Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:367-377.)
© 2002 by Society for Leukocyte Biology

Occupancy of adenosine A2a receptors promotes fMLP-induced cyclic AMP accumulation in human neutrophils: impact on phospholipase D activity and recruitment of small GTPases to membranes

Nathalie Thibault*, Chantal Burelout*, Danielle Harbour*, Pierre Borgeat*,2, Paul H. Naccache*,3 and Sylvain G. Bourgoin*,2

* CIHR Group on the Molecular Mechanisms of Inflammation, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, CHUQ et Université Laval, Départements
{dagger} d’Anatomie-Physiologie et
{ddagger} Médecine, Québec, Canada

Correspondence: Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, CHUQ, 2705 Blvd. Laurier, Sainte-Foy, Québec, G1V 4G2, Canada. E-mail: Sylvain.bourgoin{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to assess in human neutrophils the implication of an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway in the inhibitory effects of A2a receptor engagement. We found that Ro20-1724, a cAMP phosphodiesterase inhibitor, in the presence of adenosine deaminase (ADA) or A2a receptor antagonists rendered transient the fMLP-induced sustained increases in cAMP levels. The role of A2a receptor stimulation was demonstrated by the ability of the A2a receptor agonist, CGS21680, to prevent ADA-mediated reduction of the persistent cAMP elevation induced by fMLP. Persistent cAMP elevation correlated with inhibition of fMLP-induced PLD activation and recruitment of Arf, RhoA, and PKC to membranes. The suppressive effect of CGS21680 or isoproterenol, a ß-adrenergic receptor agonist, was increased by Ro20-1724 or by the adenylyl cyclase activator, forskolin, and reversed, at least in part, by the inhibitor of adenylyl cyclase, 2',5'-dideoxyadenosine. The activator of protein kinase A (PKA), Sp-cAMP inhibited fMLP-induced PLD activation and translocation of Arf and RhoA to membranes. In contrast, the suppression by A2a receptor stimulation of fMLP-induced PLD activation and cofactor recruitment was antagonized by PKA inhibitors, Rp-cAMP and H89. In conclusion, A2a receptor occupancy by extracellular adenosine inhibits fMLP-induced neutrophil activation via cAMP and PKA-regulated events.

Key Words: protein kinase C • phosphodiesterase • ADA • cAMP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are the most abundant white blood cells and are the first cells to migrate at injured or infected sites. Although neutrophils are important to limit the spread of pathogens, they are capable of damaging injured tissues and exacerbating inflammation in pathological conditions such as rheumatoid arthritis and myocardial infarction. Adenosine is recognized as an endogenous anti-inflammatory agent, suppressing the activity of cells involved in immune and inflammatory reactions [1 ]. For more than a decade, adenosine released from neutrophil suspensions has been recognized to inhibit superoxide anion generation stimulated by fMet-Leu-Phe (fMLP) [2 , 3 ]. Adenosine also reduces leukocyte adhesion to the endothelium [4 ], the expression of integrins on the neutrophil cell surface [5 ], and the release of leukotriene B4 [6 ]. These effects were enhanced by inhibitors of adenosine uptake into the cells [7 8 9 ] or inhibitors of adenosine metabolism [10 11 12 ] and were abrogated by adenosine deaminase (ADA), which prevents the accumulation of adenosine in the extracellular milieu [13 14 15 16 ]. Adenosine is released in large amounts during myocardial infarction and exerts a potent protective function on the heart when elevated prior to ischemia [17 ]. The beneficial effects of adenosine on ischemia-reperfusion injury have been attributed at least in part to inhibition of neutrophil functions [18 ].

Adenosine acts by interacting with specific receptors on the cell surface [19 ]. Studies investigating the structure-activity relationship of adenosine derivatives at A1, A2a, A2b, and A3 receptors have defined the A2a receptor subtype as the adenosine receptor mediating the suppression of neutrophil functional responses [1 ]. Although A2a receptor occupancy does not affect chemoattractant-stimulated generation of inositol 1,4,5-triphosphate by phospholipase C (PLC) [20 ] or the mobilization of intracellular pools of calcium [21 ], it decreases fMLP-mediated actin polymerization [5 ]. Furthermore, we recently demonstrated that the suppression of fMLP-mediated phospholipase D (PLD) activation by adenosine was correlated with the inhibition of the translocation of protein kinase C (PKC) and of Arf1 and RhoA small GTPases to neutrophil membranes [15 ]. Although the nature of the intracellular messenger mediating the inhibition of PLD activity and small GTPases activation by adenosine remains unclear, cyclic adenosine monophosphate (cAMP) analogues and agents that stimulate cAMP accumulation have been shown to inhibit fMLP-mediated stimulation of PLD [22 , 23 ].

Several signaling pathways, including PLD activation, initiated by receptor-ligand interactions are involved in the regulation of the oxidative burst [24 ]. A2a receptors are coupled to activation of adenylyl cyclase through the GTP-binding protein Gs [25 ], and their occupancy by adenosine has been shown previously to raise cAMP levels in human neutrophils [21 , 26 ]. Inhibition of superoxide generation by adenosine [27 ] and its enhancement by inhibitors of phosphodiesterase (PDE) support the hypothesis that cAMP may contribute to suppress this neutrophil function [1 , 28 ]. However, inhibitors of cAMP-dependent protein kinase A (PKA) have been shown to reverse the inhibitory effect of cAMP analogues but not of A2a or ß-adrenergic receptor agonists on the oxidative burst [25 ]. The cAMP-independent activation of a serine/threonine phosphatase by adenosine has been suggested to uncouple fMLP receptors from the signal transduction mechanisms [29 ].

