperplasia could be observed in all sections of the bronchial tree. Since this model reflects human airway remodelling to a high degree it could be useful for the investigation of the mechanisms that underlie this special issue of human asthma.
Lymphocytes and eosinophils in airway remodelling
The actual concept of asthma pathogenesis begins with the differentiation of naïve allergen-specific T-helper cells towards TH2 cells that are characterized by the expression of the transcription factor GATA-3 and production of several typical cytokines such as IL-5 that controls the activity and the survival of eosinophils. These cells represent the main effector cell type of the allergic airway inflammation and produce a variety of cytotoxic mediators and cationic proteins such as major basic protein (MBP) and eosinophil cationic protein (ECP) that ultimately lead to epithelial damage . This further triggers several mechanisms focussing on the reconstitution of the epithelial integrity. One of the main mediators involved in this process is transforming growth factor-beta (TGF-β) that is produced by eosinophils and structural cells such as bronchial epithelial cells and smooth muscle cells . This cytokine on the one hand possesses anti-inflammatory properties that help to limit the inflammatory process in the airways. On the other hand it triggers airway remodelling since it induces pro-collagen production in fibroblasts and transformation of these cells into smooth muscle actin containing myofibroblasts [18, 19].
We utilized our model of chronic experimental asthma to assess the effect of a novel therapeutic strategy that targets eosinophils on airway remodelling and lung function. Since the infiltration of these cells into the airways depends on interaction of the C-C chemokine receptor-3 (CCR-3) and its ligands from the eotaxin family, we antagonized this chemokine receptor by using a low molecular weight compound . To mimic the human situation as closely as possible treatment with the CCR-3 antagonist started not until allergic airway inflammation has already been established, but no signs of airway remodelling could be observed. Systemic application of 30mg/kg body weight reduced the infiltration of eosinophils into the broncho-alveolar lumen and into the airway tissue by about 70%, whereas infiltration of other inflammatory cells was not effected significantly. Additionally, neither subepithelial fibrosis nor hyperplasia of smooth muscle cells or goblet cells could be observed in animals treated with the CCR-3 antagonist. Although airway eosinophilia could not be reduced entirely airway reactivity to metacholine was nearly normalized in these animals indicating that eosinophils play an important, but not an exclusive role in the processes leading to airway remodelling and AHR. Both, involvement of eosinophils in development of AHR and in development of airway remodelling remain issues of special interest and are discussed highly controversial. In models of acute experimental asthma development of AHR is usually accompanied by airway eosinophilia. Nevertheless, a substantial number of studies clearly demonstrate that factors other than airway eosinophilia are essential for the development of AHR. These factors involve TH2-type cytokines such as IL-4 and IL-13 as well as signalling via the Fc-epsilon receptor [21–23]. Furthermore, Tournoy et al. report about a model of acute experimental asthma where airway eosinophilia and development of AHR seem to be disconnected . Similar results were achieved by Siegle et al in a mouse model of chronic experimental asthma . The discussion about eosinophils and development of AHR came to a climax when the groups of Humbles and Lee reported about the results of their studies in the same issue of the science magazine. Humbles et al. used the Δdbl GATA mouse which is completely deficient for the eosinophil lineage due to deletion of a high affinity GATA site of the GATA-1 promotor in a mouse model of experimental asthma to demonstrate that eosinophils are not obligatory for allergen-induced lung dysfunction . In contrast, Lee utilized the transgenic PHIL mouse that is specifically devoid of eosinophils, to show that these are required for development of AHR .
Even though both studies seem to be contradictory regarding the role of eosinophils within the development of AHR, both studies linked eosinophils to the development of airway remodelling. Whereas Lee et al. reported the lack of goblet cell metaplasia in PHIL mice Humbles et al. clearly demonstrated that eosinophil deficient animals were protected from airway fibrosis and smooth muscle cell hyperplasia. These findings receive further support from studies that report about the profibrotic effects of eosinophils on fibroblasts in vitro[28–30] and the results concerning antagonization of CCR-3 that were produced by Fulkerson et al.  and by our group.
