A3Adenosine Receptor Signaling Influences Pulmonary
Inflammation and Fibrosis
Eva Morschl1, Jose G. Molina1, Jonathan B. Volmer1, Amir Mohsenin1, Ralph S. Pero2, Jeong-Soo Hong3,
Farrah Kheradmand3, James J. Lee2, and Michael R. Blackburn1
1Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Medical School, Houston,
Texas;2Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, Arizona;
and3Department of Medicine, Pulmonary, Baylor College of Medicine, Houston, Texas
Adenosine is a signaling molecule produced during conditions that
the regulation of pulmonary disorders through the selective engage-
of pulmonary inflammation and fibrosis. Results demonstrated that
A3R-deficient mice exhibit enhanced pulmonary inflammation that
included an increase in eosinophils. Accordingly,therewasa selective
up-regulation of eosinophil-related chemokines and cytokines in the
lungs of A3R-deficient mice exposed to bleomycin. This increase in
eosinophilnumberswasaccompanied byadecreaseinthe amountof
extracellular eosinophil peroxidase activity in lavage fluid from A3R-
deficient mice exposed to bleomycin, an observation suggesting that
the A3R is necessary for eosinophil degranulation in this model.
Despite an increase in inflammatory metrics associated with A3R-
degree of pulmonary fibrosis. Examination of fibrotic mediators
demonstrated enhanced transforming growth factor (TGF)-b1 ex-
pression, but not a concomitant increase in TGF-b1 activity. This was
associated with the loss ofexpression ofmatrix metalloprotease 9, an
activator of TGF-b1, in alveolar macrophages and airway mast cells in
the lungs of A3R-deficient mice. Together, these results suggest that
the A3R serves antiinflammatory functions in the bleomycin model,
Keywords: pulmonary fibrosis; adenosine receptors; inflammation;
eosinophil; extracellular matrix
Pulmonary fibrosis is a component of many interstitial lung
of inflammation, aberrant fibroblast proliferation, and extracel-
lular matrix deposition that result in distortion of lung architec-
ture and compromised pulmonary function (1–4). Fibrosis is also
a major component of airway remodeling responses seen in
pulmonary inflammation and fibrosis have not been completely
elucidated, emerging evidence suggests that pulmonary fibrosis
likely results from abnormal repair responses in the damaged
lung, including deregulated inflammation, angiogenesis, and
of inflammation and the production of fibrotic mediators by
inflammatory cells has become important for understanding the
a detrimental component.
Adenosine is a signaling molecule that is produced in excess at
times of cellular stress and/or damage. Consistent with this,
adenosine levels are elevated in the lungs of patients with asthma
and patients with chronic obstructive pulmonary disease (COPD)
(7, 8), as well as in the lungs of mouse models exhibiting in-
flammation and remodeling characteristic of lung diseases such as
asthma, COPD, and idiopathic pulmonary fibrosis (9–11). Extra-
of target cells (12). Among these responses is the activation of
pathways that are important in the regulation of wound healing
associated with both pro- and antiinflammatory actions that are
dictated by specific adenosine receptor subtypes and their down-
stream signaling components (17–19). Accordingly, adenosine
signaling is emerging as a major pathway involved in regulating
the balance between tissue repair and/or fibrosis.
The A3adenosine receptor (A3R) has emerged as an impor-
tant regulator of pulmonary inflammation and airway remodel-
ing. Its levels are elevated in patients with chronic lung disease
addition, there is evidence that A3R signaling can influence
inflammatory cell functions relevant to pulmonary disease. For
example, engagement of the A3R on mouse mast cells promotes
mediator release (23, 24), and engagement of the A3R on eo-
activities. Similarly, there is evidence that A3R engagement can
serve pro- and antiinflammatory roles on macrophages and
neutrophils (27–29). Thus, there is evidence for both antiinflam-
relevant to lung disease.
The ability of adenosine to regulate both inflammation and
of inflammation and fibrosis after lung injury. To test this
hypothesis, we used a model of bleomycin-induced lung injury to
examine the involvement of A3R signaling in pulmonary inflam-
mation and fibrosis. A3R-deficient mice exhibited enhanced in-
flammation in response to bleomycin exposure as compared with
Pulmonary fibrosis is associated with many interstitial lung
diseases. The current study suggests that A3 adenosine
receptor signaling regulates key aspects of pulmonary
inflammation and fibrosis.
