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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3859-3867
IMMUNOBIOLOGY
A2A receptor dependent and A2A receptor
independent effects of extracellular adenosine on murine thymocytes in
conditions of adenosine deaminase deficiency
Sergey Apasov,
Jiang-Fan Chen,
Patrick Smith, and
Michail Sitkovsky
From the Biochemistry and Immunopharmacology Section, Laboratory of
Immunology, NIAID, National Institutes of Health, Bethesda, MD; and
Molecular Neurobiology Laboratory, Department of
Neurology, Massachusetts General Hospital, Harvard Medical School,
Charleston, MA.
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Abstract |
Adenosine deaminase (ADA) deficiency causes severe combined
immunodeficiency (SCID) and is accompanied by T-cell depletion and
accumulation of both intracellular and extracellular adenosine (extAdo)
and deoxyadenosine. To better understand the causes of T-cell depletion
in vivo and to discriminate between extracellular and intracellular
effects of exogenously added adenosine in vitro, we investigated
mechanisms of 2 different effects of adenosine on murine thymocytes.
These effects of adenosine include direct induction of apoptosis in
about 6% to 15% thymocytes and inhibition of T-cell receptor
(TCR)-induced activation of the majority of thymocytes with inhibited
ADA. A2A adenosine receptors, but not A2B,
A1, or A3 receptors, are shown to be mostly
responsible for extAdo-triggered signaling (cyclic adenosine
monophosphate [cAMP] accumulation) in murine thymocytes and this
prompted studies of the effects of extAdo on thymocytes from
A2AR gene-deficient mice. It is found that direct apoptotic
effects of extAdo on CD4+CD8+ double
positive (DP) thymocytes are completely accounted for by signaling
through A2AR, with no contribution of intracellular lymphotoxicity or of compensating A2BRs because only
A2AR +/+, but not A2AR  / thymocytes
were susceptible to apoptotic effects of extAdo. Studies of the effects
of cAMP-raising agents support observations of
extAdo/A2AR/cAMP-triggered apoptosis in DP thymocytes. Unexpectedly, the extAdo strongly inhibited TCR-triggered activation of
both A2AR +/+ and A2AR / thymocytes
in the presence of ADA inhibitors. This was confirmed with thymocytes
from ADA gene-deficient mice, suggesting the existence of
A2AR-independent effects of extAdo on thymocytes. The
presented data raises questions about the identity and functional role
of A2AR-expressing thymocytes in T-cell differentiation and
of the role of TCR-antagonizing effects of extAdo in conditions of ADA SCID.
(Blood. 2000;95:3859-3867)
© 2000 by The American Society of Hematology.
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Introduction |
Studies of biochemical mechanisms of intracellular and
extracellular effects of adenosine are important because of its role in
neuromodulation, cardiovascular biology, immune response, and pathogenesis of human diseases. It was understood for a long time that
the accumulation of adenosine in the absence of adenosine deaminase
activity (ADA) is lymphotoxic and causes severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus
and a decrease in the number of peripheral
lymphocytes.1-4
Mostly intracellular mechanisms of ADA SCID were discussed by
implicating the formation of intracellular deoxyadenosine, deoxyATP, and/or S-adenosyl homocysteine, as well as pyrimidine
starvation2-7 as the direct cause of intracellular
lymphotoxicity. The direct demonstration of ability of dATP and
cytochrome c to induce apoptotic program was recently described in
cell-free extracts.8 The "signaling" mechanism of
lymphotoxicity of adenosine and of T-cell depletion during ADA
SCID9,10 is based on the ability of exogenously added
extracellular adenosine (extAdo) to mediate transmembrane signaling
through purinergic receptors on T cells and to cause cell
death.9-18
The interest of immunologists to the effects of adenosine is also
driven by the unusual properties of adenosine, because extAdo promotes
both the thymocyte survival9 and the thymocyte
death.10 Only one other class of biologically active
compounds, glucocorticoids, has been shown to have similar
extracellular adenosine effects on thymocytes, leading to the
consideration of the role of steroid hormones in T-cell
development.19
The effects of extAdo, which include physiologic regulation of blood
supply to the heart,20,21 are considered to be mediated by
G protein-coupled receptors. These receptors were classified as
A1, A2A, A2B, and A3 on
the basis of biochemical, pharmacologic, and molecular biologic
characterization.22,23 It was demonstrated earlier that
A2A receptors (A2ARs) are mainly responsible
for cyclic adenosine monophosphate (cAMP) accumulation in peripheral murine T cells during incubation with extAdo.17
However, despite the importance assigned to the abilities of
A2A adenosine receptors to trigger apoptosis in many cell
types (reviewed in Jacobson et al24), it has not been
conclusively proven that A2A receptor-mediated signaling
does indeed cause apoptosis of thymocytes. Indeed,
apoptotic effects of extracellular adenosine and adenosine
analogs could easily be explained by its transport into cytoplasm,
followed by intracellular mechanisms of cytotoxicity.25-28
To discriminate between the intracellular and extracellular mechanisms
of adenosine-induced cell death and to explore the possibility of
A2AR-transduced signaling being the mechanism of thymocyte
death, we tested the effects of extracellular adenosine with T cells
that do not express A2A adenosine receptors. Cells from
A2A gene-deficient mice were also used to test whether
A2ARs are responsible for extAdo-mediated inhibition of
T-cell receptor (TCR)-triggered signaling in thymocytes in an in vitro
model of ADA deficiency.
