Myocardial Infarct–Sparing Effect of Adenosine A2A Receptor
Activation Is due to Its Action on CD4ⴙ T Lymphocytes
Zequan Yang, MD, PhD; Yuan-Ji Day, MD, PhD; Marie-Claire Toufektsian, PhD; Yaqin Xu, MD, PhD;
Susan I. Ramos, BS; Melissa A. Marshall, BS; Brent A. French, PhD; Joel Linden, PhD
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Background—We previously used adenosine A2A receptor (A2AR) knockout (KO) mice and bone marrow transplantation
to show that the infarct-sparing effect of A2AR activation at reperfusion is primarily due to effects on bone
marrow– derived cells. In this study we show that CD4⫹ but not CD8⫹ T lymphocytes contribute to myocardial
ischemia/reperfusion injury.
Method and Results—After a 45-minute occlusion of the left anterior descending coronary artery and reperfusion, T cells
accumulate in the infarct zone within 2 minutes. Addition of 10 ␮g/kg of the A2AR agonist ATL146e 5 minutes before
reperfusion produces a significant reduction in T-cell accumulation and a significant reduction in infarct size (percentage
of risk area) measured at 24 hours. In Rag1 KO mice lacking mature lymphocytes, infarct size is significantly smaller
than in C57BL/6 mice. Infarct size in Rag1 KO mice is increased to the level of B6 mice by adoptive transfer of 50
million CD4⫹ T lymphocytes derived from C57BL/6 or A2AR KO but not interferon-␥ KO mice. ATL146e completely
blocked the increase in infarct size in Rag1 KO mice reconstituted with B6 but not A2AR KO CD4⫹ T cells. The number
of neutrophils in the reperfused heart at 24 hours after infarction correlated well with the number of lymphocytes and
infarct size.
Conclusions—These results strongly suggest that the infarct-sparing effect of A2AR activation is primarily due to
inhibition of CD4⫹ T-cell accumulation and activation in the reperfused heart. (Circulation. 2006;114:&NA;-.)
Key Words: adenosine 䡲 inflammation 䡲 myocardial infarction 䡲 receptors 䡲 reperfusion 䡲 T lymphocyte
T
lymphocytes, or T cells, have been found to contribute
to liver, kidney, and gut ischemia/reperfusion (I/R)
injury.1–3 There is growing evidence that T cells may participate in the pathogenesis of I/R injury to the heart.4 –7
Lymphocyte-related cytokines are upregulated in the postischemic heart,4,6 and leukocyte adhesion molecules and chemokines, such as P-selectin glycoprotein ligand-1, CD11/
CD18, intercellular adhesion molecule-1 (ICAM-1), and
monocyte chemoattractant protein-1 (MCP-1), mediate experimental myocardial I/R injury4,8,9 and lymphocyte adhesion, migration, and signaling.10,11 T cells are known to
amplify inflammatory responses through the secretion of
cytokines including interferon-␥ (IFN-␥), interleukin (IL)-2,
IL-4, and granulocyte/macrophage colony-stimulating factor
(GM-CSF). These cytokines stimulate the chemotaxis of
neutrophils and monocytes to site(s) of injury.
studies show that immunodeficient mice have significantly
smaller infarcts than wild-type control mice,13 and inhibition
of T cells by cyclosporine A or tacrolimus can protect the
heart against I/R injury and significantly suppress neutrophil
infiltration.6,7 Thus, the activation and sequestration of lymphocytes may contribute to myocardial I/R injury.
Activation of the adenosine A2A receptor (A2AR) reduces
reperfusion injury in liver, kidney, skin, spinal cord, and
heart.14 Heart studies using in vivo animal models have
shown that A2AR activation by CGS-21680 or ATL146e
during reperfusion significantly inhibits neutrophil recruitment in addition to reducing MI.13,15,16 Our previous study
demonstrated that the infarct-sparing effect of A2AR activation in mice during myocardial reperfusion is primarily due to
its action on bone marrow– derived cells, possibly
lymphocytes.13
Mature T cells are divided into 3 major types: CD4⫹
T-helper (Th), CD8 ⫹ T-cytotoxic (Tc), and CD4 ⫹
T-regulatory (Treg) cells.17 In the present study we define the
kinetics of changes in circulating and cardiac-resident T
lymphocytes during early reperfusion after myocardial ische-
Clinical Perspective p
Clinically, the presence of a relative lymphocytopenia is an
early marker of myocardial infarction (MI).12 Tissue accumulation of these cells may be detrimental because intact animal
Received July 1, 2006; revision received August 14, 2006; accepted September 1, 2006.
From the Departments of Biomedical Engineering (Z.Y., M.-C.T., Y.X., B.A.F.), Medicine and Pharmacology (S.I.R., M.A.M., B.A.F., J.L.), and
Surgery (Z.Y.), University of Virginia Health System, Charlottesville; and Department of Anesthesiology, Chang Gung University Hospital, Taipei,
Taiwan (Y.-J.D.).