In the present study, we have evaluated the effect of adenosine A2a receptor occupancy on cAMP levels and of various pharmacological modulators of the cAMP/PKA signaling pathway on the ability of adenosine to suppress fMLP-mediated PLD activation. Together, the data indicate that extracellular adenosine markedly potentiated fMLP-induced cAMP elevation in neutrophils. Adenosine had no impact on the initial elevation of cAMP levels induced by fMLP but produced a more persistent stimulation of adenylyl cyclase. A2a receptor occupancy increased the cAMP response induced by fMLP with a concomitant inhibition of PLD activity and recruitment of RhoA and Arf to membranes. Our results suggest that adenosine regulates fMLP-induced PLD activation via a cAMP/PKA-dependent inhibition of the recruitment of small GTPases and of PKC{alpha} to neutrophil membranes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxiamido adenosine hydrochloride (CGS21680), 8-(3-chloro-styryl) caffeine (CSC), Ro20-1724, and forskolin were from RBI (Natick, MA). Dextran T-500 and Ficoll-Paque were purchased from Pharmacia Biotech (Dorval, Québec, Canada). ADA, diisopropylfluorophosphate (DFP), fMLP, cytochalasin B (CB), and all other reagents were from Sigma-Aldrich Canada (Oakville, Ontario, Canada). PKC{alpha} and RhoA antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal Arf1 antibody was described in previous studies [30 ]. Adenosine 3',5'-cyclic monophosphorothioate acetoxymethylester Sp (Sp-cAMPs-AM) and Rp (Rp-cAMPs-AM) isomers were purchased from Biolog, Life Science Institute (La Jolla, CA). 1-O-[3H]alkyl-2-lyso-phosphatidylcholine was obtained from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada).

Neutrophil purification
Venous blood was collected from healthy adult volunteers in isocitrate anticoagulant solution. Neutrophils were separated as described previously [15 ]. Briefly, whole blood was centrifuged at 180 g for 10 min, and the resulting platelet-rich plasma was discarded. Leukocytes were obtained following erythrocytes sedimentation at 1 g in 2% Dextran T-500. Mononuclear cells were removed by centrifugation on Ficoll-Paque cushions, and contaminating erythrocytes in the neutrophil pellets were removed by a 20-s hypotonic lysis in water. Neutrophils were resuspended in Hanks’ balanced salt solution (HBSS), pH 7.4, containing 0.8 mM Ca2+ but no Mg2+.

cAMP measurements
Neutrophils (107 cells/ml) were preincubated with 10 µM Ro20-1724 for 20 min at 37°C. Where indicated, 0.1 U/ml ADA was also added to the cell suspensions to prevent the rapid accumulation of adenosine in the extracellular milieu with time as described previously [8 , 15 ]. Then, cell suspensions (1.5x107 cells) were preincubated 5 min with 10 µM CB and the indicated concentrations of adenosine analogues or an equal volume of the vehicle. Cells were stimulated with 0.1 µM fMLP for the indicated times. Incubations were stopped by centrifuging the cell suspensions at 5000 g for 1 min and mixing the cell pellets with 0.5 ml ice-cold acidified ethanol (99% ethanol, 20 mM HCl) and kept at 4°C. Samples were sonicated for 20 s and centrifuged at 13,000 g for 45 min at 4°C. The supernatants were collected, dried under nitrogen, and stored at -20°C until used for the cAMP measurements. cAMP measurements were performed by Solution Recherche (Sainte-Foy, Québec, Canada) using a radioimmunoassay purchased from Diagnostic Products Corporation (Los Angeles, CA).

PLD measurements
Neutrophils were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 cells) for 90 min as described previously [15 ]. Neutrophils were washed and resuspended at 107 cells/ml. Cell suspensions (0.5 ml) were preincubated at 37°C for 5 min and pretreated for 5 min with 10 µM CB in the presence or absence of 0.1 U/ml ADA and the indicated concentrations of adenosine analogues. Neutrophils were stimulated with 0.1 µM fMLP for 10 min in the presence of 1% ethanol. Incubations were stopped by adding 1.8 ml chloroform/methanol/HCl (50:100:1, vol/vol/vol) and unlabeled phosphatidylethanol (PEt) as a standard. Lipids were extracted and dried under nitrogen. The lipid extracts were dissolved in 40 µl chloroform/methanol (2:1, vol/vol) and spotted on prewashed silica gel 60 thin-layer chromatography plates. [3H]PEt was separated from the other lipids using the solvent mixture chloroform/methanol/acetic acid (65:15:2, vol/vol/vol). Lipids were visualized by Coomassie brilliant blue staining (0.03% dye, 35% methanol, and 200 mM NaCl), and the different lipid classes were scraped off the plates. Radioactivity in PEt was monitored by liquid scintillation counting, and the results were corrected for background radioactivity and quenching.

Translocation assays
Neutrophils (5x107 cells/ml) were treated with 1.1 mM DFP for 30 min at 24°C. Cell suspensions were centrifuged and resuspended in HBSS at 107 cells/ml. The cells were preincubated 5 min at 37°C and treated with 10 µM CB. Where indicated, 0.1 U/ml ADA and/or adenosine analogues were added to the cell suspensions 5 min prior to stimulation. Neutrophils were stimulated with 0.1 µM fMLP for 2 min at 37°C. Incubations were stopped by diluting the cells 1:5 with ice-cold HBSS, and the samples were processed as described previously [15 ]. Briefly, cell suspensions were centrifuged as indicated and resuspended at 1.6 x 107 cells/ml in ice-cold KCl-Hepes relaxation buffer [50 mM Hepes, 100 mM KCl, 5 mM NaCl, 1 mM MgCl2, 0.5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 2.5 mM phenylmethylsulfonyl fluoride (PMSF), adjusted to pH 7.2]. Cell suspensions were sonicated for 20 s and centrifuged for 7 min at 700 g. Unbroken cells and nuclei were discarded, and the supernatants were ultracentrifuged at 180,000 g for 45 min in a Beckman TL-100 ultracentrifuge using a TL-100.4 rotor. Membrane pellets were washed once and resuspended in a small volume of buffer A containing 0.25 M Na2HPO4, 0.3 M NaCl, 2.5% sodium dodecyl sulfate (SDS), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 2.5 mM PMSF, and samples were assayed for protein content with Pierce Coomassie brilliant blue protein assay. Protein samples (30–60 µg) were resolved on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Western blots were performed using Arf (1/2000), RhoA (1/1000), or PKC{alpha} (1/1000) antibodies, and proteins were revealed using the enhanced chemiluminescence (ECL) detection system as described previously [15 ].

