Anti-Inflammatory Preconditioning by Agonists of
Adenosine A1 Receptor
Sigal Nakav1, Cidio Chaimovitz2, Yuval Sufaro1, Eli C. Lewis1, Gad Shaked3, David Czeiger3, Moshe
Zlotnik2, Amos Douvdevani1,2*
1Department of Clinical Biochemistry, Soroka Medical University Center and Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2Department of Nephrology, Soroka
Medical University Center and Ben-Gurion University of the Negev, Beer-Sheva, Israel, 3Department of General Surgery, Soroka Medical University Center and Ben-Gurion
University of the Negev, Beer-Sheva, Israel
Background: Adenosine levels rise during inflammation and modulate inflammatory responses by engaging with four
different G protein-coupled receptors. It is suggested that adenosine exhibits pro-inflammatory effects through its A1
receptor (A1R), and anti-inflammatory effects through A2Areceptor (A2AR). Therefore, understanding of the mechanisms that
govern adenosine receptor regulation may advance treatment of various inflammatory disorders. We previously reported
that peak A1R expression during leukocyte recruitment, is followed by a peak in A2AR during inflammation resolution.
Principal Findings: Here, we examined whether A1R activation sequentially induces A2AR expression and by this reverses
inflammation. The effect of adenosine on A1R mediated A2AR expression was examined in peritoneal macrophages (PMW)
and primary peritoneal mesothelial cells (PMC) in vitro. Induction of A2AR was inhibited by pertussis toxin (PTX) and partly
dependent on A2AR stimulation. Administration of A1R agonists to healthy mice reduced A1R expression and induced A2AR
production in PMC. Mice that were preconditioned with A1R agonists 24 hours before E. coli inoculation exhibited
decreased TNFa and IL-6 sera levels and reduced leukocytes recruitment. Preconditioning was blocked by pretreatment
with A1R antagonist, as well as, or by late treatment with A2AR antagonist, and was absent in A2AR2/2mice.
Conclusions: Our data suggest that preconditioning by an A1R-agonist promotes the resolution of inflammation by
inducing the production of A2AR. Future implications may include early treatment during inflammatory disorders or
pretreatment before anticipated high risk inflammatory events, such as invasive surgery and organ transplantation.
Citation: Nakav S, Chaimovitz C, Sufaro Y, Lewis EC, Shaked G, et al. (2008) Anti-Inflammatory Preconditioning by Agonists of Adenosine A1 Receptor. PLoS
ONE 3(5): e2107. doi:10.1371/journal.pone.0002107
Editor: Patricia Bozza, Instituto Oswaldo Cruz and FIOCRUZ, Brazil
Received September 6, 2007; Accepted March 23, 2008; Published May 7, 2008
Copyright: ? 2008 Nakav et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant of the Israeli Science Foundation (Grant #558/06) and by the ‘Dr. Montague Robin Fleisher Kidney Transplant Unit
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Over the past few years, a vast number of investigations have
reported the involvement of adenosine in the anti-inflammatory
process [1,2]. Adenosine is an endogenous purine nucleoside that
is constitutively present in the extracellular spaces at low concent-
rations. However, in metabolically-stressful conditions such as
tissue damage, ischemia and inflammation, adenosine dramatically
increases its extracellular levels. Extracellular adenosine levels
have been observed to increase by dephosphorylation of ATP in
non-immune and immune cells  and then to be released
through the action of specialized nucleoside transporters .
Extracellular adenosine interacts with at least four different
receptor subtypes [4–6]. The A2Areceptor (A2AR) interacts with
the G protein Gsand the A2Breceptor (A2BR) interacts with the G
proteins Gsand Gqto induce adenylyl cyclase activity and elevate
cAMP levels. In contrast, ligation of adenosine to the A1receptor
(A1R) or to the A3 receptor (A3R), through interaction with
members of the Gi/Gofamily, inhibits adenylyl cyclase activity
and decreases cAMP levels . A1R exerts a pro-inflammatory
response by enhancing phagocytosis , promoting chemotaxis
[9,10] and enhancing neutrophils adherence to endothelium dur-
ing inflammatory process . In contrast, engagement of A2AR
inhibits neutrophils adherence to endothelium during inflamma-
tion  and inhibits the activation of neutrophils, monocytes
platelets and T-cells [13–15]. In animal models, A2AR-agonists
can prevent lethal response to bacterial LPS and sepsis [16,17].
Since each of these receptor subtypes has a unique physiological
profile and a particular affinity to its ligand, the inflammatory state
is determined by both extracellular adenosine concentrations and
by the distribution and expression levels of its receptor subtypes. It
has been shown that the expression of adenosine receptors is
regulated by factors that are involved in the inflammatory
response, such as LPS , pro-inflammatory cytokines [19–21],
growth factors [22,23] and glucocorticoids . Recently, we have
shown in a model of peritonitis that shortly following inoculation,
A1R mRNA and protein levels are upregulated on peritoneal
mesothelial cells (PMC), reaching a peak in the initial phase of the
inflammatory process . Interestingly, concomitant with the
resolution phase of peritonitis, we observed a decrease in A1R
PLoS ONE | www.plosone.org1May 2008 | Volume 3 | Issue 5 | e2107
expression levels and an elevation of adenosine and A2AR levels.
