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IMMUNOBIOLOGY
Differential requirement for A2a and A3 adenosine receptors for the
protective effect of inosine in vivo
Gregorio Gomez and Michail V. Sitkovsky
Inosine is an endogenous nucleoside with
immunosuppressive properties that is
known to inhibit the accumulation of
proinflammatory cytokines and protect
mice from endotoxin-induced inflammation and lung tissue damage. There are
no known receptors specific for inosine,
but A3 adenosine receptors (A3Rs) have
been shown to bind inosine, resulting in
mast cell degranulation and increased
vascular permeability. The present study
specifically addresses the requirement
for A2aR and/or A3R for the protective
effect of inosine in 2 experimental in vivo
models of inflammatory disease. The data
show that A3R is essential for protection
against ConA-induced fulminant hepatitis since only A3R-expressing mice were
protected by inosine whereas wild-type
and A2aR-deficient mice exhibited severe
liver damage even after administration of
inosine. In addition, we show in a model
of LPS-induced endotoxemia that inosine
protected both A2aRⴚ/ⴚ and A3Rⴚ/ⴚ mice
from inflammation, but not A2aA3R
double-null mice, indicating that in this
model both A2aR and A3R were used by
inosine. Thus, we demonstrate that A2a
and A3 adenosine receptors are differentially utilized by inosine for the downregulation of tissue damage under different inflammatory conditions in vivo.
(Blood. 2003;102:4472-4478)
© 2003 by The American Society of Hematology
Introduction
Inflammatory processes are crucial for defense against pathogens,
but inappropriate and/or prolonged inflammation contributes to the
pathogenesis of major diseases.1 Therefore, identification of endogenous molecules that are capable of inhibiting activated immune
cells is important in order to better understand the physiologic
mechanisms that regulate inflammation2 and to develop novel
targets for anti-inflammatory drugs. The demonstration that the
extracellular adenosine A2aR participates in an endogenous immunosuppressive loop that serves to down-regulate inflammation in
vivo by a nonredundant mechanism3 heightened the interest in
naturally occurring purines in the regulation of inflammation and
tissue protection.
It is known that adenosine possesses anti-inflammatory properties and that adenosine analogs can protect mice from a variety of
inflammatory diseases such as septic shock4,5 and rheumatoid
arthritis6 by inhibiting the release of free radicals and proinflammatory cytokines from different immune cells. Relevantly, the selective A2aR agonist CGS21680 was shown to protect liver from
ischemia/reperfusion injury in rats and from ConA-induced tissue
damage in mice.3,53 The attractiveness of an adenosine-mediated
anti-inflammatory pathway has been enhanced by the realization
that widely used anti-inflammatory drugs, such as methotrexate and
sulfasalazine, seem to exert their effects by inducing the release
of endogenous adenosine, which then signals through adenosine
receptors.7-10 Thus, the triggering of a “natural” anti-inflammatory pathway by ligands of adenosine receptors represents an
attractive immunomodulation strategy for the treatment of
inflammatory diseases.
Inosine is an anti-inflammatory endogenous nucleoside that can
be considered a natural trigger of adenosine receptors. It is formed
as a product of adenosine deamination11 and is released into the
extracellular space at times of cellular stress (eg, systemic inflammation12 and hypoxia13) when metabolism of adenosine is high.
Indeed, while normal interstitial concentrations of inosine have
been reported to be in the micromolar range, concentrations of
more than 1 mM in ischemic tissues and in patients with sepsis
have been documented.14-18 Inosine is now known to exert wideranging anti-inflammatory effects that include inhibition of proinflammatory cytokine and chemokine production, enhancement of
anti-inflammatory cytokine interleukin-10 (IL-10) production, and
protection from endotoxin-induced inflammation and lung tissue
damage19-22 as well as skeletal muscle reperfusion injury23 in mice.
In humans, inosine has shown promise as a therapeutic agent
against multiple sclerosis (MS)24 and Tourette syndrome,25 perhaps
by indirectly increasing uric acid levels and acting as a dopamine
agonist, respectively. Thus, inosine may have wide-ranging biologic effects with potentially beneficial implications in humans,
underscoring the need to understand the molecular mechanisms of
inosine action in vivo.