Statistics
Data are expressed as means ± SE. Data were analyzed using the Student’s paired t-test (two-tailed) or the Mann-Whitney test (nonparametric test) to determine the level of significance between the treated samples and the appropriate controls (*P<0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of adenosine A2a receptor agonists and antagonists on fMLP-induced PLD activation in the presence and absence of type IV PDE inhibitor
Neutrophils were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine, and [3H]PEt, the transphosphatidylation product catalyzed by PLD in the presence of ethanol, was assessed. Figure 1 shows that the removal of extracellular adenosine by a pretreatment of neutrophil suspensions with 0.1 U/mL ADA or by blocking adenosine receptors with the selective A2a receptor antagonist CSC increased fMLP-mediated PLD activation significantly. The PLD responses were increased tenfold by a pretreatment of the cell suspensions with CB (Fig. 1A) . In contrast, the selective A2a receptor agonist CGS21680 decreased fMLP-mediated PLD activation. Figure 1A and 1B , also shows that the amounts of [3H]PEt formed in response to stimulation with fMLP were not reduced significantly by the selective inhibitor of type IV PDE, Ro20-1724 [31 ]. However, the ability of CGS21680 to inhibit fMLP-induced PLD activation was enhanced by the addition of 10 µM Ro20-1724 to neutrophil suspensions (Fig. 1A) , whereas the stimulatory effect of the selective A2a receptor antagonist CSC was not impaired by a 5 min preincubation of the cells with this inhibitor of PDE (Fig. 1B) .



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  		Figure 1. Effect of Ro20-1724 on CGS21680-mediated inhibition and CSC-induced increase in PLD activity. Labeled neutrophils were prewarmed at 37°C for 5 min. (A) Where indicated, the cell suspensions were pretreated for an additional 5 min with 0.1 U/mL ADA, 1 µmol/L CSC, 10 µmol/L Ro20-1724, or 1 µmol/L CGS21680, with or without Ro20-1724, in the absence ({square}) or the presence ({blacksquare}) of 10 µmol/L CB. (B) The samples were pretreated with 10 µmol/L CB and incubated for 5 min in the presence of 1 µmol/L CSC or 10 µmol/L Ro20-1724 alone or in combination. Cells were stimulated with 0.1 µmol/L fMLP for 10 min in the presence of 1% ethanol. PLD activity 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 ± SE of two sets of independent experiments (n=3 for each set).

 
Because the engagement of A2a receptors has been shown to inhibit PLD activation by blocking the membrane recruitment of Arf, RhoA, and PKC{alpha} [15 ], we conducted experiments to assess whether Ro20-1724 increased the ability of CGS21680 to suppress the recruitment of these PLD1 activation cofactors to membranes. As shown in Figure 2 , Arf1 and RhoA were recruited to membranes in response to stimulation with fMLP. A small increase in the amount of membrane-associated PKC{alpha} was also dectected when the blots were overexposed (not shown; see also [15 ]). As shown for PLD activity, the removal of extracellular adenosine by ADA (lane 3) or the addition of the A2a receptor antagonist CSC (lane 5) markedly increased the fMLP-induced recruitment of PKC{alpha}, RhoA, and Arf1 to membranes. In the absence of ADA, CGS21680 (lane 4) or Ro20-1724 (lane 6) alone decreased slightly the membrane recruitment of RhoA and Arf1 induced by fMLP, but their combination reduced the levels of membrane-associated RhoA and Arf to basal levels (lane 7). As estimated by densitometric analyses of the blots (not shown) and illustrated (see Fig. 4 ), the stimulatory effect of the A2a receptor antagonist CSC on the translocation of PLD1 activation factors was not attenuated significantly by Ro20-1724 (lane 8).



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  		Figure 2. Effect of Ro20-1724 on CGS21680-mediated inhibition and CSC-mediated increase of fMLP-induced translocation of PKC{alpha}, RhoA, and Arf1 to membranes. Neutrophils were prewarmed at 37°C for 5 min. Where indicated, the cell suspensions were treated with 10 µmol/L CB in the presence (lane 3) or the absence (lanes 1, 2, and 4–8) of 0.1 U/mL ADA; 10 µmol/L Ro20-1724 (lanes 6–8); 1 µmol/L CGS21680, with (lane 7) or without (lane 4) Ro20-1724; or 1 µmol/L CSC, with (lane 8) or without (lane 5) Ro20-1724 for 5 min. Neutrophils were stimulated with 0.1 µmol/L fMLP for 2 min (lanes 2–8) and neutrophil membranes were prepared as described in Materials and Methods. The samples were probed for PKC{alpha}, RhoA, and Arf1. The data are from one experiment representative of three similar experiments.

 


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  		Figure 4. Effect of cAMP-elevating agents on fMLP-mediated translocation of PKC{alpha}, RhoA, and Arf1 to membranes. Neutrophils were prewarmed at 37°C for 5 min and incubated with 10 µmol/L Ro20-1724 (lanes 2, 4, 6, and 8) or 10 µmol/L forskolin (lanes 3, 4, 7, and 8), alone or in combination (lanes 4 and 8), for 25 min at 37°C. Cells suspensions were incubated with 10 µmol/L CB and in the presence of 0.1 U/ml ADA (lanes 1–8) for an additional 5 min prior to stimulation with 0.1 µmol/L fMLP for 2 min (lanes 5–8). Reactions were stopped, and neutrophil membranes were prepared as described in Materials and Methods. The samples were probed for PKC{alpha}, RhoA, and Arf1. The data are from one experiment representative of three similar experiments. Quantitation by densitometry of Arf and RhoA in neutrophil membranes is shown in the lower left and right panels, respectively (n=3). *, P < .05 using the Student’s paired t-test.

