Review

Monthly Focus: Pulmonary-Allergy, Dermatological, Gastrointestinal & Arthritis

Adenosine A2A agonists in

development for the treatment of
inflammation

1. Introduction

Courtney M Lappas, Gail W Sullivan & Joel Linden

2. Adenosine A2A receptor

agonists and inflammatory cells

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3. Conclusions
4. Expert opinion

Departments

of Pharmacology, Box 801394, University of Virginia, Charlottesville VA 22908, USA

Extracellular adenosine binds specifically to a family of four G protein-coupled cell-surface adenosine receptors (ARs). As the activation of the A2AAR
modulates the activity of multiple inflammatory cells including neutrophils,
macrophages and T lymphocytes, the receptor is considered to be a promising pharmacological target for the treatment of inflammatory disorders.
Although adenosine binds nonselectively to all four AR subtypes, A2AAR
selective agonists have been developed and shown to inhibit multiple manifestations of inflammatory cell activation including superoxide anion generation, cytokine production and adhesion molecule expression. A2AAR agonists
are also vasodilators, but the inhibition of inflammation occurs at low doses
that produce few or no cardiovascular side effects. Therefore, the selective
activation of the A2AAR by these compounds holds significant potential in the
treatment of inflammation.
Keywords: adenosine, inflammation, T lymphocytes, macrophages, neutrophils
Expert Opin. Investig. Drugs (2005) 14(7):797-806

1. Introduction
1.1 Adenosine

The ubiquitous purine nucleoside adenosine is released by various cells, including

fibroblasts, epithelial cells, endothelial cells, platelets and muscle cells, or is derived
from the extracellular metabolism of released purine nucleotides. The production of
adenosine occurs primarily as a result of the breakdown of ATP in response to cellular energy demands, with the rate-limiting step in adenosine production being the
dephosphorylation of AMP by 5-nucleotidase, which exists as a soluble cytoplasmic
enzyme and also as an ectoenzyme, CD73 [1,2]. Other sources of adenosine are the
signalling molecule cAMP, and (S)-adenosyl-homocysteine. Adenosine is found in
intracellular and extracellular spaces due to its production in both regions and its
transport across the cell membrane by two distinct types of specific transporters.
Equilibrative nucleoside transporters (ENTs) mediate the equilibration of adenosine
concentrations by facilitated transport, whereas the concentrative nucleoside transporters (CNTs) allow for the preservation of adenosine concentration gradients
across the cell membrane [3-5]. The local concentrations of adenosine vary according
to tissue and physiological state with levels increasing dramatically during conditions of oxidative or ischaemic stress, exercise or inflammation [6-8]. Levels of adenosine return to baseline via the degradation by adenosine deaminase (resulting in the
production of inosine) or uptake into cells, and phosphorylation by adenosine
kinase (forming AMP) [9-11].
Ashley Publications
www.ashley-pub.com

1.2 Adenosine

receptors

Extracellular adenosine binds specifically to a family of G protein-coupled cell-surface

receptors called adenosine receptors (ARs). The AR family is comprised of four

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Adenosine A2A agonists in development for the treatment of inflammation

genetically and structurally similar subtypes including the

Gi-coupled A1AR and A3AR, and Gs-coupled A2AAR and
A2BAR. In some cells, the A2B receptor also couples to Gq. All
four receptor subtypes have seven membrane-spanning -helices with an extracellular N-terminus and an intracellular C-terminal tail, and although structural information about ARs has
been difficult to obtain due to the large size of the receptors and
their interactions with membrane lipids, studies utilising
mutated receptors indicate that essential receptorligand interactions occur in helices three, five, six and seven [12,13]. The
expression of ARs varies widely according to tissue and subtype,
as do the effects elicited by receptor activation. Because of their
involvement in the regulation of many physiological processes,
ARs are interesting pharmacological targets in the treatment of
multiple pathophysiological conditions including CNS disorders, cardiovascular disease, ischaemiareperfusion injury and
inflammatory diseases. However, due to their varying downstream signalling pathways, the four AR subtypes have the
potential to mediate opposing physiological effects, so the therapeutic relevancy of AR activation has been coupled to the development of subtype-specific agonists or antagonists. Although
adenosine itself is used therapeutically to terminate supraventricular tachyarrhythmias and as a coronary vasodilator for
pharmacological stress imaging, it has limited therapeutic value
because of its short half-life and non-specific binding to all four
AR subtypes. However, modifications of the molecule at three
distinct sites have resulted in analogues demonstrating
significantly increased selectivity and potency [14].
1.3 Adenosine A2A receptor agonists and
inflammation

