J Appl Physiol 95: 938945, 2003.
First published June 6, 2003; 10.1152/japplphysiol.00336.2003.
Responses to ozone are increased in obese mice
S. A. Shore, Y. M. Rivera-Sanchez, I. N. Schwartzman, and R. A. Johnston
Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115
Submitted 4 April 2003; accepted in final form 25 May 2003
Shore, S. A., Y. M. Rivera-Sanchez, I. N. Schwartz-
man, and R. A. Johnston. Responses to ozone are increased
in obese mice. J Appl Physiol 95: 938945, 2003. First pub-
lished June 6, 2003; 10.1152/japplphysiol.00336.2003.Epi-
demiological data indicate an increased incidence of asthma
in overweight adults and children. Ozone (O3) is a common
trigger for asthma. Accordingly, the purpose of this study
was to compare O3-induced airway hyperresponsiveness and
airway inflammation in lean, wild-type (C57BL/6J) mice and
mice that are obese as a consequence of a genetic defect in the
gene encoding the satiety hormone leptin (ob/ob mice). The
ob/ob mice eat excessively and weighed more than twice as
much as age- and gender-matched wild-type mice. Airway
responsiveness to intravenous methacholine was measured
by forced oscillation. In air-exposed controls, baseline pulmo-
nary resistance was greater, and the dose of methacholine
required to double pulmonary resistance was lower in ob/ob
than wild-type mice. Exposure to O3 (2 parts/million for 3 h)
caused AHR and airway inflammation in both groups of mice,
but responses to O3 were enhanced in ob/ob compared with
wild-type mice. Administration of exogenous leptin did not
reverse the enhanced inflammatory response observed in
ob/ob mice, but augmented airway inflammation in wild-type
mice. The inhaled dose of O3 per gram of lung tissue was
greater in ob/ob than wild-type mice. Our results indicate
that O3-induced airway responses are enhanced in ob/ob
mice and suggest that inhaled O3 dose may be one factor
contributing to this difference, but other aspects of the obese
phenotype may also contribute. Our results also indicate that
the hormone leptin, which is increased in the obese, has the
capacity to increase airway inflammation.
inflammation; polymorphonuclear leukocytes; pulmonary
mechanics; leptin
EPIDEMIOLOGICAL DATA INDICATE an increased incidence of
asthma, wheezing, and bronchial hyperreactivity in
overweight or obese adults and children (9, 17, 20, 29,
45). It is possible that asthmatic subjects are more
prone to obesity because of reduced exercise. However,
it is more likely that obesity leads to asthma, because
longitudinal studies controlling for potential confound-
ers, including physical activity, indicate that the rela-
tive risk of incident asthma increases progressively
with increasing obesity and that obesity antedates
asthma (7, 8). Furthermore, morbidly obese asthmatic
subjects studied after dieting or gastric surgery not
only demonstrate a marked reduction in body weight,
but also decreased severity of asthma and decreased
asthma symptoms (13, 48).
Address for reprint requests and other correspondence: S. A. Shore,
Physiology Program, Harvard School of Public Health, 665 Huntington
Ave., Boston, MA 02115 (E-mail: sshore@hsph.harvard.edu).
938 8750-7587/03 $5.00 Copyright 2003 the American Physiological Society
translational physiology
Ozone (O3) is a trigger for asthmatic episodes. The
number of hospital admissions for asthma increases on
days of high-ambient O3 concentrations (14, 51). O3
exposure causes airway hyperresponsiveness (AHR)
and airway inflammation in both humans and animals
(11, 18, 23, 34, 44, 46, 60), and these effects are likely
to worsen asthmatic episodes. The purpose of this
study was to determine whether obesity enhances the
AHR and/or airway inflammation induced by O3. To
that end, we used mice with a genetic defect in the gene
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encoding leptin, a satiety hormone produced in adipo-
cytes (ob/ob mice). Because of their inability to produce
leptin, ob/ob mice eat excessively and are markedly
obese (25). Our results indicated that O3-induced AHR
and airway inflammation are increased in ob/ob com-
pared with lean wild-type mice. To determine whether
differences between ob/ob and wild-type mice were the
direct result of their leptin deficiency, rather than
some other aspect of the ob/ob mouse phenotype, we
also acutely administered exogenous leptin to wild-
type and ob/ob mice before air or O3 exposure and
examined effects of this hormone on bronchoalveolar
lavage (BAL) cells and cytokines.