The coordinated kinetics of adenosine and its receptors led to the
hypothesis that adenosine differentially regulates its own receptors.
Since the two receptors, A1R and A2AR, have opposing biological
effects, and A1R domination precedes the elevation of A2AR, we
sought to examine whether A1R activation would be one of the
factors that trigger the anti-inflammatory phase, and whether this
action is mediated by upregulation of the A2AR.
To test our hypothesis, we examined the effect of adenosine
receptor agonists and antagonists in vivo in a model of peritonitis
induced by E. coli inoculation. This model has particular clinical
significance because peritonitis is commonly caused by patholog-
ical processes of the gastrointestinal tract or as a complication of
abdominal surgery. In vitro, we examined the regulation of the
receptors on the cell surface of PMW, which are the first line of
cellular defense against bacterial invasion in the peritoneum ,
and on PMC, the cells that line the peritoneal membrane and
therefore play an important role in transferring inflammatory
signals from the peritoneal cavity to the blood vessels [26–30]. We
demonstrate that A1R activation triggers the switching of adeno-
sine receptor subtype from A1R to A2AR. By the anti-
inflammatory effects of the ligation of adenosine to the A2AR,
the described receptor subtype switch alters the progression of
inflammation toward resolution.
Materials and Methods
Mice, bacterial strains and drugs
CD1 female mice aged 10 to 12 weeks (Harlan, Jerusalem,
Israel) were maintained in the animal laboratory of the Soroka
Medical Center. Experiments were conducted with the permission
of the Israel Committee for Animal Experiments. A2AR2/2mice
whose phenotype is well established in the literature were
graciously kindly donated by Catherine Ledent (Universite ´ Libre
de Bruxelles) .
Escherichia coli (E. coli) were grown in Luria-Bertani broth
(Conda Laboratories, Madrid, Spain) and harvested during the log
phase. Bacteria aliquots in Luria-Bertani broth containing 30%
glycerol were stored frozen at 270uC. Adenosine (Adenocor) was
purchased from Sanofi Winthrop (Auckland, NZ). A2AR antago-
nist 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazin-5-ylamino]ethyl) phe-
nol (ZM241385) was purchased from Tocris Cookson (Ellisville,
MS). Pertussis toxin (PTX) and other Adenosine receptor agonists
and antagonists were purchased from Sigma (Rehovot, Israel):
A1R agonists N6-cyclohexyadenosine (CHA) and 2-Chloro-N6-
cyclopentyladenosine (CCPA); A1R antagonist 8-cyclopentyl-1, 3-
dipropylxanthine (DPCPX); A2AR agonist 2-p-(carboxyethyl)
(CGS21680); A3R antagonist 9-Chloro-2-(2-furanyl)-5-((phenyla-
cetyl)amino)-[1,2,4]triazolo[1,5-c] quinazoline (MRS1220); A2BR
1,3-di(n-propyl)xanthine hydrate (MRS1754).
Induction of peritonitis and treatment protocol
Peritonitis was induced in mice by intraperitoneal (i.p.)
inoculation of a sub-lethal dose of E. coli (3.66109CFU).
Adenosine agonists and antagonists were injected i.p. before E.
Sera and peritoneal lavage fluids collection, leukocyte
counting and cytokine detection
At different time points after E. coli inoculation, animals were
anesthetized. 1 ml syringe flushed with heparin was used to draw
intracardial blood sample. The samples were stored on ice before
centrifugation at 1,000 g at 4uC for 10 minutes. The cell-free
supernatants were collected and frozen at 220uC until assayed by
ELISA. Peritoneal lavage was performed with 5 ml phosphate
buffer saline (PBS) containing 2% BSA and 5 mM EDTA. After
centrifugation at 400 g for 10 minutes, the cell-free supernatants
were removed and frozen at 220uC until analysis. TNFa and IL-6
levels were determined by commercial ELISA kits (Biolegend, San
Diego, CA and R&D Systems, Minneapolis, MN, respectively).
Cells were washed once, and total leukocytes were counted after
trypan blue staining using an improved Neubaur hemocytometer.
Cell counts and ELISA were performed blindly on coded samples.
Scraping of mice PMC
Following treatment, animals were anesthetized and PMC were
scraped from the peritoneal membrane. The cells were stored on
ice before centrifugation at 400g and 4uC for 10 minutes. Cells
were harvested with lysis buffer for analyzing mRNA levels or with
RIPA (150 mM NaCl, 50 mM Tris HCl pH-7.4, 1% NP-40,
0.25% Na deoxycholate, 1 mM EGTA) including protease
inhibitor cocktail (Sigma) for analyzing protein levels.
