Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 www.elsevier.com/locate/ypupt COPD corner series Murine models of COPD G.G. Brusselle, K.R. Bracke, T. Maes, A.I. D’hulst, K.B. Moerloose, G.F. Joos, R.A. Pauwels*,! Department of Respiratory Diseases, Ghent University Hospital and Ghent University, De Pintelaan 185, B-9000 Gent, Belgium Received 20 January 2005; revised 15 May 2005; accepted 8 June 2005 Abstract Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation, that is not fully reversible, and that is associated with an abnormal inflammatory response of the airways and lungs to noxious particles and gases. The airflow limitation is caused by increased resistance of the small conducting airways and by decreased elastic recoil forces of the lung due to emphysematous destruction of the lung parenchyma. In vivo animal models can help to unravel the molecular and cellular mechanisms underlying the pathogenesis of COPD. Mice represent the most favored animal species with regard to the study of (both innate and adaptive) immune mechanisms, since they offer the opportunity to manipulate gene expression. Several experimental approaches are applied in order to mimic the different traits of COPD in these murine models. Firstly, the tracheal instillation of tissue-degrading enzymes induces emphysema-like lesions in the lung parenchyma, adding further proof to the protease-antiprotease imbalance hypothesis. Secondly, the inhalation of noxious stimuli, including tobacco smoke, sulfur dioxide, nitrogen dioxide, or oxidants such as ozone, may also lead to COPD-like lesions in mice, depending on concentration, duration of exposure and strainspecific genetic susceptibility. Thirdly, in transgenic mice, a specific gene is either overexpressed (non-specific or organ-specific) or selectively depleted (constitutively or conditionally). The study of these transgenic mice, either per se or in combination with the above mentioned experimental approaches (e.g. the inhalation of tobacco smoke), can offer valuable information on both the physiological function of the gene of interest as well as the pathophysiological mechanisms of diseases with complex traits such as COPD. Keywords: COPD; Emphysema; Murine; Cigarette smoke; Inflammation; Apoptosis 1. Why do we need murine models of COPD/pulmonary emphysema? Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality throughout the world [1]. Since COPD is currently listed as the fifth Abbreviations BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; FEV1, forced expiratory volume in one second; IL (e.g. IL-1, IL-6, IL-8), interleukin; IFN-g, interferon-g; LPS, lipopolysaccharide; LBP, LPS binding protein; MMP(s), matrixmetalloproteinase(s); NF-kB, nuclear factor-kB; ROS, reactive oxygen species; RT-PCR, reverse transcriptase-polymerase chain reaction; TEAC, trolox equivalent antioxidant capacity; TLR (e.g. TLR-4), toll-like receptor; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial cell growth factor; VEGFR-2, vascular endothelial cell growth factor receptor-2. * Corresponding author. Tel.: C32 9 240 26 04; fax: C32 9 240 23 41. E-mail address: guy.brusselle@ugent.be (R.A. Pauwels). ! Prof Dr Romain A. Pauwels deceased on 3/01/2005. 1094-5539/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2005.06.001 leading cause of death in the world, and is also an important cause of chronic disability and permanent impairment, COPD represents a major economic and social burden worldwide [2]. COPD is defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as ‘a disease state characterized by airflow limitation that is not fully reversible, and that is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases’ [3,4]. Cigarette smoking is by far the most important risk factor for COPD. Pipe and cigar smokers also have greater COPD morbidity and mortality rates than nonsmokers, although their rates are lower than those for cigarette smokers. Passive exposure to cigarette smoke (i.e. environmental tobacco smoke) may also contribute to respiratory symptoms and COPD. However, only a susceptible minority (approximately 15–20%) of tobacco smokers develop clinically significant COPD, suggesting that genetic factors must modify each individual’s risk [5]. COPD is characterized by an accelerated decline in lung function, expressed as the forced expiratory volume in one second (FEV1) and its ratio to the 156 G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 forced vital capacity (FVC), namely FEV1/FVC. The reason why only a minority of smokers experiences this excessive decline in FEV1 over time is unknown. Therefore, although the major environmental risk factor for COPD—tobacco smoke—is well known since many years, the host factors that are involved in the pathogenesis of COPD have not yet been identified (besides the rare hereditary deficiency of a-1 antitrypsin) [6]. Since COPD arises from an interaction between genetic host factors and environmental exposures, the study of these gene-environment interactions is of critical importance in order to elucidate the pathogenesis of this abundant and devastating disease. Mice are the experimental tool of choice in mammalian research for the following reasons: (1) recently, both the human and mice genomes have been sequenced, revealing that only approximately 300 genes appear to be unique to one species or the other [7]; (2) more than 10,000 genetic markers are mapped in the mouse, providing useful landmarks for genetic studies; (3) the ability to alter the genetic constitution of the mouse, either by inserting new genes or increasing the gene expression levels by transgenesis, or by removing or altering genes through gene replacement techniques (e.g. knock-out mice); (4) many hundreds of inbred strains and specialized stocks (e.g. mutants) are available; (5) thorough knowledge of anatomy, biology and physiology of the mouse, especially with regard to the immunological system; and fast breading at relatively low cost. The extensive knowledge of the mouse biology and the huge genetic resources, including the ability to genetically manipulate the mouse, offer thus an unprecedented capacity to explore biological systems under physiological and pathological conditions [8]. The mouse has become our experimental surrogate, when experiments in human subjects are either technically impossible or morally inconceivable. The major goals of this research, using murine models of COPD, are twofold: first, to understand the cellular and molecular mechanisms involved, and from this knowledge derive benefit for both the quality of life and the prevention and treatment of this disease; secondly, to develop new specific drugs for COPD, in order to improve the clinical and functional status of patients with COPD, and—most importantly—in order to prevent the accelerated decline in lung function and the ensuing morbidity and mortality. 