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production via adenosine A2a receptor-dependent and independent mechanisms
1
* Inotek Corp., Beverly, Massachusetts 01915, USA;
Department of Surgery, UMD-New Jersey Medical School, Newark, New Jersey 07103, USA; and
Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129, USA
1Correspondence: Department of Surgery, UMD-New Jersey Medical School, 185 South Orange Ave., University Heights, Newark, NJ 07103, USA. E-mail: haskoge{at}umdnj.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production is only
partly mediated by A2a receptor ligation. Studies with
adenosine receptor antagonists or the adenosine uptake blocker
dipyridamole showed that adenosine released endogenously also decreases
IL-12. Although adenosine increases IL-10 production, the inhibition of
IL-12 production is independent of the increased IL-10. The mechanism
of action of adenosine was not associated with alterations of the
activation of the p38 and p42/p44 mitogen-activated protein kinases or
the phosphorylation of the c-Jun terminal kinase. Adenosine failed to
affect steady-state levels of either IL-12 p35 or p40 mRNA, but
augmented IL-10 mRNA levels. In summary, adenosine inhibits IL-12
production via various adenosine receptors. These results support the
notion that adenosine-based therapies might be useful in certain
autoimmune and/or inflammatory diseases.—Haskó, G., Kuhel,
D. G., Chen, J.-F., Schwarzschild, M. A., Deitch, E. A.,
Mabley, J. G., Marton, A., Szabó, C. Adenosine inhibits
IL-12 and TNF- production via adenosine A2a
receptor-dependent and independent mechanisms.
Key Words: inflammatory mediators • cytokines • T lymphocytes • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. IL-12 is an important link between innate and
adaptive immunity, as it is secreted on stimulation of
antigen-presenting cells and activates interferon 
(IFN-)
production, proliferation, and cytolytic activity of natural killer
cells and T lymphocytes. In turn, IFN- promotes IL-12 production and
macrophage activation, which provides the basis of an autoregulatory
positive feedback loop resulting in a strong immune/inflammatory
response directed against the antigen.
T helper lymphocytes can acquire distinct highly polarized cytokine
profiles (1
, 2)
. Early in the immune response, IL-12 also
plays a critical role in directing the development of Th1 vs. Th2 cell
differentiation characterized by an increased production of IFN- and
IL-2 (Th1 cytokines) and suppression of IL-4 (Th2 cytokine) formation.
IL-12 production and a Th1 cytokine response are indispensable in the
defense against intracellular pathogens (3
, 4)
. But
excessive production of IL-12 and Th1 cytokines can lead to autoimmune
diseases such as multiple sclerosis (5)
, inflammatory
bowel disease (6)
, insulin-dependent diabetes mellitus
(7)
, and rheumatoid arthritis (8)
as well as
contribute to other inflammatory states such as septic shock
(9)
, and the generalized Schwarzman reaction
(10)
.
The purine nucleoside adenosine is released into the extracellular
space by a wide variety of stimuli and is one of the best-characterized
endogenous immunomodulator molecules. Adenosine binds to four different
types of G-protein-coupled cell surface molecules, termed the
A1, A2a,
A2b, and A3 adenosine
receptors (11
12
13)
. Ligation of these receptors located on
immune cells has important effects on a variety of immune cell
functions including antigen presentation (14)
,
phagocytosis (15)
, negative selection (16)
,
or target cell killing (17)
. An important feature of the
regulation of immune processes by adenosine is that adenosine alters
immune cell production of soluble mediators, such as cytokines
(14
, 18
19
20
21
22
23
24
25
26)
, free radicals (20)
, and
arachidonic acid metabolites (27)
. Such an interference
with cell-to-cell communication within the immune system can modify the
outcome of the immune response. For example, adenosine or ligands of
the different adenosine receptors have been shown to ameliorate the
course of a variety of immune-mediated diseases such as rheumatoid
arthritis (24
, 28)
, endotoxin shock (21
, 29)
,
nephritis (30)
, and uveitis (31)
.
Because the immune stimuli, which induce the production of IL-12, also
cause the release of adenosine (32
33
34)
, it is important
to explore whether and how adenosine affects IL-12 expression. In the
present study, we demonstrate that adenosine, applied exogenously or
released endogenously, inhibits IL-12 production.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
, 4)
-triazolo[1,5-c]quinazoline (MRS-1220), and dipyridamole
were obtained from Research Biochemicals Inc. (Natick, Mass.).
The selective A2a antagonist
4-(2-[7-amino-2-(2-furyl)-{1,2,4}-triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol
(ZM241385) was purchased from Tocris Cookson (Bristol, U.K.).
Adenosine, adenosine deaminase, and lipopolysaccharide (LPS)
(Escherichia coli 055:B5) were purchased from Sigma (St.
Louis, Mo.); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was obtained from Fisher Scientific (Pittsburgh, Pa.).
The monoclonal Ab against mouse IL-10 and its control Ab were purchased
from R&D (Minneapolis, Minn.). IFN- was obtained from Genzyme
(Cambridge, Mass).
Preparation and treatment of peritoneal macrophages
Male BALB/c and A2a receptor-deficient
mice and wild-type littermates were injected intraperitoneally with 2
ml of 2% thioglycollate and peritoneal cells were harvested 3–4 days
later. The generation of the A2a
receptor-deficient mice was described previously (35)
.