Preparation of cultured PMC and PMW
To prepare PMC, the peritoneum was removed from eight
newborn (two-week old) mice and isolated, as previously described
. To assess the purity of mesothelial cells, samples of each
PMC preparation were morphologically inspected, as previously
described . Cells were grown in M199 and supplemented with
10% heat-inactivated FCS, 2 mmol/l L-glutamine and 100 U/ml
penicillin and 100 mg/ml streptomycin (Biological Industries, Bet
Haemek, Israel). Experiments were performed on cells from the
second to fourth passages. To prepare PMW, mice were injected
intraperitoneally with 3 ml of 3% thioglycollate (Difco, Sparks,
MD). After 3 days, peritoneal cells were collected by lavage and
seeded onto 12-well plates in RPMI supplemented with 10% heat-
inactivated FCS, 2 mmol/l L-glutamine and 100 U/ml penicillin
and 100 mg/ml streptomycin. Non-adherent cells were subse-
quently removed by washing with PBS. In experiments, to
simulate the graduate increase in adenosine levels found in vivo,
cells were treated with increasing doses of adenosine or CHA with
or without DPCPX (9 hours with 0.1 mM or 3 hours with 0.1 mM
and then 6 hours with 1 mM or 3 hours with 0.1 mM, then
3 hours with1 mM and then 3 hours with 10 mM).
Total RNA was extracted from PMC or PMW using the
Versagene RNA cell kit (Gentra systems, Minneapolis, MN).
cDNA was prepared as previously described . Quantitative
real time PCR (QPCR) assays were carried out for b-actin,
GAPDH, A1R, A2AR, macrophage inflammatory protein-2 (MIP-
2) and monocyte chemotactic protein-1 (MCP-1) with the
following primers: b-actin sense: 95-GGG TCA GGA GGA
TTC CTA TG-93, b-actin antisense: 95-GGT CTC AAA CAT
GAT CTG GG-93, GAPDH sense: 95-CAA TGC ATC CTG
CAC CAC CAA-93, GAPDH antisense: 95-GTC ATT GAG
AGC AAT GCC AGC-93, A1R sense: 95-TAC ATC TCG GCC
TTC CAG GTC G-93, A1R anti sense: 95-AAG GAT GGC CAG
TGG GAT GAC CAG-93, A2AR sense: 95-ATT TGT GCC AGC
CAG GAA GCC-93, A2AR antisense: 95-GCA TCC GGG ACT
TTA AAC CAC AGA-93, MIP-2 sense: 95-CTC CTC AGT GCT
GCA CTG GT-93, MIP-2 antisense: 95-TCC CGG GTG CTG
TTT GTT T-93, MCP-1 sense: 95-CTC ACC TGC TGC TAC
TCA TTC-93, MCP-1 anti sense: 95-GCT TGA GGT GGT
TGT GGA AAA-93. cDNAs were diluted69, mixed with primers
(0.2 mM) and Thermo start master mix (ABgene, Surrey, UK).
PLoS ONE | www.plosone.org2May 2008 | Volume 3 | Issue 5 | e2107
Reaction was carried out in Rotor-Gene real time PCR machine
(Corbett-Research, Northlake, Australia).
Western blotting analysis
Cell lysates was centrifuged at 13,000 g for 30 minutes and then
supernatants were collected for total protein determination by the
BCA protein assay kit (Pierce, Rockford, IL). 30 mg of total protein
from each sample was subjected to 10% SDS-PAGE under
reducing conditions and after heating. The gels were blotted onto
a PVDF membrane (Bio-Rad, Hercules, CA) and probed with the
following specific antibodies: rabbit anti-adenosine A2AR (Santa
Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-A1R (Alpha
Diagnostic International, San Antonio, TX) or goat anti-b-actin
(Santa Cruz Biotechnology). The membrane was then probed with
goat anti-rabbit immunoglobulins Ig-conjugated to peroxidase
agent (Santa Cruz Biotechnology) or with donkey anti-goat IgG
conjugated to peroxidase agent (Jackson Immuno Research
laboratories, West Grove, PA). Antigen-antibody complexes were
subsequently visualized by the EZ-ECL Chemiluminescence
Detection kit for HRP (Biological Industries).
Data are presented as mean6SEM. Statistical analysis was
performed by t-test or ANOVA followed by Tukey post test. P
values below 0.05 were considered significant.
Adenosine receptors exhibit unique expression kinetics
in peritoneal leukocytes following bacterial inoculation
It has been shown that adenosine is upregulated during peri-
tonitis . We therefore examined the regulation of adenosine
receptors in peritoneal leukocytes and found that the A1R and
A2AR are upregulated during the first 48 hours of peritonitis. How-
A1R mRNA levels were maximal at 6 hours after inoculation and
returned to basal levels at 24 hours, while A2AR mRNA levels
gradually increased and reached maximum at 24 hours.
Adenosine induces the expression of A2R in a dose-
Since both adenosine and adenosine receptors are upregulated
upon bacterial inoculation , we wanted to elucidate whether
the regulation of adenosine receptors is adenosine-dependent. In
order to simulate the gradual and accumulative increase of
adenosine that is observed in vivo, we treated cultured PMCs with
multiple and increasing concentrations of adenosine (0.1, 1 and
10 mM at 3 hours intervals). As shown in Figure 2, adenosine
induced the expression of A2AR mRNA levels in a dose dependent
manner. However, there was no change in A1R mRNA levels
upon treatment with the different concentrations of adenosine.