Importantly, inosine has no known specific receptors(s); however, it has been shown to utilize cyclic adenosine monophosphate
(cAMP)–inhibiting Gi-coupled A3Rs. In functional and radioligandbinding in vitro assays, Jin et al26 showed that inosine in the range
of 10 ␮M to 50 ␮M binds to and activates A3Rs on stably
transfected HEK-293 cells, RBL-2H3 rat mastlike cells, perivascular mast cells of hamster cheek pouch arterioles, and in guinea pig
lung membranes. In addition, Tilley et al27 have used mice lacking
either A3Rs or mast cells to show that A3Rs are required for the
inosine-mediated potentiation of bone marrow mast cell degranulation and promotion of plasma protein extravasation.
From the Laboratory of Immunology, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD.
Allergy and Infectious Diseases, National Institutes of Health, 10/11N311, 10 Center
Dr-MSC 1892, Bethesda, MD 20892-1892; e-mail: [email protected].
Submitted December 2, 2002; accepted August 19, 2003. Prepublished online as
Blood First Edition Paper, August 28, 2003; DOI 10.1182/blood-2002-11-3624.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Michail V. Sitkovsky, Laboratory of Immunology, National Institute of
© 2003 by The American Society of Hematology
4472
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
The question, however, remained as to whether the antiinflammatory effect of inosine was mediated by A3R activation
alone or if inosine also signaled through other subtypes of
adenosine receptors. Indeed, while the competitive binding studies
with recombinant rat and guinea pig adenosine receptors subtypes
have indicated that inosine does not bind A1Rs or A2aRs on mast
cells,26 it was suggested by Hasko and colleagues19 that inosinemediated suppression of tumor necrosis factor ␣ (TNF␣) production from lipopolysaccharide (LPS)–stimulated peritoneal murine
macrophages was significantly abrogated in the presence of
pharmacologic antagonists of A1 or A2a receptors. Although it was
not determined in this study if inosine acted directly or indirectly
by altering adenosine levels, these data supported the possibility
that inosine could trigger immunosuppressive signaling through A1
or A2a receptors.
To determine which adenosine receptors, A2aR or A3R, were
responsible for the effects of inosine, we studied acute inflammatory damage in in vivo models of autoimmune and viral hepatitis
and endotoxin-induced sepsis using mice genetically deficient in
A2aR or A3R and A2aA3R double-deficient mice. In addition,
studies of these acute inflammation models allowed us to compare
the effect of inosine on the pathogenesis of diseases with very
different initiating stimuli (polyclonal T-cell receptor [TCR] activator ConA vs Toll-like receptor activating LPS) and different types
of immune cells.
In mice, ConA-induced liver damage in mice is a welldescribed in vivo inflammation model of viral and autoimmune
hepatitis that is mediated by T cells, natural killer (NK) T cells,
macrophages, neutrophils, and a variety of cytokines and chemokines including TNF␣, IL-4, interferon ␥ (IFN␥), and macrophage
inflammatory protein-2 (MIP-2),28-34 whereas endotoxin-induced
shock involves mostly the activation of toll-receptors on macrophages.35-37 Importantly, these different cells may differ in their
repertoire of expressed adenosine receptors.
The experimental data provided in this study show that A3R
was essential for the inhibition of ConA-induced liver injury by
inosine since the accumulation of proinflammatory cytokines (eg,
TNF␣) and alanine transaminase was inhibited only in A3Rexpressing mice, whereas A2aR-deficient mice had elevated levels.
This was supported by histologic evidence of inosine inhibiting
hepatocyte apoptosis in A3R⫹/⫹ but not A3R⫺/⫺ mice. In addition,
the data show that LPS-induced TNF␣ production was inhibited by
inosine in wild-type, A2aR⫺/⫺, and A3R⫺/⫺ mice. However,
inosine was ineffective in blocking TNF␣ production in LPSchallenged A2aA3R⫺/⫺ double-knockout mice, indicating that both
A2aR and A3R were used by inosine to inhibit endotoxin-induced
inflammation.