Besides the fact that TH2 cells induce goblet cell hyperplasia and increased mucus production in asthmatic patients by secreting IL-13 , very little is known about the contribution of T cells to airway remodelling. Few recent studies focussed on this special issue and presented data that in fact suggest the participation of TH2 cells in the development of airway fibrosis and smooth muscle hyperplasia. Komai et al. investigated the effect of a neutralizing anti-CD4 antibody in a mouse model of chronic experimental asthma . The i.p. administration of this antibody during the chronic OVA aerosol challenge did not only result in diminished airway inflammation and reduced amounts of TH2-type cytokines in broncho-alveolar lavage fluids, but also decreased goblet cell hyperplasia and airway fibrosis. This was further accompanied by the observation that neutralizing CD4 downsized the level of TGF-β in BAL fluids as well. Additional experiments from the same group focussed on the question whether typical TH2-type cytokines such as IL-4 and IL-5 may contribute to airway remodelling. For this purpose IL-4 deficient animals were sensitized to OVA and chronically exposed to an OVA aerosol. Compared to wild type animals, IL-4 deficient animals did not develop allergic airway inflammation and did not reveal any signs of airway remodelling as indicated by lack of goblet cell hyperplasia and subepithelial fibrosis. Similar results could be observed in animals that were deficient for the alpha chain of the IL-5 receptor. In contrast, OVA-sensitized IL-5 transgenic (tg) mice that systemically overexpress IL-5 showed even higher levels of TGF-β and enhanced subepithelial fibrosis than wild type animals after chronic OVA aerosol challenge .
Although all these studies reported about reduced airway remodelling after abolishing CD4 positive T cells or TH2-type cytokines one could argue that all these effects might be indirect since these lowered effects were always associated with diminished airway eosinophilia. However, a study by Ramos-Barbon gave evidence that TH2 cells can directly induce airway remodelling . In contrast to all previously mentioned studies these experiments were carried out in the rat, since this species is from an anatomical point of view more suitable to mimic changes of the airway smooth muscle layer. In this study CD4 positive T cells from OVA-sensitized rats, that were challenged chronically with OVA aerosol and revealed signs of airway remodelling including airway smooth muscle cell (ASMC) hyperplasia, were adoptively transferred to naïve rats. After OVA aerosol challenge these CD4 positive T cells infiltrated the airway tissue and could be localized in close contact to muscle cells of the airways and airway vessels. This was further associated with increased airway smooth muscle mass indicating ASMC hypertrophy. By using immuno-histological staining methods Ramos-Barbon et al. were able to clearly demonstrate that ASMCs from adoptively transfered animals exhibited increased proliferation and reduced apoptosis. All the data presented in this study support the idea that airway smooth muscle remodelling, as an important feature of asthma, is driven by antigen-specific T helper cells that require direct myocyte-T cell contact.
Animal models of experimental asthma have elucidated the central role of TH2 cells and their typical cytokines in the acute inflammatory process resulting in the formation of asthma. To answer the question whether these cells are further involved in pathophysiological processes ultimately leading to airway remodelling and chronification of the disease improved models of chronic experimental asthma that are able to reflect the chronic aspects of this disease as close as possible had to be established. Recent studies used such models to demonstrate that TH2 cells play a critical role in asthma chronification especially in airway remodelling as well. Therefore, these cells still represent promising target cells for therapeutic intervention in asthmatic patients. After embryonic development the pleiotropic transcription factor GATA-3 remains to be expressed in bone marrow derived cells such as eosinophils, basophils, mast cells and especially in T cells [35, 36]. During T helper cell development GATA-3 expression promotes TH2 responses through at least three different mechanisms and therefore represents a crucial event in T cell polarization [37–40]. Furthermore, overexpression of GATA-3 leads to increased airway remodelling in a mouse model of chronic experimental asthma . So due to its central role within the pathology of asthma GATA-3 represents an interesting target for new anti-allergic strategies, that might even be able to interfere with processes that ultimately lead to airway remodelling.
Wegmann M, Renz H: Animal models of experimental asthma. Ernst Schering Res Found Workshop 2005, 50: 69–87.
Epstein MM: Do mouse models of allergic asthma mimic clinical disease? Int Arch Allergy Immunol 2004, 133: 84–100. 10.1159/000076131
Wills-Karp M: Murine models of asthma in understanding immune dysregulation in human asthma. Immunopharmacology 2000, 48: 263–268. Review 10.1016/S0162-3109(00)00223-X
Braun A, Quarcoo D, Schulte-Herbruggen O, Lommatzsch M, Hoyle G, Renz H: Nerve growth factor induces airway hyperresponsiveness in mice. Int Arch Allergy Immunol 2001, 124: 205–207. 10.1159/000053711
Hamid Q, Song Y, Kotsimbos TC, Minshall E, Bai TR, Hegele RG, Hogg JC: Inflammation of small airways in asthma. J Allergy Clin Immunol 1997, 100: 44–51. 10.1016/S0091-6749(97)70193-3
Bai TR, Cooper J, Koelmeyer T, Pare PD, Weir TD: The effect of age and duration of disease on airway structure in fatal asthma. Am J Respir Crit Care Med 2000, 162: 663–669.
