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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 519-527
HEMATOPOIESIS
Adenosine receptor occupancy suppresses chemoattractant-induced
phospholipase D activity by diminishing membrane recruitment of small
GTPases
Nathalie Thibault,
Danielle Harbour,
Pierre Borgeat,
Paul H. Naccache, and
Sylvain G. Bourgoin
From the MRC Group on the Molecular Mechanisms of Inflammation,
Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier
Universitaire de Québec, Pavillon C.H.U.L. et Université
Laval, Départements de Physiologie et Médecine,
Québec, Canada.
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Abstract |
Adenosine (Ado) is an important autocrine modulator of neutrophil
functions. In this study, we determined the effects of endogenous Ado
on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in
neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or
the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor
agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore,
inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC
or CGS15943. The removal of Ado by ADA or the blockade of its action by
CSC or CGS15943, markedly increased the membrane recruitment of
cytosolic protein kinase C (PKC), RhoA, and ADP-ribosylation
factor (ARF) in response to fMLP. As shown for PLD activity, the
stimulatory effect of Ado receptor antagonists on PLD cofactors
translocation was abolished by a pretreatment of the cells with ADA.
Moreover, the membrane translocation of both PKC, RhoA, and ARF in
response to fMLP was attenuated by CGS21680 and this effect of the A2a
receptor agonist was antagonized by CSC or CGS15943. These data
demonstrate that Ado released by neutrophils in the extracellular
milieu inhibits PLD activation by blocking membrane association of ARF,
RhoA, and PKC through Ado A2a receptor occupancy.
(Blood. 2000;95:519-527)
© 2000 by The American Society of Hematology.
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Introduction |
Adenosine (Ado)1 is an ubiquitous
nucleotide autacoid that mediates a large spectrum of biologic effects
by activation of at least 4 cell surface receptors designated as A1,
A2a, A2b, and A3.1 Ado is well-known to modulate the
functional responsiveness of inflammatory cells including
neutrophils.2 Several sources have reported that Ado acts
on neutrophils by binding 2 different classes of cell surface
receptors, the A1 and A2 adenosine receptor subtypes. Acting through A1
receptors, Ado has been reported to increase chemotaxis,3
phagocytosis of immune complexes,4 adherence to
endothelium,5 and to enhance the expression of  2
integrins on neutrophil membranes.6,7 In contrast,
occupancy of A2a receptors has opposite effects, decreasing
phagocytosis,4 adherence to endothelial cells,5
leukotriene synthesis,8 and fMet-Leu-Phe(fMLP)-stimulated
respiratory burst.3,9,10 Neutrophils express the A2a
receptor and the order of potency of Ado analogues provides
pharmacologic evidence that the A2a subtype mediates these inhibitory
effects.11,12 Thus, Ado is viewed as a potent endogenous
anti-inflammatory agent. Different drugs including
methotrexate13,14 and sulfasalazine15 cause Ado
accumulation at inflamed sites and inhibition of neutrophil migration
through occupancy of A2a receptors. A2 receptors are linked to
heterotrimeric Gs proteins and regulate adenylyl cyclase activation,
whereas A1 receptors are coupled to Gi proteins.2 The
occupancy of A2a receptors stimulated an increase in the levels of
intracellular cAMP that could only be measured after stimulation of
neutrophils with fMLP.16 However, there is little
experimental support for a role of cAMP as the second messenger
mediating the inhibition of neutrophil functions by Ado.2
Phospholipase D (PLD) activities are present in many cell types,
including human granulocytes.17 PLD catalyzes the
hydrolysis of phosphatidylcholine to yield phosphatidic acid, which is
further metabolized to diacylglycerol by lipid phosphate
phosphohydrolases.18 PLD-derived second messengers promote
the respiratory burst,19 secretion,20 the
number of cell surface 2 integrins,21 and are required
for phagocytosis.22 Two PLD isoforms have recently been
cloned, PLD123 and PLD2.24,25 PLD1 has a low
basal activity but is stimulated several times by ADP-ribosylation
factors (ARFs),26,27 RhoA,28 and
PKC ,29-31 in presence of phosphatidylinositol
4,5-bisphosphate (PIP2). In contrast, PLD2 shows a high basal activity
but PLD cofactors other than PIP2 are not required for its
activity.24,25 In granulocytes, PLD is stimulated by
RhoA,32 ARF,26,33 and PKC.34
Furthermore, neutrophil activation with chemotactic peptides promotes
the recruitment of RhoA, ARF, and PKC to membranes, which in turn
stimulates PLD activity. These observations together with
immunoblotting analyses of PLD isoforms led to the conclusion that PLD1
is expressed and activated in neutrophils after their stimulation with
chemotactic peptides.35,36
The effects of A2a receptor occupancy on fMLP-induced intracellular
signals are still incompletely characterized.2 A2a receptor
occupancy does not affect Ca++ mobilization from
intracellular stores,37 phospholipase C-mediated inositol
1,4,5-triphosphate formation,38 and the rapid rise in the
levels of diacylgylcerol stimulated by fMLP.16 However, it
diminishes Ca++ influx,39 and the late and
sustained accumulation of diacylglycerol.16 Because the
sustained generation of diacylgylcerol induced by fMLP is secondary to
dephosphorylation of PLD-derived phosphatidic acid,40 it is
possible that Ado modulates neutrophil functions by interfering with
the PLD signaling pathway. In this study, we examined the role of
endogenous Ado removal and of Ado analogues on fMLP-stimulated PLD
activity. We found that pretreatment of the cell suspensions with Ado
deaminase (ADA) or A2a receptor antagonists before stimulation with
fMLP markedly increased PLD activity, whereas A2a receptor agonists
suppressed PLD activity. The inhibitory effects of A2a receptor
agonists on fMLP-induced PLD activity were reversed by CSC, a specific
A2a receptor antagonist. Furthermore, we demonstrated that the
occupancy of Ado A2a receptors interferes with PLD activation by
diminishing the membrane recruitment of the PLD1 activators, ARF, RhoA,
and PKC.