(Received in original form November 16, 2007 and in final form April 18, 2008)
This work was funded by NIH Grants HL70952 (M.R.B.), AI43572 (M.R.B.), and
U19A1070973 (F.K.), and by the Mayo Foundation. E.M. was supported by an
American Academy of Allergy, Asthma and Immunology Strategic Training in
Allergy Research (ST*AR) Award.
Correspondence and requests for reprints should be addressed to Michael R.
Blackburn, Ph.D., Department of Biochemistry and Molecular Biology, University
of Texas Health Science Center at Houston, Medical School, 6431 Fannin,
Houston, TX 77030. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2007-0419OC on June 27, 2008
Internet address: www.atsjournals.org
Vol 39. pp 697–705, 2008
was little difference in the degree of fibrosis seen in the lungs of
wild-type and A3R-deficient mice exposed to bleomycin. Exami-
nation of fibrotic mediators revealed enhanced levels of trans-
forming growth factor (TGF)-b1 production; however, there was
is needed for eosinophil degranulation in this model, which may
also contribute to the effects seen on fibrosis. Collectively, these
studiesindicatethatthe A3Rservesantiinflammatoryfunctions in
role in regulating the levels of fibrotic mediators that may help to
influence the severity of ensuing fibrosis.
MATERIALS AND METHODS
A3R-deficient (A3R2/2) mice congenic on a C57Blk6 background were
obtained from Marlene A. Jacobson (Merck Research Labs, West Point,
PA) (23), and wild-type C57Blk6 mice (A3R1/1) were obtained from
Harlan (Indianapolis, IN). Animal care was in accordance with institu-
tional and NIH guidelines, and all procedures were approved by the
University of Texas Health Science Center at Houston Animal Welfare
Committee. Mice were housed in ventilated cages equipped with micro-
isolator lids and maintained under strict containment protocols. No
with avertin (250 mg/kg, intraperitoneally), and 3.5 U/kg bleomycin
saline or saline alone was instilled intratracheally. Endpoints were
examined at 1, 3, 7, 14 and 21 days after challenge.
Histology and Immunostaining
Mice were anesthetized, and lungs lavaged three times with 0.4 ml PBS.
Total cell counts were determined using a hemocytometer, and cellular
differentials (300 cells/sample) were conducted on cells cytospun onto
microscope slides and stained with Diff-Quick (Dade Behring, Newark,
DE). Lungs were then infused with 10% buffered formalin at 25 cm of
pressure and fixed overnight at 48C. Fixed lungs were dehydrated and
embedded in paraffin. Sections (5 mm) were collected on microscope
slides, rehydrated through graded ethanols to water then stained with
hematoxylin and eosin (H&E; Shandon-Lipshaw, Pittsburgh, PA),
Masson’s trichrome or 0.1% tolidine blue.
For immunostaining, rehydrated slides were quenched with 3%
Denmark] or Zymed pepsin [Zymed, San Francisco, CA]), and endog-
enous avidin and biotin blocked with a Biotin Blocking System (Dako
Corp). Slides were incubated with primary antibody: a-smooth muscle
actin (a -sma, 1:1,000 dilution [Sigma, St. Louis, MO] overnight at 48C
after processing sections with Mouse on Mouse Kit [Vector Laborato-
1,1:1,000dilution,1hatroom temperature), or goatanti-mouseMMP-9
(1:50 dilution, 1 h at room temperature; R&D Systems, Minneapolis,
appropriate secondary antibodies, then developed with 3,39-diamino-
benzidine (Sigma-Aldrich) and counterstained with methyl green.
Quantification of peribronchial eosinophils was assessed by analysis of
10 fields at 320 magnification. Eosinophils were counted using Image
Pro Plus 4.0 image analysis software (Media Cybernetics, Silver Spring,
9 (1:50 dilution, 1 h at room temperature; R&D Systems) followed by
CA). Slides were coverslipped with Vectashield (Vector Laboratories)
mounting medium containing DAPI to visualize nuclei.