 
Effect of ADA, A2a receptor agonists and antagonists on the increases of cAMP levels induced by fMLP
The inhibition of PLD activation and PLD1 cofactor translocation to membranes by CGS21680 and its enhancement by Ro20-1724 was consistent with the suppressive effects of adenosine being mediated by elevation of cAMP levels [21 , 26 ]. To assess the role of cAMP as the intracellular messenger for adenosine inhibition of PLD activation, we measured the effect of the removal of adenosine from the cell suspensions and of selective A2a receptor agonists and antagonists (CGS21680 and CSC, respectively) on fMLP-mediated cAMP elevation. In these experiments, neutrophils were incubated with 10 µmol/L Ro20-1724 for 25 min at 37°C, and as described below for the PLD and the translocation assays (see Figs. 4 and 5 ), cell suspensions were treated with 10 µM CB for 5 min prior to stimulation with fMLP. The presence of the PDE inhibitor was absolutely required to detect significant changes in cAMP levels (unpublished results). Ro20-1724 increased the basal levels of cAMP from 1.5–2.5 to 15–20 pmoles/15 x 106 neutrophils. As shown in Figure 3A and B, the basal levels of cAMP were not altered by the removal of extracellular adenosine with ADA. In the presence of Ro20-1724 but no ADA, fMLP augmented the cAMP levels to 50–60 pmoles by 30 s, and this level was maintained for up to 5 min, the last time sampled. In contrast, fMLP-induced cAMP accumulation was transient when neutrophils were pretreated with 0.1 U/mL ADA (Fig. 3A) . The initial increase of cAMP formation at 30 s was not attenuated by the removal of extracellular adenosine with ADA. However, under those conditions, the levels of intracellular cAMP returned to the basal levels within 90 s after the addition of fMLP, indicating that adenosine receptor occupancy is required to promote a sustained elevation of cAMP levels in response to fMLP stimulation of neutrophils.



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  		Figure 5. Effect of 2',5'-ddADO on CGS21680 and isoproterenol-mediated inhibition of fMLP-induced PLD activity. [3H]-Labeled neutrophils were prewarmed at 37°C for 5 min and incubated with the indicated concentrations of 2',5'-ddADO for 5 min at 37°C. Cells were stimulated with 0.1 µmol/L fMLP for 10 min in the presence of 1% ethanol. PLD activity was measured as described in Materials and Methods. The data are expressed as a percentage of the levels of [3H]PEt formed in fMLP-stimulated cells in the presence of CB. * and {ddagger}, P < .05 as compared with fMLP-stimulated neutrophils in the absence or the presence of CGS21680, respectively, using the Mann-Whitney test. (A) Neutrophils were treated with 10 µmol/L CB (no ADA) and incubated with or without 1 µmol/L CGS21680 for an additional 5 min prior to stimulation. The data are means ± SE of six independent experiments, except for the highest concentration of 2',5'-ddADO (n=2). (B) Neutrophils were treated with 10 µmol/L CB (no ADA) and incubated with or without 1 µmol/L isoproterenol for an additional 5 min prior to stimulation. The data are means ± SE of six independent experiments, except for the highest concentration of 2',5'-ddADO (n=2). (C) Neutrophils were treated with 10 µmol/L CB (no ADA) and incubated with or without 0.1 µmol/L CGS21680 for an additional 5 min prior to stimulation. The data are means ± SE of three independent experiments, each performed in duplicate. (D) Neutrophils were treated with 10 µmol/L CB and 0.1 U/ml ADA and incubated with or without 0.1 µmol/L CGS21680 for an additional 5 min prior to stimulation. The data are means ± SE of three independent experiments, each performed in duplicate.

 


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  		Figure 3. Effect of ADA, CGS21680, and CSC on fMLP-induced cAMP formation. Neutrophils were prewarmed at 37°C for 5 min and treated with 10 µmol/L Ro20-1724 for 25 min at 37°C. (A) Cell suspensions were incubated with 10 µmol/L CB in the absence ({circ}) or the presence (•) of ADA (0.1 U/mL ADA) for an additional 5 min and stimulated with 0.1 µmol/L fMLP (arrow). (B) Neutrophils were incubated with 10 µmol/L CB and 1 µmol/L CGS21680 in the absence ({circ}) or the presence (•) of ADA (0.1 U/mL ADA) for an additional 5 min prior to stimulation with 0.1 µmol/L fMLP (arrow). (C ) Cell suspensions were incubated with 10 µmol/L CB (no ADA) in the absence ({circ}) or the presence (•) of 1 µmol/L CSC for an additional 5 min prior to stimulation with 0.1 µmol/L fMLP (arrow). At selected times, the reactions were stopped. cAMP was extracted and measured as described in Materials and Methods. The data are means ± SE of three independent sets of experiments, each performed in duplicate (n=3 for each set). *, P < .05 using the Student’s paired t-test.

 
Because the occupation of adenosine A2a receptors is associated with an inhibition of fMLP-induced neutrophil functional responses (Figs. 1 and 2) , we reasoned that the nonmetabolizable A2a receptor agonist CGS21680 should suppress the inhibitory effects of ADA on fMLP-induced sustained increases in cAMP levels. As expected, the addition of CGS21680 to ADA-treated neutrophils restored the persistent accumulation of cAMP induced by fMLP (compare Fig. 3A with B). In addition, the amounts and the kinetics of the elevation of cAMP were not altered by the addition of CGS21680 to cell suspensions not treated with ADA (Fig. 3B) . In the absence of ADA, basal cAMP levels were reduced slightly by the selective A2a receptor antagonist CSC, and the sustained increases in cAMP levels induced by fMLP were abolished (Fig. 3C) . The kinetics of cAMP elevation were similar to those of ADA-treated cells (Fig. 3A) , and cAMP levels peaked at 30 s and returned to near basal levels within 90 s of the addition to fMLP (Fig. 3C) . Taken together, these data indicate that the occupancy of A2a receptors by agonists exerts a permissive effect on fMLP-induced cAMP elevation in neutrophils.