The experiments reported here provide the genetic evidence for
signaling and the A2AR-mediated mechanism of
adenosine-induced apoptosis in thymocytes and for thymocyte
subset-specific expression of A2ARs in thymocytes. The
observation of TCR-inhibiting effects of extAdo, even in experiments
with A2AR-deficient thymocytes, suggest that effects of
extAdo on thymocytes in conditions of ADA deficiency involve both
A2AR-dependent and A2AR-independent pathways.
These results may provide a clue as to whether T-cell activation
defects in conditions of ADA deficiency are partially responsible for
blocks in thymocyte differentiation, which, in turn, may lead to severe
peripheral T-cell depletion that is observed in patients with ADA SCID.
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Materials and methods |
Animals
Mice were maintained in a pathogen-free environment at NIH animal
care facilities. Mice were 6 to 10 weeks old, and 2 to 3 animals were
used in each experiment. A2AR gene-deficient mice (/) were generated by gene targeting as
described.40 Littermates were screened for wild type (+/+),
heterozygous (+/), and homozygous (/) mutations
using Southern blot procedures. ADA gene-deficient mice were developed
using a 2-stage genetic engineering transgenic strategy41
to rescue the ADA-deficient fetuses from perinatal lethality. The
ADA-deficient mice were bred and maintained in pathogen-free NIH animal
facilities and screened by both ADA zymograms of blood samples and
Southern blot analysis of tail DNA for genotypes. The measurements of
ADA activity in samples of blood from littermates born to ADA
(+/) heterozygous parents were performed by zymogram analysis41 using G493 agarose gel and a
temperature-controlled electrophoresis chamber made by Innovative
Chemistry (Marshfield, MA). ADA +/+ wild type and ADA (+/)
heterozygous littermates were used as control mice for ADA-deficient
animals. Only ADA +/+ and ADA / littermates were used for
in vitro studies of T-cell activation.
Cells and medium
Thymocytes were isolated from adult thymus ex vivo and incubated in
RPMI-1640 (Biofluids, Rockville, MD), supplemented with 5% dialyzed
fetal calf serum (FCS) (heat inactivated) and 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mmol/L sodium pyruvate, 1 mmol/L HEPES,
nonessential amino acids, and 5 × 10 5 mol/L
2- mercaptoethanol.
Reagents
Adenosine and adenosine analogs were prepared freshly as 100 mmol/L
stock solution with pH adjusted to 7.1 and were purchased either from
Sigma ImmunoChemicals (St Louis, MO) or from RBI (Natick, MA).
Forskolin and DBMX were purchased from Alexis (San Diego, CA).
Dideoxyforskolin and dbcAMP were obtained from Sigma (St Louis, MO).
mAbs were purchased from PharMingen (San Diego, CA).
Analysis of thymocytes
A single-cell suspension of murine thymocytes was isolated by
standard procedures. Cells were washed and incubated at 37°C in a
5% CO2 incubator. Cells
(0.5-1 × 106/mL) were cultured in a total of 0.2 mL
of medium in 96-well plates. Control incubation was performed in
parallel at 4°C at the same cell concentration. Adenosine and
adenosine analogs were added at various concentrations as indicated in
figure legends. After incubation for 16 to 18 hours, or as indicated,
cells were harvested and analyzed by flow cytometry.
Flow cytometric quantitation of live, apoptotic, and dead cells was
performed according to a modified flow cytometry
procedure.29 The assay intended to quantitate spontaneous
thymocyte death and to determine the proportion of cells that were
killed as a result of adenosine-induced cytotoxicity.
Briefly, cells from the short-term culture were gently pipetted and
transferred from 96-well plates (200 mL) directly into polystyrene
tubes (12 × 75 mm; Falcon, Becton Dickinson Labware, Lincoln
Park, NJ), and 200 mL of FACS buffer (phosphate-buffered saline [PBS]
with 2% FCS and 0.05% sodium azide) was added to each sample. Each
sample had equal volume and was analyzed at the same flow rate and for
the standard time (20 seconds) or triplicates. Propidium iodide
solution (1 µg/mL final concentration) was added to each tube for 10 seconds before FACS analysis. Live, dead, and apoptotic cells were
estimated by counting cell numbers in appropriate gates using a
forward/side-scatter dot plot in linear scale and propidium iodide (PI)
staining in log scale.9 Data are presented as a percentage
of surviving cells from total cell input. Statistical analysis of
triplicate sample measurements was performed as described
earlier.9 Standard deviations of triplicate measurements
within the same experiment usually were lower than 1%.
Fluorimetric measurements of apopotosis in cell culture were also
performed using the Annexin V binding assay as described.30 Briefly, 0.6 to 1 × 106 cells from 96-well plates
were resuspended in 100 mL of buffer containing 10 mmol/L HEPES pH 7.3, 150 mmol/L NaCl, 5 mmol/L KCL, 1 mmol/L MgCl2, 1.8 mmol/L
CaCl2, and incubated with 0.3 µg/mL of fluorescein
isothiocyanate (FITC)-conjugated Annexin V and with 5 µg/mL propidium
iodide for 15 minutes. After incubation, samples were diluted 4 times
with buffer containing 1.8 mmol/L CaC12 and analyzed by
flow cytometry. Annexin V-FITC was purchased from Trevingen
(Gaithersburg, MD). Flow cytometry data acquisition and analysis were
performed on FACScan using FACScan research software and CellQuest
programs (Becton Dickinson, San Jose, CA).
