586579

research-article2015
PRF0010.1177/0267659115586579PerfusionBoros et al.

Review

Perfusion
1­–8
Adenosine regulation of the immune © The Author(s) 2015
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DOI: 10.1177/0267659115586579
reperfusion injury prf.sagepub.com

D Boros, J Thompson and DF Larson

Abstract
It is clinically established that adenosine has negative chronotropic, antiarrhythmic effects and reduces arterial blood
pressure. Adenosine addition to cardioplegic solutions used in cardiac operations is clinically well tolerated and has
been shown to improve myocardial protection in several studies. However, the mechanism of action remains unclear.
Therefore, it is important to define the effect of adenosine on the inflammatory cascade as immune cell activation occurs
early during ischemia reperfusion injury. Adenosine appears to mediate the initial steps of the inflammatory cascade via
its four G-coupled protein receptors: A1, A2A, A2B, and A3, expressed on neutrophils, lymphocytes and macrophages.
The adenosine receptor isotype dictates the immune response. More specifically, the A1 and A3 receptors stimulate
a pro-inflammatory immune response whereas the A2A and A2B are immunosuppressive. As the adenosine receptors
are important for cardiac pre-conditioning and post-conditioning, adenosine may regulate the inflammatory responses
initiated during ischemia-mediated immune injury related to myocardial protection.

Keywords
reperfusion; cardioplegia; adenosine; lymphocyte; neutrophil; macrophage

Introduction
Myocardial preservation is associated with periods of
ischemia followed by reperfusion, known as ischemia/
reperfusion injury (I/R). The ischemic phase causes a
pattern of metabolic responses that result in a reduction
of cellular adenosine triphosphate (ATP), a transition
from aerobic to anaerobic energy utilization and an
accumulation and secretion of anaerobic metabolic
products. Reperfusion can produce an array of events,
such as damage to cellular and organelle membranes,
oxidative stress, endothelial damage and vasoconstric-
tion and cellular and non-cellular pro-inflammatory
immune responses. Multiple factors elicit the pro-
inflammatory response. In an early event, the plasma
membrane phospholipase A2 (PLA2) forms arachidonic
acid products that stimulate neutrophil adhesion, caus-
ing neutrophils to release proteases and elastases.1
Cytokines, chemokines and complement are released
from the injured myocardial cells, including myocytes,
endothelial cells and fibroblasts. These secretory factors
attract and activate neutrophils, lymphocytes and mac-
rophages to the site of I/R. Following initiation of the
immune response, endothelial cells express adhesion
molecules, providing selective attachment of immune
cells and allowing access to vulnerable myocytes. Notably,
one of the factors released during I/R is adenosine.2
Adenosine is a nucleoside produced as a metabolic
byproduct of ATP breakdown from precursor adenosine
monophosphate (AMP). Adenosine is up-regulated dur-
ing myocardial ischemia in response to an imbalance
between myocardial oxygen supply and demand.
Following up-regulation, the nucleoside exerts protec-
tive effects by increasing the supply of energy to the
Sarver Heart Center, College of Medicine, The University of Arizona,
Tucson, AZ, USA
Corresponding author:
Douglas F. Larson, Ph.D.
Sarver Heart Center, College of Medicine
The University of Arizona
Tucson, AZ 85724
USA.
Email: dflarson@u.arizona.edu
Presented at the 34th Annual Seminar of The American Academy of
Cardiovascular Perfusion, Los Angeles, California 24-27 January 2013.
2 Perfusion
myocardium and lowering energy demand.3 Due to its
cardioprotective effects, adenosine has been used as an
additive to cardioplegic solutions for over two decades.4
Although there have been several studies supporting
improved myocardial protection with adenosine supple-
mentation during cardiopulmonary bypass, the mecha-
nism of action remains unclear. Since immune activation
is a key event in myocardial I/R, this review will focus on
the effect of adenosine on immune cell types, neutro-
phils, lymphocytes and macrophages, as these immune
cell types are the most clinically relevant in the inflam-
matory response induced by myocardial I/R.