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Materials and methods |
Materials
2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxiamido
adenosine hydrochloride (CGS21680), 8-(3-chlorostyryl)caffeine (CSC), 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX), 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine
(CGS15943), 2-chloro-N-cyclopentyladenosine (CCPA),
5'-N-ethylcarboxamidoadenosine (NECA) were from RBI (Natick, MA).
Dextran T-500 and Ficoll-Paque were purchased from Pharmacia Biotech
(Dorval, Québec, Canada). Anti-PKC and anti-RhoA antibodies
were obtained from Santa Cruz (Santa Cruz, CA). The polyclonal
anti-ARF1 antibody was described in previous studies.33
ADA, di-isopropylfluorophosphate (DFP), fMLP, and all other reagents
were from Sigma-Aldrich Canada (Oakville, Ontario, Canada). ADA was
dialyzed against 0.9% NaCl prior use to remove ammonium salt.
Neutrophil purification
Venous blood was collected from healthy adult volunteers in
isocitrate anticoagulant solution. Neutrophils were separated as
described previously.35 Leukocytes were obtained after
erythrocyte sedimentation in 2% Dextran T-500 and by centrifugation on
Ficoll-Paque cushions. Contaminating erythrocytes in the neutrophil
pellets were removed by a 20-second hypotonic lysis in water.
Neutrophils were resuspended in Hanks' balanced salt solution (HBSS),
pH 7.4, containing 0.8 mmol/L Ca++ but no Mg2+
to reduce nonspecific aggregation of the cells.
Analysis of Ado
Neutrophils were washed in HBSS and cell suspensions
(2 × 107/mL) were incubated at 37°C. After
selected times, incubations were stopped by adding 100 µL of 22% TCA
(2% final). NECA (10 ng/sample) was added as an internal standard and
the denatured cell suspensions stored at  20°C for at least
30 minutes. Denatured samples were centrifuged at 2000g for 10 minutes and Ado was extracted from supernatants with Sep Pak Cartridges
as described previously.41 The samples were analyzed by
liquid chromatography-mass spectrometry with nebulizer-assisted
electrospray ionization in the positive mode and by monitoring the
transitions m/z 309 and m/z 268 of protonated parental ions to m/z 136 of protonated adenine, corresponding to the loss of the carbohydrate
moieties from NECA and Ado.41 Ado was quantitated by
extrapolating the measured Ado/NECA ratio on a calibration curve
generated from standard solutions containing NECA (1 ng) and Ado (0-4 ng).
Phospholipase D measurements
Neutrophils were prelabeled with
1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 cells) for 90 minutes as described
previously.35 The cells were then washed and resuspended at
8 × 106 cells/mL. Samples of the cell suspensions
(0.5 mL) were preincubated at 37°C for 5 minutes and pretreated
with 10 µmol/L cytochalasin B (CB) in the presence or the absence of
ADA or of Ado analogues for 5 minutes before stimulation. Ethanol
(final concentration 1.0% vol/vol) was added immediately preceding the
addition of fMLP (10-7 mol/L, 10 minutes). Where indicated,
neutrophils were stimulated with monosodium urate (MSU) crystals
(3 mg/mL, 15 minutes) but without CB. The incubations were
stopped by adding 1.8 mL cold chloroform/methanol/HCl (50:100:1,
vol/vol/vol) and unlabeled phosphatidylethanol (PEt) as a standard. The
lipids were extracted, dried under nitrogen, and spotted on prewashed
silica gel 60 thin-layer chromatographic (TLC) plates. PEt was
separated from the other lipids with the solvent mixture
chloroform/methanol/acetic acid (65:15:2, vol/vol/vol). Lipids were
visualized by Coomassie Brilliant Blue staining and the different lipid
classes were scraped off the plates. Radioactivity in PEt was monitored
by liquid scintillation counting, and the results corrected for
background radioactivity and quenching.
Translocation assays
Neutrophils (4 × 107 cells/mL) were treated with
1.1 mmol/L DFP for 30 minutes at 24°C. The cell suspensions were
centrifuged and resuspended in HBSS (107 cells/mL). The
cells were preheated 5 minutes at 37°C, preincubated at 37°C
for 5 minutes in the presence of 10 µmol/L CB with either the
indicated concentrations of ADA, of Ado analogues or an equal volume of
the vehicles. Cells were stimulated with 10-7 mol/L fMLP
for 2 minutes, incubations were stopped by diluting the cells 1:5 with
ice cold HBSS, and the samples processed as described
previously.35 Briefly, cell suspensions were centrifuged as
indicated and resuspended at 1.6 × 107 cells/mL in
ice cold KCl-HEPES relaxation buffer (100 mmol/L KCl, 50 mmol/L HEPES, 5 mmol/L NaCl, 1 mmol/L MgCl2, 0.5 mmol/L EGTA, 2.5 µg/mL aprotinin, 2.5 µg/mL leupeptin, and 2.5 mmol/L PMSF, pH 7.2). Cell suspensions were sonicated 2 × 20 seconds and centrifuged 7 minutes at 700g. Unbroken cells and nuclei
were discarded and the supernatants ultracentrifuged at
180 000g for 45 minutes. Membrane pellets were washed once and
resuspended in a small volume of buffer A containing 0.25 mol/L
Na2HPO4, 0.3 mol/L NaCl, 2.5% sodium dodecyl
sulfate (SDS), 2.5 µg/mL aprotinin, 2.5 µg/mL leupeptin,
and 2.5 mmol/L PMSF and samples were assayed for protein content.