Correspondence to Dr Zequan Yang, Departments of Biomedical Engineering and Surgery, University of Virginia Health System, Box 800759,
Charlottesville, VA 22903. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.106.649244
1
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Circulation
November 7, 2006
Animal Groups and Protocols
Group/Protocols
End Points
n
Mortality⫹Exclusion
Wild-type C57BL/6
Sham; thoracotomy
CBC and IHC
5
Control; I/R: 45 min/60 min
CBC and infarct size
14
⫹ATL; I/R: 45 min/60 min
CBC and infarct size
15
Control; I/R: 45 min/⬇60 min
IHC
16
⫹ATL; I/R: 45 min/15 min
IHC
4
Control; I/R: 40 min/60 min
Infarct size
10
⫹CD4 mAb; I/R: 40 min/60 min
Infarct size
8
⫹CD8a mAb; I/R: 40 min/60 min
Infarct size
9
Control; I/R: 45 min/24 h
Infarct size and IHC
9
⫹ATL; I/R: 45 min/24 h
Infarct size and IHC
12
1⫹2
Rag1 KO; I/R: 45 min/24 h
Infarct size and IHC
11
2⫹1
⫹B6 CD4; I/R: 45 min/24 h
Infarct size and IHC
9
⫹B6 CD4/ATL; I/R: 45 min/24 h
Infarct size and IHC
9
⫹A2AKO CD4; I/R: 45 min/24 h
Infarct size and IHC
6
Rag1 KO
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⫹A2AKO CD4/ATL; I/R: 45 min/24 h
Infarct size and IHC
7
⫹IFN-␥ KO CD4; I/R: 45 min/24 h
Infarct size and IHC
8
Total (16 groups)
1 exclusion
152
3⫹4
ATL indicates ATL146e; CBC, complete blood count; and IHC, immunohistochemistry. ATL146e was
administered at a dose of 10 ␮g/kg IP.
mia, and we explore the effects of T-lymphocyte subtypes
(CD4 and CD8) in mediating myocardial inflammatory responses. The results indicate that A2AR-mediated protection
of the heart is accompanied by a decrease in the rapid
accumulation and activation of CD4⫹ T cells during reperfusion after myocardial ischemia.
cardioprotective effects of this compound are entirely due to A2AR
activation because protection is absent in mice treated with the
A2AR-selective antagonist ZM241385 or lacking the A2AR gene.13
Methods
Mononuclear spleen cells were generated from murine spleens
according to an established protocol.1 Spleen cells at a concentration
of 5⫻107 cells per milliliter were then used for CD4⫹ T-lymphocyte
isolation with a SpinSep murine CD4⫹ kit (Stem Cell Technologies,
Vancouver, Wash) according to the manufacturer’s protocol. Greater
than 94% of the enriched cells expressed CD4 as assessed by flow
cytometry.
The authors had full access to the data and take full responsibility for
its integrity. All authors have read and agree to the manuscript as
written.
This study conformed to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health
(NIH publication No. 85-23, revised 1985) and was conducted under
protocols approved by the University of Virginia Animal Care and
Use Committee.
Animals
This study used a total of 239 (8- to 14-week-old) male mice of
different strains, including C57BL/6 (B6) mice, A2AR knockout
(KO) mice, Rag1 KO (B6.129S7-Rag1tm1Mom) mice, and IFN-␥ KO
(B6.129S7-Ifngrtm1Agt) mice. Among these, 78 mice were used as
CD4⫹ T lymphocyte donors to Rag1 KO mice at a ratio of 2 to 1 to
create chimeric mice. Nine B6 mice were used to evaluate CD4⫹ and
CD8⫹ T-lymphocyte populations after treatment with antibodies by
flow cytometry. The remaining mice (B6, Rag1 KO, and chimeras,
totaling 152) were assigned to 16 groups and underwent either
myocardial I/R injury (15 groups) or sham operation (1 group)
(Table). B6, Rag1 KO, and IFN-␥ KO mice were purchased from
Jackson Laboratory (Bar Harbor, Me). A2AR KO mice, derived
from breeders originally supplied by Dr Jiang-Fan Chen (Harvard
Medical School, Boston, Mass), were bred to be congenic with B6
mice with the use of microsatellite-assisted breeding congenics.
Some groups of mice were treated with the A2AR agonist ATL146e
(Adenosine Therapeutics, LLC, Charlottesville, Va), injected intraperitoneally at a dose of 10 ␮g/kg 5 minutes before reperfusion. The
Chimeric Mice Created by Adoptive Transfer
of Lymphocytes
Isolation of CD4ⴙ T Lymphocytes by Negative Selection
Adoptive Transfer of Lymphocyte Subpopulations
Rag1 KO mice were anesthetized with pentobarbital (50 mg/kg). The
left external jugular vein was exposed with a small skin incision. T
cells (5⫻107; in a volume of ⬇200 ␮L) were injected. Flow
cytometry confirmed effective reconstitution of T lymphocytes in the
peripheral blood and spleens of Rag1 KO mice. Five days after the
reconstitution, chimeras underwent myocardial I/R injury.