Measurements of cyclic adenosine monophosphate
DBA-2 thymuses were harvested, and thymocytes were isolated and
resuspended at 4 × 106 cells/mL in the culture
media (RPMI-1640) at 4°C. Incubations of cells
(4 × 105 thymocytes per assay) with various agents
were performed in 1.5 mL Eppendorf tubes in a final volume of 200 µL,
containing media alone, or adenosine (0 to 125 µmol/L), CGS21680 (0 to 10 µmol/L), and/or ZM241385 (0 to 1 µmol/L). Controls, such as
the complete reaction mix at time zero or no cell mixture, were used
with every experimental set. The reactions were initiated by the
addition of adenosine or adenosine analogs and incubation lasted as
indicated at 37°C in an Eppendorf Thermomixer Model 5436 (Brinkman
Instruments, Westbury, NY). Thymocyte reaction mixtures
were gently resuspended every 5 minutes, and reactions were terminated
by the addition of 25 µL of 1N HCL, followed by freezing
the samples on dry ice. The cAMP levels were determined using a cAMP
enzyme immunoassay (EIA) kit from Amersham (Buckinghamshire, England),
according to manufacturer's instructions. The lower limit of detection
of cAMP detection using the nonacetylation EIA system by Amersham Pharmacia Biotech is 12.5 fmol. Cell
incubations and extractions were performed in the absence of cAMP
phosphodiesterase inhibitors to avoid complications with interpretation
of results, due to the possibility of their effects on adenosine receptors.
Northern blot studies
Northern blot studies were performed with thymocyte clones that were
derived from p53 gene-deficient thymoma, spontaneously formed in p53
gene-deficient mice. These cells were used to provide a clean source of
cells without contaminating thymic epithelial or other cells.
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Results |
A2A (not A1, A3, or
A2B) receptors responsible for extAdo-induced cAMP
accumulation in murine thymocytes
In our earlier studies9,10 and preliminary experiments
(data not shown), we observed the thymocyte death after exposure to
extracellular adenosine both in the presence or absence of adenosine
deaminase inhibitors. The induction of death of thymocytes by
exogeneous adenosine could be explained by both intracellular lymphotoxicity3 and by signaling through adenosine
receptors.9-18 The exposure of thymocytes to adenosine
leads to accumulation of cAMP (Figure 1A),
thereby suggesting the possible signaling mechanism of apoptotic
effects of extAdo. Indeed, the accumulation of intracellular cAMP was
shown to be apoptotic for thymocytes.31
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| Fig 1.
Adenosine-induced accumulation of cAMP is not observed in
mouse thymocytes with inactivated A2AR gene.
Ex vivo thymocytes from wild type mice (A, +/+) or from
A2AR deficient (B,/) mice were incubated for
30 minutes with extracellular adenosine in the presence or absence of
A2AR antagonist ZM241385 and cAMP accumulation was measured
as described in "Materials and methods." Insert in panel B
demonstrates the typical result of Southern blot DNA screening for +/+,
+/, and / A2AR mice.
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Our earlier biochemical studies pointed to A2A receptors as
being mostly responsible for cAMP-accumulation in mouse peripheral T
cells,17,32,33 but it remained to be conclusively proven whether A2A receptors are also responsible for cAMP
accumulation in thymocytes. Experiments in Figures 1 and
2 provide both pharmacologic and genetic
evidence that extAdo-induced cAMP accumulation in murine thymocytes is
accounted for by signaling through A2A receptors. It is
shown that, not only extAdo, but also the selective agonist of
A2A receptors CGS21680 are able to trigger cAMP
accumulation in thymocytes (Figure 2A).
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| Fig 2.
A2AR are responsible for adenosine-induced
accumulation of cAMP in murine thymocytes.
(A,B) Thymocytes from wild type (+/+) but not from A2AR
gene deficient mice (/) respond to exposure to
A2AR agonist CGS21680 by cAMP accumulation. Ex vivo
thymocytes from wild type mice (A, +/+) or from A2AR
deficient (B,/) mice were incubated 30 minutes with
A2AR agonist CGS21680 in the presence of A2AR
antagonist ZM241385, and cAMP accumulation was measured as described in
"Materials and methods." (C) Northern blot analysis of mRNA
expression for A1, A2A, and A3
receptors in mouse thymocytes (lane 1) and thymocyte cell line
established from p53 / mice (lane 2). Samples in lane 3 represent mRNA from brain tissues (positive control for A1
receptor mRNA) and the mastocytoma cell line P815 (positive control for
A3 receptor mRNA).
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Because both A2A and A2B receptors for
extracellular adenosine could be responsible for cAMP accumulation as
observed here, we attempted to discriminate between these receptors by
using selective antagonist of A2A receptor ZM241385. It is
shown in Figures 1A and 2A that ZM241385 does completely block
adenosine-induced accumulation of cAMP, suggesting the involvement of
A2A receptors in the observed effects of adenosine. Further
pharmacologic evidence for the predominant expression of
A2AR was provided by the demonstration of cAMP increases in
thymocytes after incubation with selective agonist of A2AR
adenosine agonist CGS21680 (Figure 2A, B).
The inability of thymocytes to respond to extAdo by cAMP accumulation
in the presence of A2AR antagonists provided strong pharmacologic evidence against the functioning of A2B
receptor, which is the only other known cAMP-inducing receptor for
extAdo (Figures 1A and 2A). Importantly, even much higher
concentrations of adenosine, NECA or CGS21680 (up to 50 µmol/L, data
not shown), were not sufficient to induce cAMP accumulation in
A2AR / thymocytes. Taken together, these data
support the conclusion that observed effects of extAdo are mediated by
A2A receptors.