Protein samples (30-60 µg) were resolved on a 12% SDS-PAGE and
transferred to Immobilon PVDF membranes (Millipore Corporation,
Bedford, MA). Immunoblotting was performed with the anti-ARF (1/3500),
anti-RhoA (1/1000), or anti-PKC (1/5000) antibodies and proteins
were revealed with the ECL detection system.
Statistics
Data are expressed as means ± SD. Data were analyzed with the
Student paired t test (2-tailed) to determine the level of
significance, *P < .05 and **P < .01, between
the treated samples and the appropriate controls.
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Results |
Effect of ADA on fMLP-induced PLD activity and recruitment of
PKC, RhoA, and ARF to membranes
Engagement of Ado receptors has been reported to reduce the
generation of the second, slow phase of diacylglycerol accumulation in
fMLP-activated neutrophils.16 Although this late and
sustained inhibition of diacylglycerol generation has not been fully
characterized, it is now accepted that the bulk of diacylglycerol is
produced by the PLD and the lipid phosphate phosphohydrolase
pathways.42 Thus, we first evaluated the effects of
extracellular Ado removal by ADA on fMLP-induced PLD activity. PLD
activity was assayed by measuring the levels of [3H]PEt
formed in presence of 1% ethanol. As shown in Figure
1A, ethanol-treated but otherwise
unstimulated neutrophils produced little [3H]PEt
(0.031% ± 0.01% of total counts). Moreover, the addition of 0.1 U/mL ADA to neutrophil suspensions had no effect on basal PLD activity
(0.042% ± 0.01% of total counts, data not shown). The amount of
[3H]PEt formed reached 2.4% ± 0.3% of total
counts after 10 minutes' stimulation with fMLP. The removal of
extracellular Ado by ADA further increased the levels of
[3H]PEt formed in response to fMLP stimulation by
2.03- ± 0.23-fold (P < .01, n = 6) compared with
cells stimulated in the absence of ADA. Although both fMLP and MSU
crystals stimulated PLD activity in neutrophils, we previously
demonstrated that these 2 agonists act through different signal
transduction pathways or PLD isoforms.35 To determine
whether the effects of Ado receptor occupancy are specific to the
activation of PLD by fMLP, we examined the effects of ADA on MSU
crystal-induced [3H]PEt formation. As illustrated in
Figure 1B, the addition of ADA to cell suspensions was without effect
on MSU crystal-induced [3H]PEt formation.
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| Fig 1.
Effect of ADA on fMLP- and MSU-crystals-induced PLD
activity.
Neutrophils labeled with
1-O-[3H]alkyl-2-lysophosphatidylcholine were prewarmed at
37°C for 5 minutes. (A) The cell suspensions were pretreated with
10 µmol/L CB and incubated for an additional 5 minutes in the absence
or presence of 0.1 U/mL ADA. Cells were stimulated with 0.1 µmol/L
fMLP in the presence of 1% ethanol for 10 minutes. The amount of
radioactivity incorporated into PEt was measured as described in
"Materials and Methods." The levels of [3H]PEt
formed are expressed as the percentage of the radioactivity in the
total lipid extracts. The data are the means ± SEM of 6 separate
experiments. (B) Cell suspensions were stimulated with 3 mg/mL MSU
crystals in the presence of 1% ethanol for 15 minutes, without CB. The
data are the means ± SEM of 4 separate experiments.
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Because previous studies have demonstrated that the PLD1 isoform
expressed in human neutrophils is sensitive to activation by PKC,
RhoA, and ARF1,35,36 we conducted experiments to assess whether extracellular Ado interferes with the fMLP-induced
translocation of PLD cofactors. The amounts of PKC, RhoA, and ARF
associated with membranes obtained from control and fMLP-stimulated
neutrophils in the presence or absence of ADA were analyzed by
immunoblotting. As shown in Figure 2, low
basal levels of membrane-associated ARF and RhoA (but not of PKC)
were detected in unstimulated neutrophils. The addition of 0.1 U/mL ADA
to remove Ado had no effect on the basal membrane levels of ARF, RhoA,
and PKC as estimated by immunoblotting (data not shown). In
comparison, when neutrophils were stimulated with fMLP for 2 minutes,
there was a detectable increase in the amount of membrane-associated
ARF and RhoA. Furthermore, PKC was also recovered in membrane
fractions derived from fMLP-stimulated cells. Neutrophil pretreatment
with ADA before stimulation with 0.1 µmol/L fMLP resulted in a
significant enhancement of ARF, RhoA, and PKC translocation,
compared with cells stimulated in the absence of ADA. In contrast to
fMLP stimulation, MSU crystal-induced translocation of ARF and PKC
but not of RhoA to membranes (Figure 2A). When neutrophils were
stimulated with MSU crystals in the presence of 0.1 U/mL ADA, there was
a little increase in the amount of membrane-associated ARF but not of
RhoA and PKC. Taken together the data from Figures 1 and 2 indicate
that endogenous Ado accumulating in neutrophil suspensions exerts
suppressive effects on fMLP but not MSU- crystal-induced PLD activity.
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| Fig 2.
Effect of ADA on fMLP-stimulated translocation of PKC,
RhoA, and ARF1 to membranes.
Unlabeled neutrophils were prewarmed 5 minutes at 37°C. (A) 10 µmol/L CB was added and the cell suspension incubated for an
additional 5 minutes in the presence or absence of 0.1 U/mL ADA.
Neutrophils were stimulated with 0.1 µmol/L fMLP for 2 minutes. (B)
Cell suspensions were stimulated with 3 mg/mL MSU crystals for 15 minutes, without CB. Incubations were stopped and neutrophil membranes
prepared as described in "Materials and Methods." The samples
were analyzed for PKC, RhoA, and ARF1 by immunoblotting. The data
are from 1 experiment representative of 3 similar experiments.
