TOXICOLOGICAL SCIENCES 92(2), 545–559 (2006)
doi:10.1093/toxsci/kfl016
Advance Access publication May 12, 2006
Modulators of Cigarette Smoke–Induced Pulmonary
Emphysema in A/J Mice
Thomas H. March,1 Julie A. Wilder, Dolores C. Esparza, Patsy Y. Cossey, Lee F. Blair, Lois K. Herrera,
Jacob D. McDonald, Matthew J. Campen, Joe L. Mauderly, and JeanClare Seagrave
Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108
Received March 15, 2006; accepted May 10, 2006
Mice develop pulmonary emphysema after chronic exposure to
cigarette smoke (CS). In this study, the influence of gender,
exposure duration, and concentration of CS on emphysema,
pulmonary function, inflammation, markers of toxicity, and
matrix metalloproteinase (MMP) activity was examined in A/J
mice. Mice were exposed to CS at either 100 or 250 mg total
particulate material/m3 (CS-100 or CS-250, respectively) for 10,
16, or 22 weeks. Evidence of emphysema was first seen in female
mice after 10 weeks of exposure to CS-250, while male mice did
not develop emphysema until 16 weeks. Female mice exposed to
CS-100 did not have emphysema until 16 weeks, suggesting that
disease development depends on the concentration and duration
of exposure. Airflow obstruction and increased pulmonary compliance were observed in mice exposed to CS-250 for 22 weeks.
Decreased elasticity was likely the major contributor to airflow
obstruction because substantial remodeling of the conducting airways, beyond mild mucous cell hyperplasia, was lacking. Exposure to CS increased the number of macrophages, neutrophils,
lymphocytes (B cells and activated CD4- and CD8-positive T cells),
and activity of MMP-2 and -9 in the bronchoalveolar lavage
fluid (BALF). Treatment with antioxidants N-acetylcysteine or
epigallocatechin gallate (EGCG) did not decrease emphysema
severity, but EGCG slightly decreased BALF inflammatory cell
numbers and lactate dehydrogenase activity. Inflammation and
emphysema persisted after a 17-week recovery period following
exposure to CS-250 for 22 weeks. The similarities of this model
to the human disease make it promising for studying disease
pathogenesis and assessing new therapeutic interventions.
Key Words: A/J mice; animal models; cigarette smoke; pulmonary emphysema; morphometry; pulmonary function.
Chronic obstructive pulmonary disease (COPD) is currently
the fourth leading killer of adults in the United States and will
likely be the third leading cause of death worldwide by 2020
(Viegi et al., 2001). The disease is insidious with most patients
presenting with symptoms only after irreversible lung damage
1
To whom correspondence should be addressed at Lovelace Respiratory
Research Institute, 2425 Ridgecrest Drive, SE Albuquerque, NM 87108. Fax:
(505) 348-8567. E-mail: [email protected].
has occurred, and death often occurs following acute exacerbations (Rodriguez-Roisin, 2000). Cigarette smoking is by far
the biggest cause of COPD; however, not all smokers go on to
develop clinically significant reduction in lung function. Some
studies have suggested that part of this differential susceptibility is due to ‘‘dose’’ of cigarette smoke (CS), as the disease
usually manifests with greater severity after many decades of
smoking (Cosio and Guerassimov, 1999; Viegi et al., 2001).
Other predispositions for the disease, such as genetic differences in extracellular matrix repair and turnover, detoxification, and inflammatory responses, are also being studied.
The incidence of pulmonary emphysema as part of the COPD
complex (including chronic bronchitis) is difficult to assess
because most COPD patients have both emphysema and
bronchitis/bronchiolitis to some degree. Emphysema is primarily
a pathologically described inflammatory disease characterized by
the destruction of parenchyma distal to the terminal bronchioles
without significant fibrosis (Snider, 1986). Palliative treatments
include bronchodilators for improving airflow along with corticosteroids and antibiotics for preventing or treating exacerbations
(Groneberg and Chung, 2004). Reversal of emphysema has been
shown in animal models of the disease by treatment with retinoic
acid (Massaro and Massaro, 1997); however, this has not been
recapitulated in the clinical environment (Mao et al., 2002).
Prevention rather than treatment of emphysema in animals has
been attempted more often, and animals have been treated with
a variety of anti-inflammatory and antiproteolytic drugs either
prior to or concurrently with induction of the disease (Churg et al.,
2003; Martorana et al., 2005; Selman et al., 2003). Whether or not
such drugs might help patients with moderate to severe airflow
obstruction remains to be determined.
Modeling of emphysema in the past used intratracheal
instillation of proteolytic enzymes, such as pancreatic and
neutrophil-derived elastases, to induce the lesion in a variety of
animal species. This proteolytic process leads to destruction of
the extracellular matrix and is purportedly similar to that induced
by inflammatory responses to CS. This type of model has helped
to bolster the hypothesis that emphysema develops from an
imbalance in proteinase and antiproteinase activity (Snider,
1986). More recently, models have been produced by chronically
Ó The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: [email protected]
546
MARCH ET AL.
exposing animals to CS, and strain differences among mice as
well as transgenic animals have provided some insights into the
disease pathogenesis (Groneberg and Chung, 2004). The A/J
mouse develops emphysema more readily following CS exposure than other strains of mice (March et al., 2005).
The primary purpose of this study was to characterize the
time-course for development of emphysema in A/J mice
exposed to CS. We postulated that longer duration of CS
exposure would be associated with development of more severe
disease. A tangential study was also done to determine the
disease response after lowering the concentration of CS over
the same time-course. Further, because there is some indication
that women may be more susceptible to CS-induced COPD
(Langhammer et al., 2003; Viegi et al., 2001), the influence of
gender on the disease process was evaluated. Other assessed
parameters included pulmonary function, inflammation, matrix
metalloproteinase (MMP) activity, and development of mucous
cell hyperplasia in the conducting airways. The ability of CS
from two different types of research cigarettes (filtered vs.
nonfiltered) to produce emphysema was also tested. Additionally, the preventive effect of orally administered antioxidants
on development of the disease was assessed. Finally, mice were
chronically exposed to CS and then held for a ‘‘recovery’’
period in order to assess pulmonary carcinogenesis (Witschi,
2005) and persistence of the emphysema lesion.
MATERIALS AND METHODS
Animals, exposures, and treatments. All procedures were conducted
under protocols approved by an Institutional Animal Care and Use Committee
in facilities accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care International. The study design is depicted in Table 1
and represents CS exposures conducted over a 2-year period. Male and female
A/J mice (Jackson Laboratories, Bar Harbor, ME) at 8–10 weeks of age were
exposed whole body to diluted and slightly aged mainstream CS generated by an
AMESA Type 1300 smoking machine from 1R3 nonfiltered research cigarettes
(27.1 mg total particulate material [TPM] yield per cigarette; University of
Kentucky Tobacco Research and Development Center [UKTRDC], Lexington,
KY) or to filtered air (FA, control) as described (March et al., 2005). Mice were
acclimated to exposure during the first week by delivering CS targeted at 100 mg
TPM/m3 (CS-100) and exposed following acclimation at a concentration of
250 mg TPM/m3 (CS-250) for up to 22 weeks. A group of male mice was exposed to FA or CS-100 during acclimation and to CS-250 for a total of 22 weeks
of exposure and then held unexposed for an additional 17 weeks during a
recovery period (housed in shoe box cages) in a protocol designed primarily to
assess carcinogenicity (Witschi, 2005). Mice in this study were used for
assessment of both emphysema and the carcinogenicity of CS and, thus, to allow
a direct comparison to previous studies (Witschi, 2005), only males were used.
Only female mice were used in additional experimental groups because
preliminary data has suggested that female mice of different strains might be
more susceptible to CS-induced emphysema (March et al., 2005). One group of
female mice was acclimated to CS exposure at a low concentration of 50 mg
TPM/m3 during the first week and then further exposed to CS-100 for 10, 16, or
22 weeks. Two additional groups of female mice, one exposed to CS-250 and
the other exposed to FA for 16 weeks, were treated with the green tea–derived
polyphenol antioxidant epigallocatechin gallate (EGCG; Sigma, St Louis, MO)
by administration of the compound in the drinking water at 50 lg/ml (estimated
dose equivalent to that received by a heavy drinker of green tea; Muto et al.,
1999). Lastly, a group of 8- to 10-week-old female A/J mice was exposed for
the first week to CS-100 and further for a total of 10 weeks to CS-250 from
filtered 2R4F research cigarettes with a nominal yield of 11.7 mg TPM per
cigarette (UKTRDC, Lexington, KY). Emphysema in these mice was
compared to that in mice exposed to CS-250 from the nonfiltered 1R3 research
cigarettes. In addition to monitoring CS TPM, particle sizes were characterized,
and the vapor phase of CS atmospheres from each of the two types of cigarettes
was compared for concentrations of carbon monoxide, nitrogen oxides, and
total hydrocarbons as described (McDonald et al., 2004). A subset of animals
exposed to CS-250 from the filtered research cigarettes was treated with the
antioxidant thiol N-acetylcysteine (NAC) in the drinking water at 1 mg/ml
TABLE 1
Study Design for A/J Mice Exposed to CS
Gender
Duration (weeks)
Emphysema/COPD assessment
Histopathology, morphometry
Pulmonary function
Airway mucosubstances
Inflammation and toxicity assessment
BALF cell counts
BALF MMPs
BALF protein, LDH, and AP
BALF and tissue lymphocytes
CS-250, nonfiltered
cigarettes
FA
Exposure
Male
Female
Male
X
X X
X
X X X X
X
X
X
X X X
X X X
X X X
X
X X
X X
X X
X
CS-250, filtered
cigarettes
Female
Female
Female
22 þ
16 þ
22 þ
16 þ
10 16 22 Recov. 10 16 EGCG 22 10 16 22 Recov. 10 16 EGCG 22
X X X
X
CS-100, nonfiltered
cigarettes
X X
X
X
X
X
X
X
X
X
10
16
22
10
10 þ
NAC
X
X
X
X
X
X
X
X
X
X X X X
X X X X
X X X X
X
X X
X X
X X
X
Note. Procedures performed for individual groups are indicated by cells containing an X. Numbers of animals assessed are reported in the results.