Eosinophil Peroxidase Assay
Eosinophil peroxidase (EPO) activity present in 75 ml of undiluted
lavage fluid samples was detected after incubation in a 96-well plate
with equal volumes of substrate solution (50 mM Tris-HCl, pH 8, 0.1%
Triton X-100, 8.8 mM H2O2, 10 mM o-phenylenediamine [OPD], 6 m M
KBr) at 378C for 30 minutes. Duplicate samples also containing 10 mM
of the peroxidase inhibitor resorcinol (1,3-Benzenediol; Sigma-Aldrich)
were used as controls for the specificity of these reactions. The reaction
was stopped with 50 ml 4N H2SO4, and OD values were read at 490 nm
and EPO content given as OD/ml.
Analysis of mRNA
Mice were killed and the lungs were rapidly removed and frozen in
liquid nitrogen. RNA was isolated from frozen lung tissue using TRIzol
Reagent (Life Technologies, Inc., Gaithersburg, MD). RNA samples
were then DNase-treated (DNaseI; Invitrogen) and subjected to
quantitative RT-PCR. The primers, probes and procedures for the
PCR reactions were as described previously (30, 31). Values were
normalized to 18S ribosomal RNA and are presented as mean nor-
malized transcript levels using the comparative Ct method (2DDCt) (32).
MMP, Tissue Inhibitor of Metalloproteinase-1,
Gelatinolytic activity in bronchoalveolar lavage (BAL) fluid was
assessed by zymography based on standard methods (33). Briefly, BAL
After electrophoresis, SDS was removed by washes in 2.5% Triton X-
100, before incubation at 378C for 24 hours in developing buffer (50 mM
the MMP inhibitor 10 mM of 1910-phenantroline (Sigma). Gels were
0.3% wt/vol Coomassie blue R-250. MMP-9 and MMP-2 bands were
identified by their molecular weight, and 10 ml plasma from wild-type
C57Bl/6 mice served as positive controls. Protein levels for MMP-9 and
tissue inhibitor of metalloproteinase (TIMP)-1 were assessed in BAL
Piscataway, NJ) and enzyme-linked immunosorbent assay (TIMP-1,
R&D Systems). Active TGF-b1was measured using a mouse immuno-
assay kit (R&D Systems).
The Sircol assay (Biocolor Ltd., Carrick, UK) was used to measure
collagen in whole lung homogenates. Snap-frozen lungs were homog-
enized in 2 ml 0.5 M acetic acid containing pepsin (1:10 pepsin/tissue;
Sigma-Aldrich) and then incubated overnight shaking at room tem-
perature. Homogenates were spun at 10,000 rpm, and supernatant
assayed for pepsin-soluble collagen. Ashcroft scores were determined
on Masson’s Trichrome–stained lung sections using a minor modifica-
tion of the method outlined by Ashcroft and coworkers (34). Twenty
fields per slide were examined using a two-person randomized blind
study. At least six mice per group were examined.
Values are expressed as means 6 SEM. As appropriate, groups were
compared by ANOVA, with follow-up comparisons between groups
being conducted using Student’s t test with a P value of , 0.05 denoting
Pulmonary Inflammation in A3R2/2Mice Treated
A3R2/2mice were exposed to bleomycin, and pulmonary inflam-
mation was assessed 14 days later to examine the contribution of
A3Rsignaling to the pulmonary inflammation seen after bleomycin
and A3R2/2mice treated with saline (Figures 1A and 1B), suggest-
ing no obviousbasalphenotypein thelungsofA3R2/2mice.There
was pronounced reorganization of the alveolar airways of A3R1/1
mice exposed to bleomycin (Figure 1C), with extensive fibrosis and
A3R2/2mice exposed to bleomycin also exhibited severe reorgani-
698AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 392008
zation of the alveolar spaces, with fibrosis and what appeared to
be an enhancement of inflammatory cell foci (Figure 1D). These
findings suggest that A3R2/2mice exhibit enhanced pulmonary
inflammation after bleomycin exposure.