The A2AAR is expressed at low levels in the lung, heart and

blood vessels, and at notably higher levels in the spleen, thymus
and brain [13]. Peripherally administered agonists do not efficiently cross the bloodbrain barrier. Activation of the A2AAR
on cells of the immune system plays a role in terminating
inflammation. The activity of virtually all inflammatory cells,
including but not limited to macrophages, monocytes, T lymphocytes, platelets and polymorphonuclear leukocytes (PMNs),
is modulated by the activation of A2AAR with multiple manifestations of inflammatory cell activation including cytokine production and inhibition of adhesion molecule expression [12,1520]. Although the activity of inflammatory cells is vital for a host
response to infection, inappropriately high or prolonged activity
also results in host tissue destruction. It is, therefore, necessary
that inflammatory responses are tightly regulated. Experimental
evidence suggests that adenosine released from macrophage- or
neutrophil-damaged tissue acts on A2AARs expressed on these
and other inflammatory cells to inhibit further activity. In this
manner, signalling by extracellular adenosine through the
A2AAR may act as an endogenous regulator of inflammation.
For these reasons, the development of selective A2AAR agonists is
of great interest and many groups have successfully synthesised
such compounds and demonstrated their anti-inflammatory
potential. The modification of the 5-hydroxyl group of
798

adenosine yields the potent, albeit non-selective, AR agonist 5N-ethylcarboxamine (NECA). Most A2AAR agonists are 2-substitutions of NECA. These compounds include the 2-substituted amine, 2-(4-[2-carboxyethyl]phenethylamino)-5-Nethylcarboxamidoadenosine (CGS-21680; moderate affinity
and high selectivity) [21], the 2-alkynyls including 5-(6-amino-2hex-1-ynyl-purin-9-yl)-3,4-di-hydroxy-tetrahydro-furan-2-carboxilic acid ethylamide (HENECA; high potency and low selectivity) [22], the potent and selective 2-(4-substituted-cyclohexyl)
propynyl NECAs including 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]prop-2-ynyl)-cyclohexanecarboxylic
acid
methyl
ester
(ATL-146e) [19], and the hydrazines including 2-(6-amino-2[N-cyclohexylmethylene-hydrazino]-purin-9-yl)-5-hydroxymethyl-tetrahydro-furan-3,4-diol (binodenoson; MRE-0470;
Figure 1) [23]. This review focuses on the effects of selective
A2AAR agonists on the function of specific inflammatory cells
including T lymphocytes, macrophages and neutrophils.
2. Adenosine