These data provide the first genetic evidence that inosine
differentially utilizes A2a and A3 adenosine receptors to exert its
anti-inflammatory properties, and indicate that different adenosine
receptors are employed by inosine in T-cell–dependent versus
T-cell–independent acute inflammation. Accordingly, we propose
that use of A2a and A3 adenosine receptors by the endogenous
nucleoside inosine should be considered in comparative studies of
mechanisms of acute inflammation with different etiology. The A2a
adenosine receptors have been conclusively implicated in the
down-regulation of inflammation in vivo3 and the data reported
here point to the need to include inosine among those endogenous
molecules that influence inflammatory responses by directly or
indirectly triggering A2a and/or A3 adenosine receptors in vivo.
ADENOSINE RECEPTOR USAGE BY INOSINE
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Materials and methods
Mice
Eight- to twelve-week-old, age-matched, male mice were used. All mice
were on a C57BL/6 background. The A2aR⫺/⫺ and A3R⫺/⫺ mice were
backcrossed at least 10 times and the A2aA3R⫺/⫺ double-knockout mice
were backcrossed 2 times. A2aR and A3R gene-deficient mice are described
in Chen et al38 and Salvatore et al,39 respectively. A2aA3R double-deficient
mice were generated by A2aR⫺/⫺ ⫻ A3R⫺/⫺ breeding. IL-10⫹/⫹ and
IL-10⫺/⫺ mice were purchased from Taconic (Germantown, NY). All mice
were maintained under specific pathogen-free conditions at the National
Institutes of Health (NIH) animal care facilities.
Reagents and drugs
Inosine, LPS (from Escherichia coli, serotype 055:B5), and ConA were
purchased from Sigma (St Louis, MO). The A2aR agonist CGS21680 and
A3R and A1R agonist 2-Cl-IB:MECA were purchased from Tocris Cookson (Ballwin, MO).
LPS study
Mice were injected intraperitoneally with LPS (10 mg/kg) with or without
inosine (100 mg/kg) in sterile phosphate-buffered saline (PBS). Inosine was
injected 10 minutes before LPS. For pharmacologic activation of A2aR or
A3R, the agonists CGS21680 and 2-CL-IB:MECA were injected intraperitoneally at 0.5 mg/kg 10 minutes before injection of LPS. Blood was
collected from the retro-orbital vein 1 hour after LPS injection and TNF␣
levels were determined from the isolated serum.
Liver injury study
Mice were injected intravenously (tail vein) with the indicated dose of
ConA (15 mg/kg or 20 mg/kg), inosine (100 mg/kg), or a mixture of ConA
plus inosine in sterile PBS. Blood was collected at 1 hour and 24 hours from
the retro-orbital vein and TNF␣ and alanine transaminase (ALT) levels
were determined from the isolated serum. Mice were killed 24 hours after
injection and liver samples were taken for tissue histopathologic evaluation.
TNF␣ and ALT determination
TNF␣ was measured in the serum collected 1 hour after the indicated
challenge using enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN). Detection limits were less
than 5.1 pg/mL for TNF␣. Serum ALT was measured from serum collected
24 hours after challenge with ConA, inosine, or ConA plus inosine using
kits purchased from Sigma. The assays were performed according to the
manufacturer’s instructions.
Histology
Samples of liver tissue from dead mice were taken and fixed in formalin. In
situ staining of single-strand breaks in nuclear DNA to detect apoptotic cells
(TdT assay) and haematoxylin and eosin (H&E) staining were done at
Molecular Histology (Montgomery Village, MD).
Results
Inosine utilizes only A3 adenosine receptors to inhibit ConAinduced liver damage in a fulminant hepatitis model in vivo
To determine whether A2a or A3 receptors or both are required for
the inosine-mediated inhibition of inflammation in vivo, we
compared the effect of inosine on ConA-challenged wild-type,
A2aR⫺/⫺, A3R⫺/⫺, and A2aA3R⫺/⫺ mice. Given that inosine had
been shown in published studies to inhibit inflammatory responses
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4474
GOMEZ and SITKOVSKY
in vivo when administered at 100 mg/kg, we chose this concentration of inosine in these and subsequent experiments. Following
injection of ConA and/or inosine the levels of ALT and of the
proinflammatory cytokine TNF␣ in the serum were analyzed
as a measure of intensity of the inflammatory process and of
liver damage.
