Adiponectin Deficiency Increases Allergic Airway
Inflammation and Pulmonary Vascular Remodeling
Benjamin D. Medoff1,2*, Yoshihisa Okamoto3*, Patricio Leyton4, Meiqian Weng1,2, Barry P. Sandall1,
Michael J. Raher4, Shinji Kihara5, Kenneth D. Bloch4, Peter Libby3, and Andrew D. Luster1
1Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology,2Pulmonary and Critical Care Unit, and
4Cardiovascular Research Center and Anesthesia Center for Critical Care Research, Massachusetts General Hospital and Harvard Medical School,
Charlestown, Massachusetts;3Division of Cardiovascular Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston,
Massachusetts; and5Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
Obesity is associated with an increased incidence and severity of
asthma, as well as other lung disorders, such as pulmonary hyper-
tension. Adiponectin (APN), an antiinflammatory adipocytokine,
to obesity-related inflammatory diseases. We sought to determine
the effects of APN deficiency in a murine model of chronic asthma.
Allergic airway inflammation was induced in APN-deficient mice
(APN2/2) using sensitization without adjuvant followed by airway
in inflammation and lung remodeling. APN2/2mice in this model
type mice, with greater accumulation of eosinophils and monocytes
in the airways associated with elevated lung chemokine levels.
Surprisingly, APN2/2mice developed severe pulmonary arterial
muscularization and pulmonary arterial hypertension in this model,
whereas wild-type mice had only mild vascular remodeling and
comparatively less pulmonary arterial hypertension. Our findings
demonstratethat APN modulates allergic inflammation and pulmo-
nary vascular remodeling in a model of chronic asthma. These data
provide a possible mechanism for the association between obesity
and asthma, and suggest a potential novel link between obesity,
inflammatory lung disease, and pulmonary hypertension.
Keywords: asthma; obesity; pulmonary hypertension
30% of the adult population in the United States is considered
obese (1). Obesity is associated with a higher incidence of
hypertension, diabetes, cardiovascular disease, and pulmonary
disorders, such as asthma and pulmonary hypertension (2–5).
Recent experimental evidence suggests that obesity may directly
contribute to the pathogenesis of these disorders through the
metabolic and immune activity of adipose tissue (6). Consistent
with this, adipocytes secrete multiple bioactive mediators (adi-
pocytokines), which may influence energy homeostasis, inflam-
mation and tissue remodeling (6–8). Adiponectin (APN), the
most abundant adipocytokine, has a wide range of metabolic,
antiinflammatory, and antiproliferative activities (9). Of note,
individuals with obesity have low plasma APN levels, suggesting
that decreased APN levels may contribute to the increased
inflammatory state in obesity. Supporting this hypothesis, mice
with a deletion of the APN gene (APN2/2) are predisposed
to inflammatory diseases, such as diabetes and atherosclerosis
(9–11), and develop enhanced organ remodeling and vascular
smooth muscle cell (SMC) proliferation in disease (9–11).
Asthma is a complex syndrome, broadly defined by inflam-
mation of the airways associated with airways hyperresponsive-
ness (AHR) and mucus hypersecretion (12). In addition, chronic
asthma is associated with remodeling of the airways and associ-
ated vasculature (13, 14). There is accumulating evidence in the
literature that obesity can increase both the incidence and
severity of asthma in patients, and is associated with worse
asthma outcomes (2, 15–19). Studies have demonstrated that
adipose tissue expresses multiple adipocytokines, which may
influence airway inflammation and airway remodeling (7, 20).
In addition, experimental studies in animals have also supported
a link between obesity and asthma (3, 21, 22). Taken together,
these data suggest that obesity may actually contribute to the
pathogenesis of asthma; however, the mechanisms for this in-
teraction are not well defined.