In Vivo Depletion of CD4ⴙ or CD8ⴙ T
Lymphocytes in B6 Mice
Two groups of B6 mice were depleted of either CD4⫹ or CD8⫹ T
lymphocytes with the use of selective antibodies as reported by
others.1,18 Anti–CD4 monoclonal antibody (GK1.5) or anti–CD8a
monoclonal antibody (53 to 6.7) (eBioscience, San Diego, Calif) was
injected intraperitoneally on 2 consecutive days at a dose of 0.2 mg/d
per mouse. Two days after the second injection, 3 animals from each
group were used to test for successful depletion by flow cytometry.
The rest underwent myocardial I/R injury.
Flow Cytometry Analysis
Whole-blood samples were collected under anesthesia by puncturing
the right ventricle. Splenocytes were isolated and purified as de-
Yang et al
scribed above. One million splenocytes (in 100 ␮L) or 100 ␮L of
whole blood was treated with 1 ␮g of anti-CD8 fluorescein isothiocyanate–labeled or anti-CD4 phycoerythrin-labeled antibodies in 50
␮L phosphate-buffered saline/10% fetal calf serum for 30 minutes on
ice. Flow cytometry data acquisition and analysis were performed on
a FACScan with the use of CellQuest software (Becton Dickinson,
San Jose, Calif).
Myocardial I/R
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Mice were subjected to 40 or 45 minutes of coronary occlusion
followed by up to 24 hours of reperfusion and then euthanized to
count peripheral white blood cells and to evaluate MI size and/or
leukocyte infiltration (Table). A standard protocol was used, as
detailed previously.13
In the CD4- and CD8-depletion studies, the I/R protocol was
modified from 45 minutes/24 hours to 40 minutes/60 minutes.
Reperfusion time was reduced from 24 hours to 1 hour to focus the
study on early inflammatory events and to increase throughput by
reducing time to follow-up. This modification was supported by
recent longitudinal cardiac magnetic resonance imaging studies
undertaken in the murine model by our laboratory showing no
significant increase in infarct size between 1 and 24 hours after
reperfusion (data not shown).
Adenosine A2A Receptor on CD4ⴙ T Cells and MI
3
omy, at 40 minutes after LAD occlusion and 60 minutes after
reperfusion from mice with MI, and at corresponding time
points from sham mice. A total of 150 ␮L of whole blood
were withdrawn from each mouse. No significant changes
were found in hemoglobin levels or red blood cell counts
throughout the procedures or among the different groups.
Normally, the total white blood cell (WBC) count was
4470⫾450/␮L. Lymphocytes were predominant and accounted for 76.5⫾3.2% of murine WBC, whereas neutrophils
accounted for 20.1⫾3.2% and monocytes for 2.9⫾0.3%. No
significant changes in red blood cells or platelets were found
throughout the sham procedures (hemoglobin, 11.7 to 13.6
g/dL; platelets, 660⫾1⫻103/␮L). There was a trend toward
leukocytosis at 40 minutes after LAD occlusion and a trend
toward leukocytopenia 60 minutes after reperfusion in the MI
group, but the changes did not achieve statistical significance
Immunohistochemistry of Neutrophils and
CD3ⴙ T Cells
A standard protocol was used, as detailed previously.13 CD3 was
used here as a general marker of T lymphocytes, that is, nearly all
CD4⫹ and CD8⫹ T cells also express CD3.
Peripheral Blood Cell Count
Perioperatively, blood (30 to 40 ␮L) was obtained by puncturing the
left external jugular vein at each time point. Cell counts were
performed with a HemaVet Hematology System (CDC Technologies, Oxford, Conn).
Measurement of Plasma IFN-␥ by Enzyme-Linked
Immunosorbent Assay
Plasma levels of IFN-␥ were measured according to the manufacturer’s protocol (BD Bioscience, San Diego, Calif).
Statistical Analysis
All data are presented as mean⫾SEM. Infarct sizes and risk region
sizes were compared by 1-way ANOVA followed by the Student t
test for unpaired data with Bonferroni correction among Rag1 KO
groups or among B6 groups. Square roots of tissue cell counts were
compared by 1-way ANOVA. Serial changes of peripheral leukocytes were analyzed by repeated-measures ANOVA with Bonferroni
correction.
Results
Exclusion and Mortality
Of the 152 mice that underwent myocardial I/R injury, 3 mice
died early after left anterior descending coronary artery
(LAD) occlusion or reperfusion. Four mice were excluded
because of either inordinately small risk regions (⬍25% of
left ventricular [LV] mass, 2 mice) or technical failure of
2-3-5-triphenyl tetrazolium chloride (TTC) or phthalo blue
staining (2 mice) (Table).