METHODS
Animals. This study was approved by the Harvard Medical
Area Standing Committee on Animals. Male and female
mice, aged 812 wk at the time of study, were purchased
from Jackson Laboratory (Bar Harbor, ME). The ob/ob mice
have a single base pair mutation in codon 105 of the leptin
gene that results in a premature stop codon (61). In the
absence of leptin, the mice eat excessively and are severely
obese as early as at 4 wk of age. The increased body weight is
entirely the result of an increase in fat mass. Because the
mutation is expressed on a C57BL/6J background, wild-type
C57BL/6J mice were used as controls.
O3 exposure. Exposure to O3 [2 parts/million (ppm) for 3 h]
or filtered room air was conducted in a stainless steel cham-
ber with a Plexiglas door on the front (145 liters in volume).
O3 was generated by passing dry 100% oxygen through ul-
traviolet light and mixing it with filtered room air in the
chamber. Chamber atmosphere was drawn continuously via
a sampling port, and O3 concentration was measured by an
O3 chemiluminescent analyzer (model 49, ThermoElectron
Instruments, Hopkinton, MA), which was calibrated by an
ultraviolet photometric O3 calibrator (model 49PS, Thermo-
Electron Instruments). During air or O3 exposures, mice
were placed in individual wire mesh cages within the cham-
ber and were awake during the exposure.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
http://www.jap.org
 OBESITY AND AIRWAY RESPONSIVENESS
Protocol. The ob/ob and wild-type mice were exposed to O3
(2 ppm for 3 h) or to filtered air. Twenty-four hours later,
mice were anesthetized and instrumented for the measure-
ment of pulmonary mechanics by the forced oscillation tech-
nique, and airway responsiveness to intravenous methacho-
line was measured. We chose to examine airway responsive-
ness 24 h after the cessation of O3 exposure based on
previous reports using this species (36, 60). BAL was per-
formed either 4 or 24 h after the cessation of O3 exposure in
a separate cohort of mice. We used the 24-h time point to
correspond to that used for measurements of pulmonary
mechanics and because BAL polymorphonuclear leukocytes
(PMN) and protein peak at this time. We used the 4-h time
point to capture O3-induced changes in BAL cytokines, which
peak earlier than PMN and protein and then decline (see
below).
We also examined the effect of exogenous saline or leptin
administration on O3-induced airway inflammation to deter-
mine whether exogenous leptin could reverse the effects
observed in leptin-deficient ob/ob mice. For these experi-
ments, C57BL/6J and ob/ob mice were divided into two
groups. One group received two intraperitoneal injections of
leptin (2.5 g/g) on day 1, spaced 3.5 h apart. On day 2, this
group again received two injections of leptin. The first was
administered 0.5 h before initiation of O3 (2 ppm for 3 h) or
air exposure. The second injection was administered just
after cessation of the exposure. Mice were killed, and BAL
was performed 4 h after cessation of O3. The second group
was treated identically, except that the mice were given
injections of saline rather than leptin. The dose of leptin was
chosen based on published data indicating its efficacy in
reducing food consumption (5, 37, 38). To confirm that leptin
levels were still elevated at the time the BAL was performed,
blood was drawn at the end of the experiment by cardiac
puncture, and the serum was stored at 20C until assayed
for leptin by radioimmunoassay (Lincor, St. Louis, MO).
A final series of experiments was performed to determine
whether differences in O3-induced AHR or airway inflamma-
tion between wild-type and ob/ob mice could be the result of
differences in the inhaled dose of O3 resulting from differ-
ences in minute ventilation (VE) during exposure (47). For
these experiments, VE, tidal volume (VT), breathing fre-
quency (f), and end-expiratory pause (EEP) were measured
under baseline conditions and/or during O3 exposure in mice
placed in nose-only-exposure plethysmographs, as previously
described (46, 47). Some of these mice were killed at least 1
wk after O3 exposure, and the lungs and the heart were
excised and weighed.