Many different cytokines (eg, IFN␥, IL-4, TNF␣) were shown to be
involved in ConA-induced liver injury in experiments using different
gene-deficient and transgenic mice.28,31,40 As shown in Figure 1A-B,
ConA challenge resulted in severe liver damage in wild-type mice as
assessed by high levels of ALT and TNF␣ in the serum. Inosine, which
alone did not induce liver damage, completely inhibited ALT and TNF␣
accumulation in wild-type mice when coinjected with ConA. The
observed inhibition of liver damage by inosine was dependent on the
presence of A3 adenosine receptors as evidenced by experiments where
the effect of inosine on ConA-induced liver damage and inflammation
was compared between A2aR⫺/⫺, A3R⫺/⫺, and A2aA3R⫺/⫺ mice. It
was found that both the extent of liver damage (ALT levels) and TNF␣
levels were inhibited by inosine regardless of the absence of the A2aR
(Figure 1C-D), indicating that A2a receptors were not required for
inhibition by inosine in this model. In contrast, mice deficient in A3R
were resistant to the anti-inflammatory effect of inosine, and high levels
of ALT and TNF␣ were detected in these mice after challenge with
ConA (Figure 1E-H), thus indicating that A3Rs were necessary and
required for inhibition of liver damage by inosine.
Since inosine had been shown to induce production of the
anti-inflammatory cytokine IL-10,19 which was reported to have
protective effects over ConA-induced hepatitis,40,41 we compared
IL-10⫹/⫹ and IL-10⫺/⫺ mice for susceptibility to inhibition of liver
injury by inosine. Figure 2 shows that ALT accumulation was
inhibited by inosine in both IL-10⫹/⫹ and IL-10⫺/⫺ mice, indicating
that inhibition of liver injury by inosine in vivo was independent of
IL-10. Thus, inosine inhibited ConA-induced fulminant hepatitis
through an IL-10–independent but A3R-dependent mechanism.
Inosine protects liver tissue by inhibiting ConA-induced
hepatocyte apoptosis
The A3R-dependent anti-inflammatory effect of inosine in the
ConA model of hepatitis was further demonstrated by histopathologic examination of tissue sections of liver taken from A3R⫹/⫹ and
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
Figure 2. Inosine inhibits ConA-induced liver injury by an IL-10–independent
mechanism. IL-10⫹/⫹ (A) and IL-10⫺/⫺ (B) mice were injected with ConA (20 mg/kg),
inosine (100 mg/kg), or both, and serum ALT was determined 24 hours after injection.
This figure shows that inosine inhibited ConA-induced ALT regardless of the lack of
IL-10 production in IL-10⫺/⫺ mice. Representative results from one of 3 separate
experiments.
A3R⫺/⫺ mice. Figure 3 shows ConA induced severe liver damage
in both A3R⫹/⫹ and A3R⫺/⫺ mice, but that only A3R⫹/⫹ mice were
rescued from liver damage by inosine as assessed by H & E
staining. It is shown that livers from A3R⫹/⫹ and A3R⫺/⫺ mice
sustained severe damage from ConA (Figure 3A,D) but no gross
damage from inosine alone (Figure 3B,E). Moreover, Figure 3C,F
shows that liver tissue from A3R⫹/⫹, but not A3R⫺/⫺, mice was
protected from ConA-induced damage in vivo by inosine.
Since the apoptosis of hepatocytes had been suggested to be
involved in ConA-induced liver injury,33 we analyzed the effect of
inosine on hepatocyte apoptosis in livers from ConA-challenged
A3R⫹/⫹ and A3R⫺/⫺ mice by in situ staining of single-strand
breaks in nuclear DNA (TdT assay). Figure 4 demonstrates that
livers of both A3R⫹/⫹ and A3R⫺/⫺ mice had large numbers of
apoptotic cells 24 hours after ConA challenge (Figure 4A,D), but
not with inosine treatment alone. In addition, it shows that
hepatocyte apoptosis was inhibited in A3R⫹/⫹ mice that had been
coinjected with inosine as shown in Figure 4C. In contrast,
ConA-induced hepatocyte apoptosis was not inhibited by inosine in
A3R⫺/⫺ mice (Figure 4F).