Changes in Peripheral Blood Cells and Platelets
During Early Reperfusion
Fifteen B6 mice were divided into 3 groups (5 each) and
underwent either sham thoracotomy or 45-minute LAD occlusion followed by 60-minute reperfusion (MI⫾ATL146e
groups). Whole-blood samples were taken before thoracot-
Figure 1. Effects of MI on circulating leukocytes. A, The total
number of WBC was not significantly changed after MI. B, The
number of circulating neutrophils (NE) was significantly elevated
during ischemia and reperfusion. C, Circulating lymphocytes
(LY) were significantly reduced during reperfusion, and this
reduction was blocked by ATL146e (ATL). *P⬍0.05 vs corresponding baseline; †P⬍0.05 vs sham or ATL146e at same time
point.
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Circulation
November 7, 2006
(Figure 1A). Circulating neutrophils were significantly increased both during ischemia and during reperfusion in the
MI groups (Figure 1B). Significant lymphocytopenia was
observed on reperfusion in the MI group. ATL146e, administered 5 minutes before reperfusion, significantly attenuated
the reperfusion-induced lymphocytopenia (Figure 1C). These
findings suggest that neutrophils are recruited, probably from
bone marrow, within 40 minutes of reperfusion after MI. This
recruitment is not affected by ATL146e. Lymphocytes in
peripheral blood are decreased at 60 minutes after reperfusion, and this effect is blocked by ATL146e (Figure 1C).
Kinetics of Leukocyte Extravasation Into
Myocardium During Early Reperfusion
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Sixteen B6 mice underwent 45 minutes of LAD occlusion
and 60 minutes of reperfusion. Four hearts were removed
respectively at 2, 15, 30, and 60 minutes after the start of
reperfusion. An additional 4 hearts were harvested from
sham-operated mice. In sham animals, few (0 to 1) neutrophils or T lymphocytes were observed per high-power field.
In mice with myocardial I/R injury, there was a rapid increase
in neutrophil infiltration into the ischemic region that was
statistically significant within 2 minutes after reperfusion
compared with sham animals (shown at 0 minutes; P⬍0.05;
Figure 2). Neutrophil accumulation progressed between 2 and
30 minutes after reperfusion. The number of CD3⫹ T lymphocytes in the previously ischemic myocardium was also
significantly increased within 2 minutes after the initiation of
reperfusion, but no further increase was noted during the first
hour of reperfusion. In ATL146e-treated mice, the infiltration
of both neutrophils and CD3⫹ T lymphocytes was significantly reduced compared with control at 15 minutes after
reperfusion (Figure 3A).
MI size was also evaluated during early reperfusion. As
shown in Figure 3B, there was no significant difference in
risk region (percentage of LV mass) between vehicle-treated
and ATL146e-treated groups; however, infarct size (percentage of risk region) was significantly reduced in ATL146etreated mice after 45 minutes of LAD occlusion and 60
minutes of reperfusion.
Plasma IFN-␥ at 60 minutes after reperfusion was significantly reduced in ATL146e-treated mice compared with
vehicle controls (58⫾13 versus 166⫾26 pg/mL; P⬍0.05),
but this was still higher than that in sham controls (15⫾5pg/
mL; P⬍0.05 versus ATL146e group).
MI in CD4- or CD8-Depleted Mice
Flow cytometry confirmed that distinct monoclonal antibodies targeting CD4 or CD8 selectively depleted peripheral
blood of CD4⫹ or CD8⫹ T lymphocytes in B6 mice (Figure 4,
top 2 panels) and significantly reduced lymphocytes in spleen
(Figure 4, bottom 2 panels). We examined infarct size in
control mice and mice depleted of CD4⫹ or CD8⫹ T cells.
After 40 minutes of LAD occlusion and 60 minutes of
reperfusion, the risk region (percentage of LV mass) ranged
from 32% to 45% and was comparable between the 3 groups
(P⫽NS). In control mice, which have a normal complement
of CD4⫹ and CD8⫹ T lymphocytes, infarct size was 50.7⫾2.3
(percentage of risk region). In mice without CD8⫹ T lymphocytes, infarct size was 52.4⫾2.4 and did not differ from that
Figure 2. Time course of accumulation of leukocytes into the heart after reperfusion evaluated by
immunostaining of paraffin sections. Top, The
number of neutrophils and CD3⫹ T lymphocytes
per high-power field is plotted against time. Time 0
sections were obtained from sham-operated mice.
Significant accumulation of T lymphocytes was
detected 2 minutes after reperfusion, but no further
increment was noted during 60 minutes of reperfusion. Significant accumulation of neutrophils was
observed at 2 minutes of reperfusion and rapidly
increased over time. Bottom, Representative
immunohistochemical images of neutrophils and
CD3⫹ T lymphocytes in the previously ischemic
myocardium from each time point during reperfusion. *†P⬍0.05 vs corresponding sham at time 0.