There is also an increased incidence of pulmonary hyperten-
sion in the obese, with a prevalence of 5% in people with a body
to relate partially to the obesity–hypoventilation syndrome (a
syndrome notable for a combination of obesity, sleep-disordered
breathing, and hypercapnia) and obstructive sleep apnea; how-
independent of these diagnoses (23–25). Even more provocative
are autopsy data from patients with obesity, which demonstrate
a high incidence (72%) of pulmonary hypertensive changes
(including extensive muscularization of medium and small pul-
monary arteries) in this population (26, 27). Although these data
strongly suggest an association between obesity and pulmonary
hypertension, the exact mechanisms linking these disorders
Recent studies have suggested that APN can influence the
development of lung inflammation and, possibly, pulmonary
hypertension. In a murine model of acute asthma, treatment of
mice with APN attenuated airway inflammation and AHR (21).
More recently, it wasdemonstrated that APNis detectablein the
airways of normal mice at levels 5-fold less than serum. Further-
more, APN-deficient (APN2/2) mice develop spontaneous em-
The research presented in this article provides a mechanism
that might explain the increased incidence of asthma in the
obese. Furthermore, the data suggest a novel mechanism
by which obesity may influence the development of pul-
monary hypertension in the presence of inflammatory lung
(Received in original form October 28, 2008 and in final form December 22, 2008)
* These authors contributed equally to this work.
This work was supported by National Institutes of Health grants HL072775 and
HL088297 (B.D.M.), AI40618 (A.D.L.), HL34636 (P.L.), and HL074352 (K.D.B.),
the Donald W. Reynolds Foundation (P.L.), and the American Heart Association
and Eli Lilly (International Fellowship) (Y.O.).
Correspondence and requests for reprints should be addressed to Benjamin D.
Medoff, M.D., Massachusetts General Hospital, CNY 8301, 149 13th Street,
Charlestown, MA 02129. E-mail: email@example.com or firstname.lastname@example.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2008-0415OC on January 23, 2009
Internet address: www.atsjournals.org
Vol 41. pp 397–406, 2009
physema, and lung macrophages from these mice express higher
levels of inflammatory mediators than macrophages from wild-
type mice (28). Potential effects of APN on the pulmonary
vasculature have also recently been recognized. Male apolipo-
protein E–deficient mice on a high-fat diet develop pulmonary
hypertension associated with lower APN levels compared with
wild-type mice (29). Treatment of these mice with the peroxi-
some proliferator–activated receptor-g activator, rosiglitazone,
resulted in higher plasma APN levels and complete regression
of pulmonary hypertension and pulmonary artery remodeling.
affect the severity of inflammation in asthma and influence
pulmonary vascular disease. Interestingly, pulmonary hyperten-
sion and pulmonary arterial remodeling have recently been
reported in murine models of allergic airway inflammation (30–
for the development of pulmonary hypertension (30, 32–34).
However, whether obesity influences the development of pulmo-
nary hypertension in the setting of inflammatory lung disease has
not been studied. Here, we investigated the effects of APN
deficiency in a murine model of chronic allergic airway inflam-
mation, and demonstrate that APN modulates allergic airway
inflammation and pulmonary vascular remodeling.
MATERIALS AND METHODS
APN2/2mice were backcrossed seven generations onto a C57BL/6
background (35). Wild-type C57BL/6 control mice were obtained from
Taconic (Hudson, NY), so we cannot fully rule out subtle differences in
the phenotype of these mice arising from genetic differences. Mice were
used at 6 to 8 weeks of age, and were age and sex matched for all
experiments. There were no baseline differences in weight (data not
were approved by the Institutional Animal Care and Use Committee of
Massachusetts General Hospital.
Acute allergic airway inflammation was induced in mice as previously
described (36). Briefly, mice were immunized with two intraperitoneal
injections of 10 mg of chicken ovalbumin (OVA) (Sigma-Aldrich, St.
Louis, MO) bound to 1 mg of alum (Sigma-Aldrich) in 0.5 ml PBS on
Days 1 and 7. Starting on Day 14, mice were challenged by aerosol
20 minutes daily for 3 days. Chronic allergic airway inflammation was
induced in mice using a modified protocol (37). Briefly, mice were
immunized with three intraperitoneal injections of 50 mg of OVA in 0.1
ml PBS on Days1, 4, and 7. Starting on Day 12, mice were challengedby
intranasal injection with 20 mg OVA in 30 ml PBS or PBS alone (control
the last challenge.
designed cage rack, as previously described (38).