Taken together, these experiments show that inosine inhibited
T-cell–triggered inflammatory processes in liver and protected
mice from tissue damage by signaling through A3 receptors to
inhibit hepatocyte apoptosis. Importantly, they also show that
inosine did not utilize A2a receptors in the inhibition of the
pathogenesis of acute liver inflammation studied here.
Figure 1. A3 adenosine receptors are required for the
inhibition of ConA-induced ALT and TNF␣ by inosine. C57BL/6 (A-B), A2aR⫺/⫺ (C-D), A3R⫺/⫺ (E-F), and
A2aA3R⫺/⫺ (G-H) mice were injected with ConA, inosine
(100 mg/kg), or both, and the level of TNF␣ and ALT in
the serum at 1 hour and 24 hours, respectively, after
challenge was determined. Because excessive inflammation is observed in the absence of the A2aR,3 A2aR⫺/⫺
and A2aA3R⫺/⫺ mice were injected with 15 mg/kg,
whereas the C57BL/6 and A3R⫺/⫺ mice were injected
with 20 mg/kg ConA. This figure shows that inosine
inhibited ConA-induced hepatitis only in mice expressing
the A3R. Representative results from one of 3 separate
experiments.
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
Figure 3. Inosine protects A3Rⴙ/ⴙ liver tissue from ConA-induced damage in
vivo. Tissue sections of liver taken from A3R⫹/⫹ (A-C) and A3R⫺/⫺ (D-F) mice 24
hours after injection with ConA (A,D), inosine (B,E), and ConA plus inosine (C,F) were
stained with haemotoxylin and eosin (H&E). This figure shows that inosine protected
A3R⫹/⫹, but not A3R⫺/⫺, liver from ConA-induced damage. Original magnification, ⫻ 10.
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4475
inosine (100 mg/kg) followed by an injection of LPS (10 mg/kg).
The mice were bled 1 hour after LPS injection and the concentration of TNF␣ in the serum was measured. As expected, LPSstimulated TNF␣ was inhibited in wild-type mice as shown in
Figure 5A,C. In addition, we found that wild-type mice when
injected with 60 mg/kg of inosine alone had 85% mortality by 24
hours whereas 15% of mice injected with LPS plus inosine died
within the same time frame (data not shown). Thus, inosine
protected mice over a broad range of inflammatory insult. Importantly, the accumulation of TNF␣ was inhibited even in the absence
of A2aR or A3R in single-gene knockouts (Figure 5B,D), suggesting that inosine used both A2a and A3 receptors subtypes to inhibit
endotoxin-stimulated TNF␣ production in vivo.
To test this, we developed and studied A2aA3R⫺/⫺ doubleknockout mice. As predicted from the hypothesis that both A2a and
A3 receptors are involved in mediating the anti-inflammatory
signaling by inosine, the A2aA3R⫺/⫺ mice were unresponsive to
the inhibitory effect of inosine and maintained high levels of
LPS-induced TNF␣ production in vivo as compared with wild-type
littermates (Figure 5E-F). These results thus prove that inosine
utilized both A2a and A3 receptor subtypes to inhibit endotoxininduced inflammation in vivo in this model of LPS-induced
endotoxemia.
Inosine acts through both A2a and A3 adenosine receptors
to inhibit endotoxin-induced inflammation in vivo
To determine if the mechanism by which inosine inhibited LPSinduced inflammation was also dependent only on A3R, we
compared the effect of inosine on wild-type, A2aR-null, A3R-null,
and A2aA3R–double-null mice in an in vivo model of LPS-induced
endotoxemia. In these experiments, mice were pretreated with
Figure 4. Inosine inhibits ConA-induced hepatocyte apoptosis in vivo. Tissue
sections of liver taken from A3R⫹/⫹ (A-C) and A3R⫺/⫺ (D-F) mice 24 hours after
injection with ConA (A,D), inosine (B,E), and ConA plus inosine (C,F) were assessed
for apoptosis by staining single-strand breaks in nuclear DNA to detect apoptotic cells
(TdT assay). This figure shows that inosine protected A3R⫹/⫹, but not A3R⫺/⫺, liver
from ConA-induced damage by inhibiting hepatocyte apoptosis. Original magnification, ⫻ 10.