Yang et al
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Figure 3. Effect of ATL146e on myocardial accumulation of leukocytes. A, ATL146e (ATL) significantly reduced the infiltration of
both neutrophils and CD3⫹ T lymphocytes into the previously
ischemic myocardium 15 minutes after reperfusion. B, MI size
(IF) was found to be significantly smaller in ATL146e-treated
mice than in control mice. RR indicates risk region. *P⬍0.05 vs
control group.
in control mice (P⫽NS). However, in mice without CD4⫹ T
lymphocytes, infarct size was significantly reduced to
35.3⫾3.7, a ⬎30% reduction compared with control or
CD8-depleted mice (P⬍0.05 versus either group; Figure 5).
Role of A2ARs on CD4ⴙ T Lymphocytes in
Mediating MI
We next sought to learn more about the mechanisms of T
cell–mediated myocardial injury by using adoptive transfer of
various T-cell populations into Rag1 KO mice (Table). In B6
control mice, 45 minutes of LAD occlusion created a MI size
of 61.0⫾2.3 (percentage of risk region) evaluated at 24 hours
after reperfusion. ATL146e treatment reduced the infarct size
to 39.3⫾4.7 (a 36% of reduction from B6 control; P⬍0.05).
In Rag1 KO control mice, the infarct size (40.5⫾3.3) was
comparable to that of ATL146e-treated B6 mice but was
significantly smaller than that of B6 control mice (P⬍0.05).
After reconstitution with CD4⫹ T lymphocytes, infarct size
was significantly increased in Rag1 KO mice (to
52.1⫾1.6%), returning it nearly back to the level of B6
control mice. The increase in infarct size in Rag1 KO mice
Adenosine A2A Receptor on CD4ⴙ T Cells and MI
5
reconstituted with CD4⫹ T cells was completely blocked by
treating the mice with ATL146e 5 minutes before reperfusion. However, ATL146e failed to block the increase in
infarct size induced by adoptive transfer of CD4⫹ T lymphocytes from A2AR KO mice. Interestingly, there was no
significant increase in infarct size in Rag1 KO mice reconstituted with CD4⫹ T lymphocytes from IFN-␥ KO mice,
indicating that this cytokine plays an important role in
reperfusion injury (Figure 6).
Infiltration of T lymphocytes and neutrophils into the
myocardium was evaluated in mice 24 hours after MI. CD3⫹
T lymphocytes were not detectable in hearts from Rag1 KO
mice but accumulated in the previously ischemic myocardium after adoptive transfer of B6 CD4⫹ cells. This response
was blocked by the bolus administration of ATL146e just
before reperfusion. Adoptive transfer of CD4⫹ T lymphocytes
from A2AR KO mice also resulted in infiltration of T
lymphocytes during reperfusion, but ATL146e failed to block
the myocardial accumulation of these receptor-deficient T
cells. Only a few T lymphocytes were found in the myocardium of Rag1 KO mice reconstituted with CD4⫹ T lymphocytes from IFN-␥ KO mice (Figure 7, top panel).
The accumulation of neutrophils into the infarcted myocardium 24 hours after MI showed the same pattern of
response as the accumulation of T cells. Compared with B6
mice, neutrophils were significantly reduced in the previously
ischemic region in Rag1 KO mice, Rag1 KO mice reconstituted with IFN-␥ CD4⫹ T lymphocytes, and ATL146e-treated
Rag1 KO mice reconstituted with B6 CD4⫹ T lymphocytes.
ATL146e failed to reduce neutrophil accumulation in the
infarcted hearts of Rag1 KO mice reconstituted with A2AR
KO CD4⫹ T lymphocytes (Figure 7, bottom panel).
Discussion
Studies using in vivo animal models have shown that A2AR
activation by CGS-21680 or ATL146e during reperfusion
significantly inhibits neutrophil recruitment and reduces
MI.13,15,16 Bone marrow– derived cells are the primary targets
of A2AR agonists, but the identity of the specific cell type(s)
bearing the A2ARs that are responsible for mediating the
beneficial effects of A2AR activation to reduce MI size has
not been established. The present study shows that CD4⫹ but
not CD8⫹ T cells are the primary targets of A2AR agonists
and that the rapid (15 minute) accumulation of CD4⫹ T cells
in the heart after MI is inhibited by ATL146e. Although the
number of T cells entering the heart within the first 30
minutes of reperfusion after ischemia is small, these cells may
participate in an inflammation cascade involving sequential
activation of T cells, possibly tissue-resident macrophages,
and neutrophils. This cascade appears to require INF-␥
release from CD4⫹ cells because cells lacking this cytokine
are protected from MI. It is noteworthy that deletion of IFN-␥
from CD4⫹ T cells not only reduces infarct size (Figure 6) but
also reduces both T-cell and neutrophil accumulation in the
heart 24 hours after infarction (Figure 7). This suggests that
IFN-␥ release from CD4⫹ T cells in the heart participates in
attracting additional T cells and neutrophils to the heart at
later times. Neutrophil trafficking into the heart is also
inhibited by ATL146e, but it appears that the effects of
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Circulation
November 7, 2006
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Figure 4. Flow cytometry analysis of peripheral blood samples and splenocytes
from B6 mice after treatment with either
anti-CD4 or anti-CD8 monoclonal antibody. Antibody to CD3 was conjugated
with phycoerythrin (PE) to label all T lymphocytes. Antibodies to CD4 or CD8
were conjugated with fluorescein isothiocyanate (FITC). A and B show results
obtained from blood samples; C and D
show results obtained from splenocytes.