Mice were lavaged on Day 14 after exposure, and the number
of inflammatory cells was quantified in BAL fluid to better
characterize the pulmonary inflammation seen after bleomycin
exposure. There was an 8-fold increase in the number of in-
flammatory cells recovered from the airways of A3R1/1mice
exposed to bleomycin relative to that seen in A3R1/1mice
inflammatory cells was significantly higher in A3R2/2mice
exposed to bleomycin. Analysis of cellular differentials demon-
strated enhanced elevations in macrophages, neutrophils, and
eosinophils in the airways of A3R2/2mice exposed to bleomycin
(see Figure E1A in the online supplement). Examination of
inflammatory mediators demonstrated enhanced expression of
key cytokines and chemokines, including CCL2, IL-6, and
CXCL1 (Table E1). These findings demonstrate that there is
enhanced inflammation in the airways of A3R2/2mice 14 days
after bleomycin exposure.
Eosinophil Trafficking and Activation in A3R2/2Mice
Treated with Bleomycin
the pulmonary inflammation. There were no significant differ-
ences in the number of inflammatory cells recovered from the
BAL of A3R1/1and A3R2/2on Day 1 after bleomycin exposure
(Figures 2A and E1). There was a trend toward a developmental
mice treated with bleomycin; however, only eosinophil numbers
exhibited a significant increase during the later stages examined
(Figure 2A). The enrichment of airway eosinophils in A3R2/2
mice exposed to bleomycin prompted us to examine the levels of
eosinophils within the lung parenchyma. Lung sections were
stained with an antibody against mMBP-1 to identify tissue-
infiltrating eosinophils (Figure 2B). More mMBP-1–positive
cells were present within the interstitial spaces of lungs from
bleomycin-treated A3R2/2mice, a feature that was verified by
quantifying mMBP-1–positive cells in multiple tissue sections
recruitment to the lungs of A3R2/2mice treated with bleomycin,
we measured the levels of eosinophil-related cytokine and
chemokine transcripts in whole lung extracts (Figure 2D). Levels
of CCL17 (TARC) and IL-5 were found to be elevated in the
lungs of A3R1/1mice treated with bleomycin; however, their
levels were not enhanced in the lungs of A3R2/2mice. However,
transcript levels for CCL11 (eotaxin 1) and GM-CSF were
significantly increased in the lungs of A3R2/2mice treated with
mation. Mice were killed 14 days after saline or
bleomycin exposure, and the lungs were lavaged
and processed for hematoxylin and eosin staining.
(A) Lung section from an A3R1/1mouse exposed to
saline. (B) Lung section from an A3R2/2mouse
exposed to saline. (C) Lung section from an A3R1/1
mouse exposed to bleomycin. (D) Lung section from
an A3R2/2mouse exposed to bleomycin. Photo-
graphs are representative of six to eight mice from
each condition; scale bars 5 100 mm. (E) Total
bronchoalveolar lavage (BAL) cell counts presented
as mean cell counts 3 1046 SEM. *Significant
difference compared with saline treatment,#signifi-
cant difference compared with bleomycin-treated
A3R1/1mice (P , 0.05, n 5 6–8).
Pulmonary histopathology and inflam-
Morschl, Molina, Volmer, et al.: A3Adenosine Receptor Signaling in Lung Injury 699
in the BAL fluid of these mice (Table E1). These results suggest
of key mediators that lead to the enhanced recruitment of
eosinophils to the lung after bleomycin exposure.
The enhanced recruitment of eosinophils prompted us to
examine eosinophil activation. There was a marked increase in
EPO activity in the BAL fluid collected from A3R1/1mice
exposed to bleomycin (Figure 3A), suggesting substantial eosin-
ophil activation. However, despite the increase in eosinophil
numbers seen, there was no increase in EPO activity in the BAL
fluid of A3R2/2mice exposed to bleomycin. BAL cell pellets
were disrupted and EPO activity monitored in homogenates to
determine whether the lack of EPO activity in the BAL fluid
of A3R2/2mice was due to the absence of EPO protein in
eosinophils. Results demonstrated that EPO activity was equally
abundant in disrupted eosinophils from the airways of A3R1/1
and A3R2/2mice exposed to bleomycin (Figure 3B), suggesting
defects in eosinophil degranulation. These findings demonstrate
that the eosinophil degranulation associated with bleomycin
treatment is A3R dependent.
