The Journal of Immunology
A2A Adenosine Receptor Signaling in Lymphocytes and the
Central Nervous System Regulates Inflammation during
Experimental Autoimmune Encephalomyelitis
Jeffrey H. Mills,* Do-Geun Kim,* Antje Krenz,* Jiang-Fan Chen,†and Margaret S. Bynoe*
Extracellular adenosine has an important role in regulating the severity of inflammation during an immune response. Although
there are four adenosine receptor (AR) subtypes, the A2AAR is both highly expressed on lymphocytes and known as a prime me-
diator of adenosine’s anti-inflammatory effects. To define the importance of A2AAR signaling during neuroinflammatory disease
progression, we used the experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis. In EAE induc-
tion experiments, A2AAR antagonist treatment protected mice from disease development and its associated CNS lymphocyte
infiltration. However, A2AAR2/2mice developed a more severe acute EAE phenotype characterized by more proinflammatory
lymphocytes and activated microglia/macrophages. Interestingly, very high levels of A2AAR were expressed on the choroid
plexus, a well-established CNS lymphocyte entry point. To determine the contribution of A2AAR signaling in lymphocytes and
the CNS during EAE, we used bone marrow chimeric mice. Remarkably, A2AAR2/2donor hematopoietic cells potentiated severe
EAE, whereas lack of A2AAR expression on nonhematopoietic cells protected against disease development. Although no defect in
the suppressive ability of A2AAR2/2regulatory T cells was observed, A2AAR2/2lymphocytes were shown to proliferate more
and produced more IFN-g following stimulation. Despite this more proinflammatory phenotype, A2AAR antagonist treatment
still protected against EAE when A2AAR2/2lymphocytes were adoptively transferred to T cell-deficient A2AAR+/+mice. These
results indicate that A2AAR expression on nonimmune cells (likely in the CNS) is required for efficient EAE development, while
A2AAR lymphocyte expression is essential for limiting the severity of the inflammatory response.
2012, 188: 5713–5722.
trolling inflammation (2). Although adenosine is typically pro-
duced inside a cell, extracellular levels of adenosine rise as
a consequence of the catabolism of ATP that is released from
stressed or damaged cells (3, 4). This extracellular ATP is con-
verted to ADP and AMP by CD39 and then to adenosine by CD73
(5). The half-life of extracellular adenosine is on the order of
seconds, as it is removed from the extracellular space either by
adenosine deaminase, which converts it to inosine, or by cellular
reuptake via equilibrative or concentrative nucleoside transporters
(6, 7). Therefore, acute increases in extracellular adenosine levels
tend to only have local and tissue-limited effects. During inflam-
mation, increases in extracellular adenosine levels effectively turn
off the local inflammatory response to protect against excessive
cellular damage to the surrounding tissue (2). Cells that express
The Journal of Immunology,
denosine is an endogenous purine nucleoside that modu-
lates a wide range of physiological functions (1). Most
notable among its many roles is its importance in con-
one or more of the four G protein-coupled adenosine receptors
(AR) subtypes (A1, A2A, A2B, and/or A3) have the capacity to
respond to extracellular adenosine. Because adenosine has such
potent effects on inflammation, modulators of adenosine signaling
are being evaluated as potential therapeutic options for diseases
that have an inflammatory component (8). One such disease is
multiple sclerosis (MS).
MS is an autoimmune inflammatory disease of the CNS that
affects ∼2.5 million people worldwide. During MS, infiltrating
autoreactive immune cells attack and destroy myelin surrounding
the axons of nerve cells in the brain and spinal cord, resulting in
loss of neurologic function (9). The damage caused by this neu-
roinflammation can give rise to problems with vision, cognition,
sensation, and coordination and balance (10). MS disease pro-
gression can present in patients in several forms, with new
symptoms either occurring in discrete attacks (relapsing) or ac-
cumulating slowly over time (progressive). Although the cause of
MS is unknown, most MS treatment research is focused on the
prevention of disease relapse and progression (11). Therapeuti-
cally this can be accomplished in two general ways: 1) preventing/
limiting the inflammatory response of lymphocytes, or 2) pre-
venting lymphocyte infiltration in the CNS. Extracellular adeno-
sine has been shown to be involved in both.
It is well documented that extracellular adenosine has potent
anti-inflammatory properties (2). The inhibitory effects of extra-
cellular adenosine and AR signaling have been observed in lym-
phocytes (5, 12–14), neutrophils (15–17), monocytes/macrophages
(18–20), and dendritic cells (21, 22). For example, CD732/2mice
(which lack the ability to synthesize extracellular adenosine) have
been shown to undergo a more severe form of inflammatory bowel
disease (23). Likewise in the experimental autoimmune encepha-
lomyelitis (EAE) animal model for MS, adoptively transferred
*Department of Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, NY 14853; and†Department of Neurology, Boston Uni-
versity School of Medicine, Boston, MA 02118
Received for publication February 13, 2012. Accepted for publication March 28,
This work was supported by National Institutes of Health Grants R01 NS063011 (to
M.S.B.) and F32 NS 066682 (to J.H.M.).
Address correspondence and reprint requests to Dr. Margaret S. Bynoe, Department
of Microbiology and Immunology, College of Veterinary Medicine, C5 149 Veteri-
nary Medical Center, Cornell University, Ithaca, NY 14853. E-mail address: msb76@
Abbreviations used in this article: AR, adenosine receptor; EAE, experimental auto-
immune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, mul-
tiple sclerosis; Treg, regulatory T (cell).
lymphocytes from CD732/2mice cause more severe EAE (as
compared with those transferred from CD73+/+wild-type mice)
when given to T cell-deficient recipients (24). The anti-inflam-
matory effects of adenosine on immune cells are thought to be
mainly mediated by A2AAR signaling (2, 8, 25–27).