Near congenic mutant mice (N6 on C57Bl/6 background) and their
wild-type littermates were used in this study. The cells were plated on
96-well plastic plates at 1 million cells/ml and incubated in RPMI 1640
(Life Technologies (Grand Island, N.Y.) for 2 h at 37°C in a
humidified 5% CO2 incubator. Nonadherent cells
were removed by rinsing the plates three times with 5% dextrose in
phosphate-buffered saline (PBS). Cells were treated with various
concentrations of adenosine or adenosine receptor agonists 30 min
before the addition of 10 µg/ml LPS or a combination of LPS and
IFN- (100 U/ml) for 24 h; supernatants for IL-12, TNF-, and
IL-10 determination were taken 24 h after LPS. Selective
antagonists of adenosine receptors, adenosine deaminase, or
dipyridamole were added 30 min before LPS. IL-12, TNF-, and IL-10
were determined by enzyme-linked immunoassay (ELISA) as described
below.
Culture and treatment of J774.1 macrophages
The mouse macrophage cell line J774.1 was grown in RPMI 1640
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100
µg/ml streptomycin in a humidified atmosphere of 95% air and 5%
CO2. Cells were cultured in 96-well plates (200
µl medium per well) until 80% confluence. The cells were pretreated
with various concentrations of adenosine and treated with a combination
of LPS and IFN- (100 U/ml) for 24 h as for peritoneal
macrophages (see above).
Cytokine assays
Cytokine concentrations in the supernatants were determined by
ELISA kits that are specific against murine cytokines. Levels of
TNF-, IL-10, IL-12 (total), and IL-12 (p70) were measured using
ELISA kits purchased from Genzyme Co. (Boston, Mass.). Plates were read
at 450 nm by a Spectramax 250 microplate reader from Molecular Devices
(Sunnyvale, Calif.). Detection limits were 5 pg/ml for TNF-, 0.15
pg/ml for IL-10, 10 pg/ml for IL-12 (total), and 5 pg/ml for IL-12
(p70). Assays were performed as described previously (20)
and according to the manufacturer’s instructions.
Western blot analysis
Cells in 6-well plates were pretreated with adenosine or
vehicle; 30 min later the cells were stimulated with LPS (10 µg/ml)
for 15 min. After washing with PBS, the cells were lysed by the
addition of modified radioimmunoprecipitation buffer (50 mM Tris-HCl,
150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1% Nonidet P-40, 1
µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM PMSF, 1 mM
Na3VO4). The lysates were
transferred to Eppendorf tubes, centrifuged at 15,000 g, and
the supernatant was recovered. Protein concentrations were determined
using a Bio-Rad Protein Assay (Bio-Rad, Hercules, Calif.). Ten
micrograms of sample was separated on a 8–16% Tris-glycine gel
(Novex, San Diego, Calif.) and transferred to a nitrocellulose
membrane. The membranes were probed with anti-phospho-mitogen-activated
protein kinase (MAPK; p42/p44), anti-phospho-c-Jun amino-terminal
protein kinase (JNK, Promega, Madison, Wis.), anti-phospho-p38 MAP
kinase (p38 MAPK, New England Biolabs, Beverly, Mass.) and subsequently
incubated with a secondary horseradish peroxidase-conjugated donkey
anti-rabbit antibody (Boehringer, Indianapolis, Ind.). Bands were
detected using ECL Western Blotting Detection Reagent (Amersham Life
Science, Arlington Heights, Ill.).
RNA isolation and RNase protection assay
Cells in 6-well plates were treated the same way as for cytokine
protein measurements, and RNA was isolated 3 h after LPS
treatment. Macrophage monolayers were washed with PBS and total
cellular RNA was extracted from each well using a guanidinium
isothiocyanate/chloroform-based technique (TRIZOL; Life Technologies),
followed by isopropanol precipitation. The RNase protection assay was
performed using a kit obtained from PharMingen (San Diego, Calif.).
Briefly, antisense RNA multi-probe set was synthesized by in
vitro transcription of mouse cytokine template set (mCK2) using T7
RNA polymerase in the presence of [-32P] UTP
(specific activity 3000 Ci/mM, Amersham). The probe set
(3x105 cpm/µl) was hybridized with target RNA
(10 µg) at 56°C overnight in a total of 10 µl of hybridization
buffer. The free probe and single stranded target RNA were digested
with RNase at 30°C for 45 min. The proteins were digested by treating
with proteinase K for 15 min at 37°C and extracted with
phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was
removed and the protected RNA precipitated with ethanol. The pellet was
washed with 90% ethanol, air dried, and resuspended in 4 µl of
loading buffer. The sample was then heated for 3 min at 90°C and
separated on 6% sequencing gel (Novex). The gel was then vacuum dried
and exposed to X-ray film (Kodak) for 24 h at -70°C.
Measurement of mitochondrial respiration
Mitochondrial respiration, an indicator of cell viability, was
assessed by the mitochondria-dependent reduction of MTT to formazan
(20)
. Cells in 96-well plates were incubated with MTT (0.5
mg/ml) for 60 min at 37°C. Culture medium was removed by aspiration
and cells were solubilized in dimethyl sulfoxide (100 µl). The extent
of reduction of MTT to formazan within cells was quantitated by
measurement of optical density at 550 nm (OD550)
using a Spectramax 250 microplate reader.
