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
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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.
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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,
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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
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[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).
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