Measurement of pulmonary mechanics. Twenty-four hours
after exposure to air or O3, mice were anesthetized with
xylazine (7 mg/kg) and pentobarbital sodium (50 mg/kg). A
tracheostomy was performed, and a tubing adaptor (18
gauge) (Becton-Dickinson, Franklin Lakes, NJ) was used to
cannulate the trachea. The mice were then ventilated at 150
and 180 Hz in wild-type and ob/ob mice, respectively. Both
groups were ventilated at a VT of 0.3 by using a specialized
ventilator (flexiVent, SCIREQ, Montreal, Quebec). The
slightly higher frequency used for the ob/ob was chosen to
conform to their spontaneous breathing frequencies (see be-
low), whereas, during spontaneous breathing, VT did not
differ between the strains. The ventilator that we used fea-
tures a computer-controlled piston capable of producing any
desired waveform and accurately measuring delivered vol-
ume (and hence flow) by tracking piston movement, as de-
scribed by others (40). Pressure at the airway opening was
also measured by the flexiVent system. Once ventilation was
established, a wide incision was made in the chest wall
J Appl Physiol VOL 95 SEPTEMBER 2003
939
bilaterally to expose the lungs to atmospheric pressure, and
a positive end-expiratory pressure of 3 cmH2O was applied by
placing the expiratory line under water. The tail vein was
cannulated for the delivery of methacholine.
Measurements of dynamic pulmonary resistance (RL) were
made by the forced oscillation technique by using a 2.5-Hz
sinusoidal forcing function. The flexiVent system was used
both for ventilating the lungs and for delivering the oscilla-
tions. Calibration of the system with the tracheostomy tube
in place removes the mechanical properties of the tubing, so
that the data reported by the system represent the mechan-
ical properties of the lungs only. Dose-response curves to
intravenously administered methacholine were obtained as
follows. Mice were given an inflation to three times VT. One
minute later, a bolus of PBS was administered (1 l/g), and
RL was measured every eighth breath for the next 12 min,
until RL peaked and began to decline. The mouse was then
given another inflation to three times VT. The procedure was
repeated by using doses of methacholine chloride dissolved in
PBS that increased in half-log intervals from 0.03 to 3.0
mg/ml at a dose of 1 l/g. Five minutes were allowed to elapse
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between doses of methacholine. The three highest values of
RL obtained after each dose were averaged to obtain the final
values for each dose. The dose of methacholine required to
double RL (ED200RL) was obtained by log linear interpolation
between the doses bounding the point at which RL was
exactly 200% of the PBS value.
BAL. Mice were killed with an overdose of pentobarbital
sodium. The trachea was cannulated with a tubing adaptor,
and the lungs were lavaged twice with PBS (1 ml), which was
instilled and then slowly withdrawn over 30 s. The recovered
BAL fluid (8090% of that delivered) was placed on ice
until it was centrifuged at 1,200 rpm at 4C for 10 min. Cell
pellets were resuspended in PBS, and the total number of
cells was counted with a hemocytometer. Aliquots of cells
were also centrifuged onto glass slides at 800 rpm for 10 min
(Cytospin 3, Shandon, Sewickley, PA), air-dried, and stained
with Wright-Giemsa (LeukoStat, Fisher Scientific, Pitts-
burgh, PA). Cell differentiation was determined by counting
300 cells under 400 magnification. Cells were assigned as
macrophages, neutrophils, eosinophils, lymphocytes, or epi-
thelial cells based on standard morphological characteristics.
The supernatant was frozen at 70C and subsequently
analyzed for protein concentration by using the Bio-Rad dye
reagent (Bio-Rad, Richmond, CA), according to the manufac-
turers instructions. An aliquot of the supernatant was re-
centrifuged at 40,000 g at 4C for 30 min and subsequently
analyzed for eotaxin, IL-6, macrophage inflammatory pro-
tein-2 (MIP-2), and KC by using enzyme immunoassay kits
(Endogen, Woburn, MA, for IL-6; and R&D Systems, Minne-
apolis, MN, for eotaxin, MIP-2, and KC).