A2A receptor agonists and

inflammatory cells
2.1 Neutrophils

A primary response to injury or microbial infection is inflammation characterised by the local recruitment, infiltration,
accumulation and activation of neutrophils. The events leading
to neutrophil extravasation into interstitial spaces are threefold
consisting of selectin-mediated rolling of the neutrophil along
the endothelium, 2-integrin-dependent firm adhesion of the
neutrophil to the endothelium, and transmigration. The adhesion of neutrophils to the vascular endothelium is accompanied by a characteristic response of activated neutrophils, the
so-called oxidative burst [24,25]. An oxidative or respiratory
burst is a primary mechanism by which neutrophils induce tissue damage and is characterised by the release of myeloperoxidase (MPO), which converts hydrogen peroxide to
hypochlorous acid, and the production of the superoxide anion
by NADPH-dependent oxidase [26,27]. The NADPH-dependent oxidase found on the surface of macrophages and neutrophils drives the conversion of molecular oxygen to hydrogen
peroxide and the superoxide anion when activated by a
number of stimuli [28]. Activated neutrophils may also release
proteases such as elastase, gelatinase and collagenase. Although
these activities of PMNs are vital components of a successful
response to infection, neutrophil activation can lead to destructive tissue damage, particularly following iatrogenic procedures
such as tissue transplantation or balloon angioplasty, which
result in sterile inflammation. There is a vast body of work
indicating that adenosine inhibits both the adhesion of neutrophils to the endothelium as well as the generation of the
superoxide anion; however, it was not until the development of
subtype-selective agonists that the contribution of the A2AAR
to these effects was clearly delineated.
The expression of the A2AAR on PMNs has been
characterised by direct radioligand binding to membranes, and

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Lappas, Sullivan & Linden

NH2
O

NH2

O
N
H

N
H

HO
H

OH

HO

HO

OH

Binodenoson
(MRE-0470)

ATL-146e

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NH2

NH2

N
H

O
N
H

O
H

HO

O N

HO

N
H

H
HO

OH

OH

HENECA

CGS-21680

Figure 1. Prototypical adenosine A2A receptor agonist structures.

the binding potencies of multiple A2AAR agonists have been

shown to correlate well with the functional responses elicited in
intact neutrophils by these compounds [29,30]. The binding
affinities of several adenosine analogues to recombinant A2AARs
have been evaluated via competition binding assays and found
to adhere to the following order of potency: ATL-146e > acetic
acid
4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexylmethyl ester (ATL-193) > NECA > 5-(6-amino-2-[3-(4hydroxymethyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-carboxylic
acid
ethylamide
(ATL-2037) > MRE-0470 > CGS-21680. Furthermore, these
compounds trigger dose-dependent increases in intracellular
cAMP accumulation in intact human neutrophils with a similar order of potency [19]. Exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP)
induces both a transient elevation in cAMP, which is prolonged
by the co-administration of CGS-21680, and the generation
and release of the superoxide anion [31]. Early studies indicate
that the treatment of human neutrophils with NECA alone
produces no measurable elevation of intracellular cAMP,
whereas co-treatment with fMLP synergistically enhances the
concentration of the signalling molecule and addition of the
type IV phosphodiester (PDE) inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO-20-1724) additively elevates cAMP [32]. The non-selective agonist NECA has been
found to inhibit fMLP-induced superoxide anion production
in a manner that is not inhibited by either the selective protein
kinase A (PKA) inhibitor KT-5720 or the non-selective kinase
inhibitor H-7, suggesting a cAMP-independent component of
inhibition [33]. Conversely, more recent studies utilising the

A2AAR selective agonist ATL-193 demonstrate that the dosedependent inhibition of the fMLP-induced oxidative burst by
this compound is inhibited by the PKA inhibitor H-89 and
augmented by co-treatment with the type IV PDE inhibitor 4(3-cyclopentyloxy-4methoxyphenyl)-2-pyrrolidone
(rolipram), suggesting a cAMP/PKA-dependent mechanism [19].
The apparent discrepancies in these data underscore the importance of agonist selectivity in defining the signalling pathways
in cells with multiple adenosine receptor subtypes. The selective A2AAR antagonist 4-(2-[7-amino-2-(2-furyl)-(1,2,4)triazolo-(2,3-a)(1,3,5)triazin-5-yl-amino]ethyl)phenol
(ZM-241385) abrogates both the elevation of cAMP and inhibition of fMLP-induced superoxide anion production by
ATL-193. ZM-241385 also counteracts the action of the A1AR
selective agonist N6-cyclohexyladenosine (CPA) and the A3AR
selective
agonist
N6-(2-iodo)benzyl-5-N-methylcarboxamidodoadenosine (IB-MECA) to inhibit neutrophil
activation, thus indicating that these effects are mediated by the
activation of A2AAR and that the selectivity of these compounds
is limited [19,34,35]. One of several mechanisms involved in the
regulation of the oxidative burst of neutrophils is the activation
of phospholipase D (PLD). The removal of extracellular adenosine via pretreatment with adenosine deaminase or the blockade
of A2AAR with the selective antagonist 8-(3-chlorostyryl) caffeine (CSC) increases fMLP-mediated PLD activation. In contrast, CGS-26180 inhibits the activation of PLD in response to
fMLP stimulation, an effect that is enhanced by co-treatment
with Ro-20-1724 and blocked by the adenylyl cyclase inhibitor
2-5-dideoxyadenosine. Additionally, the recruitment of
PLD-activation co-factors auxin response factor (Arf )-1 and ras
homologue family member A (RhoA) to PMN membranes in