The different antibodies used to deplete
cells are indicated in separate columns.
Anti-CD4 treatment completely depletes
blood of CD4⫹ T lymphocytes and leaves
CD8⫹ T lymphocytes intact (middle column). Conversely, anti-CD8 treatment
completely depletes blood of CD8⫹ T
lymphocytes and leaves CD4⫹ T lymphocytes intact (rightmost column).
ATL146e on neutrophil trafficking are largely secondary to
effects on T lymphocytes because the A2AR agonist is not
protective in mice when the A2ARs are selectively deleted
from CD4⫹ cells.
The observation that Rag1 KO mice are protected from MI
does not in and of itself implicate T cells as regulators of
reperfusion injury because these animals also lack B cells and
have abnormal innate immune responses. The observation
that adoptive transfer of CD4⫹ cells reconstitutes severe
reperfusion injury provides clear evidence that CD4⫹ T cells
can trigger increased injury and inflammation, but this still
does not prove that these cells play such a role in wild-type
mice. Thus, it was important to confirm the role of CD4⫹ T
cells in reperfusion injury with the use of antibody depletion
as an independent means of modulating the number of CD4⫹
or CD8⫹ cells in wild-type mice. Taken together, the CD4/
CD8-depletion experiments in B6 mice and the adoptive
transfer experiments in Rag1 KO animals provide strong
evidence that CD4⫹ T cells play a central role in reperfusion
injury to the heart. The results also implicate CD4⫹ T cells as
the principal targets of protection by the A2AR agonist
ATL146e. This is based on observations that the rapid
accumulation of T cells into the reperfused heart is strongly
inhibited by ATL146e and that this T-cell accumulation is not
inhibited if T cells lack the A2AR.
We have shown previously that activation of the A2AR on
CD4⫹ T cells can reduce T-cell receptor–mediated INF-␥
production by 98%.19 IFN-␥ is produced by activated T cells
and activates antigen presenting cells such as macrophages
and dendritic cells. In the present study, additional evidence
supporting a role of T cell– derived INF-␥ in myocardial
reperfusion injury includes the following: IFN-␥ accumulates
in plasma of animals after MI; accumulation of IFN-␥ is
inhibited by ATL146e; T cells from IFN-␥ KO mice accumulate in infarcted myocardium to a lesser extent than T cells
from B6 mice; and transfer of T cells from IFN-␥ KO mice to
Rag1 KO mice fails to increase injury. Considered together,
the data strongly suggest that A2AR agonists reduce myocardial reperfusion injury largely by their effects on CD4⫹ T
cells.
CD4ⴙ T Lymphocytes Mediate Inflammatory
Responses During Reperfusion
Immune system–mediated myocardial damage has been implicated in a broad range of cardiac diseases including cardiac
transplant rejection and ischemic heart disease.20,21 An in-
Yang et al
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Figure 5. Effect of lymphocyte depletion on MI size. A, TTCand phthalo blue–stained, short-axis tissue sections of left ventricles from representative mice from various groups. Blue areas
are nonischemic tissue, yellowish-white areas are infarcted tissue, and red areas represent salvaged (viable) tissue within the
previously ischemic myocardium or risk region. B, Quantification
of risk region (RR) and infarct size (IF) in 3 groups of mice.
There was no difference in risk region among the 3 groups.
Infarct size was significantly smaller in CD4-depleted mice than
in control or CD8-depleted mice. mAb indicates monoclonal
antibody.
creasing body of clinical evidence has shown that MI is
associated with T-cell responses as identified by increased
serum levels of RANTES (regulated on activation normal T
cell expressed and secreted), soluble IL-2 receptor, IFN-␥,
and chemokines.22,23 Interestingly, in patients with acute MI,
circulating T lymphocytes, especially CD4⫹ T cells, are
significantly decreased within 24 hours, whereas there are no
significant changes in the B-cell count or in serum IgG, IgA,
or IgM levels.24 In fact, the presence of a relative lymphocytopenia is an early marker of MI.12 However, the reason for
this lymphocytopenia early after MI is poorly understood, and
the effects of lymphocytes on the myocardial inflammatory
response early after I/R injury are largely unknown. Myocardial ischemia was found to lead to local lymphocytosis
consisting predominantly of Th cells, with modest increases
in natural killer and T suppressor/cytotoxic subsets.5
Inhibition of T cells by cyclosporine A and tacrolimus has
been shown to protect the heart against I/R injury and to
significantly suppress infiltration of the neutrophils6,7 that are
often considered to be end-effectors of reperfusion injury.