Adenosine: an important signaling
molecule
Under normal physiologic conditions, intracellular ATP
levels are tightly regulated at a concentration of 5-8
mM.5-7 Following I/R injury, many cell types, including
injured myocytes, release ATP into the extracellular
space.8,9 Once released into the extracellular space, ATP
and adenosine diphosphate (ADP) are converted to
AMP via the ecto-apyrase CD39, an enzyme ubiqui-
tously expressed on cell membranes.10,11 Adenosine is
subsequently produced from AMP via the enzyme
5’-ectonucleotidase CD73.12 As an important signaling
molecule, adenosine causes vasodilation, inhibits nor-
epinephrine release from sympathetic nerve endings,
exerts anti-inflammatory effects and has negative ino-
tropic and chronotropic effects.13 In response to injury,
adenosine levels increase rapidly, with up to a 100-fold
increase occurring during systemic inflammation14 as
observed during septic shock.15
Adenosine receptors
The physiological effects of adenosine during I/R are
mediated by four distinct receptors: A1, A2A, A2B and
A3 which are expressed on neutrophils, lymphocytes
and macrophages.6 As shown in Figure 1, all four recep-
tors are members of the seven transmembrane hetero-
trimeric G-protein-coupled receptor superfamily,
defined by their ability to modulate adenylate cyclase
activity.16 The A1 and A3 receptors are Gi receptors and
inhibit adenylate cyclase activity whereas the A2A and
A2B receptors are Gs receptors and stimulate adenylate
cyclase activity.17 Due to the contrasting effects of the Gi
and Gs receptors, the adenosine receptors generate
divergent functional effects on immune cells.
A1 receptors.  The general physiological effect of A1 recep-
tor stimulation on the heart is a negative chronotropic
and dromotropic effect, including heart block, anti-
adrenergic effects, glycolysis stimulation and neutrophil
Figure 1.  Adenosine Receptors. There are 4 adenosine
receptors, A1, A2A, A2B, and A3. The A1 and A3 inhibit
adenylate cyclase activation and the A2A and A2B receptors
stimulate adenylate cyclase.
adherence. The A1 receptor also mediates the protective
effects of cardiac pre-conditioning, which is described as
short periods of ischemia and reperfusion prior to a pro-
longed ischemic insult.18 Related to the immune response,
A1 receptors induce enhanced neutrophil and macro-
phage chemotaxis, adhesion, tumor necrosis factor-alpha
(TNF-α) release and oxygen radical production.19 There-
fore, the A1 receptor has pronounced inhibitory effects on
cardiac tissue and pro-inflammatory effects on lymphoid
tissues.
A2 receptors. The A2 receptors are expressed at the
highest levels in lymphoid tissues and cells followed by
cardiac tissues, thus, the pharmacological effect of ade-
nosine predominately affects these two tissues.20 Most
importantly, both A2 receptors are activated by hypoxia
which is directly related to myocardial preservation.21,22
In general, A2A receptor stimulation of adenylate
cyclase produces vasodilation, renin release and inhibi-
tion of immune cell adherence to endothelial cells.23
A2A agonists also inhibit the immune function of neu-
trophils, macrophages and lymphocytes19,24,25 and most
importantly enhanced secretion of the immunosup-
pressive cytokine, interleukin (IL)-10.26 Like A2A
receptors, A2B receptors stimulate adenylate cyclase
activity and are expressed on neutrophils, lymphocytes
and platelets.27 The A2 receptors have been implicated
as a mechanism for cardiac post-conditioning in which
myocardial injury is reduced by brief episodes of I/R at
the onset of reperfusion following a prolonged period of
ischemia.28 In general, the A2 receptors inhibit the
immune function.
Boros et al. 3
Figure 2.  Temporal Sequence of Immune Response.
Immune cell infiltration following I/R occurs after 1 hour, 24
hours and 72 hours of reperfusion. This figure was adapted
from work by Arslan et al., using the murine model.37
A3 receptors.  Adenosine A3 receptor stimulation coun-
ters the protective effect of adenosine in the heart by
increasing oxidative stress and promoting pathological
cardiac remodeling,29 whereas, related to immune cells,
the A3 receptor has an important inhibitory role in the
regulation of macrophage and monocytes compared
with neutrophils.25,30 The A3 receptors are expressed on
macrophages31 and, similar to the A1 receptors, the A3
receptors inhibit adenylate cyclase activity.23 The mech-
anism of action mediating the anti-inflammatory effect
of an A3 agonist includes down-regulation of the NF-κB
related TNF-α signaling pathway, resulting in inhibi-
tion of pro-inflammatory cytokine production and
apoptosis of inflammatory cells.32 These low affinity A3
receptors are highly expressed in activated inflamma-
tory cells whereas low expression is associated with qui-
escent cells.