Effects of Ro20-1724 and forskolin on stimulation of PLD activity induced by fMLP in ADA-treated neutrophils
Adenosine accumulates in a time-dependent manner in neutrophil suspensions [15 ] and modulates the kinetics of the accumulation of cAMP in response to stimulation with fMLP (Fig. 3) . To further establish that cAMP elevation reduces fMLP-induced activation of PLD, we investigated the effect of Ro20-1724 and forskolin on fMLP-induced PLD activation in ADA-treated cells. The PDE inhibitor, which increased basal and fMLP-induced cAMP elevation (Fig. 3) , reduced the stimulatory effects of fMLP on the levels of PEt formation in a time- and concentration-dependent manner. A preincubation of the cells with 10 and 100 µM Ro20-1724 for 5, 15, 30, and 60 min prior to stimulation with fMLP reduced the levels of PEt formed by 15.5 ± 3.6%, 31 ± 8%, 47.3 ± 8%, and 51.2 ± 3.6% (n=3) and by 32.3 ± 2.5%, 47.4 ± 2.4%, 57.5 ± 5%, and 65.3 ± 5.4% (n=3), respectively. Forskolin also inhibited fMLP-induced PLD activation in a time- and dose-dependent manner (unpublished results). The suppressive effects of Ro20-1724 and forskolin on PLD activity were additive. Indeed, a preincubation of cell suspensions with 10 µM forskolin and Ro20-1724 alone or in combination for 30 min reduced the levels of [3H]PEt formed by 20 ± 5.9%, 17 ± 2.5%, and 38.7 ± 6.6%, respectively. The addition of a suboptimal inhibitory concentration (1 nM) of the A2a receptor agonist CGS21680 reduced the amounts of PEt formed by 22 ± 5.6% (n=3). In the presence of CGS21680, the inhibitory effects of Ro20-1724 and forskolin alone or in combination were enhanced to 43 ± 4.8%, 43 ± 9.4%, and 58 ± 5.7%, respectively. We next investigated whether the fMLP-mediated translocation of PKC{alpha}, RhoA, and Arf1 to membranes was modulated by Ro20-1724 and forskolin (Fig. 4 ). The basal levels of RhoA, recovered in membranes obtained from ADA-treated but otherwise unstimulated neutrophils (lane 1), were slightly reduced by a pretreatment of the cells with Ro20-1724 (lane 2) alone or in combination with forskolin (lane 4). The recruitment of PKC{alpha}, RhoA, and Arf1 induced by fMLP (lane 5) was not reduced significantly by Ro20-1724 (lane 6) or forskolin (lane 7). As observed for PLD activity, the inhibitory effects of Ro20-1724 and forskolin on the fMLP-mediated translocation of PKC{alpha} and small GTPases to membranes were additive (Fig. 4 , lane 8). Together, the data support the hypothesis that cAMP acts as the second messenger for inhibition of PLD activation and recruitment of PLD cofactors to membranes.

Effect of 2',5'-dideoxyadenosine (2',5'-ddADO) on CGS21680 and isoproterenol-mediated inhibition of fMLP-mediated activation of PLD
If cAMP is the second messenger for adenosine A2a receptor-mediated suppression of PLD activity, then inhibition of adenylyl cyclase should reduce the inhibitory effects of A2a receptor agonists or other receptors that stimulate the accumulation of cAMP. To determine whether inhibition of PLD activity by adenosine A2a receptor is mediated by cAMP, we studied the effect of 2',5'-ddADO, a ligand for the P-site on adenylyl cyclase, which inhibits its catalytic activity [32 ]. In these experiments, neutrophils were not treated with ADA to allow accumulation of endogenous adenosine. As shown in Figure 5 , the addition of 100, 300, and 900 µM 2',5'-ddADO to neutrophils enhanced fMLP-induced PLD activity by 44 ± 11% (n=6), 85 ± 17% (n=6), and 89 ± 36% (n=2), respectively. The increases in PEt formation were similar to those induced by the addition of ADA to cell suspensions or by blocking A2a receptors using selective antagonists prior to stimulation with fMLP (Fig. 1) . As demonstrated previously [15 ], 1 µmol/L CGS21680 reduced the amounts of [3H]PEt formed to 40.3 ± 3.1% (n=6) of the fMLP-induced response (Fig. 5A) . A treatment with 2',5'-ddADO (100, 300, and 900 µM) partially reversed the suppressive effects of CGS21680, increasing the amounts of [3H]PEt formed to 45.9 ± 3.5% (n=6), 63.5 ± 7.8% (n=6), and 58.8 ± 12.3% (n=2) of the fMLP-induced response, respectively. Other cAMP-elevating agonists also reduced fMLP-induced PLD activation. Figure 5B shows that stimulation of ß2-adrenoreceptors with isoproterenol also reduced fMLP-induced PLD activity by 45.1 ± 2.8%. 2',5'-ddADO reversed the inhibitory effect of 1 µmol/L isoproterenol (Fig. 5B) more efficiently than those of 1 and 0.1 µmol/L CGS21680 (Fig. 5A and 5C , respectively). When neutrophil suspensions were pretreated with ADA, 2',5'-ddADO has no noticeable effect on fMLP-mediated [3H]PEt formation (Fig. 5D) . However, 2',5'-ddADO (100, 300, and 900 µM) was able to partially reverse the suppressive effects of 0.1 µmol/L CGS21680 on PLD activity. These increases of [3H]PEt accumulation were statistically significant, but the levels formed in response to fMLP were twofold lower than those obtained without any CGS21680 (Fig. 5D) . These data further demonstrated that adenosine A2a receptors reduced, at least in part, PLD activation by a cAMP-dependent mechanism.