Statistical evaluation
Values in the figures, tables, and text are expressed as
mean ± SE of n observations. Statistical
analysis of the data was performed by one-way analysis of variance,
followed by Dunnett’s test, as appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although IFN- alone failed to trigger the production
of IL-12 (not shown), the combination of LPS (10 µg/ml) and IFN-
(100 U/ml) resulted in a synergistic potentiation of IL-12 p40 release
(Fig. 1B
). This combination, but not LPS alone, evoked
detectable levels of IL-12 (p70) in the peritoneal macrophages (Fig. 1C
). Adenosine pretreatment of peritoneal macrophages
stimulated with LPS caused a concentration-dependent inhibition of
IL-12 p40 production (Fig. 1A
). The effect of adenosine was
less pronounced when IL-12 was induced with LPS/IFN- (Fig. 1B
, C
). Adenosine was even less potent in
LPS/IFN--stimulated J774 cells (Fig. 1D
). Finally,
neither adenosine nor the selective adenosine receptor agonists
affected cellular viability as tested with the MTT assay (not shown).
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Effect of selective agonists and antagonists of adenosine receptors
on IL-12 production in peritoneal cells
Figure 2
shows that the adenosine receptor agonists inhibit IL-12 production in
peritoneal macrophages with the order of potency CGS-21680 >
IB-MECA > CCPA, suggesting that the suppression of IL-12 by
adenosine is predominantly an A2a
receptor-mediated process. The effect of IB-MECA was biphasic. The
inhibition of IL-12 production reached its maximum at 10 µM, but at
30 µM IB-MECA, IL-12 levels returned to the baseline, and at 100
µM, IB-MECA caused a threefold increase in IL-12.
|
In the next step, we evaluated a series of adenosine receptor
antagonists. The A2 receptor antagonist DMPX
augmented IL-12 production (Table 1
), suggesting that endogenous adenosine suppresses IL-12 production. The
effect of the nonselective A2a antagonist DMPX
was mimicked by the selective A2a antagonist
ZM241385 (1 µM), whereas the selective A2b
antagonist alloxazine (1, 10, and 50 µM) had no effect (not shown).
The A1 antagonist DPCPX also increased IL-12, but
another A1 agonist, PACPX (1, 10, and 50 µM),
failed to enhance production of this cytokine, suggesting that the
effect of DPCPX may be nonspecific. It must be pointed out that these
agents alone in the absence of LPS did not induce IL-12 production;
therefore, the possibility of LPS contamination can be excluded. The
suppressive effect of higher concentrations of DPCPX (10 and 50 µM)
is probably due to an inhibitory effect of this xanthine compound on
phosphodiesterases. The blockade of these enzymes both by xanthine and
nonxanthine inhibitors has been shown to decrease IL-12 production in
various in vitro and in vivo systems (36
, 37)
. Although MRS-1220, a potent antagonist of human
A3 receptors, was ineffective in augmenting IL-12
production (data not shown), it cannot be excluded that endogenous
adenosine via A3 receptors augments the IL-12
response, because this agent is not a potent
A3 antagonist in rodent systems
(38)
. Further evidence for an inhibitory role of
endogenous adenosine is that the adenosine uptake blocker dipyridamole
(10 µM) decreased IL-12 production (971±31 pg/ml in controls vs.
718±25 in dipyridamole-treated; P=0.0009), similarly to
adenosine receptor agonists.
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Adenosine inhibits both IL-12 and TNF- production in
A2a receptor-deficient mice
Since the order of potency of adenosine receptor agonist suggested
that A2a receptors are responsible for the
suppressive effect of adenosine on IL-12 production, we tested the
effect of adenosine in A2a receptor-deficient
mice. Because CGS-21680 and IB-MECA were the most potent agonists, we
also tested the effect of these agents on both IL-12 and TNF-
production. Although CGS-21680 completely lost its efficacy in the
A2a receptor-deficient mice (Fig. 3C
, D
), both adenosine (Fig. 3A
, B
) and IB-MECA
(Fig. 3E
, F
) continued, albeit to a lesser extent
than in the wild-type littermates, to suppress both IL-12 and TNF-
production in the A2a receptor-deficient mice.
Therefore, it can be concluded that the effect of adenosine is only
partially due to stimulation of A2a receptors.
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Receptor desensitization
Because adenosine receptors are subject to desensitization by
exposure to adenosine analogs in various experimental systems
(39)
, we examined whether the effect of adenosine
diminishes after prolonged stimulation of adenosine receptors. For this
purpose, we pretreated peritoneal cells with NECA (10 µM), a
nonmetabolizable, nonselective adenosine receptor agonist for 12 h
that has been shown to desensitize adenosine receptor-mediated
processes (40)
. Figure 4
demonstrates that after the 12 h exposure to NECA, adenosine was
no longer able to inhibit either IL-12 or TNF- release, indicating
that adenosine receptors on macrophages are subject to down-regulation.