General effect of adenosine receptor stimulation. Adenos-
ine can stimulate A1, A2A and A3 receptors with EC50
values ranging between 0.01 μM and 1 μM, while A2B
receptor activation requires a much higher adenosine
concentration, with levels surpassing 10 μM (EC50 of 24
μM).33 Under normal physiological conditions, adenos-
ine concentrations are lower than 1 μM and, therefore,
only activate A1, A2A and A3 receptors, whereas activa-
tion of the A2B receptor requires pathological condi-
tions, such as hypoxia.34 When immune cells acquire
the expression of all four adenosine receptors, they are
recruited in a sequential manner with Gi-coupled A1
receptors activated first at very low adenosine levels,
followed by the stimulation of Gs-coupled A2A and
A2B receptors and, finally, by Gi-coupled A3 receptor.35
Therefore, A1 receptors can inhibit adenylate cyclase
activation by the A2A receptors at low adenosine
Figure 3.  Temporal Sequence of Inflammatory
Cytokines. Inflammatory cytokines are up-regulated following
I/R. This figure was adapted from work by Arslan et al., using
the murine model.37
concentrations. However, at higher concentrations,
the inhibitory effect of A1 receptors is overcome by the
A2A and A2B activity.36 Predicting the effect of the
individual adenosine receptor activation is complicated;
however, based on the counter activity of A2A, A2B and
A3 on the effects of A1, there is a distinct possibility that
the A2A, A2B and A3 receptor effects would dominate
with exogenously administered adenosine for myocar-
dial preservation.
Regulation of the immune
response by adenosine
Myocardial reperfusion following an ischemic event ini-
tiates an immune response and activates the inflamma-
tory cascade. A general temporal sequence of the
immune response following I/R is presented in Figure 2
and was derived from the murine model. Neutrophils,
lymphocytes and macrophages are recruited to the
injured myocardium within the first hour of reperfusion.
Neutrophils reach their peak after 24 hours of reperfu-
sion and, subsequently, decline. Unlike neutrophils, lym-
phocytes remain constant following reperfusion, while
macrophage infiltration into the myocardium increases
at 72 hours post ischemia.37 Cytokines display a similar
up-regulation during I/R, however, peak expression
occurs earlier within the temporal sequence. Figure 3,
also derived from the murine model, demonstrates that
tissue levels of inflammatory cytokines IL-10 and TNF-α
are the highest after 1 hour of reperfusion, with IL-1
reaching maximum levels at 24 hours. As evidenced by
the model, I/R is characterized by a rapid increase in
cytokines followed by an infiltration of immune cells.37
Adenosine expression markedly increases in response to
4 Perfusion
ischemic injury2 and adenosine is a central regulator of
the immune response via its interaction with receptor
subtypes expressed on neutrophils, lymphocytes and
macrophages. Therefore, it is important to describe the
direct effect of adenosine on immune cellular function.
Neutrophils
Neutrophils are the most abundant leukocyte in
human blood14 and have a central role in initiating
inflammatory events in response to injury and stress.
Neutrophils are quickly recruited to sites of injury by
chemokines, complement proteins, cytokines and
adhesion molecules. Neutrophils release vasoactive
and cytotoxic substances, including hydroxyl radicals,
and cause adherence to the endothelium and injury to
myocytes.23
Regulation of neutrophil chemotaxis.  Chemotaxis is defined
by the directional movement of neutrophils in response
to injury and involves cell migration, cell polarization
and gradient sensing. As described above, I/R causes the
release of ATP, which is broken down to form adenos-
ine. Adenosine is both a positive and negative regulator
of neutrophil chemotaxis via selective receptor activa-
tion. The regulatory effects of adenosine on neutrophils
are achieved through both autocrine and paracrine sig-
naling.14,38 Adenosine stimulates neutrophil chemotaxis
via the A1 receptor19 and the A3 receptor.39 In contrast
to receptors A1 and A3, receptor A2B inhibits neutrophil
chemotaxis through the neuronal guidance molecule
netrin-1. In inflammatory models of lipopolysaccharide
(LPS)-induced acute lung injury, hypoxia and acute
experimental colitis, neutrophil chemotaxis was inhib-
ited by the A2B receptor.40-42 Neutrophil chemotaxis
plays a central role in inflammation and can contribute
to many different pathological states.38 Depending
on the receptor that becomes activated, adenosine can
promote or inhibit neutrophil chemotaxis.