Measurement of VE. To measure ventilation during O3
exposure, mice were placed in a Plexiglas restraining tube
that served as a head-out flow plethysmograph, as previously
described (46, 47). Different sized tubes were used for lean
and obese mice. The tube was fitted with a rubber gasket
designed to fit snugly around the animals neck. Once the
animal was in the tube, a large piston fitted with a rubber
O-ring was moved into place behind it, thus restraining the
animals movement. Air displaced at the body surface as the
animal breathed passed across a pneumotach attached to a
differential pressure transducer. The front end of the tube
was inserted into a port in the O3 exposure chamber, and the
animals were exposed nose only. The flow signal from the
pneumotach was fed to a personal computer and analyzed by
using software (BUXCO Electronics, Sharon, CT) that
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940 OBESITY AND AIRWAY RESPONSIVENESS

Table 1. Baseline RL, ED200RL, and body weight of statistically significant effect of O3 exposure or obesity
wild-type and ob/ob mice exposed to air on the total number of cells recovered from BAL. How-
or ozone (2 ppm for 3 h) ever, O3 exposure influenced the percentage of BAL
cells that were PMN (Fig. 2F). In both wild-type and
RL, cmH2O ml1 s Log ED200RL Weight, g ob/ob, O3 exposure resulted in a significant increase in
Wild type - Air 0.69 0.02 0.24 0.05 19.6 2.6 the percentage of BAL cells that were PMN measured
Wild type - O3 0.88 0.15 0.42 0.11* 17.7 1.9 at 4 h, and PMN remained elevated at 24 h. However,
ob/ob - air 0.76 0.04 0.69 0.07 50.8 8.3 O3-induced changes in BAL PMN were not different
ob/ob - O3 1.59 0.29* 0.88 0.15* 52.4 7.9
between wild-type and ob/ob mice.
Values are means SE; n 67/group. Measurements were made To determine whether differences in responses to O3
24 h after exposure. RL, pulmonary resistance; ED200RL, dose of observed in ob/ob mice were the direct result of these
methacholine (in mg/ml) required to double RL; O3, ozone. * P 0.05
compared with mice in same group exposed to air; P 0.05 com-
animals leptin deficiency or due to some other aspect of
pared with wild-type mice with same exposure. the phenotype of these mice, we examined the ability of
leptin to reverse changes in O3-induced inflammation
that were observed in ob/ob mice (Fig. 3). As described
allowed for breath-by-breath measurements of VE, VT, f,
above, exposure to O3 (2 ppm for 3 h) induced greater
and EEP.
Statistics. Differences in ventilatory parameters during O3 increases in BAL protein, eotaxin, KC, MIP-2, and IL-6
exposure were assessed by repeated-measures ANOVA. Dif- in ob/ob mice treated with saline than in wild-type
ferences in BAL cells and cytokines, baseline RL, and mice treated with saline, whereas PMN were not af-

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ED200RL were assessed by ANOVA. In each case, follow-up fected. However, no significant differences were ob-
t-tests were used to determine the significance of differences served between leptin-treated and saline-treated ob/ob
between individual groups. Differences in serum leptin were mice exposed to either air or O3. In contrast, O3-in-
assessed by unpaired t-tests. Statistical analyses were car- duced changes in BAL IL-6, KC, and protein were
ried out by using SAS software (SAS Institute, Cary, NC). greater in leptin-treated than in saline-treated wild-
RESULTS
type mice (Fig. 3). A similar trend was observed for
eotaxin and MIP-2, although the effects were not sta-
Table 1 shows baseline RL, ED200RL, and body tistically significant. After the experiments were ter-
weight in wild-type and ob/ob mice 24 h after exposure minated, blood was drawn. Serum leptin levels aver-
to air or O3. ANOVA indicated statistically significant aged 27.9 4.3 ng/ml in leptin-treated wild-type mice,
differences among the four groups for weight (P but only 5.1 1.7 ng/ml in saline-treated wild-type
0.001), baseline RL (P 0.005), and ED200RL (P mice (P 0.01).
0.001). When the air- and O3-exposed mice were The dose of O3 delivered to the lungs is the product of
grouped together, ob/ob mice weighed 51.7 2.2 g, O3 concentration, exposure time, and VE (57). VE de-
more than twice as much as wild-type mice (18.7 pends on metabolism, and, in rodents, there are
0.7 g). After air exposure, baseline RL was slightly, but changes in metabolism during O3 exposure (33, 46, 47)
significantly, greater (P 0.05) in ob/ob than wild-type and with obesity (25). To determine whether differ-
mice. The ob/ob air-exposed mice were also signifi- ences in O3 dose resulting from differences in VE might
cantly more responsive to methacholine than wild-type
mice after air exposure (Fig. 1, Table 1).