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Adenosine A2A agonists in development for the treatment of inflammation

response to fMLP is notably inhibited by CGS-21680, and this

inhibition of translocation is blocked by H-89. These observations suggest that A2AAR activation regulates the
fMLP-induced oxidative burst at least in part via the cAMP/
PKA-dependent modulation of PLD activation [31].
In addition to releasing reactive oxygen species (ROS),
activated neutrophils also release several enzymes including
collagenase, elastase and lysozyme. Because these effector
molecules are potentially toxic to host tissue, it is vital that
the activity of neutrophils is localised to areas of injury or
infection. One mechanism by which this specific accumulation is facilitated is via the modulation of adhesion-molecule
expression. The 41 integrin very late antigen (VLA)-4 is
minimally expressed by inactivated peripheral blood neutrophils, but is rapidly upregulated in response to various
chemoattractant agents. The sequential exposure of PMNs
to TNF- followed by fMLP results in a significant increase
in CD49d (the -subunit of VLA-4) expression and this
upregulation, as well as the resulting increase in neutrophil
adhesion to a vascular cell adhesion molecule (VCAM) 1coated surface, is abrogated by ATL-146e. The inhibitory
effects of ATL-146e on CD49d upregulation and cell adhesion are blocked by both ZM-241385 and H-89, indicating
the involvement of an A2AAR-specific cAMP-mediated signalling pathway [20]. It has been observed that extracellular
adenosine, although effectively inhibiting superoxide anion
generation, has little effect on PMN degranulation [36].
However, pretreatment with CGS-21680 reduces fMLPstimulated elastase release by 50%, and this inhibition is
accompanied by an accelerated clearance of Ca2+ from the
cytosol [35]. Both of these effects are antagonised by
ZM-241385, indicating A2AAR-specific agonist activity. It
was shown that the anti-inflammatory effects of cAMP-elevating agents in human neutrophils are mediated at least in
part by the upregulation of cAMP-dependent protein
kinase-modulated Ca2+-sequestering endomembrane Ca2+ATPase, and it is possible that CGS-21680 acts to inhibit
degranulation through a similar mechanism [37,38].
2.2 Macrophages

When activated by a variety of peptide or non-peptide factors

derived from tissue injury or microbial infection, macrophages secrete a broad repertoire of effector compounds
including ROS, arachidonic acid metabolites and cytokines.
In addition to serving as an important direct defence against
microbial insult, macrophages also function as antigen-presenting cells for T lymphocytes and promote the formation of
new blood vessels [39]. The exposure of macrophages to
lipopolysaccharide (LPS) initiates toll-like receptor
(TLR)-mediated signalling, which facilitates an upregulation
in the expression of the anti-inflammatory cytokine IL-10,
the angiogenic growth factor vascular endothelial growth
factor (VEGF) and multiple pro-inflammatory cytokines
including IL-12 and TNF- [40-42]. TNF- is involved in the
induction of nitric oxide (NO) synthesis in response to LPS,
800