Regulation of neutrophil adhesion to endothelial cells. In
addition to regulating neutrophil chemotaxis, adenos-
ine receptor activation has been shown to inhibit neu-
trophil adhesion to endothelial cells and subsequent
injury.43 Endothelial cells express selectins, which facili-
tate neutrophil activation and cause neutrophil integ-
rins to bind adhesion molecules on vascular endothelial
cells. Neutrophil adhesion to the endothelium is then
followed by migration toward the site of injury.14 By
inhibiting L-selectin and the expression of B2 integrins,
adenosine prevents adhesion to the endothelium.36 One
way adenosine inhibits neutrophil adhesion is through
the A2A receptor. A2A receptor activation decreased
human neutrophil CD48d expression and reduced neu-
trophil adhesion to vascular cell adhesion molecule-1
(VCAM-1).44 In a canine model for myocardial infarc-
tion, adenosine A2A receptor activation reduced the
expression of P-selectin, inhibited neutrophil migration
and resulted in a reduction in infarct size, providing
further evidence for the regulation of neutrophil adhe-
sion by the A2A receptor.23 While the adenosine A2A
receptor has been shown to inhibit neutrophil adhesion
to the endothelium, evidence suggests the adenosine A1
receptor promotes neutrophil adhesion. In vitro, the A1
receptor activation promoted neutrophil adhesion to
porcine aortic endothelial cells.26 Taken together, these
data provide support for adenosine as an important sig-
naling molecule for neutrophil adhesion.
Regulation of inflammatory mediators released by neutro-
phils.  Activated neutrophils further influence the inflam-
matory response by releasing oxygen free radicals,23
cytokines and chemokines.14 Neutrophils produce super-
oxides from nicotinamide adenine dinucleotide phos-
phate (NADPH) via NADPH oxidase.46 Superoxides can
serve as a source of damage to areas of inflammation. In
canine coronary arteries, adenosine was shown to reduce
free radical production by activated neutrophils, using an
A2A receptor agonist. The effect was eliminated using
A2A receptor antagonism, indicating that the reduction
in free radicals is mediated by the A2A receptor47 and
A2A receptor modulation could prevent subsequent
injury that may be caused by free radical production.
Adenosine A2B receptor is also involved in preventing
free radical production. During systemic inflammation,
the A2B receptor is up-regulated, with up to a 15-fold
increase. In the murine model, superoxide formation is
inhibited in native neutrophils and neutrophils treated
with TNF-α and LPS in the presence of an A2B receptor
agonist.46 Inflammatory cytokines, including TNF-α, are
also released by activated neutrophils. TNF-α is a pro-
inflammatory cytokine that is involved in regulating
immune cells. In human neutrophils, LPS stimulates
TNF-α expression in vitro. Activation of the A2A ade-
nosine receptor prevents TNF-α release in neutrophils
stimulated with LPS. In vivo, A2A receptor knockout
mice expressed elevated inflammatory cytokines TNF-α,
IL-6 and IL-1β after LPS administration.48 In neutrophils,
adenosine exerts anti-inflammatory effects via the A2A
and A2B receptors by preventing free radical production
and suppressing the release of pro-inflammatory
cytokines.
Lymphocytes
In addition to regulating neutrophil function, adenosine
is a central regulator of lymphocytes. Lymphocytes are
an important component of leukocytes in human blood
and consist of three major types: natural killer cells,
T-cells and B cells. T-cells are responsible for cell-
Boros et al. 5
mediated immunity. T-cells express T-cell receptors
(TCRs) on their surface that recognize and bind to anti-
gens displayed on antigen presenting cells (APCs), such
as dendritic cells and macrophages. Following the acti-
vation of lymphocytes, T-cells differentiate and produce
cytokines and cytotoxic activity. T-cell activation also
leads to lymphocyte adhesion to the endothelium.