Adenosine regulates A2AR expression through A1R
Since A1R is elevated shortly after bacterial inoculation
(Figure 1) and is followed by elevation of A2AR expression, we
wanted to examine whether the induction of A2AR by adenosine
may be mediated by the A1R. Therefore, we treated PMC and
PMW with 0.1, 1 and 10 mM at 3 hour intervals with A1R agonist
(CHA) or adenosine in the presence or absence of the A1R
antagonist (DPCPX, 50 nM). As shown in Figure 3A and B, CHA
upregulated mRNA levels of the A2AR while treatment with
adenosine in the presence of the DPCPX blocked A2AR
upregulation both in PMW and PMC respectively. In contrast,
stimulation with CGS, an A2AR agonist failed to induce A2AR
Ligation of adenosine to the A1R is mediated through the
interaction with members of the Gi/Go family and inhibits
adenylyl cyclase activity. To elucidate the mechanism by which
A1R induces A2AR elevation, we pretreated PMC with PTX, a Gi
inhibitor (Figure 3C). Pretreatment with PTX blocked the effect of
CHA on A2AR mRNA levels.
For effective induction of A2AR a sequential induction with
increasing doses of adenosine or CHA (0.1, 1, 10 mM) were
Figure 1. A1R and A2AR expression in peritoneal leukocytes
during inflammation in vivo. Peritonitis was induced in mice by E.
coli inoculation at a sub-lethal dose. To examine the dynamic
expression of the two high-affinity adenosine receptors, A1R and
A2AR, peritoneal lavage was performed at indicated time points. A1R
and A2AR mRNA levels in peritoneal leukocytes were analyzed by real
time PCR and normalized to b-actin levels. Data represent three
experiments and are expressed as mean6SEM. * p,0.05, between
expression levels of each receptor to expression at time 0, n=5 for each
Figure 2. Effect of adenosine on A2AR and A1R levels in vitro. To
simulate the gradual increase of adenosine that occurs during
peritonitis, cultured primary PMC were treated with multiple and
increasing concentrations of adenosine (0.1, 1 and 10 mM at 3 hour
intervals). Total RNA was extracted after 9 hours and analyzed for A1R
and A2AR mRNA levels. Results are normalized to b-actin. Data represent
five experiments and are expressed as mean6SEM fold of control.
* p,0.05, ** p,0.01 between expression levels of each receptor to
expression at time 0, n=3 for each experiment.
PLoS ONE | www.plosone.org3 May 2008 | Volume 3 | Issue 5 | e2107
necessary suggesting the involvement of an additional adenosine
receptor. CCPA, a specific A1R agonist, was less effective than
CHA, an A1R agonist with lower specificity (Figure 3D).
ZM241385, an A2AR antagonist, partially blocked the induction
of A2AR mRNA that was induce by adenosine (Figure 3D) or
CHA (data not shown), which suggests that in addition to the
requirement of A1R stimulation, A2AR ligation supports its own
induction. Treatment with adenosine in the presence of A3R
(MRS1220, 100nM) or A2BR antagonist (MRS1754, 50nM) did
not alter on A2AR mRNA levels (data not shown).
Effect of A1R agonist on the expression of A2AR and A1R
We examine whether the A1R agonist also regulates the levels of
the A2AR in vivo. We determined the mRNA and protein levels of
the A2AR and the A1R in mice that were administered an A1R
agonist (CHA, 0.1 mg/kg). We found that A2AR mRNA levels
increase ,3 fold and that A2AR protein levels increase ,2.5 fold,
compared to vehicle. In contrast, as shown in Figure 4, both A1R
mRNA and protein levels decreased in the presence of A1R
agonist by ,6 and ,2 fold, respectively.
Pretreatment with the A1R agonist reduces serum
cytokine levels and peritoneal leukocyte recruitment
Since we showed that A2AR levels are upregulated through the
activation of A1R both in vitro and in vivo, we wanted to elucidate
whether pretreatment of A1R agonist before inoculation would
upregulate the expression of A2AR and lead to advancement of the
anti-inflammatory response via A2AR. For this, mice were treated
Figure 3. A1R trigger the induction of A2AR in vitro. (A) PMW or (B) PMC were exposed to increasing concentrations of adenosine or A1R agonist
(CHA), (0.1, 1 and 10 mM 3 hours intervals) in the presence or absence of A1R antagonist (DPCPX, 50 nM, 30 min before treatment) (C) PMC were
treated with PTX for 18 hr and then with increasing concentrations of CHA. (D) PMC were treated with increasing concentrations of adenosine, A1R
agonists (CHA and CCPA) or A2AR agonist (CGS21680) in the presence or absence of A2AR antagonist (ZM241385, 50 nM). Total RNA was extracted
from cells and analyzed for A2AR mRNA levels and normalized to b-actin. CT, non-treated cells. Data represent four experiments and are expressed as
mean6SEM fold of control. ** p,0.01, *** p,0.001 from CT for B and D, n=3 for each experiment.