which in turn is necessary for VEGF expression [43,44]. In contrast, IL-10 inhibits the production of both TNF- and NO,
and inhibits various other macrophage-derived inflammatory
mediators including IFN- and IL-1 [45,46]. Contributing to
the complexity of macrophage regulation is the role played by
adenosine, which has been observed to inhibit various macrophage functions including NO production, chemotaxis and
phagocytosis; these effects are mediated by both
A2AAR-dependent and -independent mechanisms [47-49].
The intraperitoneal injection of BALB/c mice with LPS
results in a significant increase in plasma levels of TNF-,
IL-10 and NO (as measured by the presence of the NO breakdown products, nitrite and nitrate). Pretreatment with
CGS-21680 dose-dependently augments the production of
IL-10 and inhibits LPS-induced increases in TNF- and NO
generation. These effects are mimicked in a murine macrophage cell line (RAW 264.7) in which LPS-stimulated productions of TNF- and NO are inhibited by pretreatment
with CGS-21680 [50]. Data indicate that A2AAR activation
inhibits the accumulation of intracellular TNF-, rather than
the release of the cytokine, in a NF-B-independent manner
[51]. It is notable, however, that there is evidence indicating
that A2AAR activation blocks the NF-B pathway downstream of immunoreceptors by interfering with the activation
of the inhibitor of NF-B (IB) kinase (IKK) complex or by
hindering the IKKIB interaction; this activity is cAMP and
PKA dependent [52]. It has also been shown that the ability of
NF-B to bind DNA after TNF--induced activation is
inhibited by adenosine in a variety of cell types including
T lymphocytes, epithelial cells and myeloid cells [53]. The suppression of mitochondrial respiration observed after treating
RAW 264.7 cells with LPS is inhibited by pretreatment with
CGS-21680. Interestingly, the production of IL-10 in
response to LPS activation is also inhibited, rather than
enhanced, in RAW 264.7 cells by CGS-21680, thus suggesting that the augmentation observed in vivo is mediated by
some secondary factor(s) not present in vitro [50]. The release
of IL-12 by macrophages serves as an important link between
the innate and adaptive immune responses, triggering the
activation and proliferation of T lymphocytes [54]. A positive
feedback loop in which IFN- produced by activated T cells
stimulates additional IL-12 production by macrophages is
thus initiated, and although this is an important mechanism
of defence against intracellular pathogens, the unregulated
production of IL-12 and IFN- is associated with various
autoimmune and inflammatory disorders [55-57]. It has been
observed that adenosine inhibits the LPS-induced release of
IL-12 from macrophages and this effect is mimicked by adenosine analogues with the following order of potency;
CGS-21680 > IB-MECA > 2-chloro-N6-cyclopentyl
adenosine (CCPA), which is consistent with an A2AAR
response. Furthermore, the release of IL-12 in response to
LPS is enhanced by treatment with ZM-241385 or the
non-selective AR antagonist 3,7-dimethyl-1-propargyl
xanthine (DMPX), but not by alloxazine or the

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Lappas, Sullivan & Linden

A1AR-selective antagonist 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), suggesting that the inhibition