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IL-10 is not involved in the suppression of IL-12 production by
adenosine
Because IL-10 is one of the most important cytokines known to
down-regulate IL-12 production and adenosine has been shown to augment
the release of IL-10 in human monocytes (41)
, we examined
the possibility that the effect of adenosine on IL-12 is due to its
ability to increase IL-10 production. Adenosine caused a significant
augmentation of IL-10 release in mouse peritoneal macrophages
(Fig. 5A
). By using an IL-10 Ab, we confirmed that endogenous IL-10
inhibits IL-12 production, since the IL-12 concentrations were
significantly higher in the IL-10 Ab-treated cells than in the control
cells treated with the isotype control Ab (Fig. 5B
). Whereas
the anti-IL-10 Ab completely neutralized IL-10 activity in both the
adenosine-treated and control cells (Fig. 5A
), adenosine
inhibited IL-12 production to the same extent in the presence of both
the IL-10 Ab and the isotype control Ab (Fig. 5B
; 19±2% in
the control samples and 17±3% in the anti-IL-10 Ab treated cells).
Therefore, it can be concluded that the inhibition of IL-12 production
by adenosine is independent of its effect on IL-10.
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Adenosine fails to influence LPS-induced activation of the p38 and
p42/44 MAPKs or phosphorylation of JNK
Because activation of the MAPKs p38 and p42/44 and the
phosphorylation of JNK are important pathways during macrophage
activation (42)
, we tested the possibility that adenosine
exerts its effects on IL-12 and TNF- production via interfering with
the activation of these enzymes. Although LPS treatment of peritoneal
macrophages caused the activation of both p38 and p42/44 as well as JNK
as determined 15 min after LPS treatment, adenosine administered 30 min
before the LPS challenge failed to alter the activation of these
intracellular pathways (Fig. 6
).
|
Effects of adenosine on IL-12 p35, IL-12 p40, and IL-10 mRNA levels
To determine whether inhibition of IL-12 and enhancement of IL-10
protein secretion observed with adenosine receptor stimulation was at
the pretranslational level, steady-state mRNA levels were determined
under similar in vitro conditions as those described above.
First, we determined that mRNAs for both IL-12 and IL-10 were induced
by LPS as early as 3 h after stimulation (Fig. 7
). Whereas pretreatment of peritoneal macrophages with adenosine (3 to
30 µM, 30 min before LPS) resulted in a significant increase in IL-10
mRNA at 3 h after LPS, IL-12 p35 and p40 mRNA expression was not
altered by adenosine pretreatment (Fig. 7)
. Thus, the molecular level
of IL-10 augmentation but not IL-12 inhibition by adenosine is
pretranslational. This latter finding was confirmed in experiments
where adenosine was shown to inhibit IL-12 production even when added
to the cells 6 h after LPS (Fig. 8
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 33)
. This observation and the fact that exogenous
adenosine, at similar levels, has substantial anti-inflammatory and
anti-ischemic activity have led to the suggestion that this nucleoside
is an endogenous anti-inflammatory and anti-ischemic (retaliatory)
metabolite (32
, 33)
. That IL-12 contributes to the
induction of inflammatory processes has long been known (1
, 2)
, and recently this cytokine was implicated in the
pathophysiology of ischemia-reperfusion injury (43)
. Our
results showing that adenosine inhibits IL-12 production suggest that
the beneficial effects of adenosine in inflammatory processes or in
ischemia-reperfusion injury can be attributed partly to its inhibition
of IL-12 production. Because the immunomodulatory effects of adenosine
are mediated by the ligation of various adenosine receptors, it is
important to determine which receptor(s) are involved in the
suppressive effect of adenosine on IL-12 production.
To characterize the receptor subtypes involved, we performed several
studies. The pharmacological studies using various ligands of adenosine
receptors indicated that the order of potency of selective adenosine
receptor agonists was CGS-21680 > IB-MECA > CCPA and
suggested that the A2a receptor was the most
likely candidate for producing the inhibitory effect of adenosine on
IL-12 production. Next, to help confirm a role for the
A2a receptor subtype, we treated mice deficient
for the A2a receptor, hypothesizing that the
effect of adenosine would be lost in the A2a
receptor-deficient mice. To our surprise, at concentrations higher than
1 µM, adenosine maintained its inhibitory activity in the
A2a receptor-deficient mice, although the extent
of inhibition was less than in the wild-type littermate mice. Because
adenosine at 1 µM, but not at higher concentrations, lost its
efficacy in the A2a-deficient mice, it is
plausible that at lower extracellular adenosine concentrations, the
A2a receptor is the only receptor involved in the
inhibitory effect of adenosine on both IL-12 and TNF- production. On
the other hand, at higher adenosine concentrations, such as those that
may occur in pathophysiological states associated with severe cellular
distress, adenosine suppresses cytokine production via other pathways
besides the A2a receptor. Similarly to adenosine,
IB-MECA suppressed IL-12 and TNF- production in both the knockout
and wild-type mice, although it lost some of its efficacy in the
knockout animals. This also suggests that the suppressive effect of
IB-MECA has a component dependent on A2a
receptors. In sharp contrast, the effect of CGS-21680 was completely
abolished in the A2a receptor-deficient mice,
confirming the functional inactivation of A2a
receptors on the macrophages. The rank order of agonist potencies
suggests that the A3 receptor may be the other
receptor involved in the action of adenosine. This notion is supported
by the finding of a recent study in which the suppression of TNF- by
adenosine was primarily due to A3 receptor
activation (44)
.