There was no effect of O3 on baseline RL in wild-type
mice. However, in ob/ob mice, O3 caused a significant
increase in baseline RL (P 0.02). O3 exposure in-
creased airway responsiveness in both wild-type and
ob/ob mice (Fig. 1, Table 1). The effect of O3 was to
decrease ED200RL (Table 1) and to increase the maxi-
mal response to methacholine (Fig. 1). O3 exposure
resulted in a significantly greater increase in airway
responsiveness in the ob/ob mice than in the wild-type
mice (Fig. 1).
BAL protein, eotaxin, MIP-2, KC, and IL-6 concen-
trations were not significantly different in ob/ob mice
compared with wild types after air exposure (Fig. 2,
BE). O3 exposure caused a significant increase in
BAL eotaxin, MIP-2, IL-6, and KC measured 4 h after
cessation of O3 exposure that declined toward baseline
by 24 h. In contrast, BAL protein was increased at 4 h Fig. 1. Airway responsiveness to intravenous methacholine in wild-
and increased further at 24 h (Fig. 2A). There was a type and ob/ob mice exposed to air or ozone (O3) [2 parts/million
significantly greater increase in BAL protein, eotaxin, (ppm) for 3 h]. Measurements were made 24 h after exposure. Values
are means SE of data from 67 mice in each group. RL, pulmonary
KC, and IL-6 in the ob/ob mice compared with the resistance. * P 0.05, compared with air-exposed mice in the same
wild-type mice 4 h after cessation of O3 exposure. A group. # P 0.05, compared with wild-type mice in the same expo-
similar trend was observed for MIP-2. There was no sure group.

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 OBESITY AND AIRWAY RESPONSIVENESS
account for observed differences between wild-type and
ob/ob mice in the response to O3, we measured VE
and the pattern of breathing in ob/ob and wild-type mice
during O3 exposure (Fig. 4). We also measured VE and
the pattern of breathing during O3 exposure in wild-
type mice treated with exogenous saline or leptin.
We did not observe any statistically significant dif-
ferences in VE between wild-type and ob/ob mice before
O3 exposure (Fig. 4A, time 0), although there were
differences in the pattern of breathing. In particular, f
was higher in ob/ob (282 19 breaths/min) than in
wild-type mice (246 13 breaths/min) (P 0.05), but
VT was not different (0.25 0.01 and 0.26 0.02 ml in
wild-type and ob/ob mice, respectively). As previously
reported (46, 47), there was a marked decrease in VE
during O3 exposure in wild-type mice (Fig. 4A). The
ob/ob showed a similar pattern of response, and, al-
J Appl Physiol VOL
941
Fig. 2. Bronchoalveolar lavage (BAL) protein,
cytokine concentrations, and polymorphonu-
clear leukocytes in wild-type (open bars) and
ob/ob (solid bars) mice. A: protein; B: eotaxin;
C: macrophage inflammatory protein-2 (MIP-
2); D: KC; E: IL-6; and F: neutrophils. Mice
were exposed to air or O3 (2 ppm for 3 h), and
BAL was performed 4 or 24 h later. Values are
means SE of data from 67 mice in each
group. * P 0.05, compared with O3-exposed
wild-type mice.
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though VE tended to be higher in ob/ob than in wild-
type mice at all times during O3 exposure, the differ-
ences were not statistically significant. When the total
volume of air inhaled during the 3-h O3 exposure was
computed for each group, the difference was also not
statistically significant (7.77 0.92 and 9.70 0.84
liters for wild-type and ob/ob mice, respectively). These
results indicate that the total inhaled dose of O3 was
not different between wild-type and ob/ob mice. How-
ever, ob/ob mice have been reported to have smaller
lungs than wild-type controls (49), suggesting that the
inhaled dose of O3 per gram of lung tissue might, in
fact, be different in the obese mice. To examine this
possibility, we excised the lungs of a separate cohort of
ob/ob and wild-type mice at least 1 wk after O3 expo-
sure and weighed them. The weight of the lungs of
ob/ob mice (0.18 0.02 g) was less than that of age-
Fig. 3. The ob/ob and wild-type mice were treated
with saline or leptin (2.5 g/g) 2 times/day for 2
days and exposed to air or O3 (2 ppm for 3 h). BAL
was performed 4 h later. A: protein; B: eotaxin; C:
MIP-2; D: KC; E: IL-6; and F: neutrophils. Values
are means SE of data from 67 mice in each
group. * P 0.05, leptin-treated compared with
saline-treated wild-type mice. # P 0.05, saline-
treated ob/ob compared with saline-treated wild-
type mice.