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We investigated Ado accumulation in neutrophil suspensions using
experimental conditions similar to those indicated for the PLD assay.
The cells were resuspended in fresh HBSS and incubated at 37°C for
up to 45 minutes. At selected times, samples were removed for Ado
measurements. As shown in Figure 3, Ado
accumulated in cell suspensions in a time-dependent manner. The
concentrations of Ado increased from 20 nmol/L to up 120 nmol/L after
45 minutes incubation in HBSS. ADA (0.001 U/mL) had no effect on Ado
accumulation in neutrophil suspensions. However, the addition of 0.01 and 0.1 U/mL ADA after 13 minutes incubation of the cells prevented Ado accumulation in the incubation media and reduced its extracellular concentrations below 30 and 15 nmol/L throughout the experiments, respectively. The accumulation of Ado in neutrophil suspensions was
also dependent on the cell concentration, and their stimulation with
fMLP did not impact on the levels of extracellular Ado (not shown and
Krump et al41).
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| Fig 3.
Time course of Ado accumulation in neutrophil
suspensions.
Neutrophils were resuspended in HBSS at the concentration of
2 × 107 cells/mL and incubated at 37°C for 13 minutes before the addition of 0.001, 0.01, and 0.1 U/mL ADA. At
selected times, aliquots (1 mL) of the cell suspensions were denatured
and processed for measurement of Ado content by liquid
chromatography-mass spectrometry. The data are the means ± SEM of
triplicate incubations from 1 experiment representative of 3 similar
experiments.
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Occupancy of Ado A2a receptors modulates fMLP-stimulated PLD
activity
Neutrophils express Ado A1 and A2 receptors that respectively
modulate positively or negatively their functional
responses.2 To determine which receptor type is responsible
for suppressing neutrophil PLD activity, we studied the effects of 3 Ado receptor agonists, NECA, CGS21680, and CCPA. These Ado analogues
differ greatly in their affinities for receptor subtypes as determined by inhibition of ligand binding43: NECA is not specific for A1 or A2 receptors, whereas CGS21680 and CCPA are 170- and 770-fold more selective for the A2a and A1 receptor subtypes,
respectively.44 Figure 4
illustrates the inhibitory effect of NECA, CGS21680, and CCPA on
fMLP-induced [3H]PEt formation. In these experiments,
neutrophil suspensions were pretreated with 0.1 U/mL ADA to prevent
competitive binding by extracellular Ado for cell surface Ado
receptors. Both CGS21680 and NECA reduced fMLP-induced PLD activity in
a concentration dependent manner. The amounts of [3H]PEt
formed were inhibited by 19.9% ± 4.9% (P = .056),
61.4% ± 5.4% (P = .0075), 80% ± 3.8%
(P = .0023), and 84.7% ± 3.9% (P = .0021)
in the presence of 1, 10, 100, and 1000 nmol/L CGS21680, respectively.
NECA was as potent as CGS21680 in reducing fMLP-induced PLD activity,
with half maximal inhibitory effects observed at approximately 3 nmol/L. The selective A1 receptor agonist CCPA was less effective than
NECA and CGS21680. No reduction in the levels of [3H]PEt
formation was observed with up to 10 nmol/L CCPA. However, 49% ± 5% and 73.3% ± 3.9% reduction in the levels of
PEt formation were observed in presence of 0.1 and 1 µmol/L CCPA,
respectively. CGS21680 and CCPA had no effects on the basal levels of
[3H]PEt formation in unstimulated neutrophils (data not
shown). Together, the data indicate that A2a receptors are involved in the inhibition of fMLP-stimulated PLD activity by Ado receptor agonists.
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| Fig 4.
Effect of Ado receptor agonists on fMLP-induced PLD
activity.
1-O-[3H]alkyl-2-lysophosphatidylcholine-labeled
neutrophils were preincubated at 37°C for 5 minutes. The cell
suspensions were then pretreated with 10 µmol/L CB, 0.1 U/ml ADA, and
increasing concentrations of CGS21680, NECA, or CCPA for an additional
5 minutes. Neutrophils were stimulated with 0.1 µmol/L fMLP in the
presence of 1% ethanol for 10 minutes. The amount of radioactivity
incorporated into [3H]PEt was assessed as described in
"Materials and Methods" and is expressed as the percentage of the
radioactivity in the total lipid extracts. The data are the means ± SEM of 3 separate experiments.
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Effects of Ado receptor antagonists on fMLP-induced PLD
activity
Because Ado accumulates in incubation media, we hypothesized that
blocking its action with Ado receptor antagonists will increase fMLP-stimulated PLD activity, mimicking the effect of Ado removal by
ADA. The effects of 2 Ado receptor antagonists, CSC and CGS15943, were
investigated. CSC is more selective for the A2a than for the A1
receptor,45 whereas CGS15943 is a nonselective A1 and A2
receptor antagonist.1 CB-treated neutrophils were incubated with or without 0.1 U/mL ADA before stimulation with fMLP in the presence of increasing concentrations of CGS15943 (Figure
5A) or CSC (Figure 5B). As expected, the
addition of CGS15943 enhanced fMLP-induced PLD activity with a
characteristic bell-shape curve (Figure 5A). As little as 0.1 nmol/L
CGS15943 increased [3H]PEt formation by
28.7% ± 14%, but statistical significance was not reached.