PLoS ONE | www.plosone.org4 May 2008 | Volume 3 | Issue 5 | e2107
with an A1R agonist (CHA, 0.1 mg/kg) 24 hours before inocula-
tion of E. coli, after which sera were analyzed for IL-6 and TNFa
levels. As shown in Figure 5A, we found a significant reduction
both in serum IL-6 and TNFa levels 12 hours after inoculation (to
25% and 38% from vehicle, respectively).
Since PMC express an array of chemokines which cause
accumulation and activation of leukocytes in tissues, we wanted to
examine changes in the levels of CXC chemokines, MCP-1 and
MIP-2, following pretreatment with A1R agonist. As a result of
pretreatment with the A1R agonist (CHA 0.1mg/kg), MCP-1 and
MIP-2 mRNA level decreased in comparison to vehicle, as
determined 12 hours after inoculation (Figure 5B). In accordance
with reduced chemokine levels, leukocyte recruitment significantly
decreased 24 hours after inoculation to 66% from vehicle, as
determined in lavage fluid (Figure 5C).
A1R-agonist preconditioning is blocked by a selective
To ensure that the anti-inflammatory state was mediated by
selective activation of the A1R, we examined the anti-inflamma-
tory effect of low-dose CHA and an additional specific A1R-
agonist CCPA, in the presence of a specific A1R antagonist
(DPCPX). As shown in Figure 6, treatment with either CCPA (A)
or CHA (B) significantly reduced serum and lavage IL-6 and
TNFa levels. However, pretreatment with an A1R antagonist
(DPCPX, 1 mg/kg) 2 hours before administration of A1R agonist
blocked the effect of 0.02 mg/kg CHA, 0.1 mg/kg CHA (data not
shown) and 0.1 mg/kg CCPA.
Figure 4. Effect of A1R agonist on A1R and A2AR levels in vivo.
Mice were administered i.p. with the A1R agonist (CHA, 0.1 mg/kg) or
with vehicle. PMC were scraped from the peritoneal surface and
analyzed for (A) A2AR and A1R mRNA levels at 4 hours or (B+C) A2AR
and A1R protein levels at 24 hours. (B) Densitometry of protein blot
depicted in (C). A1R and A2AR mRNA levels were normalized to GAPDH
and protein levels were normalized to b-actin. Results are presented as
fold change from vehicle-treated animals. Data represent three
experiments and are expressed as mean6SEM. * p,0.05 between
conditions per receptor, n=4 for each experiment.
Figure 5. The anti-inflammatory effect of pretreatment with
the A1R agonist. Mice were treated with the A1R agonist (CHA, i.p.,
0.1 mg/kg) or vehicle 24 hours prior to bacterial inoculation. (A)
Sera levels of IL-6 and TNFa at 12 hours. (B) Chemokine mRNA levels.
12 hours after inoculation PMC were scraped from the peritoneal
membrane and total RNA was extracted, analyzed for MCP-1 and
MIP-2 mRNA levels and normalized to b-actin. (C) Total cell count at
24 hours after inoculation. Cell exudates were collected from peritoneal
lavage fluid. Data represent five experiments and are expressed as
mean6SEM for serum cytokine levels and as mean6SEM fold of control
for chemokine mRNA levels.* p,0.05, ** p,0.01, n=5 for each
PLoS ONE | www.plosone.org5 May 2008 | Volume 3 | Issue 5 | e2107
Modulation of the inflammatory response due to
pretreatment with the A1R agonist is A2AR-dependent
To prove that the modulation in the inflammatory response
(Figure 5) is mediated by A2AR, we treated animals with an A2AR
antagonist (30 min before inoculation, ZM241385, 1 mg/kg). As
shown in figure 7, blockade of the A2AR caused an increase in
serum and lavage IL-6 and TNFa levels to similar levels found in
infected mice administrated with vehicle alone. As expected,
administration of A2AR agonist (30 minutes before inoculation,
CGS21680, 1 mg/kg) reduced IL-6 and TNFa levels in serum and
lavage fluids to levels comparable to those found in CHA-treated
animals. In concordance, pretreatment of A2AR2/2mice with
A1R agonist resulted in unchanged serum IL-6 and TNFa levels
(Figure 7C), as well as chemokine mRNA levels in PMC (data not
shown). However, in WT mice there was a significant reduction
both in cytokine levels and mRNA chemokine levels (data not
shown). These data suggest that the modulation of the inflamma-
tory response caused by pretreatment with A1R agonist is, indeed,
mediated by A2AR.