Adenosine regulation of lymphocyte function is accom-
plished through the adenosine receptor A2A, which is
the main adenosine receptor found on lymphocytes.49
Adenosine receptors A2B and A3 are also found on lym-
phocytes; however, these receptors are expressed at
lower levels. Upon stimulation, adenosine receptor
expression on lymphocytes increases up to 10-fold.50
Regulation of T-cell pro-inflammatory cytokine produc-
tion.  Activated T-cells differentiate and cause the pro-
duction of pro-inflammatory cytokines, which include
IL-2, interferon-γ (INF-γ) and TNF-α.49 Activation of
the adenosine receptor A2A stimulates adenylyl cyclase
activity, which leads to the production of cyclic AMP
and protein kinase A (PKA) activation.49 The PKA
pathway is a negative regulator of type 1 cytokine pro-
duction.51 In murine helper T-cells, adenosine receptor
A2A activation markedly inhibited the production of
pro-inflammatory cytokines IL-2, TNF-α52 and INF-
γ.53 This effect was also seen with adenosine A2A
receptor agonist treatment in vivo.52 Using the mitogen
concanavalin A (Con A) to induce liver injury and
inflammation in mice, adenosine selective A2A recep-
tor activation not only inhibited TNF-α secretion, but
also attenuated liver damage and inflammation. This
provides support for the A2A receptor-mediated
mechanism for inhibiting pro-inflammatory cytokine
release from T-cells and reducing damage caused by
inflammation. In contrast to the wild-type control
group, treatment with sub-threshold doses of Con A in
adenosine 2A knockout mice resulted in a higher inci-
dence of death within 48 hours and markedly higher
serum levels of TNF-α and IFN-γ.53 In activated T-cells,
adenosine is a negative regulator of pro-inflammatory
cytokines and its actions are mediated by the A2A
receptor.
Regulation of anti-inflammatory cytokine production. In
addition to adenosine’s role in down-regulating pro-
inflammatory cytokines following T-cell activation,
adenosine also stimulates the production of anti-
inflammatory cytokines such as IL-10. The adenosine
receptor A2A stimulates IL-10 secretion,54 with adenos-
ine A2A knockout mice expressing low levels of IL-10.55
In summary, adenosine attenuates the inflammatory
response through the adenosine receptor A2A by down-
regulating the production of pro-inflammatory cytokines
and up-regulating anti-inflammatory cytokines.
Macrophages
Monocytes and macrophages are phagocytes involved in
acute and chronic inflammation. In the presence of injury
or stress, monocytes and macrophages release inflamma-
tory cytokines, free radicals, nitrogen intermediates, pro-
teinases and angiogenic factors. Following activation,
macrophages present antigens to T lymphocytes. There
are two different mechanisms for activating macrophages:
classical activation and alternative activation. Adenosine
is an important regulator in both pathways.56
Classical activation.  Classical activation of macrophages
occurs in the presence of Th1 cytokine environment
and results in TNF-α and IL-12 up-regulation and IL-10
down-regulation.57 Adenosine prevents excess macro-
phage activity through the A2A receptor by reducing
classical activation. The A2A receptor, expressed on
macrophages, down-regulates pro-inflammatory medi-
ators TNF-α,58 IL-1259 and nitric oxide (NO), while
increasing anti-inflammatory cytokine IL-10 expres-
sion.60 This effect is also mediated by the A2B receptor;
however, this receptor is expressed at lower levels on
macrophages.56,61 Adenosine exerts protective effects in
response to injury and stress in tissues by regulating
classical activation of macrophages.
Alternative activation.  While classical activation occurs in
a Th1 cytokine environment, alternative activation of
macrophages occurs in the presence of the Th2 cytokine
environment and, more specifically, cytokines IL-4 and
IL-13.62 Alternative activation of macrophages stimulates
the production of arginase-1 which catalyzes the break-
down of arginine to urea and ornithine. As a precursor to
proline and collagen synthesis, ornithine can stimulate
extracellular matrix deposition and fibrosis.63 Arginase-1
also competes with inducible nitric oxide synthesis for
arginine substrate and, therefore, indirectly decreases
nitric oxide production.56 Alternatively activated macro-
phages are important for tissue remodeling and induce
tissue inhibitor metalloproteinases-1 (TIMP-1). TIMP-1
inhibits matrix metalloproteinase, which degrades extra-
cellular matrix.64 In contrast to adenosine’s negative reg-
ulation of macrophages produced by classical activation,
adenosine promotes alternative activation of macro-
phages. Adenosine increases the expression of arginase-1
and TIMP-1 and this effect is mediated by the A2B recep-
tor.56,64 The positive regulation of alternatively activated
macrophages by adenosine is an important contributor
to tissue remodeling and repair following injury.