These data are consistent with the present results and suggest
that the T-cell receptor participates in T-cell activation during
reperfusion injury. Thus, our previous results13 and other
Adenosine A2A Receptor on CD4ⴙ T Cells and MI
7
Figure 6. Effect of T-cell genotype and ATL146e on MI size in
Rag1 KO mice. Infarct size was measured by TTC staining after
45 minutes of ischemia and 24 hours of reperfusion in B6 mice
or Rag1 KO mice⫾CD4⫹ T-lymphocyte reconstitution. There
was no significant difference in risk region among these groups.
Infarct size is reduced in Rag1 KO or ATL146e-treated B6 mice
relative to B6 control mice. Adoptive transfer of B6 or A2AR KO
CD4⫹ T lymphocytes into Rag1 KO mice restores infarct size
back toward the B6 control level. The administration of ATL146e
reduces infarct size after adoptive transfer of B6 CD4⫹ T lymphocytes but not A2AR KO CD4⫹ T lymphocytes. Adoptive
transfer of IFN-␥ KO CD4⫹ T lymphocytes did not significantly
increase the infarct size in Rag1KO mice. *P⬍0.05 vs B6 control; #P⬍0.05 vs other chimeras except IFN-␥ KO chimera.
indirect evidence from the literature provide evidence of
activation and trafficking of T cells to the heart after
myocardial I/R injury. The present study confirms that T cells
play an important role in regulating the early inflammatory
responses to myocardial I/R injury.
In the present study we found that after 45 minutes of LAD
occlusion and 60 minutes of reperfusion there is no significant change in total WBC number, but neutrophils increase
from an initial fraction of 20% to 49% of total WBC, whereas
lymphocytes are reduced from 77% to 36% (Figure 1).
During this first hour after reperfusion, lymphocytes and
neutrophils migrate to the previously ischemic myocardium
(Figure 2) and other peripheral organs such as the lung (data
not shown). This pattern of inflammatory response indicates
that the mobilization of neutrophils from the bone marrow
into the blood more than compensates for the loss of blood
neutrophils sequestrated into tissues. In contrast, more lymphocytes are sequestrated by tissues than are released into the
blood stream.
A2ARs have a nonredundant role in the attenuation of
inflammation and tissue damage in vivo and are a critical part
of a physiological negative feedback mechanism for the
limitation and termination of both tissue-specific and systemic inflammatory responses.25 Stimulation of A2ARs mediates immunosuppression by inhibiting the activation of T
lymphocytes and neutrophils, predominantly by a cAMPdependent pathway.25,26 Biochemical studies have shown that
murine T lymphocytes express the A2AR that is induced on
T-cell activation.19,26 Studies of T helper cell subsets (Th1
and Th2) reveal that lymphokine-producing cells are much
8
Circulation
November 7, 2006
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Figure 7. Representative examples of the effect of T-cell genotype and ATL146e on leukocyte trafficking into the infarcted hearts of
Rag1 KO mice 24 hours after reperfusion. The infiltration of CD3⫹ T lymphocytes (top) and neutrophils (bottom) was evaluated by
immunohistochemistry in Rag1 KO hearts after 45 minutes of LAD occlusion and 24 hours of reperfusion. Neutrophils and CD3⫹ T cells
were found predominantly in previously ischemic areas. From left to right, Rag1 KO hearts had no CD3⫹ T cells and low levels of neutrophils. Reconstitution of B6 or A2AR KO CD4⫹ T lymphocytes into Rag1 KO mice resulted in accumulation of CD3⫹ T lymphocytes in
the heart and enhanced neutrophil infiltration. ATL146e significantly reduced CD3⫹ T cell and neutrophil infiltration in mice receiving B6
CD4⫹ T lymphocytes from B6 mice but not from A2AR KO mice. Rag1 KO mice reconstituted with IFN-␥ KO CD4⫹ T lymphocytes had
significantly lower CD3⫹ T lymphocytes and neutrophil infiltration at 24 hours after reperfusion (rightmost column). The results are representative of groups as shown in Figure 6.
more likely to express A2ARs than are cells that do not
produce lymphokines.27 The immunosuppressive effects of
A2AR activation have been confirmed by studies showing
that genetic inactivation of the A2AR increases the intensity
and prolongs the duration of T lymphocyte– dependent proinflammatory cytokine accumulation and tissue damage.25
ATL146e attenuates the reduction of circulating lymphocytes after MI, consistent with a role of A2AR activation in
the inhibition of lymphocyte homing (Figure 1C). Myocardial
neutrophil infiltration is also significantly reduced in Rag1
KO mice; however, neutrophil infiltration is restored by
adoptive transfer of CD4⫹ T lymphocytes into Rag1 KO mice
(Figure 7). Taken together, these results suggest that CD4⫹ T
lymphocytes are activated early during reperfusion and contribute to an early inflammatory response that contributes to
an inflammatory cascade culminating in neutrophil chemotaxis and myocardial reperfusion injury.