Pulmonary Fibrosis in A3R2/2Mice Treated with Bleomycin
A3R2/2mice were exposed to bleomycin, and fibrosis endpoints
were examined 14 days later to assess the contribution of A3R
signaling to pulmonary fibrosis. Masson’s trichrome staining
revealed extensive collagen deposition throughout the lungs of
A3R1/1mice exposed to bleomycin (Figure 4A). Extensive
collagen deposition was also seen in the lungs of A3R2/2mice
exposed to bleomycin. There were substantial increases in the
levels of collagen transcripts and protein in the lungs of A3R1/1
mice challenged with bleomycin (Figures 4C and 4D). Collagen
transcripts and protein levels were also elevated in the lungs of
A3R2/2mice exposed to bleomycin; however, the levels were
slightly lower than those measured in A3R1/1mice. Fibrosis was
ing for myofibroblasts. Results demonstrated a similar increase in
a-SMA–positive myofibroblasts in the airways of A3R1/1and
A3R2/2mice exposed to bleomycin (data not shown). Finally,
Ashcroft scoring was used as a means of assessing the overall
fibrosis seen. Consistent with our measurements of collagen, the
degree of fibrosis in the lungs of A3R1/1and A3R2/2mice were
equivalent (Figure 4B). Collectively, these results demonstrate
that there is no substantial difference in the degree of bleomycin-
induced pulmonary fibrosis in A3R1/1and A3R2/2mice.
TGF-b1 Regulation in A3R2/2Mice Treated with Bleomycin
Given the enhanced inflammation seen in the lungs of A3R2/2
mice exposed to bleomycin, it was surprising to find little
difference in the degree of pulmonary fibrosis in these mice.
The levels of the profibrotic mediator TGF-b1 were examined to
address the mechanisms involved in this discrepancy. As
of A3R1/1mice treated with bleomycin (Figure 5A). Interest-
recruitment. (A) BAL was collected
on Days 1, 3, 7, 14, and 21 after
bleomycin exposure, and cells were
cytospun onto slides, stained with
using a hemocytometer. Triangles
depict eosinophils from A3R1/1
mice exposed to bleomycin, while
squares depict eosinophils from
Data are presented as mean cell
counts 3 1046 SEM. *Significant
difference compared with A3R1/1
mice (P , 0.05, n 5 3–8). (B) Lung
sections from A3R1/1and A3R2/2
mycin were stained with mMBP-1
to mark eosinophils; scale bar 5
1–positive cells per mm2was quan-
tified in lung sections using Image-
Pro software. Data are presented as
mean eosinophils/mm26 SEM. (D)
Total cellular RNA was isolated from
the lungs of wild-type (A3R 1) or
A3R2/2(A3R 2) mice 14 days after
exposure to saline (open bars) or
bleomycin (solid bars). Quantitative
of key eosinophil related cytokines
and chemokines. Values were nor-
malized to 18S ribosomal RNA and
are presented as mean normalized
transcript levels (D2Ct) 6 SEM.
*Significant difference from saline-
difference from bleomycin-treated
A3R1/1mice (P , 0.05, n 5 6–8).
700 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 392008
whole lung RNA extracts (Figure 5A) and RNA extracts from
BAL cell pellets (Figure 5B) of A3R2/2mice treated with
bleomycin. However, there was no difference in the levels of
active TGF-b1 protein in the BAL fluid of A3R1/1and A3R2/2
mice exposed to bleomycin (Figure 5C). These findings suggest
form of this mediator.
Studies have shown that MMP-9 is an important regulator of
active TGF-b1 production in pulmonary fibrosis (35). We there-
fore hypothesized that MMP-9 production is diminished in the
(A) Eosinophil peroxidase ac-
tivity was measured in the BAL
fluid of mice 14 days after
exposure to saline or bleomy-
cin. (B) Eosinophil peroxidase
cell pellets from mice 14 days
after exposure to saline or bleo-
mycin. Eosinophil peroxidase
activity is presented as mean
optical density (OD) at 490
nm/ml 6 SEM. *Significant dif-
ence from bleomycin-treated
Figure 4. Pulmonary fibrosis in A3R1/1and A3R2/2mice. (A) A3R1/1and A3R2/2mice were killed 14 days after saline or bleomycin exposure, and
the lungs were processed for Masson’s trichrome staining. Photographs are representative of six to eight mice from each condition; scale bars 5
100 mm. (B) Ashcroft scores were determined by examining multiple sections from lungs of mice from each condition. Data are presented as mean
Ashcroft score 6 SEM (n 5 6–8 lungs per condition). (C) RNA was extracted from the lungs of mice 14 days after saline or bleomycin exposure, and
a1 procollagen levels were quantified using quantitative rtPCR. Values were normalized to 18S ribosomal RNA and are presented as mean
normalized transcript levels (D2Ct) 6 SEM (n 5 6–8). (D) Collagen protein levels were measured in whole lungs 14 days after saline or bleomycin
exposure using the Sircol assay. Data are presented as mean mg collagen per lung 6 SEM (n 5 6–8). *Significant difference from saline-treated
A3R1/1mice (P , 0.05).