The study presented here demonstrates a novel mechanism of
adenosine receptor subtype autoregulation. Since adenosine action
is mediated through at least four different receptors, each of which
exhibits a unique affinity and opposing signaling pathways, the
regulation of subtypes expression is critical for determining the
outcome of adenosine activity . Others and we have shown that
adenosine receptors are regulated by various inflammatory
mediators and multiple endogenous factors . For example,
we found that A2AR mRNA and protein levels are upregulated in
human PMC following treatment with IL-1b and TNFa, while
treatment with IFNc strongly decrease A2AR expression both
alone and in combination with IL-1b and TNFa . In the same
study, we show that following inoculation, adenosine receptor
levels on PMCs are sequentially upregulated and that adenosine is
induced following inoculation and reaches peak levels at 24 hours
. The A1R is induced during the first phase of leukocyte
recruitment and the A2AR is induced later, at the resolution phase
of peritonitis . In the present study, we obtained the same
pattern of adenosine receptor expression on peritoneal leukocytes.
Figure 6. Treatment with A1R antagonist blocked the anti-
inflammatory effect of A1R agonists. 2 hours prior to administra-
tion of A1R agonist, (A) CCPA (0.1 mg/kg) or (B) CHA (0.02 mg/kg), mice
were injected with A1R antagonist (DPCPX, 1 mg/kg) or vehicle. After
24 hours, peritonitis was induced by bacterial inoculation. At 12 hours
from inoculation, IL-6 and TNFa were analyzed in sera and lavage fluids.
Data represent two experiments and are expressed as mean6SEM.
* p,0.05, between vehicle and CHA or CCPA, n=5 for each experiment.
Figure 7. The effect of A1R agonist, in A2AR2/2and in the
presence of A2AR antagonist. Mice were administrated with A1R
agonist (CHA, 0.1 mg/kg) or vehicle 24 prior to bacterial inoculation.
30 min before inoculation the A2AR antagonist (ZM241385, 1 mg/kg) or
the A2AR agonist (CGS21680, 1 mg/kg) were administered to the same
animals or to untreated animals. (A) sera IL-6 and TNFa (12 hours) and
(B) lavage fluids IL-6 and TNFa (12 hours). (C) A2AR2/2mice or their WT
littermates were treated with the A1R agonist (CHA, 0.1 mg/kg) i.p. or
vehicle 24 hours prior to bacterial inoculation. 12 hours following
inoculation sera were collected and analyzed for IL-6 and TNFa levels.
Data are representative of three individual experiments and are
expressed as mean6SEM. * p,0.05, ** p,0.01 between vehicle and
CHA or CGS21680 and between CHA with or without ZM241385, n=5
for each experiment.
PLoS ONE | www.plosone.org6 May 2008 | Volume 3 | Issue 5 | e2107
These results suggest that both mesothelial cells and the recruited
leukocytes are highly synchronized in their response to adenosine.
Furthermore, this sequential elevation of the A1R and the A2AR
on PMC and leukocytes suggests that adenosine may regulates its
receptors. Both our in vitro and in vivo data in the current study
support this suggestion; we found that adenosine significantly
upregulates A2AR expression levels in isolated PMC in a dose
Of all adenosine receptor subtypes, A1R exhibits the highest
affinity for adenosine (Ki=10 nM) , implying that A1R is
activated at the low levels of adenosine produced during the
initiation of inflammation. This early activation of A1R receptor
may enable the induction of A2AR. The A1R agonist, CHA,
significantly induced the expression of A2AR, while treatment with
the A1R antagonist, DPCPX, or with PTX, a Giinhibitor, blocked
A2AR induction by adenosine, indicating that A1R ligation is
necessary for the induction of A2AR. Treatment with CGS21680,
an A2AR agonist, did not induce the expression of the A2AR.
However, treatment with the A2AR antagonist in the presence of
adenosine partially blocked A2AR induction. Therefore, one can
conclude that A2AR ligation by elevated levels of adenosine is
required to support the initial signal of A1R.
According to our in vitro data, mice treated with CHA exhibited
a significant 2-3 fold increase in A2AR mRNA and protein levels as
determined, in PMCs compared to untreated animals. Interest-
ingly, mRNA and protein A1R levels were significantly down-
regulated by these same treatments in PMCs (6- and 2-fold
decrease, respectively), suggesting that A1R receptor may be
responsible for the ‘‘switching’’ between the two receptor subtypes
during inflammation. In Support of our findings, Schnurr et al.
showed that in immature plasmacytoid dendritic cells (PDCs)
adenosine activates A1R, which induces chemotaxis; however, in
mature PDCs, A1R is replaced by the A2AR, which inhibits
cytokine production .
In order to understand the physiological role of the exchange
between the two receptors, we examined whether ligation of the
A1R will trigger the induction of the A2AR and lead to an
advancement of the resolution phase of the inflammatory process.
We found that preconditioning with an A1R agonist significantly
reduces the inflammatory response to bacterial challenge. CHA or
CCPA administration at 24 hours before inoculation significantly
reduced sera and peritoneal levels of the pro-inflammatory cyto-
kines TNFa and IL-6, and reduced mRNA levels of chemokines
on PMC as well as leukocyte recruitment to the peritoneum. The
anti-inflammatory effect induced by pre-treatment (24 hours) with
A1R agonist was also achieved by a specific A2AR agonist
(CGS21680) administered to animals 30 minutes before bacterial
inoculation. Pre-treatment with CHA or CCPA had no anti-
inflammatory effect in animals that were administered with the
A1R antagonist, DPCPX 2 hours before agonists or A2AR
antagonist, ZM241385 30 minutes before inoculation or when
A2AR2\2animals were examined. The marked blocking effect of
ZM241385 and the lack of effect of CHA in A2AR knockout
animals clearly indicate that the anti-inflammatory effects of the
A1R agonist are mediated by the A2AR.