Wagner EM, Liu MC, Weinmann GG, Permutt S, Bleecker ER: Peripheral lung resistance in normal and asthmatic subjects. Am Rev Respir Dis 1990, 141: 584–588.
Lange P, Parner J, Vestbo J, Schnohr P, Jensen G: A 15-year follow-up study of ventilatory function in adults with asthma. N Engl J Med 1998, 339: 1194–1200. 10.1056/NEJM199810223391703
James AL, Pare PD, Hogg JC: The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989, 139: 242–246.
Jungsuwadee P, Benkovszky M, Dekan G, Stingl G, Epstein MM: Repeated aerosol allergen exposure suppresses inflammation in B-cell-deficient mice with established allergic asthma. Int Arch Allergy Immunol 2004, 133: 40–8. 10.1159/000075252
McMillan SJ, Xanthou G, Lloyd CM: Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the Smad signaling pathway. J Immunol 2005,174(9):5774–5580.
Temelkovski J, Hogan SP, Shepherd DP, Foster PS, Kumar RK: An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 1998, 53: 849–56.
Tanaka H, Komai M, Nagao K, Ishizaki M, Kajiwara D, Takatsu K, Delespesse G, Nagai H: Role of interleukin-5 and eosinophils in allergen-induced airway remodeling in mice. Am J Respir Cell Mol Biol 2004, 31: 62–68. 10.1165/rcmb.2003-0305OC
Hegele RG, Hogg JC: The apthology of asthma – An inflammatory disorder. In Severe asthma – Pathogenesis and Clinical Management. Volume 3. Edited by: Edited by S.J. Szefler, D.Y.M. Leung. Marcel Dekker, New York; 1996:61–76.
Wegmann M, Fehrenbach H, Fehrenbach A, Held T, Schramm C, Garn H, Renz H: Involvement of distal airways in a chronic model of experimental asthma. Clin Exp Allergy 2005, 35: 1263–1271. 10.1111/j.1365-2222.2005.02306.x
Gleich GJ: The eosinophil and bronchial asthma: current understanding. J Allergy Clin Immunol 1990, 85: 422–436. 10.1016/0091-6749(90)90151-S
Moir LM, Leung SY, Eynott PR, McVicker CG, Ward JP, Chung KF, Hirst SJ: Repeated allergen inhalation induces phenotypic modulation of smooth muscle in bronchioles of sensitized rats. Am J Physiol Lung Cell Mol Physiol 2003, 284: L148–59.
Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Maruoka S, Horie T: Transforming growth Factor-beta1 induces phenotypic modulation of human lung fibroblasts to myofibroblast through a c-Jun-NH2-terminal kinase-dependent pathway. Am J Respir Crit Care Med 2001, 163: 152–157.
Zhang HY, Phan SH: Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am J Respir Cell Mol Biol 1999, 21: 658–665.
Wegmann M, Goggel R, Sel S, Sel S, Erb KJ, Kalkbrenner F, Renz H, Garn H: Effects of a low-molecular-weight CCR-3 antagonist on chronic experimental asthma. Am J Respir Cell Mol Biol 2007, 36: 61–67. 10.1165/rcmb.2006-0188OC
Cohn L, Homer RJ, MacLeod H, Mohrs M, Brombacher F, Bottomly K: Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils. J Immunol 1999, 162: 6178–83.
Corry DB, Folkesson HG, Warnock ML, Erle DJ, Matthay MA, Wiener-Kronish JP, Locksley RM: Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity. J Exp Med 1996, 183: 109–17. 10.1084/jem.183.1.109
Mayr SI, Zuberi RI, Zhang M, de Sousa-Hitzler J, Ngo K, Kuwabara Y, Yu L, Fung-Leung WP, Liu FT: IgE-dependent mast cell activation potentiates airway responses in murine asthma models. J Immunol 2002, 169: 2061–8.