Since adenosine receptor antagonists increased IL-12 secretion and the
adenosine uptake blocker dipyridamole decreased IL-12 release, it can
be concluded that adenosine is an endogenous inhibitor of IL-12. These
data are in accord with the results of a previous study in which
endogenous adenosine was shown to suppress TNF- production in human
peripheral blood mononuclear cells (45)
. Although in our
study adenosine deaminase was expected to have a similar effect as the
receptor antagonists (that is, to enhance IL-12 production by
decreasing the bioavailability of adenosine), this enzyme failed to
significantly enhance IL-12 production (not shown). A possible
explanation for this contradiction is that the product of adenosine
deamination, inosine, could offset the enhancing effect of the lower
endogenous adenosine concentrations. This idea is supported by a recent
study showing that inosine suppresses IL-12 production
(46)
.
Another interesting finding is that the A3
receptor agonist IB-MECA has a biphasic effect on IL-12 production in
the BALB/c mice, with an inhibition of up to 10 µM but augmentation
at 100 µM. It is conceivable that, similar to another
A3 receptor agonist,
N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine
(47)
, IB-MECA at higher concentrations opens ATP-gated
potassium channels, which could contribute to the enhancement of IL-12
release. In fact, our preliminary studies show that ATP-gated potassium
channel openers increase IL-12 production (unpublished observation).
Adenosine receptors have been shown to be desensitized in many in
vitro and in vivo studies (39)
. In the
current study, we showed that the effect of adenosine on cytokine
production is abrogated in macrophages when adenosine receptors are
exposed to prolonged stimulation of the adenosine receptors by NECA.
This finding has important implications for the treatment of chronic
inflammatory diseases with adenosine-based anti-inflammatory therapies,
as the efficacy of treatment might diminish with time. In this respect,
further studies will be needed to characterize which subtypes of
adenosine receptors are subject to desensitization and which subtypes
are resistant to such an effect.
Since endogenous IL-10 inhibits LPS-induced IL-12 production
(48)
and adenosine was shown to increase IL-10 release in
human monocytes (41)
, we hypothesized that the inhibition
of IL-12 by adenosine was secondary to the enhancement of IL-10
release. Indeed, similar to what was observed in human monocytes, our
data showed that adenosine up-regulates IL-10 production in mouse
peritoneal macrophages. Therefore, to study the possible association
between increased IL-10 and decreased IL-12 production, we examined the
effect of adenosine on IL-12 production after immunoneutralization of
IL-10. Our data indicate that up-regulation of IL-10 and the
suppression of IL-12 production are independent processes in the
current setting.
The mechanism of augmentation of IL-10 production, but not that of
inhibition of IL-12 protein release by adenosine, involves an effect on
the expression of mRNAs for these cytokines. In this respect, the
adenosine regulation of IL-12 in mouse macrophages is similar to that
of TNF-; that is, adenosine receptor stimulation does not affect
steady-state levels of TNF- mRNA in mouse macrophage cell lines
(21)
. On the other hand, in a recent study using the U937
human macrophage cell line, adenosine receptor ligation suppressed
TNF- mRNA levels (44)
. Because LPS-induced activation
of the MAPKs p38 and p42/44 and of JNK are important pathways that can
modulate cytokine production at the posttranscriptional step (49
, 50)
and adenosine receptor stimulation has been demonstrated to
regulate these kinases (51
, 52)
, we evaluated whether
adenosine suppression of IL-12 production could be explained by an
effect on these pathways. Our results showed that the activation of
none of these enzymes is altered by adenosine. Regarding the p42/44
MAPK pathway, our study confirms the results of the study of Sajjadi et
al. (44)
in which adenosine stimulation failed to alter
MAPK activation in human macrophages.
In summary, the current study demonstrates that adenosine suppresses
the production of IL-12 and TNF-. These activities are brought about
by stimulating A2a and other adenosine receptors.
These effects may contribute to the beneficial effects of adenosine
receptor agonists (34)
and adenosine-releasing drugs such
as adenosine kinase inhibitors (53)
, methotrexate
(54)
, sulfasalazine (55)
, or aspirin
(56)
in several autoimmune and inflammatory
diseases.
|
ACKNOWLEDGMENTS |
|---|
Received for publication May 12, 1999.
Revision received April 27, 2000.