Effect of modulators of PKA activity, Sp-cAMPs-AM and Rp-cAMPs-AM, on fMLP-induced PLD activity and translocation of PKC{alpha}, RhoA, and Arf1 to membranes
The above results suggested that the sustained elevation of cAMP resulting from the activation of adenylyl cyclase by engagement of A2a receptors reduced the ability of fMLP to turn on the PLD signaling pathway. To determine whether adenosine inhibited the stimulation of PLD activity through the activation of PKA, neutrophils were incubated with the PDE-resistant and cell-permeable activators and inhibitors of PKA [33 ], Sp-cAMPs-AM and Rp-cAMPs-AM, respectively. In these experiments, neutrophils were preincubated with the cAMP analogues for 30 min to allow their uptake, their hydrolysis by intracellular esterases, and accumulation inside the cells. Rp-cAMPs-AM was without significant effect on the levels of [3H]PEt formed when neutrophils were treated with ADA prior to stimulation with fMLP (Fig. 6A ). Concentrations of Rp-cAMPs-AM >50 µM were not tested because they produced a small inhibition of PLD activity (not shown). In contrast, when ADA was omitted, 10 and 50 µM Rp-cAMPs-AM enhanced by 1.18 ± 0.15- and 1.66 ± 0.2-fold, respectively, the stimulation of PLD activity induced by fMLP. The stimulatory effects of the PKA inhibitor on PLD activation were similar to those shown for ADA (Fig. 1) , the A2a receptor antagonist CSC (Fig. 1) and the P-site inhibitor of adenylyl cyclase 2',5'-ddADO (Fig. 6A and 6B) . In contrast, Sp-cAMPs-AM reduced fMLP-induced PLD activity in a concentration-dependent manner (Fig. 6B) . Inhibition of fMLP-mediated [3H]PEt formation by Sp-cAMPs-AM was larger when neutrophils were pretreated with ADA to remove extracellular adenosine, thereby suggesting that PKA was already activated by endogenous adenosine.



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  		Figure 6. Effect of Sp-cAMPs-AM and Rp-cAMPs-AM on fMLP-induced PLD activity. [3H]-Labeled neutrophils were prewarmed at 37°C for 5 min and incubated for 30 min with the indicated concentrations of Rp-cAMPs-AM (A) or Sp-cAMPs-AM (B). Neutrophils were treated with 10 µmol/L CB and incubated for an additional 5 min with or without 0.1 U/ml ADA prior to stimulation with 0.1 µmol/L fMLP for 10 min in the presence of 1% ethanol. PLD activity was measured as described in Materials and Methods. The levels of [3H]PEt formed are expressed as a percentage of the fMLP-induced response. The data are means ± SE of a minimum of four independent experiments. *, P < .05 when compared with the control with no Rp-cAMPs-AM or Sp-cAMPs-AM.

 
Because adenosine, A2a receptor agonists (Fig. 2) , and other cAMP-elevating agents such as forskolin and Ro20-1724 (Fig. 4) inhibited fMLP-mediated PLD activation by diminishing the recruitment of PKC{alpha}, RhoA, and Arf1 to neutrophil membranes, we investigated whether translocation of PLD1 cofactors to membranes was also regulated by PKA using Sp-cAMPs-AM and Rp-cAMPs-AM. Densitometric analyses of the blots indicate that a preincubation of cell suspensions with the inhibitor of PKA, Rp-cAMPs-AM, in the absence of ADA enhanced fMLP-induced recruitment of Arf1 and RhoA to neutrophil membranes (Fig. 7A ). In contrast, the addition of the PKA activator, Sp-cAMPs-AM, and ADA consistently reduced fMLP-mediated translocation of RhoA and Arf1 to membranes (Fig. 7B) . Together, these data suggest that PKA inhibits fMLP-induced PLD activation and PLD-cofactor recruitment.



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  		Figure 7. Effect of Sp-cAMPs-AM and Rp-cAMPs-AM on fMLP-induced translocation of PKC{alpha}, RhoA, and Arf1 to membranes. Neutrophils were prewarmed at 37°C for 5 min and incubated for 30 min with the indicated concentrations of Rp-cAMPs-AM (A) or Sp-cAMPs-AM (B). Neutrophils were incubated for an additional 5 min with 10 µmol/L CB in the absence (A) or the presence (B) of 0.1 U/ml ADA prior to stimulation with 0.1 µmol/L fMLP for 2 min at 37°C. Reactions were stopped, and neutrophil membranes were prepared as described in Materials and Methods. The samples were probed for PKC{alpha}, RhoA, and Arf1. The data are from one experiment representative of four similar experiments. Quantitation by densitometry of Arf and RhoA in neutrophil membranes is shown in the lower left and right panels, respectively (n=4). *, P < .05 using the Student’s paired t-test.

 
Effect of H89, a selective inhibitor of PKA, on fMLP-induced PLD activity and translocation of PKC{alpha}, RhoA, and Arf1 to membranes
To further examine the role of PKA in the inhibition of fMLP-induced responses by adenosine, we investigated the effect of another structurally unrelated inhibitor of PKA, H89 [34 ]. H89 had little effect on its own on basal PLD activity. The amounts of PEt formed in response to stimulation with fMLP (no ADA) were increased by 4.1 ± 0.4-fold in the presence of H89 (Fig. 8A ). When neutrophils were incubated with ADA, H89 was without effect on fMLP-induced PLD activation. Moreover, H89 reversed the inhibitory effect of CGS21680 on PLD activity. In the presence of CGS21680 (with or without ADA), H89 restored the magnitude of [3H]PEt formed to levels similar to those measured in fMLP-stimulated and ADA-treated neutrophils. Next, we investigated whether H89 impacted on fMLP-induced translocation of PKC{alpha} and small GTPases. As estimated by densitometric analyses of the blots, H89 alone slightly increased the basal amounts of PKC{alpha}, RhoA, and Arf1 associated with membranes and enhanced their translocation in response to stimulation with fMLP (Fig. 8B , lane 4). Furthemore, H89 totally reversed the inhibitory effect of CGS21680 on fMLP-induced translocation of PKC{alpha}, RhoA, and Arf1 to neutrophil membranes (Fig. 8B , lanes 5 and 6). Together, the data are consistent with the hypothesis that the inhibition of the stimulation of PLD activity and of the recruitment of small GTPases and PKC{alpha} to membranes induced by fMLP observed upon A2a receptor occupancy is mediated by an activation of PKA (Fig. 9 ).