95 SEPTEMBER 2003 www.jap.org
942 OBESITY AND AIRWAY RESPONSIVENESS
Fig. 4. Changes in minute ventilation (VE; A)
and end-expiratory pause (EEP; B) during a
3-h exposure to O3 (2 ppm) in wild-type and
ob/ob mice. Measurements at time 0 are the
mean values measured in the 20-min period
before initiation of exposure. Values are
means SE of data from 611 mice in each
group. * P 0.05, compared with wild-type
mice.
and sex-matched wild-type mice (0.28 0.05 g, P
0.05), even though the heart weights were not signifi-
cantly different (0.16 0.02 and 0.19 0.02 g for
wild-type and ob/ob mice, respectively). Consequently,
the dose of O3 delivered to the lungs of the ob/ob mice
per gram of lung tissue was greater than that delivered
to the wild-type mice and could potentially account for
at least part of the observed differences in response to
O3 (Figs. 1 and 2).
Even though there was no significant difference in
the change in VE induced by O3 in the wild-type and
ob/ob mice, changes in breathing pattern that were
induced by O3 were different in ob/ob mice compared
with wild types. In particular, the striking increase in
EEP that is characteristic of mice exposed to O3 (47)
was significantly blunted in ob/ob mice (P 0.001)
(Fig. 4B).
We did not observe any statistically significant dif-
ference in VE between saline-injected and leptin-in-
jected wild-type mice in the 30-min period after injec-
tion (49.8 2.6 and 52.8 1.3 ml/min, respectively),
nor was there a difference in any other ventilatory
parameter. O3 exposure caused a decrease in VE in
both saline- and leptin-treated wild-type mice, but
there was no significant difference in the response
between groups (data not shown). These results indi-
cate that the inhaled dose of O3 was not different in
saline- and leptin-treated wild-type mice and that dif-
ferences in dose cannot account for the increased O3-
induced airway inflammation observed in leptin-
treated wild-type mice (Fig. 3).
DISCUSSION
Our results indicate that O3-induced AHR and O3-
induced airway inflammation are enhanced in ob/ob
mice (Figs. 1 and 2). Moreover, the difference in the
inflammatory response to O3 is not corrected by acute
exogenous administration of leptin (Fig. 3), suggesting
that obesity per se, or some other aspect of the chronic
phenotype of these mice that is not corrected by acute
leptin treatment, accounts for the enhanced response
to O3. For example, the difference in response to O3
J Appl Physiol VOL
may be a consequence of differences in O3 dose, result-
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ing from the reduced lung size in the ob/ob mice.
Surprisingly, acute exogenous administration of leptin
did result in an increase in O3-induced airway inflam-
mation in wild-type mice, even though it did not affect
responses to O3 in ob/ob mice (Fig. 3).
We observed a small but significant increase in RL in
ob/ob compared with wild-type mice (Table 1). Lung
volumes are reduced in ob/ob mice (49), and it is likely
that the increase in baseline RL is the result of this
reduction in lung volume, because airway diameter is
strongly influenced by lung volume. Indeed, in obese
humans, there is a linear correlation between changes
in airway conductance and functional residual capacity
(59). In obese humans, the reductions in lung volume
associated with obesity are thought to result from
gravitational effects, resulting from the increased ab-
dominal load (58). However, we observed a decrease in
the actual mass of the lung in leptin-deficient ob/ob
mice, despite their marked increase in body weight,
suggesting that simple gravitational effects likely do
not explain all of the reduction in lung volume ob-
served in these mice. Rather, it may be that leptin is
required for lung growth (3), because leptin receptors
are present in developing prealveolar acini of fetal
lungs (2) and because leptin stimulates surfactant syn-
thesis in fetal lung cells (2, 52), as well as proliferation
of tracheal epithelial cells (53). Alternatively, reduc-
tions of the volume of the thorax as a result of the
increased abdominal mass and even the accumulation
of fat between the lungs and the rib cage may limit
lung growth and development in ob/ob mice, because
obesity is present very early on.