Morschl, Molina, Volmer, et al.: A3Adenosine Receptor Signaling in Lung Injury701
of enhanced active TGF-b1. To address this, MMP-9 levels were
measured. MMP-9 whole lung mRNA levels (Figure 6A), whole
lung protein activity (Figure 6B), and BAL fluid activity (Figure
6C) were all elevated in the lungs of A3R1/1mice treated with
bleomycin. Levels of MMP-9 RNA and activity were diminished
in the lungs of A3R2/2mice treated with bleomycin, suggesting
that the A3R regulates the production of this protease. MMP-9
activity is also regulated by the levels of protease inhibitors such
as TIMP-1. TIMP-1 protein levels were found to be elevated in
the BAL fluid of A3R1/1mice treated with bleomycin, while
(Figure 6D). Consitent with these findings, there was a reduction
in the ratio of MMP-9 to TIMP-1 activity in the BAL fluid of
Examination of the cellular source of MMP-9 in this model
using immune detection methods revealed that MMP-9 pro-
duction is limited to inflammatory components in the lungs of
both A3R1/1and A3R2/2mice treated with bleomycin (Figures
7A and 7B). Examination of isolated BAL cells revealed that all
neutrophils and a subset of alveolar macrophages produced
MMP-9 in the lungs of bleomycin-treated A3R1/1mice (Figure
7C). In contrast, MMP-9 production was detected in neutrophils
isolated from the airways of A3R2/2mice treated with bleo-
mycin; however, alveolar macrophages isolated from these mice
did not produce MMP-9 (Figure 7D). MMP-9 expression was
not detected in eosinophils in either group of mice (data not
shown). MMP-9 expression was abundant in mast cells found
in the bronchi of A3R1/1mice treated with bleomycin (Figures
7E and 7F), but was absent from mast cells in the bronchi of
A3R2/2mice (Figures 7G and 7H). Collectively, these findings
demonstrate that MMP-9 levels are diminished in the lungs of
A3R2/2mice and that A3R-dependent expression of MMP-9 in
mast cells and alveolar macrophages likely accounts for this
Regulated inflammation and matrix production are critical
components of wound healing processes. An emerging model
for the development of pulmonary fibrosis is that abnormal
was isolated from the lungs (A) or BAL cell pellets (B) of wild-
type (A3R 1) or A3R2/2(A3R 2) mice 14 days after exposure to
saline (open bars) or bleomycin (solid bars). Quantitative rtPCR
was used to measure TGF-b1 transcript levels. Values were
normalized to 18S ribosomal RNA and are presented as mean
normalizedtranscriptlevels (D2Ct) 6 SEM(n 56–8). (C) Active
TGF-b1 protein levels were determined in BAL fluid from mice
treated the same as in A and B. Values are presented as mean
pg/ml TGF-b1 6 SEM (n 5 4–6). *Significant difference from
saline-treated A3R1/1mice,#significant difference from bleo-
mycin-treated A3R1/1mice (P , 0.05).
TGF-b1 expression and activity. Total cellular RNA
teinase (MMP)-9 and tissue in-
(TIMP)-1 expression and activ-
ity. (A) Total cellular RNA was
isolated from the lungs of
wild-type (A3R 1) or A3R2/2
(A3R 2) mice 14 days after
exposure to saline (open bars)
Quantitative rtPCR was used
to quantify the levels MMP-9
transcripts. Values were nor-
RNA and are presented as
levels (D2Ct) 6 SEM (n 5 6–
8). (B) BAL fluid (20 ml from
saline-, and 10 and 5 ml from
were subjected to 10% SDS-
PAGE in gels containing 0.02%
gelatin. Gels were fixed and
stained with 50% methanol
and 10% acetic acid that con-
tained 0.3% wt/vol Coomassie
bands were identified by their
molecular weight. MMP-9 (C)
and TIMP-1 (D) activities were
determined in BAL fluid using
specific immunoassays. (E) MMP-9 and TIMP-1 BAL fluid activities were used to determine a ratio of MMP-9 to TIMP-1. Data are presented as means
6 SEM. *Significant difference from saline-treated A3R1/1mice,#significant difference from bleomycin-treated A3R1/1mice (P , 0.05, n 5 6–8).
702AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 39 2008
inflammation and matrix regulation after lung injury progresses
to an overactive wound healing response that culminates in
fibrosis. In this context, understanding the factors produced after
injury and how they influence the normal or abnormal wound
healing response could provide important mechanistic informa-
tion. Adenosine is produced in response to tissue damage, where
it is thought to regulate key features of the injury response.
Antiinflammatory and tissue-protective activities of adenosine
are well documented (17, 18, 36), and adenosine signaling path-
demonstrate that adenosine has profibrotic activities in disease
models including the lung (14, 15, 37). Thus, adenosine pro-
duction after lung injury may access pathways that regulate the
balance of inflammation and matrix production that influence
lung repair or the progression to fibrosis. Adenosine elicits many
of its activities by engaging cell surface adenosine receptors (12).
to inflammatory and fibrotic processes after injury in the lung.
This was done by subjecting A3R2/2mice to bleomycin-induced
pulmonary injury. The results of these studies demonstrate that
A3R signaling serves a largely antiinflammatory role in the
bleomycin model; however, it also serves to increase the levels
fibrotic pathways. These findings suggest that A3R signaling may
of pulmonary fibrosis.
develop enhanced pulmonary inflammation after bleomycin ex-
posure. This was characterized by an increase in the numbers of
macrophages, neutrophils,and particularly eosinophils inA3R2/2
mice exposed to bleomycin. These findings suggest that the A3R
plays an antiinflammatory role in the bleomycin model. Similar
findings were reported in a study that demonstrated increased
inflammation in A3R2/2mice subjected to an air-pouch model
of acute inflammation (38). In addition, our observations are
consistent with findings in in vivo models of injury, including
lipopolysaccharide-induced endotoxemia (39), adjuvant-induced
arthritis (40), colitis (41), and ischemic injury in the lung (42),
where A3R activation has been shown to suppress inflammatory
processes. Thus, our findings strengthen the notion that the A3R
plays an important role in regulating the degree of inflammation
for the release of EPO from eosinophils recruited to the lung.
Despite the enhanced numbers of eosinophils recruited to the
of EPO release from these cells, leading us to conclude that the
A3R is needed for eosinophil degranulation. A3R expression is
abundant on mouse (21) and human eosinophils (43), and
Ca21levels (43). However, the role of the A3R in eosinophil
can induce superoxide production (26). In contrast, studies on
cultured human peripheral blood eosinophils do not support
a role for A3R engagement in stimulating eosinophil degranula-
tion (43, 44), and there is evidence that it may even prevent
degranulation (25). These discrepancies may arise in part from
the inability of the in vitro setting to accurately capture the
environment of the diseased lung where degranulation occurs. In
allergen-recruited BAL eosinophils from transgenic mice over-
expressing IL-5 (45), and we were not able to directly stimulate
EPO release in vitro with an A3R agonist (data not shown). We
were killed 14 days after saline or bleomycin
exposure, and the lungs were lavaged and
Immunoperoxidase detection using a MMP-9
antibody was used to localize MMP-9 (brown
stain) to inflammatory cells in the lungs of (A)
A3R1/1mice exposed to bleomycin and (B)
A3R2/2mice exposed to bleomycin. The same
antibody was used in an immunofluorecence
assay to examine MMP-9 expression (green) in
BAL cells from (C) A3R1/1mice exposed to
bleomycin and (D) A3R2/2mice exposed to
bleomycin. Blue staining denotes DAPI-stained
nuclei. Mast cells were localized in bronchi of (E)
A3R1/1mice exposed to bleomycin and (G)
A3R2/2mice exposed to bleomycin using toli-
dine blue staining. MMP-9 immunoperoxidase
detection (brown stain) was used to localize
MMP-9 to mast cells in the bronchi of (F)
A3R1/1mice exposed to bleomycin and (G)
A3R2/2mice exposed to bleomycin. n, neutro-
phils; F, macrophages; arrows denote mast
cells. Scale bars: A and B, 50 mm; C and D,
10 mm; H, 10 mm (also applies to E–G).