Studies of messenger RNA (mRNA) expression revealed the strong
expression of A2A receptors in thymocytes, but not of the
other (A3 or A1) extAdo receptors (Figure 2C).
The correct size of mRNA transcripts was detected in mastocytoma cells
or brain tissues, which were used as positive controls for the
expression of A3 and A1 receptors, respectively.
Thus, the above described data suggest that A2A receptors
are responsible for extAdo-induced cAMP accumulation in
thymocytes. The genetic evidence is provided from the observation
that extAdo does not induce cAMP accumulation in
A2AR-deficient thymocytes. Figures 1 and 2 provide
the results of parallel studies of A2AR expressing (+/+)
and A2AR deficient (/) thymocytes from wild type and homozygous A2AR littermates. It is shown in
Figure 1 that, although extAdo did induce cAMP accumulation in wild
type +/+ thymocytes, no cAMP response could be detected in
A2AR-deficient (/) thymocytes (Figure 1B) in
a parallel assay. Similar data were obtained when A2AR
agonist CGS21680 was used with thymocytes from +/+ and /
mice (Figure 2A and B). These data provide genetic evidence that
A2A receptors are the only extAdo receptors responsible for
cAMP response in thymocytes. Because no cAMP accumulation was observed
in thymocytes from A2AR (/) mice, compared
with cells from wild type mice (Figures 1 and 2), these experiments provided biochemical validation of genetic deficiency in
A2AR / mice and demonstrated that
A2ARs are completely responsible for
adenosine-induced accumulation of cAMP in mouse thymocytes.
As described above, the characterization of A2AR
/ mice intended to approach the main goal of this study
and to ask whether adenosine-induced lymphotoxicity and
extAdo-mediated inhibition of TCR-triggered activation of
thymocytes will be observed in A2AR deficient mice.
ExtAdo-induced apoptosis in thymocytes mediated by signaling through
A2A receptors
The design of the experiments to test the effects of
adenosine on ex vivo thymocytes after they were incubated for 16 to 22 hours in vitro was complicated by having the apoptotic effects of added
adenosine superimposed over the background of the already "spontaneously" dying thymocytes. Indeed, at any given moment, the thymus contains several subpopulations of thymocytes in terms of
their "location" in the apoptotic pathway as a result of the processes of thymocyte selection. It was expected that after 16 to 24 hours of in vitro culturing, those cells that have received an
apoptotic selection signal through their TCR molecules will be detected
as "dead" even in the absence of added adenosine or anti-TCR mAb.
Only cells that have not received an apoptotic signal in vivo and are
"intact" at the moment of harvesting provided an opportunity to
be tested for their response in vitro to the addition of extracellular
adenosine and/or of activating anti-CD3 mAb. Accordingly, results of
assays are presented both conventionally (eg, Annexin V vs PI) to
enable the direct enumeration of apoptotic versus necrotic versus dead
cells and as a proportion/number of surviving thymocytes. Incubation of
thymocytes with extracellular adenosine or selective A2AR
agonist CGS21680 (Figures 3,
4, and 5)
causes cell death as demonstrated by using several independent assays.
It is shown that some ex vivo thymocytes die "spontaneously" when
incubated in vitro in the culture media, but the rate of their death is
significantly increased by adding extracellular adenosine.
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| Fig 3.
Comparison of extracellular adenosine, CGS21680 and
anti-CD3 mAb-induced apoptosis and cell death in mouse thymocytes.
Ex vivo thymocytes were incubated 16 hours at 37°C in 5%
CO2 incubator in the presence of adenosine or adenosine
analog CGS21680 or anti-CD3 mAb, and the extent of apoptosis or cell
death was measured by flow cytometry as described in "Materials and
methods." (A) Detection of apoptotic events in thymocytes using
Annexin V assay; (B) comparable proportion of thymocytes is lost after
extAdo-induced and TCR/CD3-induced apoptosis. Cell loss was estimated
by flow cytometry by PI staining of dead cells. (C) Demonstration of
typical experimental gate selection for evaluation of number and
proportion of live (gate R1), apoptotic (gate R2), and dead (gate R3)
after 16 hours of incubation of thymocytes in vitro with CGS21680 as
panel A. The status of cells was studied by flow cytometry after their
staining with propidium iodide (PI) and Annexin V (upper graph) and by
their site versus forward scatter (lower graph).
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| Fig 4.
Demonstration of extracellular adenosine or
CGS21680-induced thymocyte death by PI versus Annexin V staining of
cells.
Ex vivo thymocytes were incubated 16 to 20 hours at 37°C in 5%
CO2 incubator in the presence of adenosine or adenosine
analog CGS21680, and the extent of apoptosis or cell death was measured
by flow cytometry as described in "Materials and methods." (A)
Loss of live cells in the presence of adenosine or CGS21680. (B)
Effects of different concentrations of adenosine and CGS21680 on
thymocytes. CGS21680-induced apoptosis of thymocytes reached plateau at
as low as 0.3 µmol/L.
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| Fig 5.
CD4+CD8+DP thymocytes are
predominantly susceptible to extracellular adenosine-induced apoptosis.