2. Different experimental models of COPD/pulmonary emphysema in mice: introduction Several experimental models of COPD and emphysema exist in mice, based upon different approaches [9,10]. Firstly, the tracheal instillation of tissue-degrading enzymes has been used since a long time to study the development of emphysematous lung lesions [11,12]. Secondly, inhalation of tobacco smoke and other noxious stimuli in mice induces lung tissue destruction, although the development of emphysema-like lesions appears to be strain-dependent [13,14]. Thirdly, several mouse strains with naturally occurring genetic mutations develop emphysema spontaneously without external stimuli, although a number of these mutations lead to multisystem defects and are thus not restricted to pathology of the lung [15]. Gene targeted mice can also show signs of airspace enlargement, but it is crucial to distinguish airspace enlargement due to developmental abnormal lung morphogenesis from adult emphysema, which is characterized by the destruction of mature alveoli [16]. Of course, these different approaches can be combined (e.g. exposure of gene targeted mice to tobacco smoke). As far as models of exposure to cigarette smoke are discussed in this review, we will focus on the effects of chronic exposure of different mice strains to cigarette smoke, since an excellent review of the acute effects of smoking in both human and animal studies has been published recently [17]. Before describing the different murine models of COPD in greater detail, it is important to underline the differences between COPD and asthma in humans, and by consequence also in animal models which aim to mimic these chronic airway diseases (see Table 1). Asthma is defined as a Table 1 Similarities and differences between asthma and COPD Etiology Most frequent cause Onset Symptoms Airway inflammation Leukocytes T lymphocytes Inflammatory mediators Cytokines Leukotrienes Airway wall remodeling Epithelium Mucus glands Smooth muscle Lung parenchymaldestruction Lung function Airflow limitation airway hyperresponsiveness (AHR) Response to treatment Asthma COPD Sensitizing agent House dust mite Noxious agent Cigarette smoke Early in life (often childhood) Variable (from day to day) In mid-life Eosinophils CD4C Th2 cells Neutrophils and macrophages CD8CT cells IL-4, IL-5, IL-13 LTC4, LTD4 IL-8, TNF-a LTB4 Thickening of basement membrane Glandular enlargement Muscle hypertrophy Absent Squamous metaplasia of epithelium Glandular enlargement Muscle hypertrophy Present (i.e. emphysema) Completely reversible Moderate to severe increase in AHR Glucocorticoids inhibit inflammation Largely irreversible No AHR or only mild increase in AHR Glucocorticoids have little or no effect on chronic inflammation Slowly progressive G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 chronic inflammatory disorder of the lower airways, which causes an associated increase in airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness and coughing [18]. These episodes are usually associated with widespread, but variable airflow obstruction that is often reversible. In contrast, COPD is characterized by airflow limitation that is usually both progressive and not fully reversible. The airflow limitation in COPD is associated with an abnormal inflammatory response of the airways and the lungs to noxious particles or gases [3]. Although asthma and COPD are thus both characterized by chronic inflammation of the lower airways, there are important differences between asthma and COPD in risk factors, onset of the disease, symptoms, inflammatory mediators and leukocytes, airway wall remodeling, lung function and response to treatment with inhaled corticosteroids (Table 1) [19]. Moreover, pulmonary emphysema, which is defined anatomically as ‘abnormal, permanent enlargement of air spaces distal to the terminal bronchiole, accompanied by destruction of the alveolar walls’, is a crucial part of the clinical complex of COPD, but is absent in asthma [9,20]. 3. COPD/pulmonary emphysema and the protease/ antiprotease imbalance An imbalance between proteases and their inhibitors is believed to play an essential role in the development of pulmonary emphysema. This imbalance may occur either by an excessive release of proteases by inflammatory cells and lung resident cells, or by a reduced synthesis or increased breakdown of antiproteases. The protease/antiprotease hypothesis of emphysema was first proposed 40 years ago, based on the observations that smokers with a deficiency of a1-antitrypsin were at increased risk for pulmonary emphysema [6], and that intratracheal administration of papain, a plant protease, leads to emphysema in experimental animals [21]. Since the initial experiments of Gross, a variety of proteases have been instilled into the lungs of animals. In mice, the most consistent and impressive airspace enlargement has been accomplished by the intratracheal instillation of porcine pancreatic elastase [22]. Development of emphysema after instillation of human neutrophil elastase has also been described in mice [23]. These rather crude and acute instillation models can be useful to determine the capacity of a protease to cause emphysema, and to study downstream events such as alveolar repair. However, these instillation models have at least three major drawbacks: they cannot be used to explore any upstream events; they cannot give any information about which proteases are involved in the pathogenesis of emphysema, and lastly, it is difficult to extrapolate the findings on the acute effects of elastase instillation to the slowly progressive chronic onset disease in humans. 157 Transgenic mice have artificially introduced alterations in their genome, resulting in expression or overexpression of the gene product of interest. These transgenic ‘gain-offunction’ models have added further proof to the protease/antiprotease imbalance hypothesis. Mice that overexpressed human interstitial collagenase (MMP-1) in their lungs spontaneously developed pulmonary emphysema [24]. Recently, it has been shown that MMP-1 generated this emphysematous phenotype via the degradation of type III collagen [25,26]. A major disadvantage of this kind of model is that the gene of interest is also expressed throughout organ development and growth, which makes it impossible to separate developmental abnormalities from the structural injury in adult lungs that defines emphysema. This problem of constitutive expression of transgenes can be overcome by the construction of inducible transgenic expression models. Induced overexpression of interleukin 13 (IL-13) or interferon-g (IFN-g) into the lungs of mice causes a phenotype that mirrors human COPD [27,28]. In both models, the overexpression of these inflammatory cytokines was associated with an increased expression of matrix metalloproteinases (MMPs) and cysteine proteases (cathepsins). The emphysematous changes were partly inhibited after treatment with MMP-inhibitors or cysteine protease inhibitors. These data thus further establish the role of proteases in lung tissue destruction, a hallmark of pulmonary emphysema. In contrast to transgenic ‘gain-of-function’ models, targeted mutagenesis of genes has allowed investigators to generate strains of mice that lack individual proteins, and thus study the (patho)physiology of several diseases in gene-targeted ‘loss-of-function’ models. Combination of gene targeting with the cigarette smoke-exposure model can provide useful information whether a specific proteinase contributes to the pathogenesis of emphysema (Table 2). Hautamaki et al. [29] elegantly demonstrated that MMP-12 deficient mice do not develop airspace enlargement in response to long term exposure to cigarette smoke, in contrast to the wild-type animals. MMP-12 deficient mice also failed to recruit macrophages into their lungs in response to cigarette smoke. This may be related to the abrogated generation of elastin fragments, that are chemotactic for monocytes, by MMP-12 [30,31]. Given the fact that MMP-12 is expressed in human alveolar macrophages [32], these