Tournoy KG, Kips JC, Schou C, Pauwels RA: Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness. Clin Exp Allergy 2000, 30: 79–85. 10.1046/j.1365-2222.2000.00772.x
Siegle JS, Hansbro N, Herbert C, Yang M, Foster PS, Kumar RK: Airway hyperreactivity in exacerbation of chronic asthma is independent of eosinophilic inflammation. Am J Respir Cell Mol Biol 2006, 35: 565–70. 10.1165/rcmb.2006-0135OC
Humbles AA, Lloyd CM, McMillan SJ, Friend DS, Xanthou G, McKenna EE, Ghiran S, Gerard NP, Yu C, Orkin SH, Gerard C: A critical role for eosinophils in allergic airways remodeling. Science 2004, 305: 1776–9. 10.1126/science.1100283
Lee JJ, Dimina D, Macias MP, Ochkur SI, McGarry MP, O'Neill KR, Protheroe C, Pero R, Nguyen T, Cormier SA, Lenkiewicz E, Colbert D, Rinaldi L, Ackerman SJ, Irvin CG, Lee NA: Science. 2004, 305: 1773–6. 10.1126/science.1099472
Beller TC, Friend DS, Maekawa A, Lam BK, Austen KF, Kanaoka Y: Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis. Proc Natl Acad Sci USA 2004, 101: 3047–52. 10.1073/pnas.0400235101
Gharaee-Kermani M, Phan SH: The role of eosinophils in pulmonary fibrosis. Int J Mol Med 1998, 1: 43–53.
Levi-Schaffer F, Garbuzenko E, Rubin A, Reich R, Pickholz D, Gillery P, Emonard H, Nagler A, Maquart FA: Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro: a role for transforming growth factor beta (TGF-beta). Proc Natl Acad Sci USA 1999, 96: 9660–5. 10.1073/pnas.96.17.9660
Fulkerson PC, Fischetti CA, Rothenberg ME: Eosinophils and CCR3 regulate interleukin-13 transgene-induced pulmonary remodeling. Am J Pathol 2006, 169: 2117–2126. 10.2353/ajpath.2006.060617
Ford JG, Rennick D, Donaldson DD, Venkayya R, McArthur C, Hansell E, Kurup VP, Warnock M, Grunig G: Il-13 and IFN-gamma: interactions in lung inflammation. J Immunol 2001, 167: 1769–1777.
Komai M, Tanaka H, Masuda T, Nagao K, Ishizaki M, Sawada M, Nagai H: Role of TH2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma. Br J Pharmacol 2003, 138: 912–920. 10.1038/sj.bjp.0705105
Ramos-Barbon D, Presley JF, Hamid QA, Fixman ED, Martin JG: Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma. J Clin Invest 2005, 115: 1580–1589. 10.1172/JCI19711
Hirasawa R, Shimizu R, Takahashi S, Osawa M, Takayanagi S, Kato Y, Onodera M, Minegishi N, Yamamoto M, Fukao K, Taniguchi H, Nakauchi H, Iwama A: Essential and instructive roles of GATA factors in eosinophil development. J Exp Med 2002, 195: 1379–1386. 10.1084/jem.20020170
Ting CN, Olson MC, Barton KP, Leiden JM: Transcription factor GATA-3 is required for development of the T-cell lineage. Nature 1996, 384: 474–478. 10.1038/384474a0
Zhu J, Yamane H, Cote-Sierra J, Guo L, Paul WE: GATA-3 promotes TH2 responses through three different mechanisms: induction of TH2 cytokine production, selective growth of TH2 cells and inhibition of Th1 cell-specific factors. Cell Res 2006, 16: 3–10. 10.1038/sj.cr.7310002
Klein-Hessling S, Jha MK, Santner-Nanan B, Berberich-Siebelt F, Baumruker T, Schimpl A, Serfling E: Protein kinase A regulates GATA-3-dependent activation of IL-5 gene expression in TH2 cells. J Immunol 2003, 170: 2956–2961.
Kishikawa H, Sun J, Choi A, Miaw SC, Ho IC: The cell type-specific expression of the murine IL-13 gene is regulated by GATA-3. J Immunol 2001, 167: 4414–4420.
Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF Jr, Guo L, Paul WE: Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat Immunol 2004, 5: 1157–1165. 10.1038/ni1128
Kiwamoto T, Ishii Y, Morishima Y, Yoh K, Maeda A, Ishizaki K, Iizuka T, Hegab AE, Matsuno Y, Homma S, Nomura A, Sakamoto T, Takahashi S, Sekizawa K: Transcription factors T-bet and GATA-3 regulate development of airway remodeling. Am J Respir Crit Care Med 2006, 174: 142–151. 10.1164/rccm.200601-079OC
This article has been published as part of Journal of Occupational Medicine and Toxicology Volume 3 Supplement 1, 2008: Proceedings of the 6th Workshop on Animal Models of Asthma. The full contents of the supplement are available online at http://www.occup-med.com/content/3/S1.
The author declares that he has no competing interests.