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J. M. Wilson, W. G. Ross, O. N. Agbai, R. Frazier, R. A. Figler, J. Rieger, J. Linden, and P. B. Ernst The A2B Adenosine Receptor Impairs the Maturation and Immunogenicity of Dendritic Cells J. Immunol., April 15, 2009; 182(8): 4616 - 4623. [Abstract] [Full Text] [PDF]
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 F. Jeffe, K. A. Stegmann, F. Broelsch, M. P. Manns, M. Cornberg, and H. Wedemeyer Adenosine and IFN-{alpha} synergistically increase IFN-{gamma} production of human NK cells J. Leukoc. Biol., March 1, 2009; 85(3): 452 - 461. [Abstract] [Full Text] [PDF]
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 K. A. Scheibner, S. Boodoo, S. Collins, K. E. Black, Y. Chan-Li, P. Zarek, J. D. Powell, and M. R. Horton The Adenosine A2a Receptor Inhibits Matrix-Induced Inflammation in a Novel Fashion Am. J. Respir. Cell Mol. Biol., March 1, 2009; 40(3): 251 - 259. [Abstract] [Full Text] [PDF]
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 X. Huang, D. J. Moore, R. J. Ketchum, C. S. Nunemaker, B. Kovatchev, A. L. McCall, and K. L. Brayman Resolving the Conundrum of Islet Transplantation by Linking Metabolic Dysregulation, Inflammation, and Immune Regulation Endocr. Rev., August 1, 2008; 29(5): 603 - 630. [Abstract] [Full Text] [PDF]
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Y. Sun, F. Wu, F. Sun, and P. Huang Adenosine Promotes IL-6 Release in Airway Epithelia J. Immunol., March 15, 2008; 180(6): 4173 - 4181. [Abstract] [Full Text] [PDF]
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G. Hasko and P. Pacher A2A receptors in inflammation and injury: lessons learned from transgenic animals J. Leukoc. Biol., March 1, 2008; 83(3): 447 - 455. [Abstract] [Full Text] [PDF]
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 G. E. Garcia, L. D. Truong, P. Li, P. Zhang, J. Du, J.-F. Chen, and L. Feng Adenosine A2A receptor activation and macrophage-mediated experimental glomerulonephritis FASEB J, February 1, 2008; 22(2): 445 - 454. [Abstract] [Full Text] [PDF]
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 J. E. Wunderlich, B. J. Needleman, Z. Chen, J. G. Yu, Y. Wang, I. Grants, D. J. Mikami, W. S. Melvin, H. J. Cooke, and F. L. Christofi Dual purinergic synaptic transmission in the human enteric nervous system Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G554 - G566. [Abstract] [Full Text] [PDF]
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 L. Macedo, G. Pinhal-Enfield, V. Alshits, G. Elson, B. N. Cronstein, and S. J. Leibovich Wound Healing Is Impaired in MyD88-Deficient Mice: A Role for MyD88 in the Regulation of Wound Healing by Adenosine A2A Receptors Am. J. Pathol., December 1, 2007; 171(6): 1774 - 1788. [Abstract] [Full Text] [PDF]
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 B. Csoka, Z. H. Nemeth, L. Virag, P. Gergely, S. J. Leibovich, P. Pacher, C.-X. Sun, M. R. Blackburn, E. S. Vizi, E. A. Deitch, et al. A2A adenosine receptors and C/EBP{beta} are crucially required for IL-10 production by macrophages exposed to Escherichia coli Blood, October 1, 2007; 110(7): 2685 - 2695. [Abstract] [Full Text] [PDF]
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 A. Mohsenin, T. Mi, Y. Xia, R. E. Kellems, J.-F. Chen, and M. R. Blackburn Genetic removal of the A2A adenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L753 - L761. [Abstract] [Full Text] [PDF]
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M. D. Desrosiers, K. M. Cembrola, M. J. Fakir, L. A. Stephens, F. M. Jama, A. Shameli, W. Z. Mehal, P. Santamaria, and Y. Shi Adenosine Deamination Sustains Dendritic Cell Activation in Inflammation J. Immunol., August 1, 2007; 179(3): 1884 - 1892. [Abstract] [Full Text] [PDF]
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Z. H. Nemeth, D. Bleich, B. Csoka, P. Pacher, J. G. Mabley, L. Himer, E. S. Vizi, E. A. Deitch, C. Szabo, B. N. Cronstein, et al. Adenosine receptor activation ameliorates type 1 diabetes FASEB J, August 1, 2007; 21(10): 2379 - 2388. [Abstract] [Full Text] [PDF]
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A. Haschemi, O. Wagner, R. Marculescu, B. Wegiel, S. C. Robson, N. Gagliani, D. Gallo, J.-F. Chen, F. H. Bach, and L. E. Otterbein Cross-Regulation of Carbon Monoxide and the Adenosine A2a Receptor in Macrophages J. Immunol., May 1, 2007; 178(9): 5921 - 5929. [Abstract] [Full Text] [PDF]
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 A. Lokshin, T. Raskovalova, X. Huang, L. C. Zacharia, E. K. Jackson, and E. Gorelik Adenosine-mediated inhibition of the cytotoxic activity and cytokine production by activated natural killer cells. Cancer Res., August 1, 2006; 66(15): 7758 - 7765. [Abstract] [Full Text] [PDF]
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O. Levy, M. Coughlin, B. N. Cronstein, R. M. Roy, A. Desai, and M. R. Wessels The Adenosine System Selectively Inhibits TLR-Mediated TNF-{alpha} Production in the Human Newborn J. Immunol., August 1, 2006; 177(3): 1956 - 1966. [Abstract] [Full Text] [PDF]
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Z. H. Nemeth, B. Csoka, J. Wilmanski, D. Xu, Q. Lu, C. Ledent, E. A. Deitch, P. Pacher, Z. Spolarics, and G. Hasko Adenosine A2A Receptor Inactivation Increases Survival in Polymicrobial Sepsis J. Immunol., May 1, 2006; 176(9): 5616 - 5626. [Abstract] [Full Text] [PDF]