data suggested a role for MMP-12 not only in the pathogenesis of mouse but also of human emphysema. Neutrophil elastase deficient mice were significantly protected from the development of pulmonary emphysema after cigarette smoke exposure [33]. It would seem that there are many interactions between neutrophil elastase and MMPs, with each augmenting the other’s destructive capacity. Indeed, MMPs degrade a 1-antitrypsin, whereas neutrophil elastase degrades tissue inhibitors of metalloproteinases (TIMPs). Neutrophil elastase also mediates monocyte migration, whereas MMP-12 may influence neutrophil accumulation by activation of TNF-a [34]. Interestingly, 158 G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 Table 2 Gene targeted ‘loss-of-function’ models in selected knock-out (KO) mice Gene Phenotype References Macrophage Elastase (MMP-12) KO No emphysema after long term CS-exposure Impaired recruitment of macrophages into the lungs Significant protection from CS-induced emphysema Spontaneous airspace enlargement evident at 2 weeks [ degradation of collagen [ MMP activity Spontaneous development of emphysema and fibrosis [ oxidant production in macrophages activates [ NF-kB and MMP-expression Spontaneous development of emphysema Macrophage accumulation in the lungs [ MMP-12 activation [ extensive CS-induced emphysema [ apoptotic alveolar septal cells [ markers of oxidative stress [ BAL inflammation Y acute CS-induced inflammation Y acute CS-induced connective tissue breakdown 70% less emphysema after long term CS-exposure Spontaneous development of emphysema [ static compliance of the lung at postnatal week 4 29, 34 Neutrophil Elastase (NE) KO Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) KO Surfactant Protein D (SP-D) KO Integrin avb6 KO Nuclear factor erythroid-derived 2 (Nrf2) KO TNFa receptor double KO Retinoic acid receptor KO 33 37 38, 39 41 73 35, 36 82 Y, decreased; [, increased; KO, knock-out; CS, cigarette smoke. using TNFa receptor double knockout mice, Churg et al. [35] have demonstrated that TNFa and its receptors are central to acute cigarette smoke-induced inflammation and connective tissue breakdown. When these TNFa receptor double knockout mice were exposed chronically to smoke for 6 months, the ensuing airspace enlargement was reduced by 70% compared to wild type animals [36]. These studies indicate that TNFa is a critical mediator in the pathogenesis of COPD/pulmonary emphysema. Knockout of a specific gene can also lead to the development of spontaneous, age-related emphysema (Table 2). TIMP-3 deficient mice demonstrate a progressive enlargement of alveolar airspaces with increasing age [37]. Next to the possible developmental abnormalities, these mice display an increased MMP activity. Spontaneous emphysema has also been observed in mice deficient for surfactant protein D (SP-D) [38,39]. Macrophages of SP-D deficient mice have increased oxidant production, activating NF-kB with consequent MMP expression [40]. Finally, mice lacking the integrin avb6, an activator of latent TGF-b, accumulate macrophages in the lungs, show increased MMP-12 activation and develop airspace enlargement with age [41]. Interestingly, the phenotypic effects of the avb6 deletion are overcome by crossing with MMP-12 deficient mice. This suggests that under normal conditions TGF-b inhibits MMP-12 production and that in the absence of the avb6 integrin, there is diminished TGF-b activity, leading to increased production of MMP-12 and emphysema. Mahadeva and Shapiro [16] excellently reviewed the data on several naturally occurring mutant mice that spontaneously develop emphysema due to genetic abnormalities. In some of these strains of mice, the airspace enlargement is thought to be the result of a disturbance in the balance between proteases and their inhibitors. For example, the pallid mice have a deficiency in a 1-antitrypsin and thus reduced elastase inhibitory capacity, and spontaneously develop emphysema late in life [42]. These histological changes are paralleled with a decrease in lung elastin content, but there is no alteration in the BAL cell population [43]. Chronic exposure to cigarette smoke significantly accelerated parenchymal destruction in pallid mice [44,45]. This is probably related to the diminished antiprotease capacity in these mice, and correlates with the enhanced risk for pulmonary emphysema in humans with a 1-antitrypsin deficiency. Low levels of a 1-antitrypsin have also been shown in tight skin mice [46], who develop emphysematous lesions at 2–4 weeks of age [47]. Finally, also beige mice spontaneously develop emphysema at 2–4 weeks of age [47,48]. These mice were long considered defective in neutrophil elastase and cathepsin G [49,50], but recent studies have shown that beige mice are capable of releasing these enzymes at normal levels [46,51,52]. 4. Innate immunity and murine models of COPD/ pulmonary emphysema Lipopolysaccharide (LPS or endotoxin) is a strong proinflammatory compound present in the cell wall of Gram-negative bacteria. LPS contains two parts: a polysaccharide part, that is characteristic and unique for each bacterial strain and a lipid part (lipid A), which is the least variable portion of the molecule and is responsible for the endotoxic activity [53]. G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 Bacterial endotoxin was demonstrated to be present in high concentrations in tobacco (approximately 20 mg/ cigarette) and bioactive LPS could be detected in both mainstream and sidestream cigarette smoke (approximately 0.12–0.2 mg/cigarette) [54,55]. The pulmonary inflammation and airway obstruction observed in smokers and patients with COPD could thus partially be attributed to the responses to LPS. Moreover, in healthy human subjects it was demonstrated that experimental inhalation of LPS results in chest tightness, cough, dyspnea, sputum production and an acute decline in FEV1. LPS inhalation induces a pulmonary inflammation in a dose-dependent manner, with increased numbers of neutrophils in bronchoalveolar lavage (BAL) and increased concentrations of TNF-a, IL-1b, IL-6 and IL-8 in BAL fluid [56]. Several mouse models have been described in which acute pulmonary inflammation was evaluated after a single LPS exposure, but these models will not be described here since they study acute LPS lung injury instead of COPD. Perhaps more relevant to the human disease is a chronic LPS model in which mice are exposed to LPS by repetitive intratracheal instillation during 12 weeks [57,58]. This mouse model mimics several important pathological changes that are observed in COPD patients, including goblet cell metaplasia in the larger airways, thickening of the airway walls (increased smooth muscle layer) and irreversible alveolar enlargements (emphysema). The inflammation is characterized by peribronchial and perivascular lymphocytic aggregates (CD4C and CD8CT-lymphocytes, and CD19CB-cells), accumulation of macrophages and CD8CT cells in the parenchyma and altered cytokine expression (increased levels of Th1 cytokines TNF-a, IFN-g and IL-18, measured by RT-PCR). The pulmonary inflammationinduced by chronic LPS exposure persisted up to 8 weeks after the final LPS exposure, which is comparable to the persisting airway inflammation in patients with COPD despite smoking cessation. However, LPS is only one single component of tobacco, which contains more than 4.500 compounds in the particulate and vapour phases, including many other toxic agents such as carbon monoxide, nitrogen oxides, ammonia, acrolein, benzopyrenes, hydroquinone and nicotine [59]. Obviously, the chronic administration of one single component of cigarette smoke (i.e. LPS) in animals cannot mimic all the different aspects of COPD—a complex disease caused by cigarette smoke—in humans. In another model, C3H/HeJ mice which are deficient in TLR4 (the major LPS receptor), showed a reduced pulmonary accumulation of dendritic cells, neutrophils and lymphocytes upon cigarette smoke exposure compared to control mice with a functional TLR4, suggesting that LPS signaling is important in the pulmonary inflammation in COPD [60]. Importantly, despite their defective TLR4-signalling, these C3H/HeJ 159 mice still developed emphysema upon chronic cigarette smoke exposure. 