Mouse Harvest and Analysis
as previously described (36). Cells recovered from the BAL fluid were
stained with fluorescently labeled antibodies to CD4, CD8, and CD69
(BD Biosciences, San Jose, CA).
For histopathologic examination, lungs were flushed free of blood,
inflated with 10% buffered formalin to 25 cm H2O of pressure, and
prepared and evaluated as previously described (36). Sections of paraffin-
embedded lungs were preparedand stainedwith hematoxylin and eosin,
Masson trichrome, and Picrosirius red. Sections were also stained with
antibodies to a-smooth muscle actin (Abcam, Cambridge, MA), and
ing to the manufacturers’ recommended protocols. Transferase biotin-
dUTP nick end labeling (TUNEL) assay was performed using the
Apoptag Plus Peroxidase in situ apoptosis kit (Millipore, Billerica,
MA). The number of TUNEL1nuclei per 203 field was counted in
three random fields for each section. The slides were evaluated by light
microscopy, and the amount of inflammation and vascular remodeling
amount of vascular obliteration on three whole lung sections of the left
lung from each mouse evaluated. Each set of three sections was given
a score of 0–4 for inflammation (0 5 no inflammation; 1 5 , 25% of
airways with inflammation; 2 5 25–50% of airways with inflammation;
3 5 50–75% of airways with inflammation; 4 5 . 75% of airways
of medium-sized pulmonary arteries obliterated; 3 5 50–75% of
medium-sized pulmonary arteries obliterated; 4 5 greater than 75%
of medium-sized pulmonary arteries obliterated). In addition, measure-
ments were made of the vascular wall thickness (expressed as a percent-
age of the vessel diameter) forthe preacinarvessels in each lung section,
with Picrosirius red. Sections were incubated for 90 minutes in 0.1%
Sirius red F3BA (Polyscience Inc., Warrington, PA) in saturated picric
acid. Staining with Sirius red was analyzed by polarization microscopy
with image analysis software (Image-Pro Plus; MediaCybernetics,
Airway resistance and dynamic lung compliance were measured
invasively using a whole-body plethysmograph (Buxco, Wilmington,
NC), as previously described (36).
Mice were anesthetized with ketamine (100 mg/kg) and fentanyl
(250 mg/kg) intraperitoneally, then intubated and mechanically venti-
lated (10 ml/g, 110 bpm; FiO2 5 1). Pancuronium (2 mg/kg) was
administered intraperitoneally, and a PE-10 polyethylene catheter was
placed in the left carotid artery for continuous heart rate and systemic
arterial pressure monitoring. Then, a 1F high-fidelity pressure catheter
right ventricle via the jugular vein to measure right ventricular systolic
pressure (RVSP) as an estimate of pulmonary arterial systolic pressure.
All signals were recorded and analyzed using a data acquisition system
(PowerLab with Chart; AD Instruments, Colorado Springs, CO). At the
breathing room air, and the partial pressure of oxygen was measured
using a Chiron Diagnostic Blood Gas Machine (Siemens, Deerfield,IL).
Cell Culture and Stimulation
reported (39). The cells were incubated in standard medium con-
taining 2% FCS with or without 10 mg/ml of mouse recombinant APN
(BioVendor, Candler, NC) for 18 hours. The bone marrow–derived
and TNF-a (100 ng/ml) (R&D Systems, Minneapolis, MN) for 6 hours.
After the treatment, RNA was isolated for quantitative RT-PCR.
Quantification of Gene and Protein Expression
RNA was purified from the lung and analyzed by quantitative RT-PCR,
as previously described (36). Primer sequences used were selected using
the Massachusetts General Hospital PrimerBank (http://pga.mgh.har-
then used undiluted in commercial ELISA kits for CCL11 and CCL24
(R&D Systems), according to the manufacturer’s protocol. BAL and
in a commercial ELISA kit to measure the protein levels of mouse APN
(B-Bridge International, Mountain View, CA).
Hydroxyproline was assayed as previously described (40).
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