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 L. M. Kreckler, T. C. Wan, Z.-D. Ge, and J. A. Auchampach Adenosine Inhibits Tumor Necrosis Factor-{alpha} Release from Mouse Peritoneal Macrophages via A2A and A2B but Not the A3 Adenosine Receptor J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 172 - 180. [Abstract] [Full Text] [PDF]
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J. B. Volmer, L. F. Thompson, and M. R. Blackburn Ecto-5'-Nucleotidase (CD73)-Mediated Adenosine Production Is Tissue Protective in a Model of Bleomycin-Induced Lung Injury J. Immunol., April 1, 2006; 176(7): 4449 - 4458. [Abstract] [Full Text] [PDF]
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A. Fortin, D. Harbour, M. Fernandes, P. Borgeat, and S. Bourgoin Differential expression of adenosine receptors in human neutrophils: up-regulation by specific Th1 cytokines and lipopolysaccharide J. Leukoc. Biol., March 1, 2006; 79(3): 574 - 585. [Abstract] [Full Text] [PDF]
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Z. H. Nemeth, C. S. Lutz, B. Csoka, E. A. Deitch, S. J. Leibovich, W. C. Gause, M. Tone, P. Pacher, E. S. Vizi, and G. Hasko Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism J. Immunol., December 15, 2005; 175(12): 8260 - 8270. [Abstract] [Full Text] [PDF]
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 G. Gallos, T. D. Ruyle, C. W. Emala, and H. T. Lee A1 adenosine receptor knockout mice exhibit increased mortality, renal dysfunction, and hepatic injury in murine septic peritonitis Am J Physiol Renal Physiol, August 1, 2005; 289(2): F369 - F376. [Abstract] [Full Text] [PDF]
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Y.-J. Day, Y. Li, J. M. Rieger, S. I. Ramos, M. D. Okusa, and J. Linden A2A Adenosine Receptors on Bone Marrow-Derived Cells Protect Liver from Ischemia-Reperfusion Injury J. Immunol., April 15, 2005; 174(8): 5040 - 5046. [Abstract] [Full Text] [PDF]
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Y.-J. Day, L. Huang, H. Ye, J. Linden, and M. D. Okusa Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages Am J Physiol Renal Physiol, April 1, 2005; 288(4): F722 - F731. [Abstract] [Full Text] [PDF]
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J. M. Daley, J. S. Reichner, E. J. Mahoney, L. Manfield, W. L. Henry Jr., B. Mastrofrancesco, and J. E. Albina Modulation of Macrophage Phenotype by Soluble Product(s) Released from Neutrophils J. Immunol., February 15, 2005; 174(4): 2265 - 2272. [Abstract] [Full Text] [PDF]
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 A. S. Weyrich, M. M. Denis, J. R. Kuhlmann-Eyre, E. D. Spencer, D. A. Dixon, G. K. Marathe, T. M. McIntyre, G. A. Zimmerman, and S. M. Prescott Dipyridamole Selectively Inhibits Inflammatory Gene Expression in Platelet-Monocyte Aggregates Circulation, February 8, 2005; 111(5): 633 - 642. [Abstract] [Full Text] [PDF]
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 M. Sakowicz, A. Szutowicz, and T. Pawelczyk Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes Int. Immunol., February 1, 2005; 17(2): 145 - 154. [Abstract] [Full Text] [PDF]
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 A. Thiele, R. Kronstein, A. Wetzel, A. Gerth, K. Nieber, and S. Hauschildt Regulation of Adenosine Receptor Subtypes during Cultivation of Human Monocytes: Role of Receptors in Preventing Lipopolysaccharide-Triggered Respiratory Burst Infect. Immun., March 1, 2004; 72(3): 1349 - 1357. [Abstract] [Full Text] [PDF]
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M. Schnurr, T. Toy, A. Shin, G. Hartmann, S. Rothenfusser, J. Soellner, I. D. Davis, J. Cebon, and E. Maraskovsky Role of adenosine receptors in regulating chemotaxis and cytokine production of plasmacytoid dendritic cells Blood, February 15, 2004; 103(4): 1391 - 1397. [Abstract] [Full Text] [PDF]
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G. W. Sullivan, D. D. Lee, W. G. Ross, J. A. DiVietro, C. M. Lappas, M. B. Lawrence, and J. Linden Activation of A2A adenosine receptors inhibits expression of {alpha}4/{beta}1 integrin (very late antigen-4) on stimulated human neutrophils J. Leukoc. Biol., January 1, 2004; 75(1): 127 - 134. [Abstract] [Full Text] [PDF]
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G. Gomez and M. V. Sitkovsky Differential requirement for A2a and A3 adenosine receptors for the protective effect of inosine in vivo Blood, December 15, 2003; 102(13): 4472 - 4478. [Abstract] [Full Text] [PDF]
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 J. P. Headrick, B. Hack, and K. J. Ashton Acute adenosinergic cardioprotection in ischemic-reperfused hearts Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1797 - H1818. [Abstract] [Full Text] [PDF]
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N. D. Khoa, M. C. Montesinos, A. J. Williams, M. Kelly, and B. N. Cronstein Th1 Cytokines Regulate Adenosine Receptors and Their Downstream Signaling Elements in Human Microvascular Endothelial Cells J. Immunol., October 15, 2003; 171(8): 3991 - 3998. [Abstract] [Full Text] [PDF]
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Z. H. Neemeth, S. J. Leibovich, E. A. Deitch, E. S. Vizi, C. Szabo, and G. Hasko cDNA Microarray Analysis Reveals a Nuclear Factor-{kappa}B-Independent Regulation of Macrophage Function by Adenosine J. Pharmacol. Exp. Ther., September 1, 2003; 306(3): 1042 - 1049. [Abstract] [Full Text] [PDF]