5. COPD/pulmonary emphysema and the oxidant/ antioxidant imbalance Cigarette smoke contains high concentrations of reactive oxygen species (ROS) [61,62]. Increased levels of ROS in airways and lungs upon cigarette smoking originate not only directly from the oxidants in cigarette smoke, but also indirectly from the release of ROS by infiltrating macrophages and neutrophils [61,63]. This excess of ROS disturbs the balance between oxidants and antioxidants, resulting in oxidative stress [64]. Oxidative stress may be important in different aspects of the pathology of COPD, since it could amplify the inflammatory responses, induce apoptosis, impair the function of protective antiproteases and reduce the activity of corticosteroids in the treatment of COPD [65]. In different mouse models, it was demonstrated that cigarette smoke induces oxidative stress. Mice exposed to acute cigarette smoke showed a transient, but significant decrease in antioxidant capacity (TEAC: Trolox Equivalent Antioxidant Capacity) with a decrease in protein thiols and in ascorbic acid in BAL [44]. Cigarette smoke strongly affects the glutathione metabolism, which is an important feature of the antioxidant defence in the lung [66,67]. Cigarette smoke exposure also increases the concentrations of compounds resulting from lipid peroxidation, such as 8-isoprostane and 4-hydroxy-2-nonenal in plasma, BAL and lung [44,68]. Immunohistological analysis after cigarette smoke exposure demonstrated increased amounts of 8-hydroxy-2 0 -deoxyguanosine (indicator of oxidative DNA damage) in bronchiolar and alveolar epithelial cells [68]. Moreover, the cigarette smoke-induced oxidative stress was also detected systemically, e.g. in heart and liver [69,70]. Many of the observations made in cigarette smoke-exposed mice are similar to those obtained in human studies (see reviews by Barnes [65] and MacNee [61]). Cavarra and coworkers [14,44] have demonstrated that the response towards the oxidative effects of cigarette smoke in mice is strain-specific. The oxidant-sensitive strains DBA/2 and C57Bl/6J showed a significant drop of the antioxidant capacity in their BAL fluid in response to acute cigarette smoke exposure [44]. In contrast, in the ICR mice that are not oxidant-sensitive, the antioxidant capacity in the BAL fluid increased. Interestingly, both the DBA/2 and C57Bl/6J mice developed emphysema upon chronic smoke exposure, whereas the ICR mice were largely protected from the deleterious effects of cigarette smoke [14,44]. Another study demonstrated that the oral administration of an antioxidant (a-tocopherol) could reduce the cigarette smoke-induced oxidative stress [71]. An in vivo mouse model was also used to evaluate the effect of oxidative stress on the activity of human trypsin inhibitors 160 G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 [72]. Oxidative stress—applied through acute cigarette smoke exposure—reduced the antitrypsin activity of intratracheally instilled human recombinant secretory leucoprotease inhibitor (hrSLPI), suggesting that this could be an important mechanism in the impaired antiproteolytic activity in COPD. Finally, the crucial protective role of antioxidant systems has been demonstrated in mice with a targeted disruption of the Nuclear factor, erythroid-derived 2, like 2 (Nrf2), a redox-sensitive transcription factor that is involved in the regulation of many detoxification and antioxidant genes [73] (see Table 2). Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive cigarette smoke-induced emphysema compared with wild type animals. Moreover, emphysema in Nrf2-deficient mice exposed to cigarette smoke for 6 months was associated not only with increased levels of markers of oxidative stress, but also with more pronounced BAL inflammation and with an increased number of apoptotic alveolar septal cells [73]. This experimental emphysema model in Nrf2-deficient mice provides a clear link between excessive oxidative stress due to an impaired antioxidant system, and increased inflammation, apoptosis and worsened emphysema (see infra). 6. Pulmonary repair processes and airway remodeling in COPD/pulmonary emphysema Long-term exposure to toxic gases and particles, mostly cigarette smoke, is the primary cause of COPD. Host defenses against these stimuli include innate immune responses (mucociliary clearance, epithelial repair and the acute inflammatory response) and adaptive immune responses (humoral and cellular components). Both types of response are associated with a repair process that remodels damaged tissue by restoring the epithelium and microvasculature and by adding connective-tissue matrix in an attempt to return the tissue to its previous state [74,75]. Unfortunately, this repair process often contributes—in concert with the chronic inflammation—to the complex pathological changes leading to COPD [76]. Cigarette smoke is known to inhibit human bronchial epithelial cell repair processes [77], though normally the epithelium has a tremendous capacity to repair itself following injury [78]. Several groups have shown that transforming growth factor-b (TGF-b), an anti-inflammatory cytokine, is involved in airway repair [79–81]. In an elastase-induced murine model of pulmonary emphysema, the lesions stabilize after the acute phase of tissue damage and repair. Indeed, following endotracheal administration of elastase, expression of the elastin gene was induced and synthesis of elastin in the lungs was increased, resulting in lung elastin levels which were 30% higher than controls 8 weeks after challenge [22]. More recently, McGowan reported that retinoic acid receptor-g knockout mice develop characteristics of emphysema, suggesting a role for retinoic acid in the generation and repair of the pulmonary alveolus [82] (see Table 2). Although Fujita et al. reported that exogenous applied retinoic acid fails to reverse emphysema in adult mouse models [83], Ishizawa et al. [84] showed the opposite. In addition, they demonstrated that besides all-trans-retinoic acid also granulocyte colonystimulating factor (G-CSF) is able to promote lung tissue regeneration in this mouse model of pulmonary emphysema [84]. Mao et al. [85] concluded that all-trans-retinoic acid could modulate the protease/antiprotease balance in a manner that may impact on emphysema pathogenesis. These results raise the possibility that repair mechanisms following injury may be manipulated by exogenous agents, and thus might be important in the search for therapeutic agents for COPD. Importantly, not only local structural cells of the lungs are involved in the repair processes, also stem/progenitor cells derived from the circulation contribute to the repair of lung