Elevation of cAMP usually down-regulates the inflammatory
response . Since A1R is a Gicoupled receptor that suppresses
the induction cAMP, it is not surprising that this receptor had no
direct anti-inflammatory effect. High expression of A1R implies
that immediately after inoculation, decreased cAMP levels give
rise to local pro-inflammatory cytokines and leukocyte migration,
hence allowing an adequate and effective immune response to the
invading microorganisms. In contrast, the increase in A2AR at late
phases of peritonitis is probably associated with elevated cAMP
levels, which markedly decrease local pro-inflammatory cytokine
levels and leukocyte recruitment, hence restraining inflammatory
flames (Figure 8).
In summary, our study sheds light on the sequential autoreg-
ulation of adenosine receptor subtypes. The mechanism we have
describes may directly participate in the propagation of the
compensatory anti-inflammatory response syndrome (CARS),
which follows systemic inflammation in trauma patients. Whether
patients with CARS exhibit elevated adenosine levels pursuing
traumatic insult should be explored. These findings may also have
future implications for clinical treatments by combining pre-
treatment with an A1R agonist and subsequent A2AR agonist to
enhance the anti-inflammatory effect, or to promote anti-
inflammation by endogenous adenosine at the site of inflamma-
tion. As such, preconditioning with an A1R-agonist could be used
in preparation of tissue for transplantation or to induce an anti-
inflammatory and immunosuppressive state in patients before
invasive surgery and organ transplantation.
We would like to thank Valeria Frishman for excellent technical assistance.
Conceived and designed the experiments: EL AD SN CC YS. Performed
the experiments: SN YS. Analyzed the data: AD SN. Contributed
reagents/materials/analysis tools: AD GS DC MZ. Wrote the paper: EL
Figure 8. Effect of adenosine receptor subtype autoregulation on the inflammatory process. (A) Early expression of A1R after bacterial
inoculation decreases cAMP levels, enhances production of local pro-inflammatory cytokines and promotes leukocyte migration. (B) In a later phase
of peritonitis A2AR expression increase by A1R which leads to increase in cAMP levels. High cAMP markedly decreases local pro-inflammatory
cytokines and leukocyte recruitment, hence restraining inflammatory flames.
PLoS ONE | www.plosone.org7 May 2008 | Volume 3 | Issue 5 | e2107
References Download full-text
1. Cronstein BN (1994) Adenosine, an endogenous anti-inflammatory agent. J Appl
Physiol 76: 5–13.
2. Hasko G, Cronstein BN (2004) Adenosine: an endogenous regulator of innate
immunity. Trends Immunol 25: 33–39.
3. Pastor-Anglada M, Casado FJ, Valdes R, Mata J, Garcia-Manteiga J, et al.
(2001) Complex regulation of nucleoside transporter expression in epithelial and
immune system cells. Mol Membr Biol 18: 81–85.
4. Olah ME, Ren H, Stiles GL (1995) Adenosine receptors: protein and gene
structure. Arch Int Pharmacodyn Ther 329: 135–150.
5. Olah ME, Stiles GL (1995) Adenosine receptor subtypes: characterization and
therapeutic regulation. Annu Rev Pharmacol Toxicol 35: 581–606.
6. Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines.
Pharmacol Rev 50: 413–492.
7. Olah ME, Stiles GL (2000) The role of receptor structure in determining
adenosine receptor activity. Pharmacol Ther 85: 55–75.
8. Salmon JE, Brogle N, Brownlie C, Edberg JC, Kimberly RP, et al. (1993)
Human mononuclear phagocytes express adenosine A1 receptors. A novel
mechanism for differential regulation of Fc gamma receptor function. J Immunol
9. Schnurr M, Toy T, Shin A, Hartmann G, Rothenfusser S, et al. (2004) Role of
adenosine receptors in regulating chemotaxis and cytokine production of
plasmacytoid dendritic cells. Blood 103: 1391–1397.
10. Rose FR, Hirschhorn R, Weissmann G, Cronstein BN (1988) Adenosine
promotes neutrophil chemotaxis. J Exp Med 167: 1186–1194.
11. Cronstein BN, Levin RI, Philips M, Hirschhorn R, Abramson SB, et al. (1992)
Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors
and inhibited via adenosine A2 receptors. J Immunol 148: 2201–2206.
12. McColl SR, St-Onge M, Dussault AA, Laflamme C, Bouchard L, et al. (2006)
Immunomodulatory impact of the A2A adenosine receptor on the profile of
chemokines produced by neutrophils. Faseb J 20: 187–189.