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 A. A. Borisy, P. J. Elliott, N. W. Hurst, M. S. Lee, J. Lehar, E. R. Price, G. Serbedzija, G. R. Zimmermann, M. A. Foley, B. R. Stockwell, et al. Systematic discovery of multicomponent therapeutics PNAS, June 24, 2003; 100(13): 7977 - 7982. [Abstract] [Full Text] [PDF]
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M. P. Mycko, R. Papoian, U. Boschert, C. S. Raine, and K. W. Selmaj cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity Brain, May 1, 2003; 126(5): 1048 - 1057. [Abstract] [Full Text] [PDF]
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J. G. Mabley, P. Pacher, L. Liaudet, F. G. Soriano, G. Hasko, A. Marton, C. Szabo, and A. L. Salzman Inosine reduces inflammation and improves survival in a murine model of colitis Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G138 - G144. [Abstract] [Full Text] [PDF]
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T. Greenwell-Wild, N. Vazquez, D. Sim, M. Schito, D. Chatterjee, J. M. Orenstein, and S. M. Wahl Mycobacterium avium Infection and Modulation of Human Macrophage Gene Expression J. Immunol., December 1, 2002; 169(11): 6286 - 6297. [Abstract] [Full Text] [PDF]
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K. Gounaris Nucleotidase Cascades Are Catalyzed by Secreted Proteins of the Parasitic Nematode Trichinella spiralis Infect. Immun., September 1, 2002; 70(9): 4917 - 4924. [Abstract] [Full Text] [PDF]
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S. J. Leibovich, J.-F. Chen, G. Pinhal-Enfield, P. C. Belem, G. Elson, A. Rosania, M. Ramanathan, C. Montesinos, M. Jacobson, M. A. Schwarzschild, et al. Synergistic Up-Regulation of Vascular Endothelial Growth Factor Expression in Murine Macrophages by Adenosine A2A Receptor Agonists and Endotoxin Am. J. Pathol., June 1, 2002; 160(6): 2231 - 2244. [Abstract] [Full Text] [PDF]
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 M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]
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G. Hasko, E. A. Deitch, Z. H. Nemeth, D. G. Kuhel, and C. Szabo Inhibitors of ATP-Binding Cassette Transporters Suppress Interleukin-12 p40 Production and Major Histocompatibility Complex II Up-Regulation in Macrophages J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 103 - 110. [Abstract] [Full Text] [PDF]
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 Z. H. Nemeth, E. A. Deitch, C. Szabo, Z. Fekete, C. J. Hauser, and G. Hasko Lithium Induces NF-kappa B Activation and Interleukin-8 Production in Human Intestinal Epithelial Cells J. Biol. Chem., March 1, 2002; 277(10): 7713 - 7719. [Abstract] [Full Text] [PDF]
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M. D. Okusa A2A adenosine receptor: a novel therapeutic target in renal disease Am J Physiol Renal Physiol, January 1, 2002; 282(1): F10 - F18. [Abstract] [Full Text] [PDF]
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 L. LIAUDET, J. G. MABLEY, F. G. SORIANO, P. PACHER, A. MARTON, G. HASKO, and C. SZABO Inosine Reduces Systemic Inflammation and Improves Survival in Septic Shock Induced by Cecal Ligation and Puncture Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1213 - 1220. [Abstract] [Full Text] [PDF]
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N. D. Khoa, M. C. Montesinos, A. B. Reiss, D. Delano, N. Awadallah, and B. N. Cronstein Inflammatory Cytokines Regulate Function and Expression of Adenosine A2A Receptors in Human Monocytic THP-1 Cells J. Immunol., October 1, 2001; 167(7): 4026 - 4032. [Abstract] [Full Text] [PDF]
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E. PANTHER, M. IDZKO, Y. HEROUY, H. RHEINEN, P. J. GEBICKE-HAERTER, U. MROWIETZ, S. DICHMANN, and J. NORGAUER Expression and function of adenosine receptors in human dendritic cells FASEB J, September 1, 2001; 15(11): 1963 - 1970. [Abstract] [Full Text] [PDF]
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J. G. Valenzuela, Y. Belkaid, M. K. Garfield, S. Mendez, S. Kamhawi, E. D. Rowton, D. L. Sacks, and J. M.C. Ribeiro Toward a Defined Anti-Leishmania Vaccine Targeting Vector Antigens: Characterization of a Protective Salivary Protein J. Exp. Med., August 6, 2001; 194(3): 331 - 342. [Abstract] [Full Text] [PDF]
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C. Soler, R. Valdes, J. Garcia-Manteiga, J. Xaus, M. Comalada, F. J. Casado, M. Modolell, B. Nicholson, C. MacLeod, A. Felipe, et al. Lipopolysaccharide-induced Apoptosis of Macrophages Determines the Up-regulation of Concentrative Nucleoside Transporters Cnt1 and Cnt2 through Tumor Necrosis Factor-alpha -dependent and -independent Mechanisms J. Biol. Chem., August 3, 2001; 276(32): 30043 - 30049. [Abstract] [Full Text] [PDF]
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M. Thielmann, H. Dorge, C. Martin, S. Belosjorow, U. Schwanke, A. van de Sand, I. Konietzka, A. Buchert, A. Kruger, R. Schulz, et al. Myocardial Dysfunction With Coronary Microembolization: Signal Transduction Through a Sequence of Nitric Oxide, Tumor Necrosis Factor-{alpha}, and Sphingosine Circ. Res., April 19, 2002; 90(7): 807 - 813. [Abstract] [Full Text] [PDF]
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