injury [86]. Using parabiotic mice that were joined surgically and developed a common circulation, Rennard and associates very elegantly demonstrated that after acute lung injury, induced by intratracheal instillation of elastase and/or irradiation, stem/progenitor cells in the blood contributed not only to cells of hematopoietic origin (e.g. interstitial monocytes/macrophages), but also to structural lung cells (e.g. subepithelial fibroblasts and type I alveolar epithelial cells) [86]. The term ‘remodeling’ originates from asthma research and includes all chronic structural alterations in the airway, excluding acute changes due to the recruitment of inflammatory cells or to edema [87,88]. It comprises most changes in anatomy that are not rapidly reversible such as thickening of the airway wall, subepithelial fibrosis, smooth muscle hypertrophy/hyperplasia and hyperplasia of fibroblasts, myofibroblasts and goblet cells. Some murine models of COPD describe such changes indicative of airway remodeling. Transmission electron microscopy demonstrated all the morphological stages of epithelial injury and repair in the bronchi of oxidantsensitive strains of mice after 3 months of cigarette smoke exposure [14]. In particular, areas of deciliation were observed, as well as basal cells that accumulate after epithelial cell detachment and poorly differentiated epithelial cells with short microvilli covering denuded areas. In the chronic LPS-model of COPD and emphysema, airway walls of LPS-exposed mice were thickened when compared with controls, as indicated by an increased width of the smooth muscle layer [58]. Moreover, emphysematous changes and goblet cell metaplasia were observed, whilst the mRNA expression of the Th1 cytokines TNF-a, IFN-g and IL-18 was increased in the lungs of LPS-exposed animals [58]. Some transgenic mice strains with targeted expression of cytokines in the airways under the control of the Clara Cell protein 10 (CC10) promotor (e.g. CC10-IL-11 and G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 CC10-IL-6 mice) show emphysema-like airspace enlargement, thickening of airway walls and subepithelial fibrosis without exposure to a specific agent [89,90]. Interestingly, when IL-13 was overexpressed in the murine lung, an asthma-like eosinophil—and lymphocyte-rich inflammation, goblet cell hyperplasia and airway fibrosis was noted [91]. However, in contrast to what was expected, the CC10-IL-13 transgenic mice also had manifest alveolar enlargement, even if the transgene was only induced in the fully formed lung (to exclude effects of lung-specific overexpression of IL-13 on lung development) [27]. IL-13 thus generates on the one hand airway fibrosis—by inducing and activating TGF-b [92] and on the other hand causes pulmonary emphysema, by inducing the expression of a variety of cathepsins and MMPs [27]. In humans, COPD is characterized by increased mucus production in large airways (mucous gland enlargement) as well as in bronchioles (goblet cell metaplasia) [74]. In mice, submucosal glands were found only in the proximal regions of the trachea at the same density as in humans, but unlike in humans, these glands did not extend below the trachea. Epithelial mucous cells (i.e. goblet cells) in mice are mainly found in the proximal airways, but not in smaller airways such as terminal bronchioles [93]. Recently, goblet cell metaplasia/hyperplasia has been described in a murine model of COPDbased upon chronic cigarette smoke exposure [14]. Comparing two strains of mice sensitive to oxidants, Bartalesi et al. [14] demonstrated that 75% of the animals in the C57Bl/6J group showed a positive periodic acid-Schiff (PAS) reaction of their large or middle size bronchi at 3 and 6 months smoke exposure; in contrast, in the DBA/2 group there were no PASpositive animals at these timepoints. In C57Bl/6J mice, the goblet cell metaplasia correlated with a positive reaction in the airway epithelium on immunohistochemical staining for IL-4, IL-13 and MUC5AC. However, goblet cell metaplasia is a nonspecific phenomenon and can be induced by several other stimuli including ovalbumin challenge [94], LPS exposure [58,95] and intratracheal instillation of neutrophil elastase [96]. These in vivo models are valuable tools to further unravel the mechanisms involved in goblet cell metaplasia (both in asthma and COPD). 7. Apoptosis of lung structural cells: development of pulmonary emphysema without inflammation Retamales et al. [97] showed that smokers who developed severe emphysema had a severalfold increase in the numbers of macrophages, T-lymphocytes, neutrophils and eosinophils in their lungs, compared with persons who smoked similar amounts of cigarettes, but maintained normal lung function. This suggests that people who 161 develop emphysema have an amplified inflammatory response to cigarette smoke, as explicitly mentioned in the definition of COPD by GOLD [4]. Also in murine models of chronic cigarette smoke exposure, there is a clear correlation between the magnitude of the pulmonary inflammation, comprising macrophages, neutrophils and T-cells, and the development of emphysema [13,98,99] (see Fig. 1). Thus both in humans and in mice, the traditional hypothesis for the pathogenesis of emphysema is that cigarette smoke induces an (exaggerated) influx into the lungs of inflammatory cells that release ROS and proteases, causing the degradation of matrix with subsequent loss of attachment and death of structural cells. However, at least three recent studies in mice demonstrated the development of emphysema, despite a remarkable lack of inflammation [68,100,101]. The first model used a single intratracheal injection of active caspase-3 (in Chariot as a protein transfection reagent) to induce emphysematous changes [68]. This study provides direct evidence that alveolar wall apoptosis suffice to cause pulmonary emphysema, even without the accumulation of inflammatory cells [102]. In the second model, the intravascular administration of a vascular endothelial cell growth factor receptor-2 (VEGFR-2) blocker also generated non-inflammatory emphysema [100]. Vascular endothelial cell growth factor (VEGF) is indeed a growth factor required for endothelial cell survival, and blocking VEGF leads to apoptosis of endothelial cells [103]. Chronic VEGFR-2 blockade caused thus alveolar septal cell apoptosis, and airspace enlargement. Petrache et al. [101] very recently reported that ceramide, a second messenger lipid, appears to be a crucial mediator of alveolar destruction in emphysema. Indeed, intratracheal instillation of ceramide in naı̈ve mice induced emphysema, while inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis and emphysema caused by blockade of the VEGF receptors in both mice and rats [101]. These experimental observations in mice are in agreement with recent studies showing that smokers with emphysema have high levels of apoptosis compared to smokers without emphysema [104–106]. Also in smokeexposed mouse lungs, an increase in apoptotic cells (epithelial cells and macrophages) has been demonstrated, and this was associated with enhanced expression of FasL and caspases, suggesting that cigarette smoke-induced apoptosis is mediated by a Fas/FasL death receptor apoptosis pathway [107,108]. Both models put forward a new concept in the pathogenesis of emphysema, in which apoptosis and initial loss of epithelial or endothelial cells can trigger matrix destruction and alveolar space enlargement. This