13. Sullivan GW, Rieger JM, Scheld WM, Macdonald TL, Linden J (2001) Cyclic
AMP-dependent inhibition of human neutrophil oxidative activity by substituted
2-propynylcyclohexyl adenosine A(2A) receptor agonists. Br J Pharmacol 132:
14. Cooper JA, Hill SJ, Alexander SP, Rubin PC, Horn EH (1995) Adenosine
receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine
release in human platelets. Br J Clin Pharmacol 40: 43–50.
15. Koshiba M, Kojima H, Huang S, Apasov S, Sitkovsky MV (1997) Memory of
extracellular adenosine A2A purinergic receptor-mediated signaling in murine T
cells. J Biol Chem 272: 25881–25889.
16. Sullivan GW, Fang G, Linden J, Scheld WM (2004) A2A adenosine receptor
activation improves survival in mouse models of endotoxemia and sepsis. J Infect
Dis 189: 1897–1904.
17. Mazar J, Rogachev B, Shaked G, Ziv NY, Czeiger D, et al. (2005) Involvement
of adenosine in the antiinflammatory action of ketamine. Anesthesiology 102:
18. Murphree LJ, Sullivan GW, Marshall MA, Linden J (2005) Lipopolysaccharide
rapidly modifies adenosine receptor transcripts in murine and human
macrophages: role of NF-kappaB in A(2A) adenosine receptor induction.
Biochem J 391: 575–580.
19. Rogachev B, Ziv NY, Mazar J, Nakav S, Chaimovitz C, et al. (2006) Adenosine
is upregulated during peritonitis and is involved in downregulation of
inflammation. Kidney Int 70: 675–681.
20. Khoa ND, Montesinos MC, Reiss AB, Delano D, Awadallah N, et al. (2001)
Inflammatory cytokines regulate function and expression of adenosine A(2A)
receptors in human monocytic THP-1 cells. J Immunol 167: 4026–4032.
21. Trincavelli ML, Costa B, Tuscano D, Lucacchini A, Martini C (2002) Up-
regulation of A(2A) adenosine receptors by proinflammatory cytokines in rat
PC12 cells. Biochem Pharmacol 64: 625–631.
22. Arslan G, Kontny E, Fredholm BB (1997) Down-regulation of adenosine A2A
receptors upon NGF-induced differentiation of PC12 cells. Neuropharmacology
23. Navarro A, Zapata R, Canela EI, Mallol J, Lluis C, et al. (1999) Epidermal
growth factor (EGF)-induced up-regulation and agonist- and antagonist-induced
desensitization and internalization of A1 adenosine receptors in a pituitary-
derived cell line. Brain Res 816: 47–57.
24. Ren H, Stiles GL (1999) Dexamethasone stimulates human A1 adenosine
receptor (A1AR) gene expression through multiple regulatory sites in promoter
B. Mol Pharmacol 55: 309–316.
25. Topley N (1995) The cytokine network controlling peritoneal inflammation.
Perit Dial Int 15: S35–39; discussion S39–40.
26. Basok A, Shnaider A, Man L, Chaimovitz C, Douvdevani A (2001) CD40 is
expressed on human peritoneal mesothelial cells and upregulates the production
of interleukin-15 and RANTES. J Am Soc Nephrol 12: 695–702.
27. Douvdevani A, Rapoport J, Konforty A, Argov S, Ovnat A, et al. (1994) Human
peritoneal mesothelial cells synthesize IL-1 alpha and beta. Kidney Int 46:
28. Hausmann MJ, Rogachev B, Weiler M, Chaimovitz C, Douvdevani A (2000)
Accessory role of human peritoneal mesothelial cells in antigen presentation and
T-cell growth. Kidney Int 57: 476–486.
29. Man L, Lewis E, Einbinder T, Rogachev B, Chaimovitz C, et al. (2003) Major
involvement of CD40 in the regulation of chemokine secretion from human
peritoneal mesothelial cells. Kidney Int 64: 2064–2071.
30. Chung-Welch N, Patton WF, Shepro D, Cambria RP (1997) Human omental
microvascular endothelial and mesothelial cells: characterization of two distinct
mesodermally derived epithelial cells. Microvasc Res 54: 108–120.
31. Ledent C, Vaugeois JM, Schiffmann SN, Pedrazzini T, El Yacoubi M, et al.
(1997) Aggressiveness, hypoalgesia and high blood pressure in mice lacking the
adenosine A2a receptor. Nature 388: 674–678.
32. Einbinder T, Sufaro Y, Yusim I, Byk G, Passlick-Deetjen J, et al. (2003)
Correction of anemia in uremic mice by genetically modified peritoneal
mesothelial cells. Kidney Int 63: 2103–2112.
33. Stylianou E, Jenner LA, Davies M, Coles GA, Williams JD (1990) Isolation,
culture and characterization of human peritoneal mesothelial cells. Kidney Int
34. Andresen BT, Gillespie DG, Mi Z, Dubey RK, Jackson EK (1999) Role of
adenosine A(1) receptors in modulating extracellular adenosine levels.
J Pharmacol Exp Ther 291: 76–80.
PLoS ONE | www.plosone.org8 May 2008 | Volume 3 | Issue 5 | e2107