In I/R injury, a lack of microbial antigens suggests that T
lymphocytes may become activated through antigenindependent pathways.1–3,28 Antigen-independent mechanisms for T-lymphocyte activation have recently been described, involving IL-12, MCP-1, RANTES, macrophage
inflammatory protein (MIP-1), and IFN- ␥ inducible
protein-10 (IP-10).10,11,29 The early expression of B7 costimulatory molecules is observed in ischemic organs with a
nonimmune, inflammatory response in the absence of alloantigen. Blockade of T-lymphocyte CD28/B7 costimulation,
either with antibodies or by knocking out CD28, significantly
inhibits T-lymphocyte activation and reduces I/R injury.28 An
additional mechanism of T-lymphocyte activation may involve reactive oxygen species. Very recent work has shown
that reactive oxygen species directly stimulate T lymphocytes
through the T-cell receptor.30 Nitric oxide exerts immunosuppressive effects by inhibiting the proliferation of Th1 and Th2
T cells.31 Reactive oxygen species decrease the bioavailability of nitric oxide and may thus enhance T-cell activation and
proliferation through indirect mechanisms.31
Conclusion
The present study implicates CD4⫹ T lymphocytes as targets
of adenosine A2AR-mediated cardioprotection against I/R
injury. T lymphocytes accumulate in the previously ischemic
region of the myocardium within 2 minutes of reperfusion.
This rapid accumulation of T lymphocytes, the extent of
reperfusion injury, neutrophil accumulation, and IFN-␥ release are inhibited by the activation of A2AR on CD4⫹ T
lymphocytes. The results underscore the importance of CD4⫹
T lymphocytes as mediators of myocardial reperfusion injury
after MI.
Acknowledgments
The authors are grateful to the University of Virginia Cardiovascular
Research Center for providing the key facilities to fulfill the present
experiments.
Sources of Funding
This work was supported by the University of Virginia Partner’s
Award and an American Heart Association Grant-in-Aid to Dr Yang;
National Institutes of Health grants R01 HL69494 and R01 HL58582
to Dr French and grant R01 HL37942 to Dr Linden; and the Falk
Medical Research Trust.
Disclosure
Dr Linden is the president of Adenosine Therapeutics, LLC. The
remaining authors report no conflicts.
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CLINICAL PERSPECTIVE
Inflammatory responses have been implicated not only in sepsis but also in shock, trauma, pancreatitis, surgery, and
reperfusion injury. The cellular, molecular, and genetic mechanisms underlying inflammation have been investigated
extensively. However, the immune mechanisms underlying non–antigen-induced inflammation remain unclear. Compelling evidence from both animal and clinical studies indicates that leukocytes are key factors in reperfusion injury.
Reperfusion induces a vigorous inflammatory response that includes neutrophil adherence to reperfused endothelium.
Recent studies suggest that inflammation is induced early during reperfusion by CD4⫹ T lymphocytes. CD4⫹ T
lymphocytes become activated early during reperfusion and secrete proinflammatory cytokines that potentiate the
inflammatory response. Adenosine A2A receptors (A2ARs) are widely expressed on blood-borne cells (including T
lymphocytes, monocytes, neutrophils, and platelets). Stimulation of A2ARs on leukocytes mediates immunosuppression
and inhibits neutrophil oxidative activity by a Gs protein/cAMP– dependent pathway. A2ARs play an important role in
counteracting this T cell–mediated inflammatory response because tissue injury is always associated with release of
adenosine. The present study clearly shows that exogenous activation of A2ARs on CD4⫹ T lymphocytes inhibits
inflammation and reduces myocardial infarction. The cardioprotective effect of A2AR stimulation is manifest even when
the compound is given at the time of reperfusion. This has important clinical implications because it demonstrates that it
may be possible to intervene with adenosine derivatives to reduce infarct size in patients with acute myocardial infarction.
Thus, the myocardial salvage obtained by conventional clinical approaches (percutaneous coronary intervention or
thrombolysis) may be significantly improved by the simple administration of a potent A2AR agonist before reperfusion.
Myocardial Infarct-Sparing Effect of Adenosine A2A Receptor Activation Is due to Its
Action on CD4 + T Lymphocytes
Zequan Yang, Yuan-Ji Day, Marie-Claire Toufektsian, Yaqin Xu, Susan I. Ramos, Melissa A.
Marshall, Brent A. French and Joel Linden
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Circulation. published online October 23, 2006;
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