MMP-9 immunolocalization. Mice
Morschl, Molina, Volmer, et al.: A3Adenosine Receptor Signaling in Lung Injury703
thus conclude that important co-stimulators or cellular interac-
tions necessary for A3R-mediated eosinophil degranulation are
not present inculture situations.A rolefor the A3Rin eosinophil
degranulation in vivo may have important implications for
diseases such as asthma, in which both elevations in eosinophils
and adenosine are thought to be involved in disease progression
(5, 46). Indeed, a recent expression study assessing transcripts
from the lungs of allergen-treated wild-type and eosinophil-less
mice demonstrated that A3Rtranscripts weresignificantly down-
regulated in the lungs of mice lacking eosinophils (47).
In the bleomycin model, pulmonary inflammation peaks be-
tween Days 7 and 10 after exposure and then diminishes, while
fibrotic changes appear around Day 7 and progressively increase
(48). Work from our laboratory and others has demonstrated that
associated with enhanced fibrosis (11, 49, 50). Interestingly, we
found that despite enhanced pulmonary inflammation in A3R2/2
mice exposed to bleomycin, there was not an increase in standard
deposition and a-SMA staining. This observation suggests that
removing the A3R uncouples amplification pathways that link
can contribute to fibrosis by promoting the processing of latent
MMP-9 expression is decreased in the lungs of A3R2/2mice
exposed to bleomycin and that loss of expression in alveolar
macrophages and mast cells is responsible for this decrease. In
A3R2/2mice treated with bleomycin; however, there was not
a concomitant increase in the levels of active TGF-b1. These
findings have led us to propose a model in which adenosine
generated during bleomycin injury (11) can engage the A3R on
MMP-9 that in turn can lead to the activation of TGF-b1 and
contribute to the amplification of pulmonary fibrosis. This mech-
anism could account for the observation that despite increased
inflammation in A3R2/2mice exposed to bleomycin, there is not
an increase in the degree of fibrosis seen. This provides a novel
route bywhich a factorgenerated inresponsetodamage (namely,
adenosine) can impact inflammation and subsequent fibrosis.
Another consideration for the disconnection of inflammation
elevated in models of bleomycin-induced fibrosis (53, 54). More-
over, eosinophil-derived cationic proteins such as EPO have been
shown to influence the production of mediators such as MMP-9
degranulation may play an important role in regulating fibrosis.
Although we found increased numbers of eosinophils in the lungs
of A3R2/2mice exposed to bleomycin, they did not appear to be
degranulating as evidenced by the lack of EPO release. Thus, the
lack of enhanced fibrosis in A3R2/2mice exposed to bleomycin.
In conclusion, this study has characterized the contribution of
the A3R in the inflammation and fibrosis that results from
bleomycin injury. The roles of this receptor in this model are
complex. There was clear evidence for antiinflammatory influen-
ces in that A3R2/2mice exhibited exaggerated expression of
certain cytokines and chemokines in association with an excessive
difference in the degree of fibrosis seen in A3R1/1and A3R2/2
the development of fibrosis in this model. However, investigation
of TGF-b1 activationsuggests that the A3R may contributeto the
amplification of pulmonary fibrosis by regulating MMP-9, and/or
by regulating eosinophil activation. Thus, signaling through the
A3R may contribute to inflammatory and fibrotic processes that
regulate the balance between normal wound healing and the
unremitting fibrosis seen in interstitial lung diseases.
Conflict of Interest Statement: M.R.B. is a paid consultant for CV Therapeutics for
work on the A2Badenosine receptor. None of the other authors has a financial
relationship with a commercial entity that has an interest in the subject of this
Acknowledgments: The authors thank Marlene Jacobson for providing A3R-
deficient mice, and Hope Northrop for her assistance in obtaining Blenoxane
for use in this study.
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Morschl, Molina, Volmer, et al.: A3Adenosine Receptor Signaling in Lung Injury705