contrasts with the traditional hypothesis of cigarette-smoke induced pulmonary inflammation leading to protease-mediated destruction of alveolar walls. Of course, both hypotheses on the pathogenesis of emphysema are not mutually exclusive, and it is becoming progressively 162 G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 Fig. 1. highlights the different cellular and molecular mechanisms which may be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema. Cigarette smoke can induce chronic inflammation of the airways and lung parenchyma, either directly or indirectly via the bronchial or alveolar epithelium. Several chemokines such as Interleukin-8 (IL-8) and Macrophage Inflammatory Protein-3a (MIP-3a) are released by epithelial cells upon exposure to cigarette smoke, inducing the recruitment of neutrophils and monocytes/macrophages, respectively. (1) These inflammatory cells release various proteases, including neutrophil elastase (NE), cysteine proteases (cathepsins) and matrix metalloproteinases (MMPs), such as macrophage metalloelastase (MMP-12), causing an imbalance between proteases and anti-proteases such as a1-antitrypsin (a1-AT) and Tissue Inhibitor of Matrix metalloproteinase (TIMP). Besides these elastolytic proteases, macrophages can secrete many inflammatory proteins upon activation by cigarette smoke, including Tumor Necrosis Factor-a (TNF-a) and several chemokines such as CXCL9, CXCL10 and CXCL11, which could contribute to the accumulation of CD8CT-lymphocytes, since they preferentially express the chemokine receptor CXCR3. (2) These CD8CT-cells are able to cause further destruction of alveolar epithelial cells, by cytolysis and apoptosis, through the release of granzymes and perforins. (3) Many inflammatory and structural cells in the airways produce reactive oxygen species (ROS) upon activation, leading to oxidative stress when these ROS are generated in excess of the antioxidant defence mechanisms, such as glutathione, superoxide dismutase and surfactant protein-D (SP-D). Of course, these different cellular and molecular pathogenetic mechanisms interact, since oxidative stress may inactivate anti-proteases and may amplify apoptosis as well as the chronic inflammation. apparent that both excessive proteolysis (due to the protease/antiprotease imbalance) and apoptosis of epithelial and endothelial cells in the lungs interact and enforce each others destructive potential (see Fig. 1). 8. Limitations of murine models of human COPD/pulmonary emphysema In vivo murine models can offer valuable information on several aspects of the pathogenesis and treatment of COPD and emphysema. However, as for other animal species, murine models of COPD/emphysema also have several limitations. Firstly, no model mimics the entire COPD phenotype, since many models specifically mimic only one trait of the disease, eg the enlargement of the pulmonary alveoli due to injury to the lung parenchyma (i.e. pulmonary emphysema). However, the pathogenesis of the progressive and fixed airflow limitation due to chronic (small) airway obstruction [74,75], which defines COPD, has not yet been thoroughly addressed in mice. Moreover, each murine model of emphysema has its own specific disadvantages. The most important disadvantages of the protease-induced emphysema is the lack of significant inflammation and the near absence of airway changes such as mucous cell metaplasia. Although there is a significant inflammatory reaction in the cigarette smoke-induced emphysema, this murine model has also several specific limitations, since there is a lack of standardized exposures and of standardized morphometric analyses [9]. Various smoking machines and various exposure regimens are used, but the choice of the smoking regimen is often made arbitrarily [109]. Moreover, breathing smoke generated by a machine resembles passive smoking rather than active smoking. Secondly, there are considerable differences in respiratory physiology and anatomy between mice and humans. In contrast to humans, mice are obligate nose breathers and the submucosal glands in mice are restricted to the trachea. Already during the embryonic stage of lung development, there are important species differences between murine and human lung morphogenesis including pulmonary lobation and bronchial branching (at completion: six airway generations in mice versus 23 airway generations in humans) [110]. Importantly, pulmonary emphysema in humans is classified according to the distribution of the airspace enlargement within the acinus: in the panacinar G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 emphysema—as seen in patients with a1-antitrypsin deficiency—enlargement and destruction of airspaces of the acinar unit are uniform, whereas in centriacinar emphysema—as seen in cigarette smokers—the airspace enlargement occurs primarily in the three generations of respiratory bronchioles [74]. Rodents however do not have clearly defined respiratory bronchioles nor distinct lobular architecture [9]. These distinctions in anatomy have to be taken into account when extrapolating experimental data from murine models to human disease. Lastly, there are also known discrepancies in both innate and adaptive immunity between the human and murine immune system [111]. The discrepancies in innate immunity include the balance of leukocyte subsets, defensins, Toll-like receptors, chemokines and chemokine receptor expression. The differences in adaptive immunity include among other things immunoglobulin subsets, Th1/Th2 differentiation, and costimulatory molecule expression and function [111]. All these physiological, anatomical and immunological differences should be taken into account when using mice as preclinical models of human disease. Acknowledgements This work was funded by the Fund for Scientific Research Flanders (FWO / Project Grant Number 3G001103), the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) and the Concerted Research Initiative of the Ghent University (BOF/ GOA Project Number 01251504). References [1] Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: Global burden of disease study. Lancet 1997;349:1498–504. [2] Pauwels PRA, Rabe KF. Burden and clinical features of chronic obstructive pulmonary disease (COPD). The Lancet 2004;364: 613–20. [3] Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 2001;163:1256–76. [4] Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. http://www.goldcopd.com, 2004. [5] Fletcher C, Peto R. The natural history of chronic airflow obstruction. Br Med J 1977;1:1645–8. [6] Laurell CBES. The electrophoretic alpha-1 globulin pattern of serum in alpha-1 antitrypsin deficiency. Scand J Clin Lab Invest 1963;15: 132–40. [7] Waterston RH, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420: 520–62. 163 [8] Paigen K. A miracle enough: the power of mice. Nat Med 1995;1: 215–20. [9] March TH, Green FH, Hahn FF, Nikula KJ. Animal models of emphysema and their relevance to studies of particle-induced disease. Inhal Toxicol 2000;12(suppl. 4):155–87. [10] Canning BJ. Modeling asthma and COPD in animals: a pointless exercise? Curr Opin Pharmacol 2003;3:244–50. [11] Snider GL, Lucey EC, Stone PJ. Animal models of emphysema. Am Rev Respir Dis 1986;133:149–69. [12] Karlinsky JB, Snider GL. Animal models of emphysema. Am Rev Respir Dis 1978;117:1109–33. [13] Guerassimov A, Hoshino Y, Takubo Y, et al. The development of emphysema in cigarette smoke-exposed mice is strain dependent. Am J Respir Crit Care Med 2004;170:974–80. [14] Bartalesi B, Cavarra E, Fineschi S, et al. Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants. Eur Respir J 2005;25:15–22. [15] Shapiro SD. Animal models for chronic obstructive pulmonary disease. Age of Klotho and marlboro mice. Am J Respir Cell Mol Biol 2000;22:4–7. [16] Mahadeva R, Shapiro SD. Chronic obstructive pulmonary disease * 3: Experimental animal models of pulmonary emphysema. Thorax 2002;57:908–14. [17] van der Vaart H, Postma DS, Timens W, Ten Hacken NHT. Acute effects of cigarette smoke on inflammation and oxidative stress: a review. Thorax 2004;59:713–21. [18] Global Initiative for Asthma. Global Strategy for asthma management and prevention. National Institutes of Health. NIH publication no. 02-3659. 2002. [19] Barnes PJ. Mediators of chronic obstructive pulmonary disease. Pharmacol Rev 2004;56:515–48. [20] Thurlbeck WM. Measurement of pulmonary emphysema. Am Rev Respir Dis 1967;95:752–64. [21] Gross P, Pfitzer EA, Tolker E, Babyak MA, Kaschak M. Experimental emphysema: its production with papain in normal and silicotic rats. Arch Environ Health 1965;11:50–8. [22] Valentine R, Rucker RB, Chrisp CE, Fisher GL. Morphological and biochemical features of elastase-induced emphysema in strain A/J mice. Toxicol Appl Pharmacol 1983;68:451–61. [23] Lafuma C, Frisdal E, Harf A, Robert L, Hornebeck W. Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments. Eur Respir J 1991;4:1004–9. [24] D’Armiento J, Dalal SS, Okada Y, Berg RA, Chada K. Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell 1992;71:955–61. [25] Shiomi T, Okada Y, Foronjy R, et al. Emphysematous changes are caused by degradation of type III collagen in transgenic mice expressing MMP-1. Exp Lung Res 2003;29:1–15. [26] Foronjy RF, Okada Y, Cole R, D’Armiento J. Progressive adultonset emphysema in transgenic mice expressing human MMP-1 in the lung. Am J Physiol Lung Cell Mol Physiol 2003;284:L727–L37. [27] Zheng T, Zhu Z, Wang Z, et al. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J Clin Invest 2000;106:1081–93. [28] Wang Z, Zheng T, Zhu Z, et al. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J Exp Med 2000; 192:1587–600. [29] Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997;277:2002–4. [30] Senior RM, Griffin GL, Mecham RP. Chemotactic activity of elastinderived peptides. J Clin Invest 1980;66:859–62. [31] Hunninghake GW, Davidson JM, Rennard S, Szapiel S, Gadek JE, Crystal RG. Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 1981;212:925–7. 164 G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 [32] Shapiro SD. Elastolytic metalloproteinases produced by human mononuclear phagocytes. Potential roles in destructive lung disease. Am J Respir Crit Care Med 1994;150:S160–S4. [33] Shapiro SD, Goldstein NM, Houghton AM, Kobayashi DK, Kelley D, Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 2003;163: 2329–35. [34] Churg A, Wang RD, Tai H, et al. Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-{alpha} release. Am J Respir Crit Care Med 2003; 167:1083–9. [35] Churg A, Dai J, Tai H, Xie C, Wright JL. Tumor necrosis factoralpha is central to acute cigarette smoke-induced inflammation and connective tissue breakdown. Am J Respir Crit Care Med 2002;166: 849–54. [36] Churg A, Wang RD, Tai H, Wang X, Xie C, Wright JL. Tumor necrosis factor-{alpha} drives 70% of cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med 2004; 200404–22511C. [37] Leco KJ, Waterhouse P, Sanchez OH, et al. Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3). J Clin Invest 2001;108:817–29. [38] Wert S, Jones T, Korfhagen T, Fisher J, Whitsett J. Spontaneous emphysema in surfactant protein D gene-targeted mice. Chest 2000; 117:248S. [39] Wert SE, Yoshida M, LeVine AM, et al. Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. Proc Natl Acad Sci USA 2000; 97:5972–7. [40] Yoshida M, Korfhagen TR, Whitsett JA. Surfactant protein D regulates NF-kappa B and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways. J Immunol 2001;166:7514–9. [41] Morris DG, Huang X, Kaminski N, et al. Loss of integrin alpha(v) beta6-mediated TGF-beta activation causes Mmp12-dependent emphysema. Nature 2003;422:169–73. [42] Martorana PA, Brand T, Gardi C, et al. The pallid mouse. A model of genetic alpha 1-antitrypsin deficiency. Lab Invest 1993;68:233–41. [43] de Santi MM, Martorana PA, Cavarra E, Lungarella G. Pallid mice with genetic emphysema. Neutrophil elastase burden and elastin loss occur without alteration in the bronchoalveolar lavage cell population. Lab Invest 1995;73:40–7. [44] Cavarra E, Bartalesi B, Lucattelli M, et al. Effects of cigarette smoke in mice with different levels of alpha(1)-proteinase inhibitor and sensitivity to oxidants. Am J Respir Crit Care Med 2001;164: 886–90. [45] Takubo Y, Guerassimov A, Ghezzo H, et al. Alpha1-antitrypsin determines the pattern of emphysema and function in tobacco smoke-exposed mice: parallels with human disease. Am J Respir Crit Care Med 2002;166:1596–603. [46] Gardi C, Cavarra E, Calzoni P, et al. Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors. Biochem J 1994;299(Pt 1):237–45. [47] O’Donnell MD, O’Connor CM, FitzGerald MX, Lungarella G, Cavarra E, Martorana PA. Ultrastructure of lung elastin and collagen in mouse models of spontaneous emphysema. Matrix Biol 1999;18: 357–60. [48] Keil M, Lungarella G, Cavarra E, van Even P, Martorana PA. A scanning electron microscopic investigation of genetic emphysema in tight-skin, pallid, and beige mice, three different C57 BL/6J mutants. Lab Invest 1996;74:353–62. [49] Vassalli JD, Granelli-Piperno A, Griscelli C, Reich E. Specific protease deficiency in polymorphonuclear leukocytes of ChediakHigashi syndrome and beige mice. J Exp Med 1978;147:1285–90. [50] Takeuchi K, Wood H, Swank RT. Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G. J Exp Med 1986;163:665–77. [51] Cavarra E, Martorana PA, Cortese S, Gambelli F, Di Simplicio P, Lungarella G. Neutrophils in beige mice secrete normal amounts of cathepsin G and a 46 kDa latent form of elastase that can be activated extracellularly by proteolytic activity. Biol Chem 1997;378:417–23. [52] Cavarra E, Martorana PA, de Santi M, et al. Neutrophil influx into the lungs of beige mice is followed by elastolytic damage and emphysema. Am J Respir Cell Mol Biol 1999;20:264–9. [53] Schletter J, Heine H, Ulmer AJ, Rietschel ET. Molecular mechanisms of endotoxin activity. Arch Microbiol 1995;164:383–9. [54] Hasday J, Dubin W, Fitzgerald T, Bascom R. Cigarettes are a rich source of bacterial endotoxin. Chest 1996;109:63S–64. [55] Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W. Bacterial endotoxin is an active component of cigarette smoke. Chest 1999; 115:829–35. [56] Jagielo PJ, Thorne PS, Watt JL, Frees KL, Quinn TJ, Schwartz DA. Grain dust and endotoxin inhalation challenges produce similar inflammatory responses in normal subjects. Chest 1996;110:263–70. [57] Vernooy JH, Dentener MA, van Suylen RJ, Buurman WA, Wouters EF. Intratracheal instillation of lipopolysaccharide in mice induces apoptosis in bronchial epithelial cells: no role for tumor necrosis factor-alpha and infiltrating neutrophils. Am J Respir Cell Mol Biol 2001;24:569–76. [58] Vernooy JH, Dentener MA, van Suylen RJ, Buurman WA, Wouters EF. Long-term intratracheal lipopolysaccharide exposure in mice results in chronic lung inflammation and persistent pathology. Am J Respir Cell Mol Biol 2002;26:152–9. [59] Sopori M. Effects of cigarette smoke on the immune system. Nat Rev Immunol 2002;2:372–7. [60] Maes TVK, Pauwels R. The toll-like receptor 4 and cigarette-smoke induced pulmonary inflammation and emphysema in mice. Am J Respir Crit Care Med 2004;169:A717. [61] MacNee W. Oxidative stress and lung inflammation in airways disease. Eur J Pharmacol 2001;429:195–207. [62] Pryor WA, Stone K. Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann N Y Acad Sci 1993; 686(12-27):27–8. [63] Rahman I, MacNee W. Role of oxidants/antioxidants in smokinginduced lung diseases. Free Radic Biol Med 1996;21:669–81. [64] Bowler RP, Crapo JD. Oxidative stress in airways: is there a role for extracellular superoxide dismutase? Am J Respir Crit Care Med 2002;166:S38–S43. [65] Barnes PJ, Shapiro SD, Pauwels RA. Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 2003;22: 672–88. [66] Teramoto S, Uejima Y, Teramoto K, Ouchi Y, Fukuchi Y. Effect of age on alteration of glutathione metabolism following chronic cigarette smoke inhalation in mice. Lung 1996;174:119–26. [67] Cantin AM, North SL, Hubbard RC, Crystal RG. Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 1987;63:152–7. [68] Aoshiba K, Yokohori N, Nagai A. Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol 2003;28:555–62. [69] Zhang J, Liu Y, Shi J, Larson DF, Watson RR. Side-stream cigarette smoke induces dose-response in systemic inflammatory cytokine production and oxidative stress. Exp Biol Med (Maywood) 2002; 227:823–9. [70] Howard DJ, Briggs LA, Pritsos CA, Oxidative DNA. damage in mouse heart, liver, and lung tissue due to acute side-stream tobacco smoke exposure. Arch Biochem Biophys 1998;352:293–7. [71] Koul A, Singh A, Sandhir R. Effect of alpha-tocopherol on the cardiac antioxidant defense system and atherogenic lipids in cigarette smoke-inhaling mice. Inhal Toxicol 2003;15:513–22. G.G. Brusselle et al. / Pulmonary Pharmacology & Therapeutics 19 (2006) 155–165 [72] Cavarra E, Lucattelli M, Gambelli F, et al. Human SLPI inactivation after cigarette smoke exposure in a new in vivo model of pulmonary oxidative stress. Am J Physiol Lung Cell Mol Physiol 2001;281: L412–L7. [73] Rangasamy T, Cho CY, Thimmulappa RK, et al. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 2004;114:1248–59. [74] Hogg PJC. Pathophysiology of airflow limitation in chronic obstructive pulmonary disease. The Lancet 2004;364:709–21. [75] Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004;350:2645–53. [76] Siafakas NM, Tzortzaki EG. Few smokers develop COPD. Why? Respir Med 2002;96:615–24. [77] Wang H, Liu X, Umino T, et al. Cigarette smoke inhibits human bronchial epithelial cell repair processes. Am J Respir Cell Mol Biol 2001;25:772–9. [78] Knight DA, Holgate ST. The airway epithelium: structural and functional properties in health and disease. Respirology 2003;8: 432–46. [79] Romberger DJ, Beckmann JD, Claassen L, Ertl RF, Rennard SI. Modulation of fibronectin production of bovine bronchial epithelial cells by transforming growth factor-beta. Am J Respir Cell Mol Biol 1992;7:149–55. [80] Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:S12–S16. [81] Hodge S, Hodge G, Holmes M, Flower R, Scicchitano R. Interleukin-4 and tumour necrosis factor-alpha inhibit transforming growth factor-beta production in a human bronchial epithelial cell line: possible relevance to inflammatory mechanisms in chronic obstructive pulmonary disease. Respirology 2001;6:205–11. [82] McGowan SE. Contributions of retinoids to the generation and repair of the pulmonary alveolus. Chest 2002;121:206S–28. [83] Fujita M, Ye Q, Ouchi H, et al. Retinoic acid fails to reverse emphysema in adult mouse models. Thorax 2004;59:224–30. [84] Ishizawa K, Kubo H, Yamada M, et al. Bone marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema. FEBS Lett 2004;556:249–52. [85] Mao JT, Tashkin DP, Belloni PN, Baileyhealy I, Baratelli F, Roth M D. All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema. Chest 2003;124:1724–32. [86] Abe S, Boyer C, Liu X, et al. Cells derived from the circulation contribute to the repair of lung injury. Am J Respir Crit Care Med 2004;170:1158–63. [87] Caughey GH. Chairman’s summary. Mechanisms of airway remodeling. Am J Respir Crit Care Med 2001;164:S26–S7. [88] Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 2001;164:S28–S38. [89] Tang W, Geba GP, Zheng T, et al. Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest 1996;98: 2845–53. [90] Kuhn 3rd C, Homer RJ, Zhu Z, et al. Airway hyperresponsiveness and airway obstruction in transgenic mice. Morphologic correlates in mice overexpressing interleukin (IL)-11 and IL-6 in the lung. Am J Respir Cell Mol Biol 2000;22:289–95. [91] Zhu Z, Homer RJ, Wang Z, et al. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999;103:779–88. 165 [92] Lee CG, Homer RJ, Zhu Z, et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001;194:809–21. [93] Evans CM, Williams OW, Tuvim MJ, et al. Mucin is produced by clara cells in the proximal airways of antigen-challenged mice. Am J Respir Cell Mol Biol 2004;31:382–94. [94] Shahzeidi S, Aujla PK, Nickola TJ, Chen Y, Alimam MZ, Rose MC. Temporal analysis of goblet cells and mucin gene expression in murine models of allergic asthma. Exp Lung Res 2003;29:549–65. [95] Yanagihara K, Seki M, Cheng PW. Lipopolysaccharide Induces Mucus Cell Metaplasia in Mouse Lung. Am J Respir Cell Mol Biol 2001;24:66–73. [96] Voynow JA, Fischer BM, Malarkey DE, et al. Neutrophil elastase induces mucus cell metaplasia in mouse lung. Am J Physiol Lung Cell Mol Physiol 2004;287:L1293–L302. [97] Retamales I, Elliott WM, Meshi B, et al. Amplification of inflammation in emphysema and its association with latent adenoviral infection. Am J Respir Crit Care Med 2001;164:469–73. [98] Guerassimov A, Hoshino Y, Takubo Y, et al. The development of emphysema in cigarette smoke exposed mice is strain dependent. Am J Respir Crit Care Med 2004;200309–11270C. [99] D’Hulst AVK, Brusselle G, Joos G, Pauwels R. Time course of cigarette smoke-induced pulmonary inflammation in mice. European Respiratory Journal: submitted. [100] Kasahara Y, Tuder RM, Taraseviciene-Stewart L, et al. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 2000;106:1311–9. [101] Petrache I, Natarajan V, Zhen L, et al. Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice. Nat Med 2005;11:491–8. [102] Tuder RM, Petrache I, Elias JA, Voelkel NF, Henson PM. Apoptosis and emphysema: the missing link. Am J Respir Cell Mol Biol 2003; 28:551–4. [103] Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1995;1:1024–8. [104] Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J 2001;17:946–53. [105] Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel N F. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 2001;163: 737–44. [106] Yokohori N, Aoshiba K, Nagai A. Increased levels of cell death and proliferation in alveolar wall cells in patients with pulmonary emphysema. Chest 2004;125:626–32. [107] Kang MLS, Cho K, Kim Y, Tang C, Homer R, Elias J. Apoptosis and Fas/FasL alterations in cigarette smoke induced murine emphysema. Am J Respir Crit Care Med 2004;169:A841 [abstract]. [108] Rangasamy TBS, Mai K, Tuder R. Oligonucleotide microarray analysis of cigarette smoke induced pulmonary emphysema in A/J mice. Am J Respir Crit Care Med 2004;169:A [abstract]. [109] Rennard SI. Cigarette smoke in research. Am J Respir Cell Mol Biol 2004;31:479–a-480. [110] Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, Cardoso WV. The molecular basis of lung morphogenesis. Mech Dev 2000;92:55–81. [111] Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–8.