Ex vivo thymocytes were incubated 16 hours at 37°C in 7%
CO2 incubator in the absence (control, A) or presence of
100 µmol/L adenosine (B). Cells were triple stained with anti-CD4,
anti-CD8 mAb and PI, and the extent of cell death was measured by flow
cytometry as described in "Materials and methods." (C)
Adenosine/A2a receptor-mediated signaling targets
CD4interCD8interCD4lowHSAlowCD5high
thymocytes. Thymocytes were incubated 16 hours with A2AR
selective and apoptosis-inducing agonist CGS21680 (1 µmol/L) in the
presence or absence of A2AR antagonist ZM241385 (2 µmol/L) or in the media alone. Cells were then stained with Annexin
V, PI, and different mAb to important surface antigens and the
proportion of cells with a particular surface marker was estimated by
flow cytometry among live cells, which were gated on the basis of
Annexin V and PI staining. (D) Effect of cAMP and cAMP-raising agents
on apoptosis of thymocytes. Cells were incubated with different agents
at concentrations that are indicated and, after 16 hours of incubation,
the proportion of CD4+ versus CD8+ cells among
surviving cells was estimated by flow cytometry. (E) cAMP-raising
agents target for apoptosis mostly CD4+CD8+ DP
thymocytes.
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Three different assays for the detection of cell apoptosis were used in
the described studies of the effects of extracellular nucleotides on
thymocytes, including (i) detection of apoptotic events using Annexin V
assay (Figure 3); (ii) detection of dead cells by both PI and Annexin V
staining (Figure 4); and (iii) detection of dead cells by cell size on
the basis of forward versus side-scatter measurements. Similar data
were obtained in estimation of apoptotic cells in our earlier
studies.10
The analysis of multiple parallel experiments in which extAdo-induced
thymocyte death was compared with the thymocyte response to other
apoptotic stimuli revealed that extAdo or A2AR agonist CGS21680 induced a 6% to 15% cell loss in the 16- to 20-hour assay, which is comparable to the degree of cell death induced by anti-CD3 mAb
in parallel assays (Figure 3A, B). ExtAdo is found to be less potent
than CGS21680 because dose-response studies indicated that much lower
concentrations of CGS21680 were sufficient to accomplish similar levels
of apoptosis and cell loss (Figure 4). This is most likely due to the
degradation of extAdo by ADA in the absence of ADA inhibitors in this
set of experiments.
In the next series of experiments (Figure 5), we attempted to identify
the thymocyte subset, which was susceptible to
A2AR-transduced apoptotic signals. This was performed by
incubating thymocytes with or without adenosine for 18 hours, followed
by the staining of surviving cells by fluoresceinated anti-CD4 or
anti-CD8 mAb to discriminate between
CD4CD8(DN),
CD4+CD8(4+SP),
CD4CD8+(8+SP), and
CD4+CD8+(DP) thymocyte subsets. These
experiments were expected to clarify whether susceptibility to
adenosine-induced apoptosis is spread among all thymocytes subsets or
is subset specific. It is shown that 45.8% of
CD4+CD8+ thymocytes survived 18 hours of
incubation in media alone, whereas only 38.3% did so in the presence
of extAdo (Figure 5A, B). No changes in the other subsets were detected
in this and other (data not shown) experiments. Thus, the extracellular
adenosine induced cell death of mostly DP
CD4+CD8+, but not of
CD4CD8,
CD4+CD8, or
CD4CD8+ thymocytes. Adenosine analog
CGS21680 was as efficient as the adenosine, but at lower concentrations
and a similar proportion of DP thymocytes was eliminated after
incubation of thymocytes in parallel assays. These experiments with
extAdo and selective agonist CGS21680 provided suggestive pharmacologic
evidence to implicate A2A receptors in extAdo-induced
apoptosis, but the relatively high concentrations of adenosine were
required to observe cytotoxic effects of extAdo on thymocytes. The
estimation of 6% to 15% of thymocytes that are susceptible to
adenosine-induced apoptosis and are dying after 16 to 24 hours in vitro
was very consistent and was observed in many independent experiments to
further identify these cells in terms of different surface markers.
Thymocytes were treated with A2AR selective and
apoptosis-inducing agonist CGS21680 (Figure 5C) and, after 16 hours
incubation in the presence or absence of CGS21680 and A2AR
antagonist ZM241385 cells, were stained with different mAbs to
important surface antigens. This was followed by the evaluation of the
loss of cells that express particular surface markers. It is shown that
CGS21680 did induce the cell loss, whereas addition of A2A
receptor antagonist ZM241385 prevented the effect of
CGS21680. The evaluation of relative cell loss among
different markers allowed further description of cells that are
targeted by adenosine/A2A receptor signaling as
CD4interCD8interCD44lowHSAlowCD5high
thymocytes, which represent a transient stage of thymocyte
differentiation toward single positive T cells. Because cAMP-inducing
treatment of cells with adenosine and A2A receptor agonist
CGS21680 was shown to kill some thymocytes, it was important to test
(i) whether different cAMP-raising agents have a different ability to
kill thymocytes and (ii) whether cAMP increases can target equally well
all thymocyte subsets or only DP immature thymocytes. Five different
cAMP level-raising treatments were used to answer these questions. It
is shown in Figure 5D, that the direct activation of adenylate cyclase
by Forskolin or the addition of dbcAMP caused the strongest thymocyte
death (about 30% and 22%, respectively), whereas the inhibitor of
cAMP phosphodiesterase IBMX damaged about 15% of thymocytes. The
triggering of A2A receptors by CGS21680 was more apoptotic
than the triggering of adrenergic receptors by isoproterenol.
Figure 5E shows that mostly CD4+CD8+ DP
thymocytes are susceptible to the apoptotic effects of cAMP and
cAMP-raising agents. It is shown that the incubation of thymocytes with
Forskolin, IBMX, and dbcAMP, but not with the inactive analog
dideoxyforskolin, had decreased proportion of
CD4+CD8+ DP thymocytes among the surviving
cells after 16 hours in vitro incubation.