of IL-12 production by endogenous adenosine is mediated at
least in part by A2AAR activation [58].
It is important to note that although antigen presentation
to T lymphocytes is an important initiating factor in inflammatory responses, macrophages alone are not responsible for
this presentation. Rather, dendritic cells (DCs) are professional antigen-presenting cells that migrate to T-cell areas of
secondary lymphoid organs following maturation, process
antigens and activate naive T lymphocytes. The expression of
the A2AAR on human monocyte-derived DCs is upregulated
during maturation, and the activation of the receptor with the
selective agonist N6-(2-[3,5-dimethoxyphenyl]-2-[2-methylphenyl]-ethyl)adenosine (DPMA) stimulates the accumulation of intracellular cAMP and the inhibition of IL-12
production by LPS-differentiated DCs; these effects are
blocked by CSC [59-61]. Similarly, the treatment of murine
monoctye-derived DCs with CGS-21680 stimulates cAMP
accumulation and inhibits LPS-induced TNF- release [62].
Additionally, the activation of the A2AAR modulates
DC-migratory activity. Treatment with NECA delays the
migration of DCs from skin explant cultures, inhibits the
migration of epidermal and dermal DCs from the periphery
into the draining lymph node on immunostimulation and
delays the transmigration of DCs in response to the chemoattractant macrophage inflammatory protein (MIP)-3 [60].
Thus, A2AAR agonists function to modulate the activity of
multiple APCs (including macrophages and dendritic cells),
effectively limiting T-lymphocyte activation.
In contrast to the pro-inflammatory potential of TNF-,
NO and IL-12, IL-10 and VEGF are generally considered to
be protective in nature. Resting macrophages express low levels of VEGF that are dramatically upregulated in response to
several stimuli, most notably hypoxia or the combination of
IFN- and LPS [44]. It was shown that CGS-21680 and
NECA increase resting macrophage VEGF expression, and
significantly enhance both hypoxia and LPS/IFN-stimulated VEGF responses in murine peritoneal macrophages.
The augmentation of the LPS/IFN- response by CGS-21680
and NECA is absent in A2AAR-/- cells and blocked by
ZM-241385, but not by alloxazine or the A1AR selective
antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX),
which is consistent with an A2AAR-specific response. Furthermore, in macrophages collected from C3H/HeJ mice in
which mutations have been engineered into the cytoplasmic
domain of TLR4 rendering the receptor functionally inactive,
co-treatment with LPS and NECA elicits an increase in
VEGF expression that is similar to that elicited by NECA
alone, indicating that the A2AAR acts synergistically with
TLR4 to elevate VEGF expression [63]. Independent experiments utilising TLR2, -7 or -9 agonists indicate that A2AAR
activation also acts in concert with these receptors on murine
macrophages to increase VEGF [64]. A functional consequence
of such activity is the promotion of angiogenesis and wound

healing by A2AAR activation. The topical application of CGS21680 increases the rate of wound closure and the number of
microvessels in the wounds of wild-type but not A2AAR-/mice [65].
2.3 T

lymphocytes
Normally, the recognition of an antigen by the T-cell receptor
(TCR) complex on T lymphocytes initiates a cascade of signalling events resulting in T-cell activation as manifested by
the upregulation of adhesion molecules, the synthesis and
secretion of cytokines including IFN- and IL-2, cellular cytotoxicity and cell proliferation. Adenosine has been observed to
have multiple and varied effects on T-cell function; intracellular adenosine, such as accumulates in conditions of adenosine deaminase deficiency, is directly lymphotoxic resulting
in T-cell depletion, whereas extracellular adenosine interferes
with normal TCR-mediated signalling [66]. It was demonstrated that murine T cells preferentially express the A2AAR
compared with other AR subtypes, and studies utilising a
monoclonal antibody to quantify the expression of the A2AAR
on T lymphocytes show that the receptor is differentially
expressed on T-cell subsets with greater receptor density
found on CD4+ versus CD8+ cells and on lymphokine-producing cells as opposed to cells that do not actively secrete
cytokines [67-69]. Additionally, signalling through the TCR
mediates a rapid and transient upregulation of A2AAR mRNA
that is correlated with an increase in the efficacy of ATL-146e
to stimulate intracellular cAMP accumulation in murine
CD4+ T lymphocytes [16]. Furthermore, data indicate that
there is no A2AAR reserve on T lymphocytes as multiple functional responses to A2AAR agonists are inhibited half-maximally in cells from A2AAR+/- mice [16,70]. A functional
consequence of the upregulation of A2AAR expression with
T-cell activation is the inhibition of multiple T-cell effector
functions by A2AAR agonists.
The crosslinking of the TCR complex produces a dramatic
increase in IFN- production by CD4+ T cells that is inhibited by adenosine analogues with the following order of
potency: ATL-146e > 4-(3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2yl]prop-2-ynyl)piperidine-1-carboxylic acid methyl ester
(ATL-313) > NECA > CGS-21680 >> CPA > 2-chloro-N6(3-iodobenzyl)-5-N-methylcarboxamide
(Cl-IBMECA).
This order correlates well with the binding affinities of the
analogues to recombinant murine A2AAR. The suppressive
effect of ATL-146e on IFN- production is inhibited by
ZM-241385, abolished in A2AAR-/- cells and the maximum
response is reduced by 50% in A2AAR+/- cells. Furthermore,
this activity of ATL-146e is mimicked by rolipram in both
A2AAR+/+ and A2AAR-/- CD4+ T cells suggesting that the
cAMP-elevating activity of A2AAR activation mediates the
inhibition of TCR-induced IFN- production [16]. Along with
stimulating the production of the pro-inflammatory cytokine
IFN-, TCR signalling also upregulates the expression of IL-2
and the IL-2 receptor -chain (CD25), which together