Implications for ischemia reperfusion injury
Ischemia/reperfusion injury induces an up-regulation
of inflammatory cytokines, followed by infiltration of
6 Perfusion
immune cells into damaged tissue. Following cardiac
arrest, myocardial reperfusion activates neutrophils,
leading to the generation of reactive oxygen species, the
release of cytokines and the activation of complement.
The inflammatory response initiated by reperfusion is
further compounded by surgical trauma and the reac-
tion to the cardiopulmonary bypass circuit, which also
induce a systemic inflammatory response.65 Inadequate
myocardial preservation to protect against I/R injury
can lead to low cardiac output, the requirement for post-
operative inotropic support, increased time in the ICU
and other serious complications;66 therefore, strategies
to improve myocardial protection are essential for
improving clinical outcomes. Adenosine, an important
signaling molecule, has been used as an additive to car-
dioplegic solutions for over two decades. Recently,
Jakobsen et al. demonstrated that adenosine cardiople-
gia is associated with reduced postoperative atrial fibril-
lation when compared to hyperkalemic solutions. The
study was a randomized clinical trial in patients under-
going coronary bypass grafting procedures and com-
pared 1.2 mmol/L of adenosine in normokalemic
crystalloid cardioplegia to a hyperkalemic solution con-
taining 20 mmol/L of potassium.67 Prior to the work by
Jakobsen et al., Onorati et al. found that microplegia
containing adenosine and lidocaine reduced myocardial
damage and improved postoperative outcomes when
compared to standard 4:1 Buckberg cardioplegia.68
Despite the recent studies supporting the clinical bene-
fits of adenosine supplementation to cardioplegia, the
mechanism of action whereby adenosine attenuates
myocardial damage remains unclear. The effects of ade-
nosine are mediated by four G-coupled protein recep-
tors: A1, A2A, A2B, and A3 found on various cell types,
including neutrophils, lymphocytes and macrophages.23
Adenosine has an extremely short half-life, with a half-
life of approximately 1.5 seconds in human plasma.69
The rapid removal of adenosine supports a mechanism
occurring early within the I/R temporal sequence. As an
early event, inflammatory cytokines are up-regulated at
the site of tissue damage. Adenosine activation of recep-
tors A2A and A2B inhibits the release of pro-inflamma-
tory cytokines, including TNF-α,48,52 IL-2,52 INF-γ,53
IL-6,48 IL-1β48 and IL-1α.70 Furthermore, adenosine
stimulates the expression of anti-inflammatory cytokine
IL-10 via the A2A receptor.54,55,60 As inflammatory cyto-
kines mediate the recruitment and activation of immune
cells to the site of I/R, adenosine attenuates the overall
myocardial damage by down-regulating inflammatory
cytokines and up-regulating pro-inflammatory cyto-
kines, thereby, decreasing immune cell infiltration. In
order to achieve the anti-inflammatory effects of exog-
enously administered adenosine, the concentration
delivered must be sufficient to activate the adenosine
receptors. The concentration of adenosine used in the
recent study by Jakobsen et al. surpasses the described
tissue threshold concentration, activating all four ade-
nosine receptors.67 Since exogenously administered
adenosine activates receptors on key immune cell types,
the positive clinical outcomes may also be attributed, in
part, to the ability of adenosine to minimize the sys-
temic inflammation response associated with surgical
stress.
Conclusion
Based on the above understanding of adenosine, the
acute inflammatory processes triggered by I/R injury
should be diminished with adenosine-supplemented
cardioplegic solutions.
Declaration of Conflicting Interest
The authors declare that there is no conflict of interest.
Funding
Steinbronn Heart Failure Research Award to DFL.
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