To conclusively implicate the involvement of A2ARs in the
apoptotic effects of extAdo, we used thymocytes from wild type (+/+) and A2AR deficient (/) mice. Figure
6 shows the results of a representative
experiment, in which ex vivo thymocytes were tested in parallel for the
ability of extAdo to trigger cAMP accumulation and apoptosis. In
agreement with other experiments, CGS21680 and extAdo did induce the
cell loss of thymocytes from wild type (+/+) mice, but in none of the
experiments was apoptosis detected in thymocytes from A2AR
deficient (/) mice, providing genetic evidence of the
necessity of A2AR for the transmission of extAdo-initiated apoptotic signal. Because it is shown that the disruption of the A2AR gene results in the elimination of extAdo and
CGS21680-induced signaling in thymocytes, these experiments
conclusively prove the direct involvement of A2A receptors
in adenosine-induced apoptosis of thymocytes.
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| Fig 6.
A2AR deficient thymocytes are not susceptible
to extracellular adenosine and CGS21680-induced apoptosis and cell
death.
Ex vivo thymocytes from wild type mice (A2AR+/+) or from
A2AR deficient (A2AR/) mice were
incubated 20 hours at 37°C in 5% CO2 incubator in the
presence of adenosine (100 µmol/L) or adenosine analog CGS21680 (1 µmol/L), and the extent of cell death was measured by flow cytometry
as described in "Materials and methods."
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Although only about 10% to 15% of thymocytes express
apoptosis-inducing A2ARs (Figures 3 through 6), the
possibility existed that many more thymocytes do express
A2ARs with less efficient coupling to Gs
protein adenylate cyclasecAMP pathway. Accordingly, it was still possible that some thymocytes do respond to extAdo by cAMP
accumulation, which is not followed by apoptosis, but does inhibit
TCR-signaling in thymocytes. This A2AR-mediated mechanism was first to be considered and tested in explanation of an unusual ability of extAdo to block TCR-triggered but not the Fas- or
CD45-triggered apoptosis in thymocytes.9
A2A adenosine receptors not responsible for the
extAdo-mediated inhibition of TCR-induced thymocytes activation and
apoptosis in conditions of inhibited ADA
We have shown recently that the up-regulation of both CD69 and CD25
(IL-2 receptor) activation markers was inhibited by extAdo in the
presence of ADA inhibitor.9 The inhibition of TCR-induced thymocyte activation could be observed only in conditions of inhibited ADA, because neither adenosine alone (data not shown) nor ADA inhibitor, coformycin, alone were having an effect on thymocytes in the
16- to 24-hour activation assays presented here (Figure 7) and in recently published
experiments.9 However, it was important to exclude the
possibility that some unknown drug interaction or synergy in the
effects of adenosine and coformycin or
erythro-9(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA) could be the
reason for the inhibition of thymocyte activation by adenosine in the
presence of ADA.
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| Fig 7.
Inhibition of TCR-induced up-regulation of T-cell
activation marker CD69 in both A2A receptor-expressing
(+/+) and A2A receptor-deficient (/) thymocytes
by extracellular adenosine in the presence of ADA inhibitor.
Ex vivo thymocytes from A2A receptor-expressing (+/+) and
A2A receptor-deficient (/) mice were
incubated 16 hours at 37°C in 5% CO2 incubator with
immobilized anti-TCR/CD3 mAb (20 µg/mL) in the presence of
extracellular adenosine (100 µmol/L) and adenosine deaminase
inhibitor coformycin (cof, 10 µmol/L) as indicated on the graphs.
Cells were harvested and stained with Annexin V, PI (to set gate for
nonapoptotic cells), and anti-CD69-PE mAb to estimate the proportion of
activated cells. The extent of up-regulation of CD69 was measured by
flow cytometry as described in "Materials and methods."
|
|
To address this issue, we took advantage of the availability of
ADA-deficient mice41 and tested the effect of adenosine on
TCR-induced up-regulation of CD69 activation marker in the presence or
absence of exogeneously added adenosine. It is shown that anti-CD3 mAb
triggered up-regulation of CD69 in more than 60% of ADA-deficient T
cells and about 90% of ADA-expressing T cells. However, the addition
of adenosine resulted in virtually complete inhibition of TCR-triggered
activation only of ADA-deficient T cells, but not of T cells from
ADA-expressing littermates (Figure 8).
Thus, these experiments provided genetic evidence for the ability of
adenosine to block the activation of T cells by TCR cross-linking.
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| Fig 8.
Adenosine inhibits in vitro TCR-triggered activation of
ADA-deficient T cells in the absence of ADA inhibitors.
Ex vivo spleen T cells from ADA / and ADA +/+ littermates
were incubated in 96-well plates with immobilized anti-CD3 mAb (5 µg/mL mAb) or with serum-free, ADA-free media alone (0 µg/mL mAb)
in the presence or absence of adenosine (100 µmol/L), and 16 hours
later, the TCR-triggered up-regulation of T-cell activation markers
CD69 in T cells was evaluated by flow cytometry.
|
|
It was most straightforward to test whether the elimination of
A2A receptors would lead to the loss of susceptibility of
thymocytes to these effects of extAdo. To this end, the ex vivo
thymocytes were incubated with immobilized anti-CD3 mAb for 16 hours
and then the cells were stained with both Annexin V and anti-CD69 mAb
(Figure 7). Analysis of gate with Annexin V negative (live, nonapoptotic) cells reveals that the TCR-induced up-regulation of
activation marker CD69 on thymocytes from wild type
(A2AR+/+) mice was dramatically inhibited by extAdo.