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Adenosine A2A agonists in development for the treatment of inflammation

IFN- production
Apoptosis

Granule exocytosis
T cells

CD25 upregulation

FasL expression

TNF- production
IL-10 production

NO generation

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m s

VEGF expression

IL-12 production

PLD activation
(Arf1 and RhoA translocation)

CD49d
upregulation

PMNs

Elastase release

Superoxide anion generation

Figure 2. Effects of adenosine A2A receptor agonists on

inflammatory cells. The events that are inhibited by adenosine
A2A receptor agonist treatment are shown, along with the events
that are augmented (in bold).
FasL: Fas ligand; IFN: Interferon; NO: Nitric oxide; PLD: Phospholipase D;
m: Macrophage; PMN: Polymorphonuclear leukocyte; RhoA: Ras homologue
family member A; TNF: Tumour necrosis factor; VEGF: Vascular endothelial
growth factor.

function to drive T-cell proliferation. Adenosine has been

observed to inhibit IL-2 production and T-cell proliferation at
doses that do not affect cell viability, and CGS-21680 and
ATL146e inhibit the upregulation of CD25 in response to
TCR activation [16,68,71]. The effect of A2AAR activation on
CD25 expression is mimicked by the cAMP analogue dibutyryl (db)-cAMP and the PDE inhibitors rolipram and 3-isobutyl-1-methylxanthine (IBMX) [16,68]. Additionally, A2AAR
engagement activates Src homology region 2 domain-containing phosphatase (SHP)-2, resulting in signal transducer and
activator of transcription (STAT)-5 dephosphorylation and
reduced IL-2 receptor signalling. This inhibition of IL-2 signalling is manifested as a suppression of IL-2-dependent
T lymphocyte proliferation [72].
Although T helper type 2 (TH2) cells predominantly
function to activate B lymphocytes and promote antibody
production and allergic responses, TH1 cells activate macrophages and are required for the differentiation of cytotoxic T
lymphocyte (CTL) precursors into cytotoxic effector cells.
Thus, TH1 cells are vital for the induction of cell-mediated
immunity. It is notable that whereas TH1 cytokine responses
(including the production of IL-2 and IFN-) are inhibited
by A2AAR activation and cAMP elevation, the production of
TH2 cytokines (including IL-4, -5, -6 and -10) is generally
802

either enhanced by or unaffected by cAMP elevation [73-77].

Therefore, A2AAR activation may preferentially inhibit TH1driven immune responses. CTLs directly kill target cells via
perforin- and granzyme-containing granule exocytosis and/
or FasFas ligand (FasL) interactions; both of these activities
are triggered by TCR engagement. NECA, 2-CADO and
CGS-21680 inhibit TCR-mediated CTL granule exocytosis
and the resulting antigen-specific target-cell lysis. Additionally, 2-CADO inhibits the lysis of target cells by CTLs
derived from perforin-deficient mice, indicating an inhibitory effect on the FasL-based mechanism of cytotoxicity;
this effect is most likely to be mediated by the A2AAR as adenosine has been shown to inhibit TCR-induced increases in
FasL mRNA expression in a manner that is blocked by
ZM-241385 [78]. Although low doses of A2AAR agonists
inhibit T-cell responses without affecting cell viability,
higher doses of adenosine or CGS-21680 induce T-cell
selection in the thymus by inducing thymocyte death in a
dose-dependent manner. The extent of thymocyte apoptosis
induced by CGS-21680 is greater than that elicited by adenosine treatment, which is likely to be due to its extended
half-life. CD4+/CD8+ double-positive thymocytes have been
observed to be most susceptible to apoptosis that is triggered
by A2AAR activation and A2AAR-/- thymocytes are resistant
to the apoptosis-inducing effects of adenosine and
CGS-21680 [79].
3. Conclusions