Figure 5. Inosine utilizes both A2aR and A3R to inhibit LPS-induced TNF␣ in
vivo. Mice were injected intraperitoneally with 100 mg/kg inosine followed by 10
mg/kg LPS and the level of TNF␣ in the serum collected 1 hour after LPS injection
was assessed. This figure shows that inosine inhibited LPS-induced TNF␣ production in wild-type (A,C,E), A2aR⫺/⫺ (B), and A3R⫺/⫺ (D) mice, but did not inhibit TNF␣
in A2aA3R⫺/⫺ (F) mice, which lacked both receptors. Representative results from 1 of
3 separate experiments.
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GOMEZ and SITKOVSKY
Pharmacologic activation of A2a or A3 adenosine receptors
inhibits endotoxin-induced TNF␣ production in vivo
The data shown in Figures 1-5 firmly implicate A2a and A3
receptors in the anti-inflammatory effects of inosine. These receptors have been suggested to be anti-inflammatory in earlier studies
of the inhibitory effects of adenosine and pharmacologic agonists
of both A2aR and A3R on proinflammatory cytokine production
from monocytes/macrophages and mast cells.4,5,42-47 It was important to confirm these pharmacologic observations by genetic in
vivo experiments to allow the conclusive implication of individual
subtypes of adenosine receptors in the anti-inflammatory effect
of inosine.
Therefore, to verify that it was the activation of A2a or A3
receptors that caused the inhibition of LPS-induced TNF␣ production in vivo, we treated wild-type and A2aR-deficient or A3Rdeficient mice with selective pharmacologic agonists to A2aR or
A3R followed by challenge with LPS. It is shown in Figure 6 that
injection of the A2aR agonist CGS21680 or A3R and A1R agonist
2-Cl-IB:MECA into wild-type mice resulted in decreased TNF␣
levels in blood serum following lethal injection of LPS (Figure
6A,C). In addition, Figure 6 shows that CGS21680 had no effect in
A2aR⫺/⫺ mice (Figure 6D) and, likewise, 2-Cl-IB:MECA had no
effect in A3R⫺/⫺ mice (Figure 6C), confirming earlier studies by
Salvatore et al.39 Thus, the data presented here provide the genetic
proof that both A2a and A3 receptor subtypes play a role in the
negative regulation of LPS-induced TNF␣, and support our observation that inosine utilized both receptor subtypes to negatively
regulate endotoxin-induced inflammation in vivo. These data also
confirm the selectivity of tested pharmacologic agonists in the
assays of acute inflammation used in these experiments.
Figure 6. Pharmacologic activation of A2aR or A3R inhibits LPS-induced TNF␣
production in vivo. A2aR⫹/⫹ (A) and A2aR⫺/⫺ (B) mice were injected with 0.5 mg/kg
of CGS21680, and A3R⫹/⫹ (C) and A3R⫺/⫺ (D) mice were injected intraperitoneally
with the 0.5 mg/kg of 2-Cl-IB:MECA. Ten minutes later the mice were injected with 10
mg/kg LPS. The mice were bled 1 hour after LPS injection and the level of TNF␣ in the
serum was measured. This figure shows that LPS-induced TNF␣ was inhibited by
CGS21680 or 2-Cl-IB:MECA in A2aR⫹/⫹ (A) and A3R⫺/⫺ (C) mice, respectively.
However, these adenosine receptor–specific agonists did not affect A2aR⫺/⫺ and
A3R⫺/⫺ mice (B and D, respectively). Representative results from 1 of 3 separate
experiments.
BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
Discussion
In this report, we addressed the issue of which adenosine receptors
were essential for the in vivo protective anti-inflammatory effect of
the endogenous purine inosine. This was important since the
biochemical and pharmacologic evidence to support or refute the
use of adenosine receptors other than the A3R by inosine was
conflicting. Therefore, genetic in vivo testing in assays of inflammatory tissue damage was required to resolve the issue. The question
as to which adenosine receptor(s) is/are utilized functionally by
inosine was intriguing given that inosine was reported to induce
mast cell degranulation (via A3R binding),26,27,39 a proinflammatory event, yet inosine inhibited endotoxin-induced proinflammatory cytokine production from macrophages, an antiinflammatory event, and protected mice from endotoxemia.19
This ability of inosine to trigger both pro- and anti-inflammatory
actions suggested to us that inosine utilizes more than one type
of adenosine receptor.
Our study specifically addressed the requirement for A2a and/or
A3 adenosine receptors for the protective effect of inosine in 2
experimental in vivo murine models of disease. Our experimental
data clearly show that A3R was required for the inosine-mediated
inhibition of ConA-induced liver damage since wild-type and
A2aR⫺/⫺, but not A3R⫺/⫺ or A2aA3R double-knockout mice, were
rescued from inflammatory damage. The data also show that the
requirement for adenosine receptors in the LPS model studied was
different than that in the ConA model. In this case, the data show
that both A2aR and A3R were employed in protecting mice from
LPS-induced endotoxemia since both A2aR and A3R singleknockout mice, but not A2aA3R double-knockout mice, were
protected by inosine in this model. Based on these data, we
concluded that both A2a and A3 adenosine receptors were utilized
for inosine to protect tissue from excessive inflammatory damage
in vivo. Furthermore, we concluded that the type of inflammatory
stimuli determines the overall requirement for one or both receptors.
The possibility that the observed protective effect was a
consequence of an indirect action of inosine should be considered.
It was possible that the anti-inflammatory effect of inosine was a
consequence of indirect activation of A3 receptors leading to the
release of mast cell mediators including adenosine triphosphate,
which might be converted to adenosine and activate other adenosine receptors, and histamine, which has been shown to modulate
cytokine production.48 Indeed, our data showing that only A3Rexpressing mice were protected from ConA-induced liver damage
might be suggestive of such an indirect effect of inosine rather than
a direct activation of A2a receptors. However, if this were the case
then one would expect similar results in the model of LPS-induced
endotoxemia as in the ConA experiments such that only mice
expressing A3R would be rescued from inflammatory damage by
inosine. That is, if the observed effect of inosine is due to the
release of anti-inflammatory mediators from mast cells following
activation of A3 receptors by inosine then only mice expressing A3
receptors should be protected from inflammation following injection of inosine. In fact, this was not the case since A3R⫺/⫺ mice
were rescued from inflammation in our in vivo model of LPSinduced endotoxemia. Moreover, it was only when both A2a and
A3 receptors were absent that mice were unprotected from
inflammatory damage caused by LPS in this model.
It was also possible that inosine had systemic effects that
influenced the observed experimental results. Inosine had been
shown to have positive effects on the cardiovascular system and on
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BLOOD, 15 DECEMBER 2003 䡠 VOLUME 102, NUMBER 13
neuronal growth.49-51 Perhaps most interesting is the possibility that
alterations in cellular metabolic processes or of inosine itself were
responsible for our observations. In a very recent article, Scott et
al52 reported that administration of inosine or inosinic acid
suppressed clinical signs of experimental allergic encephalomyelitis (EAE) and promoted recovery from the disease in their model.
Importantly, they showed that intraperitoneal administration of
inosine or inosinic acid resulted in a transient, minor increase in
serum levels of inosine but a strong increase in uric acid. In
addition, they showed that uric acid but not inosine accumulated in
the spinal cord tissue of mice with EAE fed inosine or inosinic acid.
The authors concluded that the mode of action of inosine and
inosinic acid on EAE was due to its metabolism to uric acid. These
data suggest that metabolism of inosine might be responsible for
the observations reported here. However, if the protective effect of
inosine in our studies was due to an indirect consequence of its
effect on a nonimmune system rather than a direct activation of
adenosine receptors then one might expect the same or no
requirement for adenosine receptors under different inflammatory
conditions. In our study, there was clearly a differential requirement for both A2a and A3 receptors in the models studied.