Abbreviations: FA, filtered air exposed (control); CS, cigarette smoke exposed; Recov., recovery period of 17 weeks following exposure (mice were also assessed
for pulmonary tumor development); and þ EGCG or þ NAC, treatment with the antioxidant concurrent to the exposure.
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
(estimated 20-fold greater than effective human dose; Pela et al., 1999;
van Overveld et al., 2005) concurrent to the exposure (Table 1).
Necropsy, lavage, and tissue analysis. Lungs from mice designated for
morphometry of emphysema were inflated with 4% buffered paraformaldehyde
or 10% neutral buffered formalin at a constant hydrostatic pressure of 25 cm for
6 h as described (March et al., 2005). Lungs were fixed further by immersion in
fixative for 48–72 h. The volume of fixed whole lungs (VL) was determined, and
left lung lobes were trimmed, histoprocessed, sectioned, and stained with
hematoxylin and eosin (H&E) as described (March et al., 2005). The amount of
tissue shrinkage due to histoprocessing was determined in five randomly
selected animals per group as described (March et al., 2005).
Sections of lungs were qualitatively assessed by light microscopy for the
severity of emphysema. Emphysema was described as irregular, multifocal expansion of alveolar airspaces and alveolar ducts due to destruction of parenchymal
tissue (Snider, 1986). The irregular nature of airspace enlargement was given
emphasis in that alveolar septal destruction occurred nonuniformly, and alveoli of
normal size were often juxtaposed to greatly expanded airspaces. Morphometry
was used to quantify emphysema as described previously (March et al., 2005).
Briefly, microscopic fields were chosen by a systematic random method, and
emphysema was assessed in these fields with computer-aided measurements. The
most commonly used indicator of alveolar airspace size, the mean linear intercept
(Lm, in lm), was calculated. The Lm was corrected for histoprocessing shrinkage by
dividing it by the group-average linear shrinkage factor. Using the VL and Lm, the
total alveolar surface area (Sa, in cm2) was calculated. From point counting, the
fractional volume densities of alveolar septa (VVspt) and alveolar airspace (VVair)
were also measured and multiplied by VL to yield total volume of a particular
component for the lung. The ratio of Sa to VL was also evaluated. Additionally, in
a randomly selected subset of female mice from the 16-week time point exposed to
FA or CS-250 (Table 1), sections of 5 lm thickness from the paraffin blocks
described above were stained with alcian blue (at pH ¼ 2.5 for acid mucosubstances) in combination with periodic acid-Schiff (AB-PAS). The volume density
of epithelial mucosubstances was then determined (March et al., 1999) in crosssections of the axial airway within the two largest lung sections (comprising the
airway near the hilus).
In separate groups of animals (Table 1), mice were killed, tracheas cannulated,
left main stem bronchi temporarily clamped, and right lungs lavaged three times
with 0.5 ml phosphate-buffered saline (PBS). The bronchoalveolar lavage fluid
(BALF) was assessed for the number of inflammatory cells and the activity of
MMPs assessed in the noncellular supernatant fraction as described (March et al.,
2002; Seagrave et al., 2004). Lavage fluid was also analyzed as described (March
et al., 2002) for amount of total protein as well as the activities of lactate
dehydrogenase (LDH) and alkaline phosphatase (AP) as indicators of injury. In
another group of mice, whole lungs were lavaged three times with 0.7 ml of PBS,
and whole-lung tissue was enzyme digested as described (Wilder et al., 2001) with
the modification that tissue was treated with a proteinase mixture (Liberase
Blendzyme 3, Roche Applied Science, Indianapolis, IN) at 0.28 mg/ml in 5% FBS/
RPMI media and with DNAse (Sigma, 30 lg/ml) for 60 min. Total cells from
BALF (0.2 3 106 to 0.6 3 106 per sample) and digested tissue (1 3 106 per sample)
were counted with a hemacytometer and then incubated for 10 min on ice with
FcBlock (rat anti-mouse CD16/CD32) and subsequently immunostained for
subsets of activated T lymphocytes using CD4-FITC (GK1.5) and CD8-PerCP
(Ly2) in combination with either CD62L-PE (phycoerythrin) (Mel-14) and CD25APC (allophycocyanin) (PC61) or CD69-PE (H1.253) and CD44-APC (PGP-1,
Ly24). Antibody CD45R/B220-PerCP (RA3-6B2) was used to stain B lymphocytes in a separate tube. All antibodies were purchased from BD Biosciences (San
Diego, CA), and their appropriately labeled rat or hamster (CD69-PE only)
immunoglobulin isotype controls were used to stain pools of BALF and lung cells.
Data were collected on a BD Biosciences FACSCalibur and analyzed using
CellQuest Pro software. Gates were set to enumerate small mononuclear
leukocytes and to distinguish those cells expressing fluorescence above that
observed from isotype control–stained cells. Data were reported as the absolute
number of specifically stained cells in a given sample.
Pulmonary function testing. Pulmonary function testing was performed
on mice after 22 weeks of exposure to CS-250 essentially as described
547
(Harkema et al., 1996). The number of surviving female mice originally
designated for pulmonary function tests was few, such that statistically
powerful results from females were precluded. Thus, male mice were
anesthetized with an ip injection of xylazine and ketamine (0.01 and 0.08
mg/kg, respectively), orotracheally intubated with a 22-gauge catheter, and
maintained under inhalation anesthesia at a baseline respiration frequency of
110–120 breaths/min with 1.5–2.5% halothane in a 50:50 oxygen:nitrous oxide
mix. Mice were placed in a heated plethysmograph box (approximately 500 ml
volume). Transpulmonary pressure (Ptp) was measured with a fluid-filled 22gauge esophageal cannula connected to a differential pressure transducer
(MPX11DP, Motorola, Phoenix, AZ). Respiratory flows were measured with
a pneumotachograph composed of a differential pressure transducer (MP45,
Validyne, Northridge, CA) connected across nine layers of stainless steel, 316mesh wire cloth covering a 0.7-cm-diameter hole in the plethysmograph wall.
The Ptp and flow signals were conditioned by preamplifiers (Buxco
Electronics, Sharon, CT) and routed to a PC-based monitor and data collection
system (AcqKnowledge 3.7.2, BioPac Systems, Inc, Goleta, CA). Once stable,
the values for flow, volume, and frequency during spontaneous breathing were
measured, and tidal volume (VT), minute volume (VE), total pulmonary
resistance (RL), and dynamic lung compliance (Cdyn) were calculated. Static
lung compliance (Cstat) was measured by observing the volume needed to
inflate lungs to Ptp ¼ 20 cm H2O. The diffusing capacity for carbon monoxide
(DLCO) was then measured by a modified single-breath method as described
(Harkema et al., 1996). Following this, mice were injected with 70 lg/kg
vecuronium bromide (Ben Venue Labs, Inc, Bedford, OH) ip to arrest
spontaneous breathing. A quasistatic deflation from total lung capacity to
residual volume was performed by inflating the lung to a pressure of þ 30 cm
H2O and deflating slowly until cessation of flow occurred. Inspiratory capacity,
vital capacity, and the expiratory reserve volume were determined. Quasistatic
chord compliance (Cqs) was calculated from slopes of the steepest portion of
the volume versus Ptp curves, covering a pressure range of 3.4–8.0 cm H2O (see
the ‘‘Results’’ section). A forced expiration maneuver was done by inflating the
lung to þ 30 cm H2O and deflating to approximately 45 cm H2O. Forced vital
capacity (FVC) and peak expiratory flow rate (PEFR) were calculated, and
volumes and rates at various time points were related to the FVC (i.e., forced
expiratory volume at 0.05 s [FEV0.05] as a percentage of FVC). Two or three
forced maneuvers were made per mouse, and the average values were reported.
Mice were euthanized following the procedure.
Statistical analysis. Several comparisons were made by the general linear
models ANOVA procedure (SAS Institute, Inc, Cary, NC) often using three
independent variables (e.g., exposure, gender, and duration). Data were tested
for normal distribution (Kolmogorov-Smirnov test) and equal variance
(Levene’s median test or two-sample F-test). Where the tests failed ( p <
0.05), data were transformed by ranking prior to ANOVA. Interactions of the
independent variables were of particular interest. For example, the effect of an
interaction between exposure and duration might have suggested progressive
worsening of the disease process over time. Pairwise comparisons between
individual groups, post hoc to ANOVA, were performed using a least square
means procedure with the Tukey-Kramer adjustment for multiple comparisons,
and the significance level was set at p < 0.05. In other instances, end points
from two groups (FA control and CS exposed, generally) were compared by
Student’s two-tailed unpaired t-test or the nonparametric Mann-Whitney rank
sum test with statistical significance set at p < 0.05.
RESULTS
Histopathology and Morphometry
Effect of gender, exposure duration, and CS concentration. Comparisons were made between male and female mice
exposed either to FA or to CS-250 for 10, 16, or 22 weeks.
548
MARCH ET AL.
Similar to previous results (March et al., 2002, 2005), CS
exposure caused mild to moderate, irregular, multifocal
enlargement of airspaces throughout the parenchyma, such
that alveoli of normal size were often juxtaposed to large,
expanded airspaces (Fig. 1). The organization of parenchymal
acini was often disrupted or unrecognizable. Female mice
exposed to CS-250 had features of irregular alveolar airspace
enlargement at 10 weeks while effects in male mice were not
readily apparent until 16 weeks (Fig. 1). Female mice exposed
to CS-100 did not have substantial airspace enlargement at the
10-week time point, but at 16 and 22 weeks, emphysema in
these mice was morphologically similar to that of female mice
exposed to CS-250 (Fig. 1).