Airway responsiveness was increased in ob/ob com-
pared with wild-type mice, even in the absence of O3
exposure (Fig. 1). An association between increased
body mass index and onset of AHR has also been
observed in a longitudinal study of aging in humans
(26). Breathing at low lung volume has been shown to
augment airway responsiveness both in animals and in
humans (12). The ob/ob mice do breathe at lower lung
volumes than wild-type mice (49), but, in our study,
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 OBESITY AND AIRWAY RESPONSIVENESS
lung volume was fixed by opening the chest wall and
applying a standard amount of positive end-expiratory
pressure. Furthermore, the lower lung volume of ob/ob
mice is the result of a smaller lung. It is not clear that
small lungs per se should lead to AHR, although im-
mature animals usually have AHR compared with
adults. In both obese humans (24, 35, 56) and obese
mice (19, 55), there is chronic systemic inflammation,
even in the absence of any overt inflammatory insult.
Although we did not note any differences in BAL cyto-
kines in the ob/ob compared with wild-type air-exposed
mice (Fig. 2), it is possible that differences existed but
were below the limit of detection of the assays used.
The relationship between airway inflammation and
AHR is quite complex, and it is clear that AHR can
occur in the absence of airway inflammation (6), par-
ticularly under conditions that promote airway remod-
eling (6, 10). However, multiple stimuli that promote
airway inflammation also promote AHR (6), and it is
possible that the AHR observed in the air-exposed
ob/ob mice may be the result of low-grade inflamma-
tion.
It is unlikely that differences in tidal stretching of
airway smooth muscle account for the difference in
baseline airway responsiveness between the air-ex-
posed ob/ob and the wild-type mice. Fredberg et al. (15)
have shown that the periodic strains on airway smooth
muscle imposed by tidal lung inflations reduce the
muscles ability to shorten. The amount of tidal stretch-
ing of airway smooth muscle depends on the VT in
relation to the size of the lungs. We used identical VT
values during measurements of pulmonary mechanics
for the ob/ob and wild-type mice based on the observa-
tion that their spontaneous VT values were not differ-
ent. However, the obese mice had smaller lungs.
Hence, when normalized for the size of their lungs, VT
values were actually greater in the obese mice, imply-
ing greater tidal smooth muscle stretching, which
should have resulted in reduced, not increased, airway
responsiveness, as observed. Nevertheless, obese hu-
mans do breath with smaller VT values (42), and re-
duced tidal stretching of airway smooth muscle could
play a role in the increased airway responsiveness
observed in obese humans (26).
Our results indicate that both O3-induced AHR and
O3-induced airway inflammation are increased in
ob/ob compared with wild-type mice (Figs. 1 and 2).
Data from many investigators suggest that the mech-
anistic basis for O3-induced AHR is inflammation aris-
ing from oxidant injury to the lungs and airways,
although the precise aspect(s) of the inflammatory cas-
cade required for AHR is still not firmly established.
Thus it is likely that the increased O3-induced AHR
observed in ob/ob mice (Fig. 1) is a consequence of the
increase in airway inflammation (Fig. 2). It is unlikely
that the enhanced O3-induced airway inflammation
observed in the ob/ob mice is the direct result of leptin
deficiency, because treating ob/ob mice with leptin did
not restore O3-induced inflammation to that observed
in wild-type mice (Fig. 3). Instead, our results suggest
that the changes observed in ob/ob mice resulted from
J Appl Physiol VOL 95 SEPTEMBER 2003
943
an effect of chronic leptin deficiency, such as obesity
per se, or reduced lung size, that could not be reversed
with acute leptin treatment. Although the inhaled vol-
ume of O3 was the same in ob/ob and wild-type mice, a
greater dose of O3 per gram of lung tissue was actually
delivered to the obese mice, because their VE was
essentially the same as that of the wild-type mice, but
their lungs were smaller. Such differences in dose
could have accounted for at least part of the differences
in O3-induced inflammation and AHR. It will ulti-
mately be important to examine other murine models
of obesity in which lung growth is not impacted to
determine whether the reduced lung size of the ob/ob
mouse accounted for the differences in response to O3
or whether other aspects of the obese phenotype also
contribute. For example, chronic systemic inflamma-
tion in the obese (19, 24, 35, 55, 56) may amplify
subsequent inflammatory responses.