In addition to extracellular adenosine’s role in downmodulating
inflammation, it also has the potential to stimulate the migration of
immune cells. For example, increases in extracellular adenosine
and AR signaling promote the chemotaxis of neutrophils (28, 29)
and immature dendritic cells (30) and induce cell migration into
the lungs following injury to augment tissue repair (31). Addi-
tionally, AR signaling has been shown to be required for efficient
lymphocyte migration into the CNS during EAE. CD732/2mice
or wild-type animals that are given either broad spectrum AR
antagonists (such as caffeine) or A2AAR-specific antagonists are
protected from EAE development due to a lack of lymphocyte
migration into the brain and spinal cord (24, 32, 33). A2AAR
signaling in the brain at the choroid plexus, which is the blood to
cerebral spinal fluid barrier and the initial entry point into the CNS
for lymphocytes during EAE (34–36), may promote this lym-
phocyte migration (24).
Because A2AAR signaling on lymphocytes has been suggested
to have both anti-inflammatory (2) and promigratory effects
on lymphocytes (24), we sought to define the role of A2AAR
during the development of EAE. Utilizing A2AAR2/2mice, we
determined that absence of A2AAR expression led to the devel-
opment of a more severe form of EAE compared with wild-type
mice. This heightened disease course was due to the increased
proinflammatory phenotype of A2AAR2/2immune cells, which
overcame any protection that was imparted by the lack of A2AAR
signaling on nonimmune cells within the CNS. Our results dem-
onstrate the differential role for the A2AAR in inflammation
versus its role in CNS barrier function.
Materials and Methods
C57BL/6 mice (The Jackson Laboratory) were used as wild-type.
A2AAR2/2mice (37) were a gift from Dr. Jiang-Fan Chen (Boston
University School of Medicine, Boston, MA). Tcra2/2mice for transfer
EAE experiments were purchased from The Jackson Laboratory. Myelin
oligodendrocyte glycoprotein (MOG)-specific T cells from 2d2 TCR
transgenic mice (38) were used for suppression assay experiments. All
genetically modified mice were on the C57BL/6 background. Animals
were bred and housed under specific pathogen-free conditions at Cornell
University (Ithaca, NY). All procedures were done in accordance with
approved Institutional Animal Care and Use Committee protocols.
EAE induction and scoring
EAE was induced as previously described (39). Briefly, a 1:1 emulsion of
MOG35–55peptide (1 mg/ml in PBS) (Anaspec) and CFA (Sigma-Aldrich)
was injected s.c. (50 ml) into each flank. Pertussis toxin (20 ng; Biological
Laboratories) was given i.v. (200 ml in PBS) at the time of immunization and
again 2 d later. To induce EAE in Tcra2/2mice, wild-type and A2AAR2/2
mice were primed with CFA/MOG peptide, and after 7 d, CD4+T cells were
isolated from spleen and lymph nodes by magnetic negative separation.
CD4+cells (106) were transferred i.v. to Tcra2/2mice in a total of 200 ml in
sterile PBS, with concomitant MOG/CFA s.c. injection and pertussis toxin
i.v. injection. For SCH58261 (Tocris Bioscience) treatments, mice were
given 5 mg/kg in olive oil via s.c. injection every 3–4 d, starting at day 1 post
immunization. Mice were scored daily for EAE based on disease symptom
severity: 0, no disease; 0.5–1.0, weak/limp tail; 2.0, limp tail and partial
hindlimb paralysis; 3.0, total hindlimb paralysis; 4.0, both hindlimb and
forelimb paralysis; 5.0, death. Mice with a score of 4.0 were euthanized.
PBS, and the brains, spinal cords, and spleens were isolated and snap
frozen in Tissue-Tek OCT medium. Five-micrometer sections (brains in
a sagittal orientation) were affixed to Supefrost Plus slides (Fisher Scien-
tific), fixed in acetone, and stored at 280˚C. For immunostaining, slides
were thawed, washed in PBS, blocked with casein (Vector Laboratories) in
normal goat serum (Zymed Laboratories), and then incubated with Abs
against CD45 (YW62.3; AbD Serotec), CD4 (L3T4; BD Biosciences),
CD11b (M1/70.15; Caltag Laboratories), F480 (RM2915; Caltag Labora-
tories), IBA1 (polyclonal; Wako Pure Chemical Industries), or Foxp3
(FJK-16s; eBioscience). For brightfield microscopic visualizations, slides
were stained with a goat anti-rat biotin Ab (Jackson ImmunoResearch
Laboratories) and then incubated with streptavidin-HRP (Invitrogen), de-
veloped with an AEC kit (Invitrogen), and counterstained with hematox-
ylin (Fisher Scientific). Images were obtained on a Zeiss Axio Imager M1
microscope utilizing AxioVision software.
Fluorescence in situ hybridization
For detection of AR mRNA in the brain, we performed fluorescence in situ
hybridization using biotin-labeled A2AAR DNA oligonucleotide probes
GGCG-39; Integrated DNA Technologies) (40). Anesthetized mice were
perfused with PBS and brains were isolated and snap frozen in Tissue-Tek
OCT medium. Twelve-micrometer cryosections were mounted on Super-
frost Plus slides (Fisher Scientific) and then fixed (4% neutral buffered
paraformaldehyde) and rinsed (13 PBS). Next, the sections were equili-
brated in 0.1 M triethanolamine and acetylated in 0.1 M triethanolamine
with 0.25% acetic anhydride. The sections were dehydrated through an
ascending ethanol series and stored at room temperature. For hybridization,
the sections were rehydrated in PBS, equilibrated in 53 SSC (0.75 M NaCl,
0.075 M sodium citrate), and prehybridized for 1 h at 42˚C in hybridization
buffer (50% deionized formamide, 43 SSC, 40 mg/ml salmon sperm DNA,
20% [w/v] dextran sulfate, 13 Denhardt’s solution). The probes (300 ng/
ml) were denatured at 80˚C and added to the prewarmed (42˚C) buffer
(hybridization mix). The hybridization reaction was carried out at 42˚C for
38 h with 250 ml hybridization mix. The sections were washed in 23 SSC
(room temperature), 0.23 SSC/0.1% SDS (65˚C), and then equilibrated in
PBS. Sections were incubated with Texas Red-X–conjugated streptavidin
(S6370, 1 mg/ml; Molecular Probes) and then washed in PBS followed by
0.23 SSC/0.1% SDS (65˚C) and PBS washes. Slides were mounted with
Vectashield mounting medium with DAPI (Vector Laboratories). Images
were acquired using a Zeiss Axio Imager M1 fluorescent microscope.
and 2d2 transgenic mice. Lymphocytes were incubated with ACK buffer
(0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA [pH 7.3]) to lyse RBCs.
For wild-type and A2AAR2/2cells, CD4+cells were enriched by negative
magnetic selection by incubating the cells with Abs to CD8 (TIB-105),
IAb,d,v,p,q,r(212.A1), FcR (2.4-G2), B220 (TIB-164), NK1.1 (HB191), and
then with BioMag goat anti-mouse IgG, IgM, and goat anti-rat IgG
(Qiagen), and then by removing the Ab/bead-bound cells. These enriched
CD4+cells were incubated with a biotinylated Ab against CD25 (PC61.5;
eBioscience) and followed by anti-biotin–conjugated beads (Miltenyi
Biotec). The CD25+fraction was magnetically isolated and used as the
suppressor populations. Effector cells from 2d2 mice were first labeled
with CFSE and then cultured (3.5 3 106) with MOG (10 mg/ml) and
suppressor cells at varying concentrations. Proliferation was measured
after 72 h via CFSE loss analyzed on a FACSCanto II (BD Biosciences)
with BD FACSDiva software (BD Biosciences).
Bone marrow radiation chimeric mice
For chimeras, the bone marrow recipients were irradiated (Mark I model 68
gamma irradiator, cesiumsource) with 2 3 650 rads spacedat a 4-hinterval,
which is effectively lethal to hematopoietic/immune cells (41–43). Twenty-
four hours later, bone marrow was aseptically obtained from donor mice by
removing the femurs/tibias from both legs and flushing out the bone
marrow with a 27-gauge needle and syringe. The bone marrow containing
stem cells was washed and transferred i.v. (106cells/mouse) to irradiated
recipients. After 8–10 wk reconstitution, mice were used for EAE studies.
Isolated lymphocytes (5 3 106cells/ml) from the spleens and lymph nodes
of MOG immunized mice were treated with varying concentrations of
MOG peptide (0, 1, 5, and 25 mg/well) or the mitogen Con A. After 48 h
culture, 1 mCi [3H]thymidine was added to each well. Eighteen hours after
the addition of thymidine, cells were harvested using a Tomtec Mach III
harvester and quantified using a LS6500 multipurpose scintillation counter
5714A2A ADENOSINE RECEPTOR SIGNALING DURING EAE PROGRESSION
Isolated lymphocytes (5 3 106cells/ml) from the spleens and lymph nodes
of MOG immunized mice were treated with varying concentrations of
MOG peptide (0, 1, 5, and 25 mg/well). Supernatants were collected at
48 h and analyzed utilizing IFN-g, IL-17, TNF-a, and IL-1b ELISA kits
(eBioscience) according to manufacturer’s instructions. Cytokine meas-
urements on samples were performed on a BioTek Synergy 4 and con-
centrations were derived from a standard curve utilizing Gen5 data
were determined utilizing GraphPad Prism and Microsoft Excel software.
Statistical differences between EAE treatment groups were determined by
two-way ANOVA analysis, whereas difference between time points was
determined utilizing the Mann–Whitney U test. The Student t test was used
for other comparisons unless stated within the figure legends. Statistical
differences were determined where p # 0.05.
A2AAR2/2mice develop more severe EAE
Extracellular adenosine and AR signaling have been previously
shown to be involved in both the development and progression of
EAE (24, 32). For example, mice unable to produce extracellular
adenosine (CD732/2mice) (24) or given the A2AAR-specific
antagonist SCH58261 (Fig. 1A, Ref. 24) are protected against
EAE induction and lack CNS lymphocyte infiltrates that are as-
sociated with disease progression (24). To fully investigate the
importance of A2AAR signaling during EAE progression, we first
actively induced EAE in wild-type and A2AAR2/2mice (37) by
the MOG35–55immunization method (see Materials and Methods)
and monitored and scored them daily for clinical signs of EAE.
A2AAR2/2mice developed more severe EAE than did wild-type
control mice (Fig. 1B). Although A2AAR2/2mice exhibited
significantly more severe paralysis at days 12–16 after EAE in-
duction (Fig. 1B), no difference between the mean day of disease
onset and average maximum EAE score between wild-type and
A2AAR2/2mice was observed (Table I). These studies indicate
that genetic disruption of the A2AAR does not confer protection
against EAE, but instead promotes a more severe acute disease.
A2AAR2/2mice have more CNS lymphocyte infiltrates
compared with wild-type mice during EAE
EAE is mediated by the infiltration of autoreactive immune cells
into the CNS. The inflammatory response directed against myelin,
paralysis in mice as a result of motor neuron demyelination (34,
44). To assess CNS lymphocyte infiltration during EAE in
A2AAR2/2mice, brain and spinal cord sections were examined
for presence of CD45+(general leukocyte marker) and CD4+
T cells by immunohistochemistry (Fig. 2). Following EAE in-
duction, both wild-type and A2AAR2/2mice had distinct patches
of immune cell infiltration in the cerebellum (Fig. 2A, 2E, 2I, 2M),
hippocampus (Fig. 2B, 2F, 2J, 2N), and spinal cord (Fig. 2C, 2D,
2G, 2H, 2K, 2L, 2O, 2P). However, A2AAR2/2mice had visually
and quantitatively significantly more CD45+(Fig. 2A–H) and
CD4+(Fig. 2I–Q) cells in their brains and spinal cords compared
with wild-type mice. These results indicate that the more severe
disease observed in A2AAR2/2mice (Fig. 1B) is associated with
increased immune cell numbers in the CNS.
A2AAR2/2mice have increased dissemination and retention of
CD11b+/F480+and IBA1+cells in the CNS during EAE
Macrophage/microglia migration and activation in the CNS are
critical for the demyelination and clinical signs of EAE (45, 46). To
assess the frequency of macrophages/microglia in the CNS of
wild-type and A2AAR2/2mice with MOG35–55-induced EAE,
brain and spinal cord sections were stained with CD11b and F480
and analyzed by immunohistochemistry (Fig. 3A–P). Whereas
both wild-type and A2AAR2/2mice had a large number of
CD11b+(Fig. 3A–H) and F480+(Fig. 3I–P) cells present in their
CNS following EAE, the frequency of CD11b+/F480+cells was
dramatically higher in A2AAR2/2(Fig. 3E–H, 3M–P) compared
with wild-type mice (Fig. 3A–D, 3I–L). The most noticeable
differences in frequency between the groups were observed in the
spinal cord (Fig. 3C, 3D, 3G, 3H, 3K, 3L, 3O, 3P), with most
A2AAR2/2mice displaying heavy patches of CD11b+/F480+
cells (Fig. 3G, 3H, 3O, 3P). These data suggest that the absence of
A2AAR2/2mice are susceptible to EAE. (A) EAE was induced in
wild-type mice that were given SCH58261 A2AAR antagonist (4, n =
12) or vehicle (:, n = 13) treatment, disease activity was monitored
daily, and the mean EAE score was calculated. The results shown are
from three separate experiments. Error bars represent the SEM. (B)
EAE was induced in wild-type (n, n = 12) and A2AAR2/2mice (N, n =
13), disease activity was monitored daily, and the mean EAE score was
calculated. The results shown are representative of two separate
experiments. Error bars represent the SEM. Statistically different mean
EAE scores at each time point are indicated. *p # 0.05.
A2AAR antagonism protects against EAE, whereas
Table I.A2AAR2/2mice are susceptible to EAE development
Mean Day of
Mean Maximum EAE
11.6 6 1.1
10.5 6 0.9
2.3 6 0.3
2.7 6 0.2
Wild-type and A2AAR2/2mice were induced to develop EAE and scored daily
for EAE severity based on the five point scale assessing ascending paralysis.
aNumber of mice that achieved a score of 0.5 (weak tail) in the experimental group.
bAverage day of onset (an EAE score of 0.5; 6SEM).
cAverage of the maximum EAE score for each individual mouse (6SEM).
The Journal of Immunology5715
A2AAR signaling results in increased microglia/macrophage mi-
gration to the spinal cord during EAE.
We next determined the activation state of the macrophages/
microglia in the CNS following EAE induction in spinal cord
sections stained for the presence of Iba1 (Fig. 3Q–T), a marker for
macrophage and microglial cell activation (47). Spinal cord sec-
tions from A2AAR2/2mice with EAE had many areas that
reacted strongly with the Iba1 Ab and displayed a greater pre-
ponderance of ameboid-shaped cells (Fig. 3S–U), which are rep-
resentative of activated microglia (48). Conversely, whereas spinal
cord sections from wild-type mice with EAE did have a few areas
depicting microglia with an activated phenotype (data not shown),
most of their spinal cord had microglial cells that displayed a
predominantly ramified morphology with less Iba1 staining (Fig.
3Q, 3R, 3U), which is consistent with that of resting microglial
cells (48). This suggest that lack of A2AAR expression on
microglial cells confers a more inflammatory microglia population
in the CNS microenvironment, which is consistent with the more
severe disease observed in A2AAR2/2mice (Fig. 1B).
A2AAR2/2lymphocytes produce more IFN-g than do those
from wild-type mice
Proinflammatory cytokines, such as IFN-g and IL-17, have been
shown to have a prominent role in the inflammatory response
mediated by the infiltrating CNS lymphocytes during EAE (49).
To determine whether A2AAR2/2lymphocytes are intrinsically
more proinflammatory than those from wild-type mice, lympho-
cytes from MOG-immunized wild-type and A2AAR2/2mice
were restimulated with MOG in vitro and cytokine production was
assessed (Fig. 4A). Lymphocytes activated with MOG from
A2AAR2/2mice produced significantly more IFN-g in a dose-
dependent manner compared with those from wild-type mice (Fig.
4A). No significant difference in IL-17 (Fig. 4B), TNF-a (Fig.
4C), IL-1b (Fig. 4D), IL-4 (data not shown), or IL-10 (data not
shown) production was detected. High production of IFN-g sug-
gests that the lack of A2AAR expression on lymphocytes pro-
motes a more proinflammatory phenotype.
A2AAR2/2lymphocytes have a higher proliferative potential,
whereas regulatory T cell frequency and function are unaltered
To determine whether A2AAR2/2lymphocytes are intrinsically
more proliferative than are those from wild-type mice, lympho-
cytes were isolated from the spleen and lymph nodes of wild-type
and A2AAR2/2mice, stimulated with the mitogen Con A, and
proliferation was measured based on thymidine incorporation
(Fig. 5A). Although there was no difference in the baseline pro-
and spinal cord of A2AAR2/2mice following EAE. Representative
images taken from frozen tissue sections of the cerebellum (A, E, I, M),
hippocampus (B, F, J, N), and spinal cord (C, D, G, H, K, L, O, P) from
post-EAE–induced wild-type (A–D, I–L) and A2AAR2/2(E–H, M–P) mice
were labeled with either CD45 (A–H) or CD4 (I–P) Abs to detect lym-
phocyte infiltration in the CNS follow disease induction. Immunoreactivity
was detected with HRP anti-rat Ig plus AEC (red) against a hematoxylin-
stained nuclear background (blue). Scale bars, 100 mm. (Q) In post-EAE–
induced wild-type and A2AAR2/2mice, six anatomically similar fields
per brain (two from cerebellum, two from hippocampus, and one each
from frontal lobe and brain stem) and four fields per spinal cord per mouse
were analyzed at 310 magnification for CD4 cell infiltration. Error bars
represent the SEM (n # 10). Statistically different infiltrating CD4 cells
mean values counted per field between wild-type and A2AAR2/2mice in
each tissue are displayed. *p # 0.05, **p # 0.01.
Increased numbers of lymphocytes are observed in the brain
are observed in CNS of A2AAR2/2mice following EAE. Representative
images taken from frozen tissue sections of the cerebellum (A, E, I, M),
hippocampus (B, F, J, N), and spinal cord (C, D, G, H, K, L, O–T) from
EAE-induced wild-type (A–D, I–L, Q, R) and A2AAR2/2(E–H, M–P, S, T)
mice were labeled with either CD11b (A–H), F480 (I–P), or IBA1 (Q–T)
Abs to detect microglial/macrophage infiltration and activation in the CNS
following disease induction. Immunoreactivity was detected with HRP
anti-rat Ig plus AEC (red) against a hemotoxylin-stained nuclear back-
ground (blue). Insets show increased magnification. Scale bars, 100 mm.
(U) In EAE-induced wild-type and A2AAR2/2mice, seven fields per
spinal cord per mouse were analyzed at 310 magnification for IBA1
staining. Error bars represent the SEM (n # 5). Statistically different
IBA1+mean spinal cord counts per field between EAE-induced wild-type
and A2AAR2/2mice are displayed. ***p # 0.001.
Increased macrophage/microglial infiltration and activation
5716A2A ADENOSINE RECEPTOR SIGNALING DURING EAE PROGRESSION
liferation within control nonstimulated splenocytes, A2AAR2/2
lymphocytes that were stimulated with Con A proliferated sig-
nificantly more compared with those from wild-type mice (Fig.
5A). To determine whether lymphocytes from A2AAR2/2mice
also proliferated more in response to antigenic stimuli, lympho-
cytes from mice immunized with MOG35–55were isolated and
their recall response to MOG was tested in culture (Fig. 5B).
Similar to the results for Con A stimulation, lymphocytes reac-
tivated with MOG from A2AAR2/2mice proliferated signifi-
cantly more than did those from wild-type mice (Fig. 5B). These
results indicate that lymphocytes from A2AAR2/2mice have an
enhanced proliferative potential.
Regulatory T (Treg) cells have a critical role in autoimmune
suppression and regulation ofinflammation (51). Decreases in Treg
cell number or function have been shown to leave mice suscep-
tible to severe EAE (52). To determine whether the Treg cell
population is altered in A2AAR2/2mice, we analyzed their fre-
quency (Foxp3+) and functionality (ability to suppress effector cell
proliferation). Immunohistochemistry staining of Foxp3+cells in
the spleen and CNS showed no difference in the frequency of
Foxp3+cells between wild-type and A2AAR2/2mice with EAE
(Fig. 5C–E). Additionally, no difference was observed in the
ability of A2AAR2/2mice Treg cells (CD4+CD25+from naive
mice) to suppress effector cell proliferation compared with Treg
cells from wild-type mice (Fig. 5F). At all Treg/T effector cell
ratios, A2AAR2/2Treg cell suppressor function was similar to
that of wild-type Treg cells (Fig. 5F). These findings indicate that
the severe EAE observed in A2AAR2/2mice was not due to
a significant alteration in the Treg cell population.
A2AAR expression on immune cells limits the severity of EAE,
whereas A2AAR expression on nonimmune cells promotes more
It is well established that the A2AAR is expressed on a variety of
cells, including lymphocytes (5, 12, 53) and cells in the CNS (54,
55). Within the brain, high expression of the A2AAR was found
throughout the choroid plexus (Fig. 6A), a site shown to be in-
volved in immune cell migration into the CNS (34–36). A2AAR
expression, although less than that at the choroid plexus, was also
observed in the meninges (Fig. 6B) and near the hippocampus
(Fig. 6C) and cerebellum, albeit to a lesser degree (Fig. 6D).
Because A2AAR2/2mice are susceptible to severe acute EAE
(Fig. 1B), we next wanted to determine where the A2AAR must
be expressed (i.e., on immune cells or the CNS) to prevent this
aggravated disease phenotype. Therefore, we used the radiation
bone marrow chimera experimental model system (41–43), in
which the animal’s immune cells (which are both sensitive to
radiation and derived from stem cells in the bone marrow) are
replaced following irradiation and subsequent bone marrow
transplantation from donor animals. Gamma-irradiated naive wild-
type mice were reconstituted with bone marrow from either naive
wild-type or A2AAR2/2donor mice and then immunized with
MOG35–55to induce EAE (Fig. 6E). Wild-type mice reconstituted
with A2AAR2/2cells developed significantly more severe EAE
compared with those that received bone marrow from wild-type
donors (Fig. 6E, Table II). This exacerbated disease development
suggests that A2AAR expression on bone marrow-derived im-
mune cells is required to control the severity of EAE.
To determine the influence of A2AAR on nonimmune cells
(i.e., radiation-resistant cells), irradiated A2AAR2/2mice were re-
constituted with bone marrow from wild-type donor mice and then
induced to develop EAE (Fig. 6E). A2AAR2/2mice reconstituted
with wild-type cells were protected from EAE and developed only
mild disease compared with wild-type donor cells transferred into
wild-type recipient mice (Fig. 6E, Table II). This protection from
disease development suggests that A2AAR expression on radiation-
resistant cells (such as those in the CNS) is required for EAE pro-
gression, which is similar to the protective EAE effects of the
Error bars represent the SEM (n # 5). Statistically different values of wild-type compared with A2AAR2/2mice for each condition are displayed. *p # 0.05.
A2AAR2/2lymphocytesproduce higher levels of IFN-g. Splenocytesfrom wild-type (filledbars) and A2AAR2/2(openbars) post-EAE–induced
The Journal of Immunology 5717
To assess CNS lymphocyte infiltration during EAE in the chi-
meric mice, brain and spinal cord sections were examined for the
presence of CD4+T cells by immunohistochemistry (Fig. 6F).
Similar to EAE severity (Fig. 6E), A2AAR2/2recipient mice had
virtually no CD4+T cell infiltration in the spinal cord, whereas
mice that received A2AAR2/2donor cells had significant spinal
cord infiltration (Fig. 6F). Overall, these results indicate that lack
of A2AAR expression on hematopoietic cells (such as lympho-
cytes) promotes severe EAE, whereas lack of A2AAR on non-
hematopoietic cells (most likely in the CNS) is protective during
A2AAR antagonism protects against EAE mediated by
A2AAR2/2adoptively transferred cells
We have shown that the SCH58261 A2AAR antagonist can protect
against EAE development (Fig. 1A, Ref. 24), despite the fact that
genetic disruption of the A2AAR leaves mice prone to developing
a severe acute form of EAE (Fig. 1B), which is likely due to the
more proinflammatory nature of A2AAR2/2lymphocytes (Figs.
4, 5). Therefore, we next asked whether SCH58261 treatment can
prevent EAE when A2AAR2/2effector T cells are used to induce
disease. To test this, primed CD4+T cells from the spleen and
lymph nodes of MOG immunized wild-type and A2AAR2/2mice
were transferred into Tcra2/2(A2AAR+/+) recipient mice and
given SCH58261 or vehicle control treatments following EAE
induction. Tcra2/2mice lack endogenous T cells and cannot
develop EAE on their own (data not shown and Ref. 56). In the
vehicle control groups, A2AAR+/+tcra2/2recipient mice that re-
ceived CD4+T cells from A2AAR2/2donors developed a more
severe disease progression compared with those that received
wild-type CD4+T cells (Fig. 7). However, mice that received
SCH58261 treatment, regardless of whether wild-type or
A2AAR2/2CD4+ T cells were transferred, were protected from
EAE development (Fig. 7). These results suggest that A2AAR
antagonist-mediated blockade is effective at preventing EAE in
a lymphocyte-independent manner. Overall, our results indicate
that the A2AAR has an important role in mediating both the
proinflammatory potential of lymphocytes and the susceptibility
to CNS disease progression/lymphocyte infiltration during EAE
In this study and previously (24), we have demonstrated that
blockade of the A2AAR with an A2A-specific AR antagonist
protected mice from EAE by hindering lymphocyte entry into the
brain and spinal cord of wild-type mice. This finding was unex-
pected, as this function of adenosine, that is, mediating lympho-
cyte migration into the CNS, was previously unknown. Further-
more, the facts that adenosine suppresses the immune response
and resolves inflammation (2) are at odds with the finding that
blockade of the A2AAR, which mediates the preponderance of
adenosine’s suppressive and anti-inflammatory functions (2),
protects mice from EAE (24). The purpose of this study was to
delineate adenosine’s role in the immune response from its
function in mediating immune cell migration into the CNS via the
We show that A2AAR2/2mice developed more severe EAE
than did their wild-type counterparts. This severe disease was
characterized in the CNS by increased numbers of lymphocytes
and activated macrophage/microglia cells in the CNS parenchyma,
liferate more despite normal regulatory
T cells frequency and function. (A and B)
Splenocytes from wild-type (filled bars) and
A2AAR2/2(open bars) post-EAE–induced
mice were isolated and stimulated in culture
with (A) Con A or (B) varying concentrations
of MOG. (A and B) Cell proliferation was as-
sessed by thymidine incorporation and mea-
sured utilizing a scintillation counter. Error
bars represent the SEM (n # 5). (C–E) Frozen
tissue sections of spleen (C, D) and brain and
spinal cord (E) from EAE-induced wild-type
[(C), filled bar in (E)] and A2AAR2/2[(D),
open bar in (E)] mice were labeled with an
Ab against Foxp3 to detect Treg cells. Im-
munoreactivity was detected with HRP anti-
rat Ig plus AEC (red) against a hemotoxylin-
stained nuclear background (blue). Scale
bars, 100 mm. (E) Wild-type and A2AAR2/2
mice from EAE mice were analyzed for total
Foxp3 staining in their brain and spinal cord.
Error bars represent the SEM (n # 6). (F)
CD4+CD25+lymphocytes from naive wild-
type and A2AAR2/2mice were used as
suppressor T cells for the effector T cells
isolated from 2d2 MOG-specific TCR trans-
genic mice stimulated in vitro with MOG.
Percentage of 2d2 T cell proliferation in re-
sponse to MOG stimulation is displayed for
varying effector 2d2 cell/suppressor cell ra-
tios. Error bars represent the SEM (n $ 2).
Statistically different values of wild-type
compared with A2AAR2/2mice for each
condition are displayed. *p # 0.05.
5718A2A ADENOSINE RECEPTOR SIGNALING DURING EAE PROGRESSION
especially in the spinal cord. Furthermore, we found that the
highly proinflammatory nature of the A2AAR2/2immune cells
appears to be directly responsible for the severe EAE in the
A2AAR2/2mice (similar observations were made at both day 16
during the peak of disease [not shown] and at day 30 after EAE
induction). For example, the transfer of hematopoietic cells
lacking the A2AAR into irradiated wild-type recipients not only
caused more severe EAE than did wild-type cells transferred into
irradiated wild-type mice, but the disease was more severe than
the EAE observed in the parent A2AAR2/2mice (mean maxi-
mum EAE score, 3.9 versus 2.7). More importantly, A2AAR2/2
(recipient) mice that received wild-type (donor) bone marrow
were protected from EAE development. This suggests that
A2AAR2/2mice are susceptible to a severe acute form of EAE
due to the more proinflammatory immune cells. This is further
supported by data showing that A2AAR2/2lymphocytes are more
proliferative and produced more IFN-g than did their wild-type
counterparts. These results are consistent with the role of the
A2AAR in extracellular adenosine-mediated regulation of in-
flammation and of the immune response (2, 8, 25–27). Also im-
portant, our chimeric data suggest that the lack of A2AAR
signaling in the CNS (most likely on CNS barrier cells such as the
choroid plexus and the blood–brain barrier) confers protection
against EAE development. Therefore, the A2AAR has two ap-
parent roles during EAE progression: 1) to control the magnitude
of the inflammatory response (via expression on lymphocytes),
and 2) to allow for efficient lymphocyte entry/infiltration into the
CNS (via expression at the choroid plexus). These studies show
subsets in bone marrow chimeric mice influences EAE susceptibility. (A–D)
Fluorescence insitu hybridization ofA2AARexpression(red)inthebrainin
(A) choroid plexus, (B) meninges, (C) hippocampus, and (D) cerebellum of
F) Gamma-irradiated recipient wild-type and A2AAR2/2mice given bone
8 wk after the irradiation/reconstitution. Disease activity was monitored
daily andthemean EAEscorewas calculated. Plot legends arereadas“bone
marrow donor” into “irradiated recipient” mice. (E) Error bars represent the
SEM (n # 8). Statistical comparison between the total EAE disease course
for the chimera groups in each plot was performed via two-way ANOVA
analysis, with the resulting p values displayed. (F) In post-EAE–induced
wild-type and A2AAR2/2mice, five anatomically similar fields per brain
(two from cerebellum, two from hippocampus, and one from frontal lobe)
for CD4 cell infiltration. Error bars represent the SEM (n # 5). Statistically
different infiltrating CD4 cells meanvalues counted per field compared with
the wild-type into A2AAR2/2group are displayed. *p # 0.05.
A2AAR expression on hematopoietic and nonhematopoietic
whereas A2AAR2/2recipient mice are more resistant to EAE
A2AAR2/2hematopoietic cells produce more severe EAE,
Wild-type into wild-type
Wild-type into A2AAR2/2
7.7 6 0.4
8.0 6 0.4
9.6 6 1.6
3.0 6 0.2
3.9 6 0.4
1.1 6 0.4
Bone marrow chimeric mice were induced to develop EAE and scored daily for
EAE severity based on the five point scale assessing ascending paralysis. For wild-
type into wild-type compared with A2AAR2/2into wild-type, mean maximum EAE
score, p = 0.0566. For wild-type into wild-type compared with wild-type into
A2AAR2/2, mean maximum EAE score, p = 0.00073.
aNumber of mice that achieved a score of 0.5 (weak tail) in the experimental
bAverage day of onset (an EAE score of 0.5; 6SEM).
cAverage of the maximum EAE score for each individual mouse (6SEM).
mediated by adoptively transferred A2AAR2/2cells. EAE was induced in
T cell-deficient Tcra2/2mice that received adoptively transferred lym-
phocytes from wild-type (squares) or A2AAR2/2(circles) mice and were
given SCH58261 A2AAR antagonist (open shape) or vehicle (filled shape)
treatment (n # 5). Disease activity was monitored daily and the mean EAE
score was calculated.
The A2AAR antagonist SCH58261 protects against EAE
The Journal of Immunology 5719
that the heightened proinflammatory potential of A2AAR2/2
immune cells can mask the protective effects imparted by the
absence of A2AAR signaling on CNS barriers.
Our findings showing a role for the A2AAR in controlling the
severity of EAE are consistent with its role described in other
experimental model systems demonstrating the importance of
A2AAR signaling in limiting inflammation and tissue injury. For
example, mice lacking the A2AAR have increased liver damage in
ischemia–reperfusion liver injury (25) and are more sensitive to
bronchiolitis obliterans (26). Additionally, A2AAR expression
on bone marrow-derived cells is important in protecting against
damaged caused by inflammation in LPS-induced acute lung in-
jury (57), myocardial infarction (13), spinal cord injuries (58),
cerebral ischemia (37), and other various neuroinflammatory
injuries (59). Furthermore, the use of A2AAR-specific agonists
has been shown to be beneficial in the treatment of inflammatory
bowel disease (8) and ischemia–reperfusion liver (25) and lung
(60) injuries. These protective effects of A2AAR stimulation have
been attributed to its ability to inhibit the production of the
proinflammatory cytokines IL-12, INF-g, IL-6, and TNF-a (12,
27, 53, 61, 62). Indeed, we also show that lymphocytes from
A2AAR2/2mice with EAE produce more IFN-g than do those
from wild-type mice. We also observed that A2AAR2/2lym-
phocytes have a higher proliferative capacity than do those
expressing the A2AAR. Therefore, it was not surprising we ob-
served that A2AAR2/2bone marrow-derived cells (i.e., lympho-
cytes) were able to cause more severe EAE compared with those
from A2AAR+/+wild-type mice.
Our results also suggest that A2AAR signaling plays a major
role in regulating lymphocyte migration into the CNS. For in-
stance, mice treated with an A2AAR antagonist (24) or that lack
A2AAR expression in their CNS have significantly fewer lym-
phocytes in their CNS during EAE compared with control mice.
Consistent with our observations, other studies have also identified
extracellular adenosine signaling as having an important role in
regulating cell migration, albeit in a cell- and tissue-specific
manner. For example, extracellular adenosine has been shown to
induce chemotaxis in immature dendritic cells (30), endothelial
cells (63, 64), oligodendrocytes (65), and bronchial epithelial cells
(31). Conversely, it has also been reported that extracellular
adenosine can inhibit the migration of eosinophils (66), mast cells
(67), and microglia/monocytes (68). Furthermore, with other cell
types such as neutrophils, adenosine signaling has been shown to
have the capacity to both induce (28, 29) and inhibit (17, 69) cell
migration depending on the experimental system. Therefore, the
effects of extracellular adenosine on cell migration may not only
vary among cell types, but also depend on the circumstance in
which the AR signaling occurs.
During EAEprogression withinthe CNS,ourresults suggest that
extracellular adenosine may be functioning as a major “danger/
damage” signal (70–72). ATP released from stressed or damaged
cells in the CNS is hydrolyzed into extracellular adenosine (3, 4).
Because the CNS is an immune privileged site (73), we think that
this danger signal is a trigger that promotes lymphocyte infiltration
into the CNS (24). Our results suggest that this extracellular ATP/
adenosine danger signal is modified (ATP hydrolysis to adeno-
sine), interpreted (AR signaling), and executed by the choroid
plexus (lymphocyte entry), which possesses the enzymes (CD39
and CD73) and receptors (A2AAR) required to synthesize and
bind extracellular adenosine and has been shown to be a CNS
entry point for lymphocytes during EAE progression (34–36). Our
bone marrow chimera data provide further evidence of this, as the
lack of the A2AAR on non-bone marrow-derived cells (such as
those comprising the choroid plexus) confers a degree of protec-
tion against lymphocyte infiltration and its subsequent EAE de-
velopment. Similar results are observed during lung injury, where
AR signaling has been shown to induce cell migration to repair
damaged tissue (31).
Extracellular adenosine signaling has also been shown to play
a role in inflammation during hypoxia and ischemia–reperfusion
injuries. Hypoxia that results from ischemia typically leads to
vascular leakage, the accumulation of inflammatory cells, and
elevated serum cytokine levels (74). Additionally, just as hypoxia
can induce inflammation, inflamed tissues often become severely
hypoxic (74). Interestingly, hypoxic conditions have been reported
to promote increases in extracellular adenosine levels by both
blocking adenosine’s uptake into cells (75) and its catalysis into
AMP by adenosine kinase (76). Therefore, hypoxic states promote
ideal conditions for stimulating the A2AAR, which requires high
concentrations of extracellular adenosine to become activated (1).
The increased extracellular adenosine levels during hypoxia have
been associated with tissue protection against inflammation,
which is in part regulated by the induction of netrin-1 that ef-
fectively attenuates neutrophil transmigration (77). Interestingly,
netrin-1 expression has also been found in EAE lesions in rats
(78). However, a direct link between netrin-1 and extracellular
adenosine signaling during EAE has yet to be established.
Overall, it is evident that extracellular adenosine plays a vital
and complex role in the development of EAE. A2AAR signaling,
although important for controlling the magnitude of an inflam-
matory response, is also involved in lymphocyte entry into the
CNS. Additionally, complete ablation of the A2AAR does not
protect mice from developing EAE, whereas A2AAR blockade
with drug treatments directly inhibiting signaling is beneficial in
EAE (24). Furthermore, both acute and chronic relapsing–remit-
ting EAE can be prevented/reversed by the drug methylthio-
adenosine (79, 80), which acts both as an agonist for the A1AR
and an antagonist for the A2AAR (81). Therefore, the data pre-
sented here strongly suggest that since extracellular adenosine and
A2AAR signaling are so highly involved in lymphocyte infiltra-
tion and inflammation in the CNS, therapeutic strategies targeting
extracellular ATP/adenosine metabolism and signaling pathways
may be beneficial in the treatment of many diseases such as MS
that have a major neuroinflammatory component.
The authors have no financial conflicts of interest.
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5722 A2A ADENOSINE RECEPTOR SIGNALING DURING EAE PROGRESSION