The activation of the A2AAR by selective agonists modulates

the activity of neutrophils, macrophages and T lymphocytes,
as well as various other inflammatory cells including fibroblasts, monocytes, platelets and mast cells [80-82]. The characteristic responses of activated neutrophils, including the
generation of superoxide anion, upregulation of adhesionmolecule expression and release of elastase are inhibited by
A2AAR signalling. Similarly, the production of pro-inflammatory cytokines by stimulated macrophages and T lymphocytes
is efficaciously inhibited by A2AAR activation. In addition to
inhibiting the activity of individual cell types, the effects of
A2AAR agonist treatment also modulate the interactions
among inflammatory cells. For example, the inhibition of
IL-12 release from macrophages and IFN- release from
T lymphocytes serves to block the propagation of the positive
regulatory loop facilitating the activation of both cell types.
Along with modulating PMN, macrophage and T lymphocyte activity, A2AAR activation has also been suggested to
inhibit the production of IL-6 and -12 and IFN- by plasmacytoid dendritic cells [83]. Additionally, exposure of human
dermal microvascular endothelial cells to TNF- stimulates
an upregulation in A2AAR expression, and the A2AAR agonist
2-(2-[4-chlorophenyl]ethoxy)adenosine (MRE-0094) elicits a
dose-dependent increase in cAMP levels in TNF--treated
cells that is almost completely blocked by ZM-241385 [84].
The anti-inflammatory effects of A2AAR agonists are largely

Expert Opin. Investig. Drugs (2005) 14(7)

Lappas, Sullivan & Linden

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due to the modification of cytokine production by activated

inflammatory cells; this activity is thought, in some but not in
all cases, to be a result of the inhibition of NF-B activity
[85,86]. The signalling by extracellular adenosine through the
A2AAR may, therefore, act as an endogenous regulator of
inflammatory responses, forming a feedback loop wherein the
adenosine released from macrophage- or neutrophil-damaged
tissue serves to limit further inflammatory cell activity. This
endogenous effect can be effectively mimicked by the
administration of A2AAR-selective agonists (Figure 2).
4. Expert

opinion

The activation of the A2AAR regulates the activity of the

inflammatory cells involved in innate and adaptive immune
responses, making the receptor a promising pharmacological
target in the treatment of a variety of inflammatory disorders. Owing to its non-selective activation of the four widely
expressed AR subtypes and the resulting, potentially severe,
side effects, including hypotension and bradycardia, the systemic administration of adenosine has limited clinical
potential for inflammation. However, agents that selectively
increase the concentrations of adenosine in local areas of
injury or inflammation, or agonists that selectively activate
A2AAR, show promise in the treatment of inflammation. It
was demonstrated that the anti-inflammatory effects of the
rheumatoid arthritis drug methotrexate are mediated by the
induction of adenine nucleotide release from injured tissue
and the subsequent activation of A2AARs on local immune

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Expert Opin. Investig. Drugs (2005) 14(7)

Affiliation

Courtney M Lappas1, Gail W Sullivan2

& Joel Linden1,2
Author for correspondence
1Departments of Pharmacology, Box 801394,
University of Virginia, Charlottesville VA 22908,
USA
Tel: +1 434 924 5600; Fax: +1 434 924 2828;
E-mail: jl4v@virginia.edu
2Departments of Internal Medicine, Box 801394,
University of Virginia, Charlottesville VA 22908,
USA