However, 1 and 10 nmol/L CGS15943 enhanced by
1.78- ± 0.13-(P = .029) fold and
1.87- ± 0.16-(P = .032) fold fMLP-stimulated PLD
activity, respectively. Increasing CGS15943 concentrations up to 1 µmol/L resulted in a progressive reduction of PLD activity, but the
levels of [3H]PEt formed were still larger
(1.52- ± 0.07-fold) than those elicited by fMLP alone. These
stimulatory effects of the A1/A2 antagonist CGS15943 were abolished by
the removal of extracellular Ado with ADA. The A2a selective antagonist
CSC also enhanced fMLP-induced [3H]PEt formation in a
concentration-dependent manner (Figure 5B). The levels of
[3H]PEt formed averaged 116% ± 5.8%,
152% ± 8.7%, and 170% ± 7% of the fMLP-induced response
in the presence of 0.01, 0.1, and 1 µmol/L CSC, respectively. As
observed using CGS15943, the stimulatory effects of CSC on PLD
activation were totally abolished by a pretreatment of the cell
suspension with ADA before fMLP stimulation. The ability of MSU
crystals to stimulate PLD was not enhanced by the addition of 1 µmol/L CSC to neutrophil suspensions. The levels of
[3H]PEt formed in response to MSU crystals were
0.951% ± 0.212% and 0.885% ± 0.365% of total counts in
the absence or presence of CSC, respectively (data not shown). By using
the same cell suspensions, we found that the basal levels of
[3H]PEt formed (0.039% ± 0.006% of total counts)
were not affected by the addition of 1 µmol/L CSC to neutrophil
suspensions (0.036% ± 0.005% of total counts), whereas the
amount of [3H]PEt formed in response to fMLP increased
from 0.755% ± 0.107% in the absence to 1.98% ± 0.62% of
total counts (n = 3) in the presence of CSC. Thus, in contrast to our
observation on fMLP-stimulated neutrophils, Ado receptor occupancy does
not impact on MSU crystal-induced PLD activity.
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| Fig 5.
Effect of Ado receptor antagonists on fMLP-induced PLD
activity.
1-O-[3H]alkyl-2-lysophosphatidylcholine-labeled
neutrophils were preincubated at 37°C for 5 minutes. The cell
suspensions were then pretreated with 10 µmol/L CB and increasing
concentrations of either CGS15943 (A) or CSC (B) and incubated in the
presence or absence of 0.1 U/mL ADA for 5 minutes. Neutrophils were
stimulated with 0.1 µmol/L fMLP in the presence of 1% ethanol for 10 minutes. The amount of radioactivity incorporated into
[3H]PEt was assessed as described in "Materials and
Methods" and is expressed as the percentage of the radioactivity in
the total lipid extracts. The data are the means ± SEM of 3 separate experiments.
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Ado receptor antagonists suppress the inhibitory effect of
CGS21680 on fMLP-induced PLD activity
To confirm that Ado modulates PLD activity by engaging A2a
receptors, we determined whether the potent A1 and A2 competitive antagonists CGS15943 and the selective A2a receptor antagonist CSC
could reverse the inhibitory effects of CGS21680 (0.1 µmol/L) on
fMLP-induced [3H]PEt formation. The addition of CGS15943
reversed the CGS21680-induced inhibition of fMLP-stimulated PLD
activity (Figure 6A). When cells were
incubated with ADA, CGS15943 was without effect on
fMLP-induced [3H]PEt formation, but it abrogated
significantly CGS21680-mediated inhibition of PLD activity (Figure 6A).
The addition of 0.1 and 1 µmol/L CGS15943 restored the magnitude of
[3H]PEt formed to levels very similar to those measured
in fMLP-stimulated and ADA-treated neutrophils (79.4% ± 15.9%
and 97.6% ± 5.2%, respectively). Compared with fMLP-stimulated
but otherwise untreated neutrophils (no ADA), the amounts of
[3H]PEt formed increased from 25.4% ± 4.4%
(CGS21680 alone) to 98.8% ± 0.7%, and 129.8% ± 11.2% in
the presence 0.1 and 1 µmol/L CGS15943, respectively (data
not shown). Similarly, the A2a antagonist CSC reversed in a
concentration-dependent manner formation (not shown) the inhibition by
the A2a selective agonist (CGS21680) of fMLP-induced [3H]PEt. The data obtained with the highest concentration
of CSC tested (10 µmol/L) and ADA are shown in Figure 6B. CSC
reversed significantly the inhibitory effect of CGS21680, increasing
the levels of [3H]PEt formed from
23.6% ± 2.3% to 67.1% ± 3.5% of fMLP-stimulated neutrophils. In the absence of ADA, CGS21680 reduced PLD activity to 44.7% ± 3.5%, compared with fMLP-stimulated cells
and this inhibition was overcome by the addition of 10 µmol/L CSC to
the cell suspension (131.9% ± 12.8% of control fMLP, data not
shown).
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| Fig 6.
The Ado receptor antagonists CGS15943 and CSC reverse the
suppressive effect of CGS21680 on fMLP-stimulated PLD activity.
1-O-[3H]alkyl-2-lysophosphatidylcholine-labeled
neutrophils were prewarmed at 37°C for 5 minutes. (A) The cell
suspensions were pretreated with 10 µmol/L CB and 0.1 U/mL ADA in the
presence or absence of 0.1µmol/L CGS21680 for an additional 5 minutes. Where indicated, 0.1 and 1 µmol/L CGS15943 was also added to
neutrophil suspensions 5 minutes before stimulation with 0.1 µmol/L
fMLP for 10 minutes in the presence of 1% ethanol. (B) The
experimental conditions were similar to A. Where indicated, 10 µmol/L
CSC was added to neutrophil suspensions 5 minutes before stimulation
with 0.1 µmol/L fMLP for 10 minutes in the presence of 1% ethanol.
The amount of radioactivity incorporated into [3H]PEt was
assessed as described in "Materials and Methods" and is expressed
as the percentage of the radioactivity in the total lipid extracts. The
data are the means ± SEM of 3 separate experiments.
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Ado receptor antagonists CSC and CGS15943 reverse CGS21680-induced
inhibition of fMLP-mediated translocation of PLD cofactors to membranes
We next assessed whether the A2 antagonists also impact on the
membrane translocation of ARF, RhoA, and PKC. The effects of the
addition of the A1/A2 antagonist CGS15943 before stimulation of
CGS21680-treated neutrophils with fMLP are shown in Figure 7. CGS15943 had no effect on the membrane
levels of ARF, RhoA, and PKC in unstimulated neutrophils as
estimated by immunoblotting (data not shown). However, the addition of
CGS15943 to cell suspensions enhanced the fMLP-induced recruitment to
membrane of the small GTPases and PKC (data not shown), mimicking the
effect of extracellular removal of Ado by ADA (Figure 2). The A2a
selective agonist CGS21680 totally abolished the membrane recruitment
of PKC and decreased the amounts of membrane-associated RhoA and
ARF in fMLP-stimulated neutrophils (Figure 7, lane 3). These
inhibitions by CGS21680 were reversed by the antagonist CGS15943 in a
concentration-dependent manner (Figure 7, lanes 4-8). The inhibitory
effect of 0.1 µmol/L CGS21680 was not affected by 0.1 nmol/L CGS15943
(lane 4) but was fully reversed by a 10 nmol/L concentration of the
nonselective Ado receptor antagonist (lane 6). Higher CGS15943
concentrations (0.1 and 1 µmol/L) increased the amount of
membrane-associated ARF, RhoA, and PKC above the levels detected in
fMLP-stimulated but otherwise untreated neutrophils as estimated by
immunoblotting (compare lanes 7 and 8 with lane 3). We next
investigated the effects of the more selective A2a antagonist, CSC. As
shown in Figure 8 the addition of CGS21680
and CSC alone or in combination had no effect on the basal levels of
membrane-associated ARF, RhoA, and PKC. As observed for
fMLP-stimulated PLD activity, the inhibitory effects of CGS21680
on fMLP-induced ARF, RhoA, and PKC membrane translocation were
fully reversed by the addition of 10 µmol/L CSC either in the
presence (Figure 8A, lane 8) or absence (Figure 8B, lane 8) of ADA,
suggesting that the 2 events may be causally related. In the absence of
ADA, the translocation of PLD cofactors induced by fMLP was potentiated
by CSC (Figure 8B, lane 6). This stimulatory effect was abolished by
the removal of extracellular Ado with ADA (Figure 8A, lane 6)
indicating that Ado A2a receptor occupancy exerts suppressive effects
on fMLP-induced ARF, RhoA, and PKC membrane translocation.
View larger version (26K):
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[in a new window]
| Fig 7.
Effect of CGS15943 on CGS21680-induced inhibition of
fMLP-stimulated translocation of PKC, RhoA, and ARF1 to membranes.
Unlabeled neutrophils were prewarmed 5 minutes at 37°C. Then 10 µmol/L CB was added and the cell suspensions incubated for an
additional 5 minutes in the absence of ADA. Where indicated, 0.1 µmol/L CGS21680 (lanes 3-8) and increasing concentrations of CGS15943
(lanes 4-8) were added to the cell suspensions. Neutrophils were
stimulated with 0.1 µmol/L fMLP for 2 minutes (lanes 2-8).
Incubations were stopped and neutrophil membranes were prepared as
described in "Materials and Methods." The levels of PKC, RhoA,
and ARF1 in samples were analyzed by immunoblotting. The data are from
1 experiment representative of 3 similar experiments.
|
|
View larger version (52K):
[in this window]
[in a new window]
| Fig 8.
Effect of CSC on CGS21680-mediated inhibition of
fMLP-stimulated translocation of PKC, RhoA, and ARF1 to membranes.
Unlabeled neutrophils were prewarmed 5 minutes at 37°C. Neutrophils
were then pretreated with 10 µmol/L CB in the presence (A) or absence
(B) of 0.1 U/ml ADA for 5 minutes. Where indicated, 0.1 µmol/L
CGS21680 (lanes 3, 4, 7, 8) and 10 µmol/L CSC (lanes 2, 4, 6, 8) were
also added to the cell suspensions. Neutrophils were stimulated with
0.1 µmol/L fMLP (lanes 5-8) or DMSO (lanes 1-4) for 2 minutes.
Incubations were stopped and neutrophil membranes prepared as
described in "Materials and Methods." The levels of PKC, RhoA,
and ARF1 in samples were analyzed by immunoblotting. The data are from
1 experiment representative of 3 similar experiments.
|
|
| |
Discussion |
In this study, we demonstrated that endogenous Ado is a potent
autocrine regulator of PLD activity in ligand-activated neutrophils. Ado exerts its biologic effects through the pharmacologically distinct
receptor subtypes, A1, A2a, A2b, and A3.43,44 Both A1 and
A2 receptors are expressed on neutrophil plasma membranes.2 Several lines of evidence identify the Ado receptor that diminishes fMLP-stimulated PEt formation in neutrophils as the A2a receptor. First, NECA (nonselective) and CGS21680 (A2a selective) inhibited ligand-activated neutrophils with the same efficacy and they were 30-fold more potent than the selective A1 agonist CCPA. Moreover, inhibition of fMLP-induced PLD activity by CCPA only occurred at
concentrations known to activate the A2a receptor.46 The order of agonist potencies observed, NECA = CGS21680 > CCPA is characteristic of the involvement of A2a receptors.43,47
Secondly, the involvement of the A2 receptor is further supported by
the ability of CGS15943, which has similar affinity for both the A1 and
the A2 receptors, to fully reverse the inhibition by CGS21680 of
ligand-stimulated PLD activity. Furthermore, our data strongly suggest
the involvement of the A2a receptor, because the highly selective A2a
antagonist CSC efficiently blocked the suppressive effect of CGS21680.
In addition, the selective A1 antagonist DPCPX (see the
Appendix) did not reverse the inhibitory effect of Ado or CGS21680 at
concentrations up to 100 nmol/L (ie, 30 to 300 times higher than the Kd
of DPCPX for the A1 receptor).1 With respect to
neutrophils, pharmacologic studies with Ado receptor agonists and
antagonists support the involvement of A2a receptors in the inhibition
of neutrophil functional responses.12,41 The expression of
A2a receptors on neutrophils was supported by reverse transcriptase
polymerase chain reaction detection of its mRNA.11
The mechanism by which Ado suppresses neutrophil functional responses
remains to be defined. Although the occupancy of A2 receptors has been
suggested to uncouple chemoattractant receptors from their downstream
signal transduction pathways,48 Ado does not interfere with
the generation of inositol 1,4,5-triphosphate by phospholipase
C,38 the mobilization of intracellular Ca++, or
the early transient in diacylglycerol formation induced by fMLP.16,37 Ado has however been shown to inhibit the influx of Ca++ induced by fMLP,39 and to diminish the
late and sustained increase in diacylglycerol that follows fMLP
stimulation.16 We previously attributed this late and
sustained increase in diacylglycerol formation to the sequential
actions of a phosphatidylcholine-specific PLD and of lipid
phosphohydrolases.40 These observations led us to
hypothesize that Ado might exert a negative regulation of PLD activity
in ligand-activated neutrophils. Accordingly, we found that the
addition of Ado receptor agonists to neutrophil suspensions diminished
fMLP-induced PLD activity. It is recognized that Ado accumulates in
neutrophil suspensions in a time- and cell concentration-dependent
manner.41 Measurements of Ado in neutrophil suspensions
clearly indicate that its extracellular concentration rapidly increases
after only a few minutes of incubation up to 30 to 120 nmol/L. Given
the reported IC50 (50-100 nmol/L) for the inhibition of
neutrophil adherence to endothelial cells,5 of the
respiratory burst,9,11,49 and of leukotriene
synthesis,8 such concentrations are likely to alter
neutrophil functions, including PLD activation. Accordingly, the
addition of ADA was sufficient to suppress the accumulation of Ado in
the extracellular milieu and to reverse the inhibitory effect of
endogenous Ado on stimulated PLD activity. The concentration of Ado in
ADA-treated cell suspensions (< 20 nmol/L) is very similar to the
levels reported in plasma or in blood.41,50 Ado uptake by
red blood cells contributes to maintain the plasma Ado concentration at
low levels but pharmacologic agents such as the inhibitor of ADA
deoxycorformycin,50 the inhibitor of adenosine kinase
GP515,51 or the inhibitor of Ado uptake
dipyridamole41 have been reported to increase its
concentration in the micromolar range. Several studies have established
that GP515,52 methotrexate, and sulfasalazine53
share the capacity to increase Ado release at sites of inflammation.
Furthermore, these studies showed that increased Ado concentration at
inflammatory sites suppresses inflammation by diminishing leukocyte
accumulation in the air pouch model. These anti-inflammatory effects
could be completely reversed by the addition of ADA or inhibition of Ado action by receptor antagonists.13,15,53 Similarly, we observed that endogenous Ado exerts a negative constraint on the PLD
activation pathway that could be nullified by ADA or the receptor antagonists CSC and CGS15943. Thus, endogenous Ado suppresses fMLP-induced PLD activation and, at least in vitro, decreases the
ability of neutrophils to respond to agonists.
It is well established that A2a receptors are linked to
Gs.54 Like other Gs-linked receptors, Ado receptors
on neutrophils stimulate an increase in the intracellular levels of
cAMP.16,37,48 Although this was not investigated in this
study, it is tempting to speculate that the suppression of fMLP-induced
PLD activation by Ado is related to changes in the intracellular
concentrations of cAMP that follow the occupancy of A2a receptors.
Indeed, enhanced intracellular cAMP accumulation as the result of
ligand-induced activation, or treatment of neutrophil suspensions with
cell membrane permeable cAMP analogues have previously been shown to
reduce fMLP-stimulated PLD activity.55,56 Interestingly,
elevation of intracellular cAMP levels has been shown to inhibit
chemoattractant-stimulated RhoA activation,57 a small
GTPase that regulates human neutrophil PLD.58 The
hypothesis that inhibition of fMLP-stimulated PLD activity by Ado is
mediated by cAMP accumulation and activation of protein kinase A will
be the subject of future investigations.
Ado does not affect Ca++ mobilization from intracellular
stores37 but diminishes Ca++ influx stimulated
by fMLP.39 Because the calcium ionophore A23 187 increased
PLD activation by increasing Ca++ entry and intracellular
Ca++ concentration, it seems possible that the decreased
Ca++ influx and the reduction in the concentration of
cytosolic Ca++ that follow the occupancy of A2a receptors
impact on fMLP-stimulated PLD activity. In accord with such a role for
calcium, the addition of EGTA in the incubation media (to prevent
Ca++ influx) or the buffering of the elevation of cytosolic
Ca++ with the intracellular calcium chelator BAPTA have
been shown to reduce fMLP-stimulated PLD activity.59,60
However, the putative effect of Ca++ entry on PLD
activation in intact cells is likely to be indirect, because in vitro
Ca++ itself has no impact on PLD activation by ARF, RhoA,
and PKC.31
Two mammalian PLDs, PLD1,23 and PLD224 have
recently been cloned. Two spliced isoforms of PLD1 named PLD1a and
PLD1b exist.31 The levels of expression of PLD1a and PLD1b
proteins are cell-type and tissue-specific.61,62 We
reported using immunoblot analyses that PLD1a but not PLD1b could be
detected in human neutrophils.35 Both PLD1 and PLD2 require
PIP2 for activity.24,25 PLD1 is activated by ARF, RhoA, and
PKC in vitro.58 In contrast, PLD2 shows high basal
activity and is not activated by the same cofactors. Neutrophil PLD is
activated by RhoA, ARF, and PKC.17 These cytosolic
proteins are recruited to membranes of fMLP-stimulated cells and there
is strong evidence suggesting that they mediate fMLP-induced PLD
activity in human granulocytes.33,36 Our results demonstrate that the removal of extracellular Ado using ADA
significantly increased the levels of RhoA, ARF, and PKC recovered
in the membrane fractions obtained from ligand-activated human
neutrophils. These were the first indications that endogenous Ado may
suppress fMLP-stimulated PLD activity by inhibiting the recruitment of
RhoA, ARF, and PKC to membranes. This hypothesis is further
supported by the experiments showing that blockade of Ado action by the
antagonists CSC or CGS15943 produced very similar increases in the
levels of small GTPases and PKC associated with membranes. As shown
for stimulated PLD activity, ADA and A2 receptor antagonists have no
additive effect on the magnitude of translocation of these PLD
cofactors. In contrast, we found that the selective A2a agonist
CGS21680 reduced fMLP-stimulated translocation of ARF, RhoA, and
PKC. This suppressive effect of Ado on PLD cofactors recruitment to neutrophil membranes in ligand-activated human neutrophil was also
characteristic of the involvement of A2 receptor. This is demonstrated
by the ability of CGS15943 to reverse in a concentration-dependent manner the suppression by GGS21680 of fMLP-induced membrane
translocation of PLD cofactors. Furthermore, the Ado receptor involved
is likely to be the A2a subtype because the selective A2a antagonist
CSC efficiently abolished the inhibition by CGS21680 of ARF, RhoA, and
PKC translocation elicited by fMLP. Taken together, the results strongly suggest that the engagement of A2a receptor inhibits PLD
activation by limiting the recruitment of ARF, RhoA, and PKC to
membranes of fMLP-stimulated neutrophils. Interestingly, the magnitude
of the inhibitory effects of Ado and Ado analogues on PLD activity
correlated with the inhibition of PLD1 cofactor association to
membranes, supporting a causal link.
Our studies demonstrate a unique role for Ado in inhibiting the PLD
signaling cascade in fMLP-activated human neutrophils. To our
knowledge, the PLD signaling pathway is the only one reported to be
inhibited by Ado. The ability of the A2a receptor to suppress PLD
activity is therefore of pathophysiologic interest. Many studies have
shown that the occupancy of the A2 receptor suppresses several neutrophil functional responses,4 including actin
polymerization,63 adherence to endothelium,5
expression of CD11b/CD18,6,7 secretion,51
leukotriene synthesis,8,41 and fMLP-stimulated respiratory
burst.3,9-12 Inhibition of PLD is likely to reduce the
generation of PLD-derived phosphatidic acid (and lysophosphatidic acid
and diacylglycerol) and the functional responses associated with the
generation of these intracellular mediators. There is increasing
evidence suggesting that PLD-derived second messengers regulate
particle phagocytosis,22 the expression of 2
integrins,21 secretion,20 and the respiratory
burst.64 The suppression of neutrophil functions by Ado is
therefore compatible with an inhibition of the PLD1 isoform by
occupancy of A2a receptors. Moreover, the suppressive effect of Ado and
Ado analogues on fMLP-stimulated PLD activity is agonist specific and
cannot be generalized because PLD activation induced by MSU crystals
was not inhibited; this observation suggests that the PLD isoform
activated by MSU crystals is unlikely to be the PLD1
isoform.35
In summary, this study demonstrates the ability of endogenous Ado and
Ado analogues to inhibit the activation of PLD in neutrophil suspensions through engagement of A2a receptors. Our results indicate that the inhibition of the fMLP-induced PLD activity is functionally linked to inhibition of fMLP-stimulated recruitment of ARF, RhoA, and
PKC to membranes. Taken together, these results indicate that Ado
exerts suppressive effects on human neutrophil functions by uncoupling
chemoattractant receptors from PLD1 activation.
| |
Appendix |
The addition of 0.1 and 1 µmol/L DPCPX to cell suspensions
increased the amount of PEt formed in response to fMLP by
1.73- ± 0.12-fold and 1.98- ± 0.27-fold, respectively. The
inhibition by CGS21680 of fMLP-induced PLD activity was reversed by
DPCPX. The amounts of PEt formed increased from 34% ± 9.2% to
37.5% ± 12.9%, 53.2% ± 16.5%, and 77.2% ± 25%
of control fMLP in the presence of 0.1, 1, and 10 µmol/L DPCPX,
respectively. DPCPX (10 µmol/L) was able to reverse the inhibitory
effect of CGS21680 on fMLP-stimulated membrane translocation of ARF,
RhoA, and PKC, an effect characteristic of the involvement of A2
receptor.1
| |
Acknowledgments |
We thank Dr David Brindley for valuable discussions and critical
reading of the manuscript. In addition, we thank Serge Picard for
expert technical assistance.
| |
Footnotes |
Submitted May 18, 1999; accepted September 23, 1999.
Supported by a Senior Scholarship from the Arthritis Society of Canada
and grants from the Medical Research Council of Canada (MT-14 790) and
the Arthritis Society. Nathalie Thibault is the recipient of a
studentship from the "Fonds pour la Formation de Chercheurs et
l'Aide à la Recherche".
Reprints: Centre de recherche en Rhumatologie et Immunologie,
C.H.U.Q., Pavillon C.H.U.L, 2705 Blvd Laurier, Sainte-Foy, Québec, G1V 4G2, Canada.
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