Nevertheless, it remains to be determined if direct administration of
uric acid exerts the same protective anti-inflammatory effect as
inosine. If so, does uric acid have the same requirement for A2a and
A3 adenosine receptors as reported here for inosine?
Although the possibility exists that activation of A2a receptors
was an indirect consequence of the effect of inosine on A3
receptors, the data from the LPS model of endotoxemia clearly
show the independent participation of both A2aR and A3R in
response to inosine in vivo.
The differences in the effect of inosine on the different
inflammatory models have important mechanistic implications.
The obtained results are consistent with the hypothesis that
different inflammatory stimuli will result in recruitment of different
types of immune cells with a different repertoire of purinergic
receptors. Accordingly, it is the type of immune cells and the
expression of different subtypes of adenosine receptors that will
determine the final outcome of regulation by extracellular purines,
including inosine. For example, ConA-induced liver injury is
triggered by activation of T-cell receptors by ConA and the
resulting hepatocyte death is mediated by the complex interplay of
T cells, NK T cells, neutrophils, Kupffer cells, and cytokines such
as IFN␥, TNF␣, IL-4, IL-12, and IL-18, as well as nitric oxide and
other molecules.28-34 On the other hand, LPS-induced endotoxemic
processes are triggered by activation of Toll-like receptors mostly
on macrophages and other cells, but not T cells.35-37 It remains to be
determined in detailed future studies as to why the activation of
either A2a or A3 receptors was sufficient for inhibition of
LPS-induced inflammation, whereas only A3 receptors were re-
ADENOSINE RECEPTOR USAGE BY INOSINE
4477
quired for the inhibition of ConA-induced liver damage. At least
one explanation may be that there are differences in the time course
of up-regulation of A2aR and/or A3R on T cells versus macrophages and neutrophils during the course of ConA-induced or
LPS-induced immune cell activation in the models of acute
inflammation tested. It seems likely that excessive inflammatory
tissue damage causes accumulation of extracellular inosine and
signaling through adenosine receptors on immune cells in local
tissue environment. Since the expression of purinergic receptors on
immune cells is dependent on activation and differentiation, only
those immune cells that express sufficient numbers of adenosine
receptors will be susceptible to the protective effect of inosine. In
addition, it is possible, but has to be experimentally tested, that the
time course of up-regulation of A2a receptors will differ from that
of A3 receptors and, as a result, different immune cells will express
different combinations of A2a versus A3 receptors at different time
points in vivo.
Taken together, the data reported here indicate that inosine has
properties that are consistent with it being an endogenous metabolic “switch” that serves to modulate inflammation in vivo. The
ability of inosine to functionally activate A2a receptors as reported
here extends the anti-inflammatory effect of inosine to include
negative regulation of immune cells that do not express A3R, but
do express a sufficient number of A2a receptors as well as
enhanced inhibition of immune cells that express both A2aR and
A3R. Thus, we propose that both A2a and A3 purinergic receptors
are functionally utilized, directly or indirectly, by endogenous
inosine with potential to protect tissue from excessive inflammatory damage, and that down-regulation of tissue damage in
different inflammatory conditions is under control of the complex
interplay of different types of inflammatory cells with different
repertoire of adenosine receptors. Although the models used in this
study are experimental and might have limited similarities with
actual disease states in humans, these findings may prove important
in the understanding of molecular mechanisms of the pathogenesis
of acute inflammation–associated diseases and in the search for
pharmacologic anti-inflammatory agents for the treatment of
inflammatory/autoimmune disease such as hepatitis, arthritis,
and sepsis.
Acknowledgments
We thank Marlene Jacobson for the A3R⫺/⫺ mice; Jiang Fan Chan
for the A2aR⫺/⫺ mice; and Ricardo Orellana, Charles Caldwell,
Manfred Thiel, and Akio Ohta for technical support and helpful
discussions. We also thank Mrs Leticia Gomez for help in
preparation of the manuscript.
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2003 102: 4472-4478
doi:10.1182/blood-2002-11-3624 originally published online
August 28, 2003
Differential requirement for A2a and A3 adenosine receptors for the
protective effect of inosine in vivo
Gregorio Gomez and Michail V. Sitkovsky
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