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1. Introduction
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3. Conclusions
4. Expert opinion
Departments
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
1.2 Adenosine
receptors
797
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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
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
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
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
799
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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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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
801
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
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opinion
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