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  		Figure 8. Effect of H89 on fMLP-mediated PLD activity and translocation of PKC{alpha}, RhoA, and Arf1 to membranes. (A) [3H]-Labeled neutrophils were prewarmed at 37°C for 5 min. Where indicated, the samples were treated with 10 µmol/L CB, 20 µmol/L H89, and 0.1 µmol/L CGS21680 in the absence ({square}) or the presence ({blacksquare}) of 0.1 U/ml ADA and incubated for an additional 5 min prior to stimulation with 0.1 µmol/L fMLP for 10 min at 37°C in the presence of 1% ethanol. PLD activity was measured as described in Materials and Methods. The levels of [3H]PEt formed are expressed as a percentage of the radioactivity in the total lipid extracts. The data are means ± SE of three independent experiments. * and {dagger}, P < .05 as compared with fMLP-stimulated neutrophils in the absence or the presence of ADA, respectively, using the Student’s paired t-test. (B) Neutrophils were processed as in A and incubated in the absence of 0.1 U/ml ADA for 5 min prior to stimulation with fMLP for 2 min. Reactions were stopped, and neutrophil membranes were prepared as described in Materials and Methods. The samples were probed for PKC{alpha}, RhoA, and Arf1. The data are from one experiment representative of three similar experiments. Quantitation by densitometry of Arf and RhoA in neutrophil membranes is shown in the lower left and right panels, respectively (n=3).

 


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  		Figure 9. Model of PLD regulation by adenosine A2a receptors. Occupancy of adenosine A2a receptors (A2a-R) by adenosine or the agonist CGS21680 enhances adenylyl cyclase activation; then, cAMP activates PKA. PKA activation appears to inhibit fMLP-induced translocation of PKC{alpha} and of small GTPases, thereby reducing fMLP-induced PLD activation. PKA activity can be enhanced indirectly by stimulating adenylyl cyclase with forskolin, inhibiting the degradation of intracellular cAMP by PDE IV with Ro20-1724, or directly by the PKA activator Sp-cAMPs. The removal of extracellular adenosine with ADA or the addition of the adenosine A2a receptor antagonist CSC reduces cAMP accumulation and PKA activation. PKA activity is also reduced by the inhibition of adenylyl cyclase with 2',5'-ddADO or by the PKA inhibitors H89 and Rp-cAMPs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study provide evidence that the presence of adenosine in the extracellular milieu bathing human neutrophils negatively modulates the stimulation of the activity of PLD induced by fMLP. The data indicate that these effects are mediated by increases in the kinetics of the elevations of cAMP and in the activity of PKA. Finally, several lines of evidence suggest that the inhibition of PLD is mediated by an interference with the translocation of small GTPases, Arf, and RhoA.

Adenosine exerts its biological effects via the pharmacologically distinct receptor subtypes, A1, A2a, A2b, and A3 [35 , 36 ]. The expression of A2a receptors on neutrophils is supported by the detection of its mRNA by reverse transcriptase polymerase chain reaction [37 ]. Furthermore, pharmacological and immunological evidence has identified functional A1 and A2 receptors on neutrophil plasma membranes [38 , 39 ]. Endogenous adenosine is a potent autocrine regulator of PLD activity in chemoattractant-activated neutrophils, and our previous pharmacological studies support the involvement of A2a receptors in the inhibition of fMLP-stimulated PEt formation [15 ]. As initially described by Jackowski and Sha’afi [40 ], fMLP increases cAMP levels in neutrophils. This elevation in the levels of cAMP can be divided in two distinct phases, one short-lived and another more sustained. In the present study, we demonstrated that the removal of endogenously produced adenosine by ADA or the addition of the selective A2a receptor antagonist CSC inhibited the sustained phase of cAMP accumulation without affecting the initial transient elevation in cAMP levels. In contrast, the addition of the selective A2a receptor agonist CGS21680 to ADA-treated neutrophils restored the long-lasting cAMP elevation. The persistent increases in cAMP levels were functionally correlated to the suppression of fMLP-induced PLD activity and highlight the role of A2a receptors in regulating the long-lasting increase in cAMP levels. This is consistent with previous studies showing that intracellular cAMP elevation as the result of ligand-induced activation or neutrophil treatment with cell membrane-permeable cAMP analogues reduced fMLP-stimulated PLD activity [22 , 23 ].

Although the occupancy of A2a receptors has been suggested to uncouple chemoattractant receptors from their downstream signal transduction pathways [25 ], adenosine does not interfere with PLC-mediated inositol 1,4,5-triphosphate formation [20 ], the mobilization of intracellular Ca2+, or the early, transient increases in diacylglycerol formation induced by fMLP [21 , 41 ]. However, adenosine has been shown to inhibit the influx of Ca2+ induced by fMLP [42 ] or platelet-activating factor (PAF) [14 ] and to diminish the late and sustained increases in diacylglycerol that follow fMLP stimulation [41 ]. We attributed this late and persistent increase in diacylglycerol formation to the sequential actions of a PLD and of lipid phosphohydrolases [15 , 43 ]. Therefore, the inhibitory effects of adenosine on the late phase of accumulation of diacylglycerol are consistent with the results of the present and previous studies [15 ] describing inhibitory effects of adenosine on the stimulation of the activity of PLD in human neutrophils. A2a receptors are linked to G{alpha}s [44 , 45 ], and their stimulation by selective agonists stimulates cAMP formation in intact granulocytes [21 , 27 ] or in neutrophil membranes [38 ]. The ability of adenosine to induce long-lasting increases in the levels of cAMP in response to fMLP stimulation has been demonstrated only in the presence of nonmethylxanthine inhibitors of PDE [26 ] but not with the xanthine-based PDE inhibitors, 3-isobutyl-1-methylxanthine [46 ] or theophylline [26 ], which are also nonselective adenosine receptor antagonists [19 ]. Furthermore, stimulation of ß-adrenergic receptors, which belong to G{alpha}s-coupled receptors and are known to increase cAMP levels in neutrophils, reduced fMLP-induced PLD activity [26 , 46 ]. These suppressive effects of A2a and ß adrenergic receptors are likely mediated through activation of adenylyl cyclase, because 2',5'-ddADO, an inhibitor of adenylyl cyclase [33 ], reversed, at least in part, the suppressive effect of A2a and ß-adrenergic receptor agonists.

Depending on the cell types studied, increases in cAMP inhibit [47 48 49 ] or stimulate [50 , 51 ] PLD activity via activation of PKA. PLD1a is the major PLD isoform in human neutrophils [52 ]. PLD1 requires phosphatidylinositol 4,5-bisphosphate for activity [53 ] and is activated by Arf, RhoA, and PKC{alpha} in vitro [54 ]. These cytosolic proteins are recruited to membranes of fMLP-stimulated cells, and there is evidence suggesting that they mediate fMLP-induced PLD activity in human granulocytes [30 , 55 ]. Furthermore, the engagement of A2a receptors by selective agonists reduces PLD activity by limiting the recruitment of Arf, RhoA, and PKC{alpha} to membranes [15 ]. As summarized in Figure 9 , the cAMP-PKA pathway is likely to play a key role, because the blockade of PKA activity by H89 or Rp-cAMP increased, whereas the PKA activator, Sp-cAMP, reduced PEt formation and the levels of small GTPases and PKC{alpha} associated with membranes. It is interesting that elevations of intracellular cAMP concentrations have been shown to inhibit chemoattractant-induced activation of RhoA [56 ]. There is evidence that PKA phosphorylates RhoA [57 ] and that the phosphorylation by PKA of RhoA associated to granulocyte membranes reduced GTP{gamma}S-induced PLD activity [58 ]. However, besides the phosphorylation of RhoA, the inhibition of fMLP-induced Arf recruitment involves other signaling molecules because PKA has no effect on Arf-mediated activation of PLD in isolated neutrophil membranes [58 ]. Moreover, PKA activity has been shown to promote the association of Arf1 to Golgi membranes [59 ]. Our data do not extend this concept to neutrophils because PKA activation correlated with the suppression of Arf1 recruitment to membranes. The molecular mechanisms implicated in the cAMP/PKA-dependent inhibition of Arf1 activation or translocation to neutrophil membranes remain to be clarified.

Although adenosine increases cAMP levels, A2a receptors have also been shown to initiate some functional responses via cAMP-independent pathways in human neutrophils [3 ]. Accordingly, the inhibition of PKA by KT-5720 failed to reverse the inhibition of apoptosis [60 ] of fMLP-mediated superoxide-anion generation [25 ] or the activation of serine/threonine phosphatases [29 ] by A2 receptor agonists. In contrast, inhibition of PKA by H89 or Rp-cAMP has been shown to suppress the inhibitory effect of A2 receptor agonists on actin polymerization and phagocytosis [5 ], fMLP-induced activation of the respiratory burst [37 , 61 ], and PAF-induced leukotriene biosynthesis and translocation of 5-lipoxygenase [62 ]. The activation of PLD correlates with activation of the NADPH oxidase [24 , 63 , 64 ]. A model in which a phosphatidic acid (PA)-activated kinase is involved in the activation of the NADPH oxidase has been proposed [24 ]. Elevation of intracellular cAMP by physiological agonists (e.g., PGE2, histamine, or ß-adrenergic agonists) inhibits the respiratory burst and PLD activation in fMLP-stimulated neutrophils, consistent with a functional link [22 , 23 ]. The inhibition of PLD activation and superoxide anion generation by adenosine A2a receptor activation is consistent with a role for PA-derived PLD in NADPH activation [15 , 37 , 61 ]. However, determining the impact of adenosine A2a receptor occupancy on the activation of rac, a small GTPase required for NADPH oxidase activity [65 ], is required to critically evaluate the role of PA-derived PLD in this model.

In summary, the results of this study demonstrate that endogenous adenosine inhibits the activation of PLD through the engagement of A2a receptors, which changes the transient elevations of cAMP induced by fMLP into persistent elevation of cAMP levels. Our data also indicate that the inhibition of fMLP-induced PLD activity is functionally linked to inhibition, via a PKA-regulated mechanism, of fMLP-induced recruitment of Arf, RhoA, and PKC{alpha} to membranes. Our studies are consistent with the hypothesis that adenosine uncouples chemoattractant receptors from the PKC and small G protein-dependent activation of PLD1 activation via PKA-dependent mechanisms. These various observations are schematically depicted in the model shown in Figure 9 .


   ACKNOWLEDGEMENTS
 
This work was supported by a Senior Scholarship from the Arthritis Society of Canada to S. G. B. and grants from the Canada Institute for Health Research, MT-14790 (to S. G. B.) and MGC-36034 (to P. B., P. H. N., and S. G. B.). N. T. is the recipient of a studentship from the "Fonds pour la Formation de Chercheurs et l’Aide à la Recherche".

Received June 14, 2001; revised September 19, 2001; accepted September 24, 2001.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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