An increase in the Lm was regarded as the main morphometric indicator of emphysema because it is the best estimate
of alveolar size in this type of morphometry. For the CS-250
exposure, ANOVA demonstrated that the Lm was affected by an
interaction between the independent variables of exposure (FA
vs. CS-250), gender, and duration of exposure (10–22 weeks;
Table 2). Pairwise tests demonstrated that the CS-induced
increase in Lm was significant only in females and was different
from FA control values at each of the time points. In contrast,
CS-250 caused an increase in Lm in the lungs from male mice
only at the 16- and 22-week time points, but these increases
were not significant by pairwise tests. This suggested that the
response was more robust in females, in parallel to the
histopathologic findings (Fig. 1). Age-related enlargement of
alveoli was seen in FA- and CS-250–exposed mice of both
genders. The significant effect of an interaction between
duration and exposure in females suggested that alveolar
enlargement was different than expected from either aging or
CS exposure alone. However, the severity of the emphysema in
the females did not clearly worsen during the study period
because the CS-induced increase in Lm over mean FA control
values was similar at 10, 16, and 22 weeks (24, 25, and 22%
enlargement, respectively).
Other morphometric measures of the parenchyma included
the total volume of alveolar septa (VVspt 3 VL), alveolar
airspace (VVair 3 VL), and alveolar surface area (Sa). As seen in
previous experiments (March et al., 2002, 2005), CS-250
caused a significant increase in the VVair 3 VL of approximately
1.2-fold in males and 1.3-fold in females when averaged over
the entire exposure period (Table 2). The magnitude of increase
in VVair 3 VL was similar to the CS-induced increase in VL.
There were also increases in the volume of alveolar septa (VVspt
3 VL) associated with CS exposure, and in males this increase
was similar to the increase in VL (1.2-fold, averaged over the
whole exposure period). The VVspt 3 VL in females increased
only slightly (1.1-fold) consistent with the enlargement of VL
being primarily due to increased parenchymal airspace rather
than an increase in septal tissue. Similarly, there was a significant effect of exposure on the total alveolar surface area (Sa),
attributable primarily to the increased Sa in the CS-exposed
males at the 10-week time point (Table 2). Differences in Sa
FIG. 1. Histopathology of emphysema in male and female A/J mice
exposed to FA, CS-100, or CS-250 for 10 or 16 weeks. Males, exposed only to
CS-250, and females, exposed to both levels of CS, also had histologic evidence
of moderate emphysema at the 22-week time point (for example in males, see
Fig. 5). Photomicrographs at low magnification are from parenchymal regions
lateral to the hilus of the left lung. H&E; bars ¼ 200 lm.
were not substantial at the other time points in either gender
and in fact were not apparent in the females at the 16- and
22-week time points.
The ratio of alveolar surface area to lung volume (Sa:VL) was
assessed as an indicator of tissue loss per unit volume of lung.
The ratio was decreased at all time points for the CS-exposed
females, but only significantly at the 10-week time point (Table 2).
Note. All values are the means ± SDs for n ¼ 6–8 per group. Data for parameters marked with an asterisk were ranked prior to ANOVA because of nonnormal distribution and/or unequal variances.
Abbreviations: Lm, mean linear intercept; VL, lung volume; Sa, total alveolar surface area; VVspt 3 VL, total volume of alveolar septal tissue; VVair 3 VL, total volume of alveolar airspace (cf also
Table 1).
a
Significant effects of exposure, duration, or gender are indicated by E, D, or G, respectively (p < 0.05, three-way ANOVA). Significant interactions between the independent variables (p < 0.05) are
indicated as products (e.g., E 3 D 3 G). Individual effects of the independent variables are obvious (e.g., CS-250 decreased BW, BW increased with duration, and males had greater BW). Where three-way
interactions are present, significant differences between groups by pairwise tests are indicated by the superscript letters b–d applied to values in a row as described below (p < 0.05, least square means test); b,
different from gender- and exposure-matched previous time point; c, different from gender- and duration-matched FA control; d, different from duration- and exposure-matched male mice.
E3D3G
E, D, G
E, D3G
E3D3G
E, D3G
E, D
E, D
E, D, G
10.5b,c
0.14
107
60b
0.03
0.16
24
2.1
±
±
±
±
±
±
±
±
75.4
1.56
731
462
0.19
1.18
231
18.6
±
±
±
±
±
±
±
±
7.2c,d
0.20
149
73
0.03
0.20
13
1.6
±
±
±
±
±
±
±
±
58.0
1.43
867
609
0.15
1.10
215
18.0
±
±
±
±
±
±
±
±
7.8c
0.08
126
87c
0.02
0.09
13
1.5
±
±
±
±
±
±
±
±
50.5
1.38
945
685
0.16
1.01
185
16.7
±
±
±
±
±
±
±
±
13.4
0.10
108
75
0.02
0.06
36
1.6
±
±
±
±
±
±
±
±
79.6
1.69
750
446
0.19
1.24
218
20.8
±
±
±
±
±
±
±
±
10.1b
0.09
129
86b
0.03
0.10
13
1.2
±
±
±
±
±
±
±
±
71.3
1.50
752
501
0.20
1.12
213
20.0
±
±
±
±
±
±
±
±
3.7
0.11
94
48
0.02
0.08
20
1.4
±
±
±
±
±
±
±
±
42.8
1.36
1105
815
0.19
0.97
190
19.8
±
±
±
±
±
±
±
±
Lm (lm)*
VL (cm3)*
Sa (cm2)
Sa:VL (cm1)*
VVspt 3 VL (cm3)
VVair 3 VL (cm3)*
LW (mg)
BW (g)
Duration (weeks)
47.0
1.20
897
749
0.15
0.90
130
22.0
FA
Exposure
4.9
0.11
77
81
0.02
0.11
22
1.3
10
CS-250
60.8
1.23
722
561
0.18
0.90
139
23.6
FA
5.8b
0.06
100
67b
0.03
0.08
16
1.2
CS-250
65.0
1.39
716
520
0.17
0.99
168
23.8
FA
5.0
0.15
71
50
0.03
0.11
42
1.5
22
CS-250
40.8
1.04
912
874
0.15
0.78
137
19.5
FA
4.0
0.09
137
94
0.03
0.10
21
0.7
10
CS-250
46.5
1.14
877
740
0.15
0.86
139
20.6
FA
3.8d
0.15
112
77d
0.02
0.08
8
1.0
16
16
CS-250
Females
Males
Gender
TABLE 2
Morphometry Parameters of Emphysema in A/J Mice Exposed to CS for Varying Durations
61.6
1.31
725
554
0.15
0.97
156
21.4
FA
3.4b
0.19
110
34b
0.02
0.16
23
1.8
22
CS-250
Effectsa
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
549
The ratio for the males was decreased to a lesser degree (16
and 22 weeks, only), but the decreases were not significant.
Other morphometric parameters in the gender-duration experiment, including lung weight (LW) and body weight (BW),
were also affected by CS-250 exposure, gender, and duration
but without significant interaction among the variables (Table
2). The CS-250 exposure increased LW and decreased BW,
while gender and duration influences were as expected for
male and female mice aging over a 3-month period.
Morphometry of lungs from mice that were exposed to CS100 for 10 to 22 weeks revealed responses intermediate to those
elicited by exposure at the high concentration of CS-250.
Significant enlargement of Lm in the CS-100 group was
demonstrated, and similar to the histopathologic findings
(Fig. 1), this was apparent after 16 or 22 weeks of exposure,
but not after 10 weeks (Fig. 2A). There was no interaction of
the CS exposure with duration. Instead, Lm averaged over the
entire period in the CS-100–exposed mice was significantly
greater than that of the FA control group and significantly less
than that induced by exposure to CS-250 (p < 0.05, least square
means test). This was reiterated by VL enlargement (Fig. 2B).
The ratio of Sa:VL was also significantly decreased by CS
exposure at both concentrations, but there was no difference for
this parameter between the two CS exposure levels (Fig. 2C).
The VVair 3 VL was not increased significantly by CS-100 in
contrast to the increase apparent in the CS-250 group (Fig. 2D).
The VVspt 3 VL was not affected by CS exposure but was
affected by duration with the average value at 16 weeks lower
than at the 22-week time point (Fig. 2E). Significant CSinduced decreases in BW followed a pattern seen with the Lm;
mice exposed to CS-100 had mean weights that were between
those of the FA- and CS-250–exposed groups (Fig. 2F). In
summary, exposure to CS-100 was associated with intermediate enlargement of Lm and VL (11% greater than FA for both
parameters over the entire period) compared to that induced by
CS-250 (23 and 26% greater than FA, respectively), suggesting
induction of less severe emphysema.
Histopathologic inflammation and mucus production. Mild
to moderate pulmonary inflammation was present in all CSexposed mice and was characterized by exudates of macrophages and neutrophils among cell debris in scattered alveolar
lumina (Fig. 3). Inflammatory cells were numerous in airspaces
near broncholoalveolar duct junctions. Alveolar septa were
sometimes thickened by infiltrates of similar cells along with
other mononuclear leukocytes. Infiltrates of macrophages, lymphocytes, and fewer neutrophils were common in the perivascular interstitium; thin-walled venules deep in the parenchyma
were often affected (Fig. 3). Smaller numbers of mononuclear
leukocytes and rare neutrophils were often present in the interstitium around terminal bronchioles and associated arterioles
and lymphatic vessels. Larger airways were unaffected or only
slightly affected by inflammation; however, the number of
cells containing mucus was increased in the lining epithelium
550
MARCH ET AL.
FIG. 2. Morphometry of lungs from female mice exposed to two different concentrations of CS. Mice were exposed to FA (control), CS-100, or CS-250 for 10,
16, or 22 weeks. Bars are the means (þ SD) for n ¼ 6–8 per group. Data for FA and CS-250 (see Table 2) are also depicted here for comparison purposes. Data for
VL and Sa:VL were ranked prior to two-way ANOVA. While there are no significant interactions of the independent variables of exposure and duration, statistically
significant effects of each by itself are indicated by the letters E or D, respectively (p < 0.05, two-way ANOVA), in the upper portion of each graph. Differences due
to exposure by pairwise comparison are indicated by the labels a and b on the legend of each graph. Likewise, the effect of duration is indicated by the superscript
letters c and d on the x-axis labels (p < 0.05, least square means test; see below). a: Different from FA control; b: different from CS-100; c: different from 10-week
time point; and d: different from 16-week time point.
of sections stained with AB-PAS (Fig. 4). These cells also
had more abundant amounts of mucus per cell. Morphometry
revealed a fivefold increase in the volume density of ABPAS–positive mucosubstances in CS-exposed mice over FA
controls (Fig. 4).
Effect of a recovery period. The effect of a recovery period
on emphysema was assessed in male mice exposed to CS-250
for 22 weeks and then held without exposure for an additional
17 weeks. One purpose of this experiment was to determine the
carcinogenicity of mainstream CS in the A/J mouse in a protocol similar to that established previously (Witschi, 2005).
Mice exposed to FA for 22 weeks then held for 17 weeks had
a 17% incidence (five mice of n ¼ 29 survivors) of lung tumors
characterized as adenomas (photomicrographs not shown),
while those exposed to CS-250 for 22 weeks and held for
a 17-week recovery had a tumor incidence of 20% (six of n ¼
30; p ¼ 1.0, Fisher’s exact test, comparison to FA). The lungs
of one mouse in the FA group had two tumors while the
remaining four mice with lung tumors in the group each had
one. Mice with lung tumors in the CS-250 group each had only
one tumor. Tumor incidence in this experiment was low for
control animals, but multiplicity (number of tumors per lung)
was similar in comparison to previous work (Witschi, 2005).
Histopathologically, irregular airspace enlargement, alveolar
septal destruction, and inflammation were present in lungs
from the CS-250 plus recovery group, and the severity of the
emphysema was similar to that of mice exposed to CS-250 for
22 weeks alone (Fig. 5).
In corroboration of the histopathology (Fig. 5), morphometry demonstrated a significant increase in the Lm in mice held
for recovery (Table 3). The increase was paralleled by
significant increases in the VL and the VVair 3 VL of
approximately 1.2-fold compared to the FA-exposed control
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
FIG. 3. Inflammation in the parenchyma (A) and parabronchiolar interstitium (B) of female mice exposed to CS-250 for 16 weeks. In (A),
macrophages and neutrophils are numerous and mixed with cell debris in
alveolar lumina (filled arrow), while small numbers of lymphocytes and
macrophages are present in the interstitium around a venule (ve). Septal
fragments are evident (open arrows) as are breaks in alveolar septa (arrowheads). In (B), vacuolated macrophages with pigmented cytoplasm and
lymphocytes (arrows) are present in the interstitium juxtaposed to a terminal
bronchiole (tb) and an arteriole (ar). H&E; bars ¼ 50 lm.
group. These fold increases were similar to those in CS-250–
exposed males at the 22-week time point (Table 2). Unlike the
measures in males at the 22-week time point, the VVspt 3 VL
and Sa were not increased, and the Sa:VL was significantly
decreased. Another notable exception was that the mean BW of
mice formerly exposed to CS-250 was no different from that of
the FA group (Table 3). Thus, the recovery period allowed
regains in BW, while restoration of the damaged pulmonary
parenchyma did not occur.
Effect of antioxidant treatment and CS from filtered
cigarettes. Female A/J mice were treated with the polyphenol
EGCG concurrently with exposure to CS-250 for 16 weeks.
Emphysema was similar in all CS-exposed mice regardless of
the EGCG treatment (photomicrographs not shown). The lack
of a treatment effect was corroborated by morphometry (below,
Table 4). Female A/J mice were also exposed to CS-250 from
filtered, 2R4F research cigarettes for 10 weeks. Emphysema in
551
FIG. 4. Mucosubstances stained with AB-PAS in the epithelium of the
main axial airways of female mice exposed to (A) FA (control) or (B) CS-250
for 16 weeks. Cells in the lining epithelium are more numerous in CS-exposed
mice (arrows). The inset depicts the volume density of mucosubstances
measured by morphometry (pl/mm2 of basement membrane; mean þ SD,
n ¼ 5 per group). Mucosubstance density in the CS-250–exposed mice is
significantly greater than that in FA-exposed mice (p ¼ 0.01, rank sum test).
AB-PAS; bars ¼ 50 lm.
these animals was both histopathologically (photomicrographs
not shown) and morphometrically similar (below, Table 4) to
that induced by exposure to CS-250 from nonfiltered 1R3
research cigarettes. This suggested that equivalent development of the disease did not depend on the type of cigarette used
to generate the exposure atmospheres, as long as concentrations of particulate material were similar. A subset of animals
exposed to CS from the 2R4F cigarettes was also treated with
NAC throughout the 10-week duration. The antioxidant had no
ameliorating effect on the histopathologic evidence of emphysema (photomicrographs not shown) or the morphometry for
alveolar airspace expansion (below, Table 4).
Particulate material concentrations (target of 250 mg TPM/m3),
particle sizes, and yields of vapor-phase components from
the filtered and nonfiltered cigarettes were compared. Particulate concentrations between the two types of cigarettes were
not different (1R3s: 250 ± 23 TPM/m3, 2R4Fs: 237 ± 15 TPM/m3,
n ¼ 12 samplings each; p > 0.05, t-test). By impactor
552
MARCH ET AL.
nificantly greater (32%) vapor-phase concentrations of total
hydrocarbons were generated from the filtered cigarettes
(1R3s: 44 ± 4 ppm, 2R4Fs: 58 ± 4 ppm, n ¼ 6 each; p < 0.0001,
t-test) at the target concentration of 250 mg TPM/m3.
Emphysema was similar after exposure to CS-250 from
filtered and nonfiltered cigarettes for 10 weeks based on
enlargement of the Lm (Table 4). Additionally, neither NAC
nor EGCG prevented the increase in Lm associated with CS250 exposure (Table 4). Increases in Sa (NAC) and VVspt 3 VL
(EGCG) in CS-exposed mice were paralleled by increases in
VL. However, the alveolar airspace (VVair 3 VL) was also
significantly greater in the NAC-treated, CS-exposed mice at
1.1-fold over the mean value for mice exposed to CS-250 alone
and 1.5-fold over that of FA-exposed mice (Table 4), suggesting that much of the VL enlargement could be attributed to an
increase in alveolar airspace.
FIG. 5. Emphysema in male A/J mice exposed to FA (top) or CS-250
(bottom) for either 22 weeks (left) or for 22 weeks followed by a 17-week
recovery period (22 þ 17 weeks, right). The irregular alveolar airspace
expansion in CS-250–exposed lung parenchyma persists regardless of the
recovery period. H&E; bars ¼ 200 lm.
analysis, 66 and 83% of the particulate mass in CS-250 from
1R3 and 2R4F cigarettes, respectively, was from particles
less than 500 nm in diameter. By differential mobility
analysis in the 15- to 600-nm range, particles in the two types
of CS were of similar size (count median diameter of 240 nm
and geometric standard deviation [GSD] of 1.5 in each;
calculated mass median aerodynamic diameter ¼ 400 nm and
GSD ¼ 1.4, assuming unit density). Vapor-phase samples from
each of the cigarettes contained similar concentrations of
carbon monoxide (1R3s: 326 ± 6 ppm, n ¼ 5; 2R4Fs: 345 ± 25
ppm, n ¼ 6; p > 0.05) and nitrogen oxides (1R3s: 4.7 ± 0.5
ppm, 2R4Fs: 5.2 ± 0.4 ppm, n ¼ 6 each; p > 0.05). SigTABLE 3
Morphometry of Emphysema in Male A/J Mice Exposed to
Either FA or CS for 22 Weeks Followed by a 17-Week
Recovery Period of No Exposure
FA
Lm (lm)
VL (cm3)
Sa (cm2)
Sa:VL (cm1)
VVspt 3 VL (cm3)
VVair 3 VL (cm3)
LW (mg)
BW (g)
78.1
1.38
914
475
0.13
1.12
144
26.6
±
±
±
±
±
±
±
±
CS-250
9.7
0.13
115
75
0.02
0.09
21
1.9
96.0
1.67
988
385
0.14
1.38
170
25.3
±
±
±
±
±
±
±
±
11.8
0.15
177
56
0.03
0.12
21
1.7
a
p
0.009
0.002
0.018b
< 0.001
0.031
Note. All values are the means ± SDs for n ¼ 8 per group. For abbreviations,
see Table 2.
a
Only values for p < 0.05, CS-250 versus FA (Student’s t-test), are given.
b
Wilcoxon two-sample exact test because of nonnormal distribution.
Pulmonary Function Tests
Male mice were assessed for changes in pulmonary function
after 22 weeks of exposure to CS-250. This exposure caused
a mild decrease in forced expiratory flow rate and an increase in
pulmonary compliance (Table 5, Fig. 6). Notably, PEFR was
reduced, the FVC was increased, and both the FEV0.05 and the
FEV0.05/FVC ratio were significantly decreased in the CS-250
group. Because histopathology demonstrated no obstruction of
the conducting airways, the decreased airflow might have been
primarily due to decreased elastic recoil (increased compliance; Table 5, Fig. 6), which has been suggested as typical for
rodent models of ‘‘pure’’ emphysema (Costa et al., 1992). The
mean DLCO in animals exposed to CS-250 was not different
from that of FA-exposed controls. This suggested that the
exposure caused no net loss in gas exchange tissue; this was
consistent with the morphometry results on Sa (above).
However, DLCO normalized to the volume necessary for lung
inflation at 20 cm H2O pressure was 15% less in the CSexposed mice (data not shown, p ¼ 0.03, t-test).
BALF Analysis
Inflammatory cells. The number of BALF inflammatory
cells was increased at all time points by CS exposure when
comparing gender and duration effects of exposure to CS-250
for 10 to 22 weeks (Table 6). There were no differences
between genders in the inflammatory response at any time
point. Differential cell counts showed that most of the inflammatory cells were macrophages and neutrophils in approximately equal numbers. Few lymphocytes were observed.
The CS-250 exposure was associated with 90- to 1800-fold
elevations in BALF neutrophils, and lymphocytes were elevated 14- to 80-fold compared to FA-exposed mice. Macrophages were increased by CS-250 exposure three- to eightfold
over mean control values. The effect of exposure interacted
with that of duration on the neutrophil and lymphocyte counts,
553
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
TABLE 4
Morphometry of Emphysema in Female A/J Mice Exposed to FA or CS from Filtered or Nonfiltered Research Cigarettes
and the Effect of Antioxidant Treatments
Exposure
FA
CS-250
CS-250
CS-250
FAa
CS-250a
FA
CS-250
Cigarette type
—
Nonfiltered
Filtered
Filtered
—
Nonfiltered
—
Nonfiltered
Duration (weeks)
10
10
10
10
16
16
16
16
None
None
None
NAC
None
None
EGCG
EGCG
Antioxidant
treatment
Lm (lm)
VL (cm3)
Sa (cm2)
Sa:VL (cm1)
VVspt 3 VL (cm3)
VVair 3 VL (cm3)
LW (mg)
BW (g)
39.3
0.71
644
904
0.18
0.44
144
20.2
±
±
±
±
±
±
±
±
3.1
0.09
93
80
0.04
0.05
28
0.8
44.1
0.89
711
800
0.21
0.57
188
18.0
±
±
±
±
±
±
±
±
3.1d
0.07d
104
84
0.04
0.08d
28
1.0d
44.0
0.84
716
850
0.22
0.57
177
17.4
±
±
±
±
±
±
±
±
3.2d
0.08d
79
74
0.04
0.07d
29
1.7d
45.2
0.96
795
827
0.24
0.65
176
17.8
±
±
±
±
±
±
±
±
pb
4.5d
0.003
0.08d,e < 0.001
90d
0.03
96
0.04
0.07d,e < 0.001
23
0.9d
< 0.001
46.5
1.14
877
740
0.15
0.86
139
20.6
±
±
±
±
±
±
±
±
3.8
0.15
112
77
0.02
0.08
8
1.0
58.0
1.43
867
609
0.15
1.10
215
18.0
±
±
±
±
±
±
±
±
7.2
0.20
149
73
0.03
0.20
13
1.6
52.9
1.22
794
653
0.13
0.90
142
20.9
±
±
±
±
±
±
±
±
8.1
0.08
116
99
0.02
0.06
14
1.2
58.8
1.53
946
621
0.17
1.20
208
18.4
±
±
±
±
±
±
±
±
8.3
0.07
125
80
0.02f
0.05
23
1.3
Effects, pc
E, 0.002
E, < 0.001
E, 0.01
E 3 T, 0.02
E, < 0.001
E, < 0.001
E, < 0.001
Note. All values are the means ± SD for n ¼ 6–9 per group. For abbreviations compare also Table 2.
Data from Table 2 for comparison purposes.
b
Significant effect of exposure-treatment group is indicated with the p value when < 0.05 (one-way ANOVA). Significant differences between groups by
pairwise comparison are indicated by the superscript letters d and e applied to the values as described below (p < 0.05, least square means test); d, different from
FA-exposed controls; and e, different from filtered CS-250 plus no NAC treatment.
c
Significant effects of CS exposure (E) are indicated with the p value when < 0.05 (two-way ANOVA), and the exposure effect is obvious (e.g., CS increased
Lm compared to FA regardless of EGCG treatment). There is no effect of treatment with EGCG (T) on any of the parameters, except for a significant interaction
of treatment with exposure (E 3 T) on VVspt 3 VL. In this instance, significant differences between groups by pairwise comparison are indicated by the
superscript letter f as described below (p < 0.05, least square means test); f, different from FA-exposed controls plus EGCG treatment.
a
generally because of a decrease in cell numbers (in females)
from 16 to 22 weeks, but neutrophil numbers were similar at
each of the time points.
Treatment with the antioxidant EGCG was associated with
a significant reduction in BALF total inflammatory cells and
macrophages without an interaction with exposure, i.e., cell
numbers averaged from both the CS- and FA-exposed groups
were lessened by EGCG treatment in comparison to groups that
received no treatment (Fig. 7A). The slight reduction of
neutrophils by EGCG treatment in BALF from CS-exposed
mice was not significant in comparison to untreated CSexposed mice (Fig. 7A). The CS-induced elevation of lymphocytes was not affected by EGCG treatment. Cells in BALF
from male A/J mice exposed to CS-250 for 22 weeks and then
held for a 17-week recovery period were also counted. The CSinduced elevations in neutrophils and lymphocytes were
persistent after the recovery period (Fig. 7B), albeit at much
lower levels when compared to the male mice exposed to CS250 for 22 weeks (Table 6).
BALF lymphocyte subsets. Exposure to CS-250 for 16
weeks increased the number of BALF mononuclear inflammatory cells as measured by flow cytometry, but the corollary was
not evident in the lung tissue (Table 7). Total cells in the lung
tissue digests (inflammatory cells, including neutrophils, and
other cell types) were significantly increased by CS-250
exposure. The insignificant reduction in lung tissue mononuclear leukocytes may have been in part due to the infiltration
of neutrophils as observed in histologic sections. The CSinduced increase in BALF mononuclear cells was paralleled by
increases in all the CD4- and CD8-positive T lymphocytes
including activated cells (CD69 and CD25 positive, low
expression of CD62L) and memory cells (CD44 positive, high
expression). In lung tissue, total CD4- and CD8-positive cells
were slightly but insignificantly decreased, and only some of
the subsets of activated lymphocytes were significantly increased. Interestingly, the CD4:CD8 ratio in cells from lung
tissue was decreased by 17% ( p ¼ 0.0495); in the BALF an 11fold increase in the same ratio was induced by CS exposure
( p ¼ 0.0460, rank sum tests). The B220-positive B lymphocytes were significantly elevated only in the BALF after exposure to CS-250 (Table 7).
BALF protein and MMPs. Exposure of both genders of
mice to CS-250 for 10 to 22 weeks elevated total protein and
increased activities of LDH and AP in the cell-free BALF
supernatant (data not shown). The BALF protein was affected
by a significant interaction between the independent variables
of exposure, duration, and gender ( p ¼ 0.02, ANOVA on
ranked data). However, protein concentrations among the CSexposed mice were approximately threefold greater than those
for FA controls in both genders and at all time points. Similarly,
554
100
101
3
2
±
±
±
±
±
±
±
±
42
41
4
1
3620
2239
1301
81
±
±
±
±
447
307
303b
30b
260
258
1
1
±
±
±
±
83
82
1
1
2060
1132
908
19
±
±
±
±
755
447
307b,c
14b,c
480 ± 75
477 ± 70
4±5
0
3838
2227
1579
32
±
±
±
±
997
631
418b
42b
392
389
2
1
FA
CS-250
FA
CS-250
FA
CS-250
Note. All values (thousands of cells/ml BALF) are the means ± SDs for n ¼ 5–6 per group. Values were ranked prior to ANOVA. For abbreviations, see Table 1.
a
Significant effects of exposure or duration are indicated by E or D, respectively (p < 0.05). There are no significant effects of gender (p > 0.05, three-way ANOVA). Significant differences associated
with either E or D alone are obvious here, where CS exposure elevates total cells and macrophages regardless of gender and duration, and the duration effect is associated with a decline in total cells and
macrophages from 16 to 22 weeks. Significant effects of an interaction between E and D (p < 0.05) are indicated as products (E 3 D), and for these, differences between groups by pairwise comparison
are indicated by the superscript letters b and c applied to values in a row as described below (p < 0.05, least square means test); b, different from gender- and duration-matched, FA-exposed controls; and
c, different from gender- and exposure-matched previous time point.
554
365
222b,c
6b,c
±
±
±
±
±
±
±
±
233
226
6
1
563
406
365b
45b
±
±
±
±
CS-250
16
FA
418
412
2
1
544
385
376b
21b
±
±
±
±
3995
2143
1824
28
493 ± 118
492 ± 118
1±2
0
Total cells
Macrophages
Neutrophils
Lymphocytes
FIG. 6. Pulmonary function in male mice exposed to CS at 250 mg TPM/
m3 (CS) or FA (control) for 22 weeks. (A) Quasistatic deflation curves for two
individual mice showing change in volume and Ptp. Quasistatic compliance
(Cqs ¼ Dvolume/DPtp) is from the steepest, most linear portion of the sigmoid
curve over a DPtp range that varied among all mice (minimum ¼ 3.4, maximum
¼ 8.0, and mean range ¼ 5.0 ± 1.1 [SD] cm H2O). Aside from increased Cqs,
the volume necessary to inflate the lung to capacity (Cstat) is greater in the CSexposed mouse (1.4 vs. 0.8 ml in FA; group data in Table 5). (B) Mean flow rate
versus mean flow volume (± SD) for FA-exposed (n ¼ 9) and CS-exposed (n ¼
7) mice during forced expiration maneuvers. The PEFR for each group is
indicated by asterisk and is significantly less in the CS-exposed mice (see Table
5). Other measurements in the graph were made at 50, 75, and 90% of the FVC.
CS-250
CS-250 caused approximately twofold elevations over FA
control mean values in the activity of LDH without any striking
differences between the genders and time points despite a
significant effect of a three-way interaction ( p ¼ 0.003, ranked
data). The AP activity was approximately three- to fourfold
FA
Note. All values are the means ± SDs. Abbreviations: VT, tidal volume; VE,
minute volume; f, breathing frequency; RL, total pulmonary resistance; Cdyn,
Cstat, and Cqs, dynamic, static, and quasistatic compliance, respectively; DLCO,
carbon monoxide diffusing capacity; IC, inspiratory capacity; ER, expiratory
reserve; VC, vital capacity; Ptp, transpulmonary pressure; and FEV0.05, forced
expiratory volume in 0.05 s (cf also Table 2).
a
Only values for p < 0.05, CS-250 versus FA, are given (Student’s t-test).
b
Values of the CS-250–exposed mice for Cstat and those values below are for
n ¼ 7, i.e., measurements on spontaneous breathing were made on only six of
the mice.
Exposure
0.030
0.049
0.035
< 0.001
10
0.026
0.045
3533
1992
1462
79
FA
58
50
9
1
1634
1087
534
14
CS-250
22
0.023
0.012
22
0.052
3.3
3.6
0.061
0.019
0.008b
0.004
0.14
0.06
0.17
0.032
1.2
0.21
16.4
0.19
15.1
16
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
10
0.211
11.6
54.4
0.170
0.059
0.052
0.027
1.17
0.20
1.37
0.104
5.4
1.36
41.8
0.60
44.7
Duration
(weeks)
0.054
2.9
2.5
0.089
0.007
0.006
0.006
0.24
0.10
0.20
0.013
0.9
0.19
9.9
0.10
6.3
Females
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
Males
0.196
10.6
55.4
0.176
0.041
0.042
0.026
0.98
0.16
1.13
0.078
4.8
1.12
56.1
0.78
69.4
pa
Gender
VT (ml)
VE (ml/min)
f (breaths/min)
RL (cm H2O-min/ml)
Cdyn (ml/cm H2O)
Cstat (ml/cm H2O)
DLCO (ml/min/mm Hg)
IC (ml)
ER (ml)
VC (ml)
Cqs (ml/cm H2O)
Ptp range for Cqs (cm H2O)
FVC (ml)
PEFR (ml/s)
FEV0.05 (ml)
FEV0.05/FVC (%)
CS (n ¼ 6)
TABLE 6
Inflammatory Cells in the BALF of Male and Female A/J Mice Exposed to CS for up to 22 Weeks
FA (n ¼ 9)
Effectsa
TABLE 5
Pulmonary Function in Male A/J Mice Exposed to
CS-250 for 22 Weeks
E, D
E, D
E3D
E3D
MARCH ET AL.
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
FIG. 7. Inflammatory cells in BALF from (A) female mice exposed to FA
(control) or CS-250 for 16 weeks and treated concurrently with EGCG
(þEGCG) or not (No Rx, data also present in Table 6) and (B) male mice
exposed to FA or CS-250 for 22 weeks and then held for a 17-week recovery
period. Bars are the means (þ SD) for n ¼ 6 per group. In (A), there is no effect
of an interaction between exposure and EGCG treatment for total cells,
macrophages, or lymphocytes (p > 0.05, ANOVA on ranked data), but
significant effects of exposure are present and indicated by the label a on the
legends (p < 0.05, least square means test, see below). Likewise, there is an
effect of EGCG treatment on total cells and macrophages which is indicated by
the superscript letter b on the x-axis labels (p < 0.05, least square means test;
see below). For neutrophils, there is an interaction between exposure and
treatment (p < 0.05, ANOVA), and bars are labeled with the letters a or c to
indicate significant pairwise differences (p < 0.05, least square means test; see
below). In (B), there are significant differences between FA and CS-250 for
both neutrophils and lymphocytes (p < 0.05, rank sum test; see below),
suggesting persistence of inflammation following the 17-week recovery period.
a: Different from FA control; b: different from No Rx; and c: different from
CS-250 groups and from FA plus No Rx.
greater in most CS-exposed groups and was affected by
interactions between exposure and duration ( p ¼ 0.001, ranked
data) and between exposure and gender ( p ¼ 0.006). Similar to
total protein and LDH, there were no large differences in AP
activity among the groups that might indicate strong gender or
duration influences despite some statistically significant pairwise differences among the groups.
Part of the CS-induced elevation of proteins in the BALF
supernatant was due to increases in MMPs. The activities of
555
MMP-2, a proenzyme form of MMP-2, and MMP-9 were
measured by zymography. Due to variation between the assays,
duration effects could not be analyzed, but exposure to CS-250
elevated MMP-2 activity four- to eightfold in both genders at
all time points (data not shown, p < 0.0001, two-way ANOVA
on ranked data for exposure and gender effects). The proenzyme form of MMP-2 was also elevated four- to sevenfold
over FA control values by exposure to CS-250 at the three time
points (data not shown, p < 0.0001, two-way ANOVA on
ranked data). The greatest increase in CS-induced activity
occurred with MMP-9, where mean CS-250 values were up to
35-fold greater than FA control values (data not shown, p <
0.0001, ANOVA, ranked data). Significant effects of gender
were evident for MMP-9 activity at 16 weeks (females greater
than males) and for all the analyzed MMP activities at
22 weeks (males greater than females), although none of these
gender-related differences were large.
Protein and enzyme activities were also measured in BALF
supernatant from female mice exposed to CS-250 for 16 weeks
and treated concurrently with EGCG and results compared to
those from the mice described above. The CS exposure
increased protein concentration and activities of LDH, AP,
and the MMPs without regard to the EGCG treatment (data not
shown; p < 0.0001 for the exposure effect, two-way ANOVA
on ranked data). The EGCG treatment reduced LDH activity
slightly in BALF from both FA- and CS-exposed mice (7 and
21% reductions, respectively; p ¼ 0.001 for the treatment
effect, ANOVA on ranked data) but did not affect total protein
concentration or AP activity (data not shown). These results
suggested that EGCG might protect cells from some CSinduced toxicity. In contrast, EGCG treatment increased the
activities of MMP-2 ( p < 0.0001) and the proenzyme form of
MMP-2 ( p < 0.05 for the treatment effect, ANOVA on ranked
data) in both FA- and CS-exposed mice by four- to sevenfold
(data not shown).
DISCUSSION
The major conclusions from these studies are that CSinduced emphysema occurs more readily in female mice than
in males, the emphysema severity is dependent on the
concentration of CS TPM, and the morphology of emphysema
in mice is corroborated by functional changes that are
characteristic of COPD. Mucous cell hyperplasia and hypertrophy are present in the epithelium of large, conducting
airways of CS-exposed mouse lungs, and inflammatory cell
infiltrates and elaboration of MMPs in lungs from CS-exposed
mice are similar to those observed in human smokers with
COPD (Finkelstein et al., 1995; Hunninghake and Crystal,
1983; Jeffery, 1999; Ohnishi et al., 1998; Segura-Valdez et al.,
2000). Further, allowing mice to recover from CS exposure is
not associated with reversal of emphysema, and CS-induced
pulmonary inflammation persists in recovery. The induction of
556
MARCH ET AL.
TABLE 7
Lymphocyte Subsets in Lung Tissue Digests and BALF from Female Mice Exposed to CS for 16 Weeks
Lung tissue cells
FA
Total cells (3 105)
Mononuclear cells (3 105)
CD4þ T cells (total, 3 105)
Plus CD62Lþ (low)
Plus CD69þ
Plus CD25þ
Plus CD44þ (high)
CD8þ T cells (total, 3 105)
Plus CD62Lþ (low)
Plus CD69þ
Plus CD25þ
Plus CD44þ (high)
CD4:CD8 ratio
B220þ B cells (3 105)
49.17
27.38
8.77
3.20
0.84
0.52
5.32
4.35
0.84
0.24
0.05
1.83
2.03
7.85
±
±
±
±
±
±
±
±
±
±
±
±
±
±
CS-250
6.83
3.89
0.85
0.16
0.22
0.14
0.64
0.74
0.38
0.03
0.01
0.57
0.20
1.91
56.33
18.21
6.84
3.50
1.59
0.75
5.61
4.01
1.26
0.90
0.23
2.40
1.69
4.44
±
±
±
±
±
±
±
±
±
±
±
±
±
±
18.39
8.89
3.72
1.39
0.26
0.22
2.30
2.05
0.15
0.32
0.14
0.56
0.08
2.41
BALF cells
pa
0.048
0.050
0.048
0.050
0.050
0.050
FA
13.36
0.157
0.002
0.004
0.005
0.002
0.014
0.017
0.018
0.007
0.005
0.012
0.16
0.014
±
±
±
±
±
±
±
±
±
±
±
±
±
±
CS-250
5.07
0.119
0.001
0.003
0.003
0.002
0.003
0.008
0.007
0.006
0.008
0.009
0.07
0.016
24.23
1.876
0.426
0.408
0.231
0.158
0.707
0.267
0.221
0.128
0.055
0.279
1.80
0.228
±
±
±
±
±
±
±
±
±
±
±
±
±
±
10.08
0.771
0.124
0.116
0.046
0.037
0.220
0.126
0.096
0.040
0.018
0.100
0.71
0.023
pa
0.050
0.046
0.050
0.046
0.050
0.050
0.050
0.050
0.050
0.050
0.050
0.046
0.050
Note. Whole lungs were lavaged and enzyme digested. Total cells were counted by light microscopy before immunostaining and analysis by flow cytometry.
Values are the means (± SD) of cells found either in the whole volume of BALF or within the whole lung digest for n ¼ 3 per group. Abbreviations: FA, filtered
air exposed (control). For CD (cluster of differentiation) definitions, see text.
a
For each sample type, values from CS-250–exposed mice were compared to those from FA-exposed control mice. Only values for p 0.05 are given (rank
sum test).
this lesion by CS makes the mouse a relevant model to study
human emphysema (Groneberg and Chung, 2004).
Unpublished studies in our institution have suggested that
female mice of different strains are more susceptible than male
mice to CS-induced emphysema, and enhanced susceptibility
of women to CS-induced COPD has also been suggested
(Langhammer et al., 2003; Viegi et al., 2001). Our previously
published work, however, demonstrated no effect of gender on
the induction of emphysema in CS-exposed A/J mice (March
et al., 2005). Results presented here (Table 2) are seemingly
contradictory, but in the previous work, the effect of gender
was assessed at only one time point (March et al., 2005), and
males in the present study required longer exposure than
females in order to develop significant (and slightly less severe)
emphysema. Because of the duration effect, the morphometric
analysis was better able to discern the influence of gender on
CS-induced emphysema.
Structural changes in the pulmonary parenchyma of the CSexposed mice of this study were supported by functional
changes typical of COPD, including increased compliance and
mild decrements in expiratory flow. Other investigators described decreased pulmonary ‘‘elastance’’ (increased compliance) in the AKR mouse following exposure to CS for
6 months, but no elastance change was observed in similarly
exposed A/J mice or mice of several other strains (Guerassimov
et al., 2004). Other investigations also showed no correlation
between compliance and pathologic features of emphysema in
CS-exposed A/J mice (Foronjy et al., 2005). A reason for the
contrary findings depicted here is not apparent, but the
exposure procedures here may have induced emphysema at
a severity detectable by function tests.
Indicators of pulmonary inflammation in CS-exposed mice,
particularly the CS-induced elevation in CD8-positive T lymphocytes, were similar to findings in people. Numerous CD8positive T lymphocytes have been found in the interstitium and
mucosa of large and small conducting airways as well as in the
parenchyma of human smokers with COPD, and the number of
these cells has been correlated with the severity of decrements
in FEV over time (Cosio et al., 2002; O’Shaughnessy et al.,
1997; Saetta et al., 1998, 1999). The CD8-positive cells have
been speculated to be either directly responsible for tissue
destruction or the orchestrators of other types of destructive
inflammatory cells in a process analogous to an autoimmune
disease (Cosio et al., 2002).
Neutrophil and macrophage numbers in lung tissue, sputum,
or BALF from humans with COPD vary depending on methodology, and such discrepancies have led to confusion about
roles of specific cell types in the pathogenesis of the disease
(Cosio et al., 2002). Some studies have demonstrated increased
neutrophils in the BALF of COPD patients (Hunninghake and
Crystal, 1983; Martin et al., 1985; Thompson et al., 1989),
while neutrophils in histopathologically examined parenchymal tissue of human COPD patients have not always correlated
well with severity of emphysema (Finkelstein et al., 1995;
Saetta et al., 1999). In the current experiments, there were no
large differences among the CS-exposed groups in the number
of BALF inflammatory cells over the 10- to 22-week duration
(Table 6). Subsets of T cells, however, were measured only at
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
16 weeks. If infiltrates of CD8-positive cells confer the
progressive worsening of the disease, as implicated in humans,
then infiltrates of such cells early in the exposure of animals
could serve as a biomarker for identifying when the balance is
tipped toward production of the disease.
The lack of an increase in macrophages and neutrophils from
10 to 22 weeks might be similar to a response demonstrated
previously in CS-exposed mice (Gairola, 1986), where BALF
total cells and neutrophils gradually increased from 1 to 9
weeks of exposure to reach a plateau in cell numbers from 10 to
16 weeks. This inflammation plateau may be related to the
pathogenesis of the emphysema lesion because there was no
progressive enlargement of parenchymal airspace from the 10to the 22-week time point. Perhaps this apparent inflammation
plateau allowed reparative processes to occur or that the
inflammation severity was not sustained above a threshold that
would allow progression of emphysema, and additional
airspace enlargement from 10 to 22 weeks was mostly related
to normal aging processes as indicated in FA-exposed mice. In
similarity, progressive worsening of the disease does not
always occur in human smokers with COPD (Groneberg and
Chung, 2004).
Interestingly, emphysema and low levels of inflammation
persisted in male mice held for a 17-week recovery period
following 22 weeks of CS exposure. This is similar to findings
in people who have quit smoking (Hogg et al., 2004).
Persistence of emphysema following recovery has also been
demonstrated in a CS-induced guinea pig model (Wright and
Sun, 1994). Inflammation in the ‘‘recovered’’ lung suggested
that ongoing tissue destruction might be responsible for
persistence of emphysema. Obviously, proinflammatory stimuli remained in the lungs after the recovery period. Persistent
inflammatory cell infiltration and the subsequent release of
proteinases might have been induced by chemotactic peptides
from the damaged parenchymal extracellular matrix (Houghton
et al., 2006). Whether or not the persistent inflammation
counteracted any reparative process requires further study.
Other biomarkers that might be predictive of emphysema
pathogenesis could include quantitative measures of MMP
activity. Proteolysis of the extracellular matrix in the parenchyma might change during development of the lesion. There
was no apparent progression of the disease over the limited
observation period. However, MMP activity might have
reached a critical level during the initial development of
parenchymal damage. Past results indicate that activity of
MMPs in BALF is increased to a similar extent as observed
here after 6 weeks of CS exposure (Seagrave et al., 2004), and
MMPs at earlier time points need evaluation.
The effect of the CS concentration on emphysema development was a major interest in this study. Deposition in the
respiratory tract of a mouse exposed to CS-250 was estimated
to be 450 mg TPM/kg BW/week (March et al., 2005). This is
equivalent to a person smoking approximately 15 packs of high
tar-yielding cigarettes/day (e.g., 1R3 research cigarettes)
557
assuming that the entire particulate yield of a cigarette is
inhaled by the smoker. With this massive concentration, mice
develop the lesion after a much shortened exposure period
relative to humans. Reducing the concentration of CS was done
in an attempt to generate a concentration-response effect in
mice. Histopathologically, mice exposed to CS-100 had little or
no emphysema after 10 weeks of exposure, while their
counterparts exposed to CS-250 readily developed the lesion.
With longer duration, the lower concentration did cause
emphysema (Figs. 1 and 2). There was a significant trend by
regression analysis for increasing Lm with increasing CS
concentration and duration (data not shown). However, the
exposure caused a plateau of the emphysema response in mice
exposed to CS-250 for 10–22 weeks, i.e., mice exposed to CS250 had a similar increase in the Lm above FA controls at all
time points. Thus, exposing female A/J mice for more than
10 weeks (6 h/day, 5 days/week) at 250 mg TPM/m3 is not
necessary, while exposing them at 100 mg TPM/m3 requires
16 weeks or more to attain significant emphysema.
The shortcoming of the A/J mouse model was that it did not
mimic all features of COPD or emphysema in humans. The
histopathologic findings of emphysema were generally mild to
moderate in severity. There was no apparent loss of gas
exchange tissue as measured morphometrically by Sa and VVspt
3 VL and physiologically by DLCO. However, in this and past
experiments (March et al., 2002, 2005), CS did not, in most
instances, cause any increase in Sa or VVspt 3 VL despite
significant increases in VL. This suggested that overt hyperplasia and hypertrophy of parenchymal tissue did not occur as
might be expected in rodents following inhalation exposure to
toxicants (Costa et al., 1992). Alveolar septa within enlarged
lungs may have been stretched because of decreased elastic
recoil, and this might have given the false impression of
increased septal tissue. The morphometry was inadequate to
discern tissue stretching because the intercept counting method
did not account for alveolar septal thickness.
Another shortcoming of the model was the lack of substantial histopathologic change in the conducting airways. The
lungs from CS-exposed mice lacked one of the major
components of COPD, namely inflammation of the large
conducting airways (chronic bronchitis). There were minimal
inflammatory cell infiltrates around terminal bronchioles and at
broncholoalveolar duct junctions, but extensive inflammation
and remodeling of the small airways were also lacking. Such
small airway disease is regarded as the major contributor to
airflow obstruction in people with COPD (Hogg et al., 2004).
Anatomic differences might explain some of this lack of
correlation because mice lack cartilaginous support structures
and submucosal mucous glands in the largest intrapulmonary
conducting airways and have fewer generations of airway
branches preceding the parenchyma. Despite the lack of
inflammation, there was a significant increase in mucosubstances in mice exposed to CS for 16 weeks (Fig. 4). The
increase was not substantial given that it did not result in any
558
MARCH ET AL.
demonstrable airway obstruction; however, the mucous cell
hyperplasia and hypertrophy paralleled those seen in humans
with COPD.
Treatment of CS-exposed mice with the antioxidants EGCG
or NAC did not prevent the development of emphysema in CSexposed mice. The green tea–derived polyphenol EGCG decreased inflammatory cells in the BALF to a modest extent. In
COPD patients, treatment with the mucolytic sulfhydryl antioxidant NAC has decreased pulmonary inflammation (van
Overveld et al., 2005), but in the current studies inflammation
in CS-exposed mice treated with NAC was not quantified. In
corroboration of the present work, NAC was ineffective in
preventing exacerbations and lung function decline in COPD
patients (Decramer et al., 2005), while the opposite effect was
demonstrated in other work (Pela et al., 1999). Neither doses for
the antioxidants in the present studies were optimized nor were
any pharmacokinetic analyses performed to determine effects of
CS exposure on these drugs, and the high concentrations of CS
used may have overwhelmed any protective effect of these
compounds. Thus, the effect of antioxidants on COPD in general
and emphysema in particular requires further clarification.
The pathologic and physiologic changes in CS-exposed mice
of the current set of studies were similar to findings in people
with COPD. Emphysema is a pathologically defined disease,
while its functional consequence of expiratory airflow obstruction is manifested in the clinical syndrome of COPD. The
progression of some aspects of the disease in people can be
monitored with pulmonary function, but the pathogenesis of
parenchymal tissue destruction is not as easily evaluated.
Several important candidate biomarkers of cellular and biochemical changes reflecting the pathogenesis of the disease were
characterized in this study. The biomarkers of inflammation,
particularly CD8-positive T-cell infiltrates, and of tissue destruction, such as expression of MMPs, may prove to be useful
surrogates for the histopathology of emphysema. Through better
characterization in animals, these and other markers may predict
the course of the disease and may be useful in future studies of
COPD in animals and humans exposed to CS.
Jeffery, P. K. (1999). Inflammation in chronic obstructive lung disease. Am. J.
Respir. Crit. Care Med. 160, S3–S4.
ACKNOWLEDGMENTS
Langhammer, A., Johnsen, R., Gulsvik, A., Holmen, T. L., and Bjermer, L.
(2003). Sex differences in lung vulnerability to tobacco smoking. Eur. Respir.
J. 21, 1017–1023.
The authors wish to thank the excellent technical staff for production of this
article including those in the Animal Care, Aerosol Science, and Technical
Communications Units. This work was funded by the Tobacco Master
Settlement through a cooperative research agreement with the University of
New Mexico.
REFERENCES
Churg, A., Wang, R. D., Xie, C., and Wright, J. L. (2003). a-1-Antitrypsin
ameliorates cigarette smoke-induced emphysema in the mouse. Am. J.
Respir. Crit. Care Med. 168, 199–207.
Cosio, M. G., and Guerassimov, A. (1999). Chronic obstructive pulmonary
disease. Inflammation of small airways and lung parenchyma. Am. J. Respir.
Crit. Care Med. 160, S21–S25.
Cosio, M. G., Majo, J., and Cosio, M. G. (2002). Inflammation of the airways
and lung parenchyma in COPD. Chest 121, 160S–165S.
Costa, D. L., Tepper, J. S., and Raub, J. A. (1992). Interpretations and limitations
of pulmonary function testing in small laboratory animals. In Treatise on
Pulmonary Toxicology, Volume I: Comparative Biology of the Normal Lung
(R. A. Parent, Ed.), pp. 367–399. CRC Press, Inc., Boca Raton, FL.
Decramer, M., Rutten-van Molken, M., Dekhuijzen, P. N., Troosters, T.,
van Herwaarden, C., Pellegrino, R., van Schayck, C. P., Olivieri, D., Del
Donno, M., De Backer, W., et al. (2005). Effects of N-acetylcysteine on
outcomes in chronic obstructive pulmonary disease (Bronchitis Randomized
on NAC Cost-Utility Study, BRONCUS): A randomised placebo-controlled
trial. Lancet 365, 1552–1560.
Finkelstein, R., Fraser, R. S., Ghezzo, H., and Cosio, M. G. (1995). Alveolar
inflammation and its relation to emphysema in smokers. Am. J. Respir. Crit.
Care Med. 152, 1666–1672.
Foronjy, R. F., Mercer, B. A., Maxfield, M. W., Powell, C. A., D’Armiento, J.,
and Okada, Y. (2005). Structural emphysema does not correlate with lung
compliance: Lessons from the mouse smoking model. Exp. Lung Res. 31,
547–562.
Gairola, C. G. (1986). Free lung cell response of mice and rats to mainstream
cigarette smoke exposure. Toxicol. Appl. Pharmacol. 84, 567–575.
Groneberg, D. A., and Chung, K. F. (2004). Models of chronic obstructive
pulmonary disease. Respir. Res. 5, 18.
Guerassimov, A., Hoshino, Y., Takubo, Y., Turcotte, A., Yamamoto, M.,
Ghezzo, H., Triantafillopoulos, A., Whittaker, K., Hoidal, J. R., and Cosio,
M. G. (2004). The development of emphysema in cigarette smoke-exposed
mice is strain dependent. Am. J. Respir. Crit. Care Med. 170, 974–980.
Harkema, J. R., Mauderly, J. L., and Griffith, W. C. (1996). Pulmonary function
alterations in F344 rats following chronic ozone exposure. Inhal. Toxicol. 8,
163–183.
Hogg, J. C., Chu, F., Utokaparch, S., Woods, R., Elliott, W. M., Buzatu, L.,
Cherniack, R. M., Rogers, R. M., Sciurba, F. C., Coxson, H. O., et al. (2004).
The nature of small-airway obstruction in chronic obstructive pulmonary
disease. N. Engl. J. Med. 350, 2645–2653.
Houghton, A. M., Quintero, P. A., Perkins, D. L., Kobayashi, D. K., Kelley,
D. G., Marconcini, L. A., Mecham, R. P., Senior, R. M., and Shapiro, S. D.
(2006). Elastin fragments drive disease progression in a murine model of
emphysema. J. Clin. Investig. 116, 753–759.
Hunninghake, G. W., and Crystal, R. G. (1983). Cigarette smoking and lung
destruction. Accumulation of neutrophils in the lungs of cigarette smokers.
Am. Rev. Respir. Dis. 128, 833–838.
Mao, J. T., Goldin, J. G., Dermand, J., Ibrahim, G., Brown, M. S., Emerick, A.,
McNitt-Gray, M. F., Gjertson, D. W., Estrada, F., Tashkin, D. P., et al. (2002).
A pilot study of all-trans-retinoic acid for the treatment of human
emphysema. Am. J. Respir. Crit. Care Med. 165, 718–723.
March, T. H., Barr, E. B., Finch, G. L., Nikula, K. J., and Seagrave, J. C. (2002).
Effects of concurrent ozone exposure on the pathogenesis of cigarette
smoke-induced emphysema in B6C3F1 mice. Inhal. Toxicol. 14, 1187–1213.
March, T. H., Bowen, L. E., Finch, G. L., Nikula, K. J., Wayne, B. J., and
Hobbs, C. H. (2005). Effects of strain and treatment with inhaled all-transretinoic acid on cigarette smoke-induced pulmonary emphysema in mice.
COPD: J. Chronic. Obstruct. Dis. 2, 289–302.
March, T. H., Kolar, L. M., Barr, E. B., Finch, G. L., Ménache, M. G., and
Nikula, K. J. (1999). Enhanced pulmonary epithelial replication and axial
airway mucosubstance changes in F344 rats exposed short-term to mainstream cigarette smoke. Toxicol. Appl. Pharmacol. 161, 171–179.
MODULATORS OF CS-INDUCED PULMONARY EMPHYSEMA IN A/J MICE
Martin, T. R., Raghu, G., Maunder, R. J., and Springmeyer, S. C. (1985). The effects
of chronic bronchitis and chronic air-flow obstruction on lung cell populations
recovered by bronchoalveolar lavage. Am. Rev. Respir. Dis. 132, 254–260.
Martorana, P. A., Beume, R., Lucattelli, M., Wollin, L., and Lungarella, G.
(2005). Roflumilast fully prevents emphysema in mice chronically exposed
to cigarette smoke. Am. J. Respir. Crit. Care Med. 172, 848–853.
559
T-lymphocytes in the peripheral airways of smokers with chronic
obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 157, 822–826.
Seagrave, J. C., Barr, E. B., March, T. H., and Nikula, K. J. (2004). Effects of cigarette
smoke exposure and cessation on inflammatory cells and matrix metalloproteinase activity in bronchoalveolar lavage fluid of mice. Exp. Lung Res. 30, 1–15.
Massaro, G. D., and Massaro, D. (1997). Retinoic acid treatment abrogates
elastase-induced pulmonary emphysema in rats. Nat. Med. 3, 675–677.
Segura-Valdez, L., Pardo, A., Gaxiola, M., Uhal, B. D., Becerril, C., and
Selman, M. (2000). Upregulation of gelatinases A and B, collagenases 1
and 2, and increased parenchymal cell death in COPD. Chest 117, 684–694.
McDonald, J. D., Barr, E. B., and White, R. K. (2004). Design, characterization,
and evaluation of a small-scale diesel exhaust exposure system. Aerosol Sci.
Technol. 38, 62–78.
Selman, M., Cisneros-Lira, J., Gaxiola, M., Ramı́rez, R., Kudlacz, E. M., Mitchell,
P. G., and Pardo, A. (2003). Matrix metalloproteinases inhibition attenuates
tobacco smoke-induced emphysema in guinea pigs. Chest 123, 1633–1641.
Muto, S., Yokoi, T., Gondo, Y., Katsuki, M., Shioyama, Y., Fujita, K., and
Kamataki, T. (1999). Inhibition of benzo[a]pyrene-induced mutagenesis by
() epigallocatechin gallate in the lung of rpsL transgenic mice.
Carcinogenesis 20, 421–424.
Snider, G. L. (1986). Experimental studies on emphysema and chronic
bronchial injury. Eur. J. Respir. Dis. 69(Suppl. 146), 17–35.
Thompson, A. B., Daughton, D., Robbins, R. A., Ghafouri, M. A., Oehlerking,
M., and Rennard, S. I. (1989). Intraluminal airway inflammation in chronic
bronchitis. Characterization and correlation with clinical parameters. Am.
Rev. Respir. Dis. 140, 1527–1537.
Ohnishi, K., Takagi, M., Kurokawa, Y., Satomi, S., and Konttinen, Y. T. (1998).
Matrix metalloproteinase-mediated extracellular matrix protein degradation
in human pulmonary emphysema. Lab. Investig. 78, 1077–1087.
O’Shaughnessy, T. C., Ansari, T. W., Barnes, N. C., and Jeffery, P. K. (1997).
Inflammation in bronchial biopsies of subjects with chronic bronchitis:
Inverse relationship of CD8þ T lymphocytes with FEV1. Am. J. Respir. Crit.
Care Med. 155, 852–857.
Pela, R., Calcagni, A. M., Subiaco, S., Isidoro, P., Tubaldi, A., and Sanguinetti,
C. M. (1999). N-acetylcysteine reduces the exacerbation rate in patients with
moderate to severe COPD. Respiration 66, 495–500.
Rodriguez-Roisin, R. (2000). Toward a consensus definition of COPD
exacerbations. Chest 117, 398S–401S.
Saetta, M., Baraldo, S., Corbino, L., Turato, G., Braccioni, F., Rea, F.,
Canallesco, G., Tropeano, G., Mapp, C., Maestrelli, P., et al. (1999). CDþve
cells in the lungs of smokers with chronic obstructive pulmonary disease.
Am. J. Respir. Crit. Care Med. 160, 711–717.
Saetta, M., Di Stefano, A., Turato, G., Facchini, F. M., Corbino, L., Mapp,
C. E., Maestrelli, P., Ciaccia, A., and Fabbri, L. M. (1998). CDþ
van Overveld, F. J., Demkow, U., Gorecka, D., de Backer, W. A., and
Zielinski, J. (2005). New developments in the treatment of COPD:
Comparing the effects of inhaled corticosteroids and N-acetylcysteine.
J. Physiol. Pharmacol. 56(Suppl. 4), 135–142.
Viegi, G., Scognamiglio, A., Baldacci, S., Pistelli, F., and Carrozzi, L. (2001).
Epidemiology of chronic obstructive pulmonary disease (COPD). Respiration 68, 4–19.
Wilder, J. A., Collie, D. D., Bice, D. E., Tesfaigzi, Y., Lyons, C. R., and
Lipscomb, M. F. (2001). Ovalbumin aerosols induce airway hyperreactivity
in naive DO11.10 T cell receptor transgenic mice without pulmonary
eosinophilia or OVA-specific antibody. J. Leukoc. Biol. 69, 538–547.
Witschi, H. (2005). A/J mouse as a model for lung tumorigenesis caused by
tobacco smoke: Strengths and weaknesses. Exp. Lung Res. 31, 3–18.
Wright, J. L., and Sun, J. P. (1994). Effect of smoking cessation on pulmonary
and cardiovascular function and structure: Analysis of guinea pig model.
J. Appl. Physiol. 76, 2163–2168.