One of the most interesting results of this study was
Downloaded from http://jap.physiology.org/ by 10.220.33.1 on November 23, 2017
the observation that, in wild-type mice, exogenous lep-
tin increased O3-induced cytokine and protein release
into BAL fluid (Fig. 3). To our knowledge, this is the
first report of proinflammatory effects of leptin in the
lung in vivo, although leptin has also been shown to
enhance inflammatory and/or immune responses in
other organs. For example, treatment of mice with
exogenous leptin has been shown to augment the in-
flammatory and fibrogenic response of the liver to
hepatotoxic chemicals (21). Leptin treatment has also
been shown to render mice more susceptible to the
development of autoimmune encephalomyelitis after
immunization with myelin-derived peptides (32). The
tertiary structure of leptin resembles that of many
cytokines, and the leptin receptor has close homology
to class I cytokine receptors, along with JAK/STAT
signaling capabilities (50). Hematopoietic cells express
leptin receptors, and bone marrow cells cultured in the
presence of leptin demonstrate growth of granulocyte-
macrophage colonies (16, 54). The leptin receptor is
also expressed in CD4 T cells, and both mouse and
human T cells respond to leptin with increased prolif-
erative responses to mitogenic stimuli and increased
production of some cytokines (28, 31). Peritoneal mac-
rophages also express leptin receptors and respond to
leptin administration with an increase in LPS-stimu-
lated TNF-, IL-6, and IL-12 production (16, 27). In
human monocytes, leptin induces the expression of
activation markers, causes proliferation, and increases
IL-6 and TNF- expression (43). Leptin receptors are
also present on endothelial cells and cause activation of
activator protein-1 and NF-B, transcription factors
important in the induction of many inflammatory
proteins (4). We do not know the precise cell types
that are the target of leptins effects on O3-induced
pulmonary inflammation. However, both macro-
phages and airway epithelial cells have been shown
to be targets of O3 (1, 39), both cell types express
leptin receptors (27, 53), and both cell types express
cytokines after O3 exposure (22).
In summary, our results demonstrate increased air-
way responsiveness in ob/ob mice. Whereas the re-
www.jap.org
944 OBESITY AND AIRWAY RESPONSIVENESS
duced lung size of these mice may contribute to part of
this difference in responsiveness, it is possible that
low-grade systemic inflammation also plays a role. Our
results also demonstrate that both O3-induced AHR
and O3-induced airway inflammation are enhanced in
ob/ob mice. Differences in inhaled O3 dose consequent
to differences in lung size may account for some of
these changes. As such, the effects of leptin on lung
growth may confound use of this leptin-deficient model
of obesity for understanding the relationship between
obesity and asthma. Other models in which obesity
develops without attendant effects on lung size may
prove more useful. However, the results suggest the
possibility that the obese may represent an at risk
population in terms of their susceptibility to air pollut-
ants.
Our results also indicate that leptin has the capacity
to augment O3-induced airway inflammation. Given
that leptin is produced in proportion to adipocyte mass
and that leptin concentrations are four to six times
greater in severely obese compared with lean human
subjects (30, 41), it is possible that differences in serum
leptin may also promote allergic airway inflammation
and asthma. In this respect, it is interesting to note
that the association between obesity and asthma is
stronger in females than in males (9, 20). Similarly, for
equivalent body mass index, leptin levels are higher in
women than in men (41).
The authors thank David Chism and Dr. G. G. Krishna Murthy for
excellent technical assistance and Dr. Jeffrey Fredberg for helpful
comments and suggestions.
DISCLOSURES
This work was supported by National Institutes of Health Grants
HL-33009, HL-66879, and ES-00002.
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