Remarkably, the same effects of extAdo were observed with thymocytes
from A2AR/ mice (Figure 7) in a parallel
experiment using littermates of A2AR+/+ and
A2AR/mice. Thus, it appears that
A2ARs are not required for the inhibition of TCR-triggered
activation of thymocytes by extAdo. It remains to be conclusively
determined whether such TCR-antagonizing effects are mediated by a
novel adenosine receptor or by non-cAMP-mediated signaling through
A2B receptors or by intracellular toxic effects of
exogeneously added extAdo.
Taken together, the results of the experiments presented here suggest
that sustained increases in concentrations of extAdo in conditions of
ADA deficiency may signal to T cells in normal extracellular
environment and cause elimination of about 6% to 15%
A2AR-expressing thymocytes. Moreover, the adenosine is able to block the TCR-triggered activation even in thymocytes that lack
expressed A2A receptors.
| |
Discussion |
The effects of extracellular adenosine on lymphocytes that were
described earlier9,10,17,33,34 and in this report, put this
physiologically abundant compound among a very small and select group
of molecules that have a potential to modulate normal immune response
in vivo and to cause immune pathologies, including T-cell depletion and
T-cell defects observed in conditions of ADA SCID.9
The attractive features of the described effects of adenosine on
thymocytes include its paradoxical abilities to induce the apoptotic
death of a small subset of CD4+CD8+ thymocytes
(Figures 3 through 5) and to inhibit the TCR-triggered activation and
TCR-induced apoptosis (Figures 7 through 9) of the majority of
CD4+CD8+ DP thymocytes. The experiments with
A2AR / thymocytes described here provide
strong evidence that the apoptosis-inducing effects of extAdo are
mediated by A2A receptors (Figures 3 through 5) and suggest
that the TCR-antagonizing effects of extAdo are A2A receptor independent (Figures 7 through 9).
These data provide the formal proof of earlier biochemical and
pharmacologic findings that, of all 4 classes of adenosine receptors,
the A2ARs are responsible for cAMP accumulation in murine17 and human32 peripheral T-lymphocytes
and in murine thymocytes (Figures 1 and 2). The experiments described
in Figures 3 and 4 suggest that the increases in extracellular
adenosine levels in the conditions of ADA deficiency may result in
different effects on murine thymocytes. These effects include both
cytotoxic and cytoprotective effects of extAdo as well as the
intracellular toxicity of adenosine catabolites after translocation of
extAdo by nucleoside transporters. The adenosine-triggered
lymphotoxicity, in turn, could be explained by both intracellular
toxicity and by extracellular signaling through adenosine receptor or
by both mechanisms, and it was important to evaluate the relative
contribution of these 2 different mechanisms.
Strong lymphotoxicity of intracellular adenosine, especially in the
conditions of deficiency of ADA, was known for several decades,2-7 but it has been difficult to estimate the
contribution of extracellular signaling into the overall effects of
adenosine. The attempts to interpret apoptotic effects of so-called
"slow-hydrolyzable" analogs of adenosine as supporting the
"signaling" mechanism of apoptosis10,13-17 were
weakened by observations of these analogs being intracellularly
toxic.25-28
Findings of A2AR being responsible for adenosine-induced
cAMP accumulation in murine T cells17 and thymocytes
(Figures 1 and 2) and the availability of A2AR-deficient
mice provided a valuable opportunity to directly test the involvement
of adenosine receptors in adenosine-triggered cytotoxicity. It is shown
(Figure 6) that the disruption of the gene for A2AR results
in the elimination of adenosine-induced apoptosis of thymocytes,
thereby confirming that there are no functional A2AR
receptors in these mice and that their loss was not compensated by
expression of other (eg, A2B) adenosine receptors in
A2AR / mice. These data both provide the
evidence of extAdo/A2A receptor-mediated thymocyte death
and validate the use of adenosine analog CGS21680 as a selective
agonist of A2AR and as a selective apoptosis-inducing drug
in thymocytes.
Because the apoptotic effects of exogeneous adenosine and adenosine
receptor agonist CGS21680 on thymocytes are completely accounted for by
signaling through A2ARs, these data demonstrate that
intracellular toxicity of adenosine and adenosine catabolites was not a
contributing factor in tested conditions of incubations. Interestingly,
the A2AR-mediated apoptotic effects of extracellular adenosine affect only 6% to 15% of normal thymocytes. The relative small size of this subset may not, however, reflect its significance in
the overall process of the positive and negative selection of
thymocytes, and experiments are underway to determine the functional consequences of elimination of this subset by extAdo or adenosine analogs treatment with CGS21680.
The differentiation of thymocytes involves several stages, including
transitions from CD4CD8(DN) to
CD4+CD8+(DP) to
CD4+CD8(SP) and
CD4CD8+(SP) thymocytes that mature into
peripheral CD8+ T cells or CT4+ T cells
(reviewed by Robey and Fowlkes35 and Cibotti et
al36). It is interesting that mostly immature
CD4+CD8+(DP) thymocytes are susceptible to
effects of cAMP-raising agents (Figure 5), thereby reflecting
differences in intracellular signaling pathways between immature double
and single positive thymocytes. This, in turn, suggests that the
A2AR-mediated signaling could be involved in T-cell
selection processes that use apoptotic and intracellular cAMP-pathway
by selectively triggering Gs-coupled A2ARs with a subset-specific expression. These observations
support the view that expression of apoptotic-signal transducing
A2ARs is differentiation dependent. The exact
phenomenologic and functional identity of DP cells that are susceptible
to A2AR-mediated apoptosis is yet to be determined in
future studies using different functional and surface markers of T
cells, but data shown in Figure 5C suggest that this
CD4interCD8interCD44lowCD5hi
subset of DP thymocytes represents a transitional stage toward their
maturation. The use of A2AR / mice provided
genetic evidence for selectivity of a well-known A2AR
agonist, CGS21680 in elimination of a numerically minor (10%-15%)
individual subset of thymocytes (Figure 5C).
Especially dramatic are the effects of extAdo on thymocytes
with inhibited ADA (Figures 7 through 9). The addition of extAdo to
TCR-activated thymocytes prevents the TCR-induced up-regulation of cell
surface markers (Figure 8) and rescues them from TCR-induced apoptosis
(Figures 7 and 9). This "apoptosis rescue" assay is of special
value for studies and interpretation of the effects of adenosine on
thymocytes in conditions of ADA deficiency, because, in contrast to
direct apoptotic effects of extAdo (Figures 3 through 6), the
TCR-antagonizing effects of the adenosine require the inhibited ADA,
suggesting that there is a need in sustained concentrations of added
extAdo to observe protection of TCR-activated thymocytes from apoptosis.
The requirement in the low activity of ADA to observe the
effects of extAdo on the majority of all TCR-activated thymocytes leads
to a view that, if this mechanism does function in vivo in normal
animals, it could be most likely operational only in the
microenvironment with low ADA activity, which was described in the
studies of distribution of ADA activities in the thymus.7 This requirement is satisfied in the conditions of ADA SCID. Thus, the
described experiments revealed 2 novel types of signaling effects of
extracellular adenosine on thymocytes with one of them being unique for
conditions of ADA deficiency.
First, in the presence of normal ADA activity, the addition
of adenosine causes the elimination of about 10% of DP
CD4+CD8+ thymocytes (Figures 3 through 5). This
observation has the implication for both in vitro studies (eg, by
enabling the selective elimination of A2AR-expressing
subset in the investigation of normal thymocytes differentiation) and
for the understanding of the mechanisms of ADA deficiency (Figure
9). Second, in the absence of ADA activity, the TCR-triggered activation of large proportion of TCR-triggered thymocytes could be inhibited by adenosine (Figures 7 and 9), thereby
interfering with the processes that are needed for the survival of
thymocytes. As a result of such effects of adenosine, the thymocytes
that would normally be selected for further maturation could be dying
from "neglect" in an ADA-deficient environment.
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| Fig 9.
Adenosine acting through A2A receptors may
directly trigger the apoptosis of murine thymocytes and inhibit
TCR-triggered activation by both A2A receptor-dependent and
A2A receptor-independent mechanisms.
Receptor X is postulated to stimulate yet-to-be-determined TCR
inhibiting pathway only in conditions of inhibited ADA. These effects
of adenosine on TCR-triggered activation are not affected by inhibition
of adenosine transport.
|
|
The molecular mechanisms of extAdo inhibition of TCR-signaling are not
yet well understood. Originally, we expected that these effects could
be mediated by the A2A receptors, but this was not the
case, because the A2AR / thymocytes were as
susceptible to the inhibition of TCR-triggered activation of thymocytes
by extAdo as the wild type, A2AR-expressing thymocytes
(Figures 7 and 8). It remains to be established whether
A2ARs receptors for extAdo in thymocytes are responsible
for these TCR-antagonizing effects of extAdo, but the existing data are
consistent with the model (Figure 9) in which about 10% to 15% of DP
thymocytes are targets for A2AR-mediated signaling, which
can cause their apoptosis. The unexpected ability of
adenosine to block activation, even of A2AR-deficient
thymocytes and of A3 receptor-deficient thymocytes (data
not shown) could be reconciled by postulating the existence of a novel
adenosine receptor X. This receptor is predicted to have much lower
affinity for adenosine than known adenosine receptors because much
higher levels of adenosine (most likely achieved only on conditions of
inhibited ADA) are required to observe its effect. These properties of
the receptor may help in designing strategies of its expression cloning.
The possible clue as to what kind of signaling may be involved in
TCR-antagonizing effects of extAdo is provided by studies of
neuroprotective and cardioprotective37 effects of adenosine since it was shown that adenosine agonists were having cytoprotective effects when added during ischemia and epilepsy.38,39
Neuroprotective and cardioprotective effects of extracellular adenosine
were explained by short-term functional antagonism between the
adenosine receptor and other receptors or channels-mediated signaling.20 The alternative explanation of A2B
adenosine receptor functioning through non-cAMP-based mechanism still
remains to be investigated.
Independent of the exact molecular mechanism of the effects of
adenosine in conditions of ADA deficiency, the ability of adenosine to
directly kill 10% to 15% of thymocytes and to block TCR-induced activation in a majority of thymocytes should be taken into account in
explanations of pathogenesis of ADA SCID.
| |
Acknowledgments |
We thank Dr Diana L. Marquardt (University of California, San Diego)
for A1 cDNAs, Dr David Grandy (Vollum Institute for
Advanced Biomedical Studies, Portland, OR) for A3 cDNA,
Brenda Rae Marshall for editorial help, and Shirley Starnes for help in
preparation of the manuscript.
| |
Footnotes |
Submitted September 27, 1999; accepted February 14, 2000.
Reprints: M. Sitkovsky, Biochemistry and Immunopharmacology
Section, Laboratory of Immunology, NIAID, National Institutes of
Health, Bethesda, MD 20982-1892.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction