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(Journal of Leukocyte Biology. 2003;73:530-539.)
© 2003 by Society for Leukocyte Biology

Arachidonic acid activates phospholipase D in human neutrophils; essential role of endogenous leukotriene B4 and inhibition by adenosine A2A receptor engagement

Sonya Grenier*, Nicolas Flamand*, Julie Pelletier*, Paul H. Naccache*,{dagger}, Pierre Borgeat*,{ddagger} and Sylvain G. Bourgoin*,{ddagger}

* Canadian Institutes for Health Research Group on the Molecular Mechanisms of Inflammation, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ (CHUL) et Départements
{ddagger} d’Anatomie-Physiologie et de
{dagger} Médecine, Faculté de Médecine, Université Laval, Québec, Canada

Correspondence: Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ (CHUL), Local T1-49, 2705 Boulevard Laurier, Québec, Qc, Canada, G1V 4G2. E-mail: sylvain.bourgoin{at}crchul.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report in human neutrophils (PMN) that phospholipase D (PLD) was stimulated by micromolar concentrations of arachidonic acid (AA) and nanomolar concentrations of leukotriene B4 (LTB4), and eicosapentaenoic acid was inactive. The stimulatory effect of AA occurred only when adenosine was eliminated from PMN suspensions or when PMN were incubated with adenosine A2A receptor antagonists. The mechanism of AA-induced PLD activation was investigated. The results show that AA- and LTB4-induced PLD activation were inhibited by the LTB4 receptor 1 (BLTR1) antagonist CP 105,696, whereas the LTA4 hydrolase inhibitor SC57461A and the LT biosynthesis inhibitor MK-0591 inhibited AA- but not LTB4-mediated PLD activation. The AA-induced ARF1 and RhoA translocation to PMN membranes was inhibited by CP 105,696 and SC57461A. These results provide evidence of a requirement for an autocrine-stimulatory loop involving LTB4 and BLTR1 in the translocation of small GTPases to membranes and the activation of PMN PLD by AA.

Key Words: phospholipase A2 • 5-lipoxygenase • chemotaxis • inflammation • LTB4 receptors • adenosine deaminase • adenosine receptors • CGS-15943 • CGS-21680 • chlorostyryl caffeine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of human polymorphonuclear neutrophils (PMN) by soluble agonists and phagocytic stimuli increases the levels of intracellular-free calcium and causes cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid (AA) [1 ]. Although AA is considered an important PMN activator, the molecular mechanisms through which it regulates PMN functions are still poorly understood and contentious. Conversely, in cell-free systems and in intact cells as well, AA can stimulate the activity of protein kinase C (PKC) [2 , 3 ] and of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [4 ], the release of calcium from intracellular pools [5 ], and the phosphorylation and activation of p38 mitogen-activated protein kinase [6 ]. The above responses are mediated by AA itself and not by biologically active metabolites of AA, as they were unaffected by the addition of cyclooxygenase or lipoxygenase (LO) inhibitors. Conversely, AA promotes a number of biological processes, such as chemotaxis [7 ], aggregation [8 ], adhesion [9 ], and degranulation [8 ], in a manner that is sensitive to inhibitors of AA metabolism. Indeed, 5-LO metabolites mediate AA-induced activation of downstream signaling cascades, including those dependent on PLC [10 ], phosphatidylinositol 3-kinase [11 ], and extracellular signal-regulated kinase [6 ]. These effects are mediated by pertussis toxin (PT)-sensitive G-proteins.

The major AA metabolites synthesized by activated PMN are the products of the stereospecific biotransformation of AA by the 5-LO. 5-Hydroperoxyeicosatetraenoic acid, the first metabolite of the 5-LO pathway, can be reduced to 5-hydroxyeicosatetraenoic acid (5-HETE) and/or metabolized further by the 5-LO to leukotriene A4 (LTA4) [12 ]. LTA4 is then rapidly converted to LTB4 by the LTA4 hydrolase. LTB4 is a biologically active compound [12 ] that acts through interaction with the specific cell-surface receptors BLTR1 and BLTR2 [13 14 ].

Adenosine acting via occupancy of adenosine A2A receptors (A2AR) is a potent suppressor of PMN functions [15 , 16 ]. It is well documented that PMN suspensions release adenosine to levels that can strongly inhibit AA release and LTB4 production by formyl-Met-Leu-Phe (fMLP)- or platelet-activating factor (PAF)-stimulated cells [15 , 17 ], as well as the biosynthesis of LTB4 induced by exogenous AA [18 ]. Furthermore, adenosine inhibits fMLP-mediated stimulation of PLD activity and translocation of the small GTPases, adenosine 5'-diphosphate-ribosylation factor (ARF)1 and RhoA, which are direct activators of PLD [19 ]. LTB4 is one of the most potent leukocyte chemoattractants [14 ] and has been shown to stimulate the activity of PLD in PMN [20 ].

In the present study, we sought to characterize the putative effect of AA on PLD activation and in particular, to define its relationship to the biosynthesis of LTB4 and its modulation by adenosine. The results of these studies indicate that in the absence of adenosine, AA induces PLD activation through an autocrine-stimulatory loop involving LTB4. We also observed that A2AR occupancy inhibited AA- and LTB4-induced PLD activation. These studies substantiate that the pharmacological manipulation of adenosine concentrations at inflammatory sites can suppress AA- and LTB4-induced stimulation of biological responses in human PMN.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
8-(3-Chlorostyryl)caffeine (CSC), 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943), and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) were purchased from RBI (Natick, MA). The BLTR1 antagonist CP 105,696 was a generous gift from Henry J. Showell (Pfizer, Groton, CT). The LT biosynthesis inhibitor MK-0591 and the LTA4 inhibitor SC57461A were kindly provided by Dr. Robert Young (Merck Frosst, Kirkland, Québec, Canada) and Dr. Walter G. Smith (Searle and Co., Skokie, IL), respectively. Monosodium urate crystals (MSU) were kindly provided by Dr. Robert de Médicis (University of Sherbrooke, Québec, Canada) and were prepared as described previously [21 ]. Dextran T-500 and Ficoll-Paque were purchased from Pharmacia Biotech (Dorval, Québec, Canada). Anti-RhoA antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). AA, adenosine deaminase (ADA), di-isopropyl-fluorophosphate (DFP), eicosapentaenoic acid (EPA), fMLP, LTB4, and all other reagents were obtained from Sigma-Aldrich Canada (Oakville, Ontario). ADA was dialyzed against 0.9% NaCl before use to remove ammonium salts. Mg2+-free Hanks’ balanced salt solution (HBSS) was obtained from Wisent Canadian Laboratories (St-Bruno, Québec). Rabbit anti-ARF1 antibodies were generated against bovine ARF1 and characterized in previous studies [22 ].

Isolation of human neutrophils
Venous blood was collected from healthy volunteers in bags containing isocitrate as an anticoagulant. PMN were isolated sterilely as described previously [19 ]. Briefly, after discarding the platelet-rich plasma, erythrocytes were removed by dextran sedimentation. Mononuclear cells were then separated from the granulocytes by centrifugation on Ficoll-Paque cushions. The granulocyte cell pellet was subjected to hypotonic lysis with sterile water to remove the remaining erythrocytes. The granulocyte suspension contained mainly PMN (>=95%) with eosinophils as the major contaminants, as assessed by Diff Quick® staining. Cell viability was always greater than 97%, as measured by trypan blue exclusion. The cells were resuspended in HBSS, supplemented with 0.8 mM CaCl2.

Stimulation of the cells
For the experiments on PLD activation, PMN (8x106 cells/ml) were preincubated at 37°C for 5 min and treated with 10 µM cytochalasin B (CB) for a further 5 min. PMN were then stimulated for 10 min with fMLP, AA, EPA, or LTB4 at the indicated concentrations (see figure legends) in the presence of 1% ethanol. Where indicated, PMN were preincubated with 1.5 nM tumor necrosis factor {alpha} (TNF-{alpha}) and 700 pM granulocyte macrophage-colony stimulating factor (GM-CSF) for 30 min at 37°C in HBSS before stimulation with 300 nM fMLP or PAF for 10 min [23 ]. For the MSU-stimulation experiments, PMN were preincubated at 37°C for 10 min and stimulated with 3 mg/ml MSU for 15 min. In the experiments where the translocation of RhoA and ARF1 was analyzed, PMN (107 cells/ml) were preincubated 5 min at 37°C and treated with 10 µM CB for a further 5 min before stimulation with 100 nM LTB4 or 2.5 µM AA for 2.5 min. Unless stated otherwise, 1 U/ml ADA was added to the cell suspensions 5 min before all stimulations to eliminate the inhibitory constraint of endogenous adenosine [15 , 19 ]. CGS-21680, CGS-15943, CP 105,696, SC57461A, as well as MK-0591 in solution in Me2SO were added to the cell suspensions at the indicated concentrations (see figure legends) 5 min before stimulation. Me2SO concentrations never exceeded 0.2% in incubation media.

Analysis of PLD activity
PMN were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 PMN) for 90 min as described previously [19 ] and stimulated as described above. All incubations were stopped by the addition of 1.8 ml cold (4°C) stop solution (CHCl3/MeOH/HCl; 50/100/1, by vol) containing unlabeled phosphatidylethanol (PEt) as a thin-layer chromatography (TLC) standard. The lipids were extracted as described previously [19 ], and the extracts were evaporated to dryness under a stream of nitrogen. The lipid residues were dissolved in CHCl3/MeOH (2:1, v/v) and spotted on prewashed silica gel 60 TLC plates. PEt was resolved from the other lipids using the solvent mixture CHCl3/MeOH/CH3COOH (65/15/2, by vol). Lipids were visualized by Coomassie brilliant blue staining, and the different lipid classes were scraped off the plates. Radioactive PEt was measured by liquid scintillation counting, and the results were corrected for background radioactivity and quenching.

Translocation of RhoA and ARF1
PMN (4x107 cells/ml) were treated with 1.1 mM DFP (30 min, 24°C) and stimulated as described above. The incubations were stopped by the addition of 5 vol cold (4°C) incubation buffer, and the cell suspensions were immediately centrifuged. Cell pellets were then resuspended at 1.6 x 107 cells/ml in cold (4°C) KCl-HEPES relaxation buffer [100 mM KCl, 50 mM HEPES, 5 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 2.5 mM phenylmethylsulfonyl fluoride (PMSF), and 2.5 µg/ml aprotinin and leupeptin, pH 7.2] and were sonicated using a Branson sonifier (2x20 s, 100% duty cycle). The sonicates were centrifuged for 7 min at 700 g. Unbroken cells and nuclei pellets were discarded, and the supernatants were ultracentrifuged (180,000 g, 45 min) at 4°C. The membrane pellets were washed once and resuspended in 150 µl buffer A [250 mM Na2HPO4, 300 mM NaCl, 2.5% sodium dodecyl sulfate (SDS), 2.5 mM PMSF, and 2.5 µg/ml aprotinin and leupeptin, pH 6.8]. Samples were assayed for protein content, and proteins were separated by SDS-polyacrylamide gel electrophoresis (12%) as described previously [19 ] and were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Membranes were blotted using the anti-ARF1 (1/3500) and the anti-RhoA (1/1000) antibodies and revealed with the enhanced chemiluminescence detection system.

Analysis of 5-LO products
Cells were primed with TNF-{alpha} and GM-CSF as described above before stimulation with 300 nM fMLP or PAF for 5 min at 37°C. In all experimental setting, 1 U/ml ADA was added to PMN suspensions 30 min before stimulation to eliminate the accumulation of extracellular adenosine [17 ]. For the determination of the 5-LO products, cell supernatants were denatured by the addition of 0.5 vol ice-cold MeOH/MeCN (1:1, v/v) containing 12.5 ng prostaglandin B2 (PGB2) and 19-OH-PGB2 as internal standards. The samples were processed and analyzed by reversed phase-high-pressure liquid chromatography (RP-HPLC) using an on-line extraction procedure [23 ]. The sum of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6(E)-LTB4, 6(E)-12-epi-LTB4, and 5-HETE was compiled and referred to as 5-LO products.

Measurement of endogenous AA release
The release of endogenous AA from thapsigargin-activated PMN was determined as described previously [24 ]. Briefly, PMN (5x106/ml) were treated with ADA (1 U/ml) and simultaneously exposed or not exposed to 100 nM CGS-21680 5 min before stimulation with 100 nM thapsigargin for the indicated times. AA was isolated from the incubation medium by RP-HPLC and analyzed by liquid chromatography–mass spectrometry [24 ].

Statistics
Data are expressed as means ± SE. Data were analyzed using the Student’s paired t-test (two-tailed) 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
 
AA and LTB4 activate PLD
We have previously demonstrated that the stimulation of human PMN by exogenous AA leads to the biosynthesis of important quantities of LTB4 [18 ], and others have shown that LTB4 stimulates PLD activity in PMN [20 ]. Therefore, a first series of experiments was performed to evaluate the putative effect of AA on PLD activity in human PMN. It is important to point out here that these experiments were performed in the presence of ADA to remove adenosine from cell suspensions, as the nucleotide is a potent inhibitor of LTB4 biosynthesis in PMN [18 ]. Figure 1A shows that the addition of exogenous AA enhanced PLD activity in a concentration-dependent manner. The PLD activity observed in these experimental conditions was maximal at 2.5 µM AA and declined at higher concentrations of the fatty acid. In contrast, EPA, an {omega}-3 fatty acid, which is efficiently metabolized to LTB5 by the 5-LO in human PMN, did not increase PLD activity over basal level (Fig. 1A) .



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  		Figure 1. Effect of AA, EPA, and LTB4 on PLD activity in human PMN. Human PMN were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine and were suspended in HBSS (8x106 cells/ml). Cell suspensions were prewarmed at 37°C for 5 min and incubated with CB (10 µM) and ADA (1 U/ml) for an additional 5 min. PMN were then stimulated for 10 min in the presence of 1% ethanol and (A) increasing concentrations of AA or EPA or (B) increasing concentrations of LTB4 as described in Materials and Methods. Incubations were stopped with the addition of 3.6 vol cold (4°C) stop solution containing unlabeled PEt as TLC standard. [3H]PEt was recovered as described in Materials and Methods, and radioactivity content was analyzed. Data are the mean ± SEM of at least three separate experiments, each performed in duplicates. *, P < 0.05 using the Student’s paired t-test.

 
A second series of experiments was performed to evaluate the efficiency of LTB4 to induce PLD activation under identical conditions (Fig. 1B) . LTB4 and PAF (data not shown) were capable of stimulating PLD activity in PMN, although they were less potent than fMLP (fMLP>=PAF>>LTB4; data not shown). The activation of PLD was up-regulated by LTB4 in a concentration-dependent manner up to the highest concentration tested (100 nM).

LT biosynthesis inhibitors and antagonists inhibit the AA-induced activation of PLD
It was previously suggested that AA-induced cell signaling may implicate the LTB4 receptor [25 ]. Experiments were performed to determine if the PLD activity induced by exogenous AA in human PMN was dependent on 5-LO activation, LTB4 biosynthesis, and stimulation of BLTR1 in our experimental conditions. As shown in Figure 2A , the enhancement of PLD activity observed in AA- or LTB4-stimulated PMN was completely abolished by preincubating the cells with CP 105,696, a selective BLTR1 antagonist [13 ]. The AA-induced PLD activity was slightly more sensitive to the inhibitory effect of the BLTR1 antagonist than was LTB4-induced activity. These results are in perfect agreement with the previously reported inhibitory effects of CP 105,696 on LTB4-induced chemotaxis [26 ] and AA-induced Ca2+ mobilization, 5-LO translocation, and LT biosynthesis in human PMN [18 ]. To further assess the cause–effect relationship between the de novo biosynthesis of LTB4 and the stimulation of the activity of PLD in AA-stimulated human PMN, two additional experiments were performed using the LTA4 hydrolase inhibitor SC57461A and the LT biosynthesis inhibitor MK-0591. As shown in Figure 2B and 2C , increasing concentrations of SC57461A or MK-0591 strikingly abrogated the stimulation of the activity of PLD induced by AA but not the activity induced by LTB4. These results confirm that the stimulatory effect of AA on the activity of PLD depends on the generation of 5-LO-derived metabolites and of LTB4 in particular.



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  		Figure 2. Effect of 5-LO pathway inhibitors and antagonist on AA-induced PLD activity. Human PMN were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine and suspended in HBSS (8x106 cells/ml). Cell suspensions were prewarmed at 37°C for 5 min and incubated with CB (10 µM) and ADA (1 U/ml) for an additional 5 min. PMN were then stimulated for 10 min in the presence of 1% ethanol and AA (2.5 µM) or LTB4 (100 nM) with increasing concentrations of (A) the LTB4 antagonist CP 105,696, (B) the LTA4 hydrolase inhibitor SC57461A, or (C) the LT biosynthesis inhibitor MK-0591, added 5 min before stimulation (with AA or LTB4) as described in Materials and Methods. Incubations were stopped with the addition of 3.6 vol cold (4°C) stop solution containing unlabeled PEt as TLC standard. [3H]PE was recovered as described in Materials and Methods, and radioactivity content was analyzed. Data are the mean ± SEM of at least three separate experiments, each performed in duplicates. The basal levels of PEt formed were 0.025 ± 0.006% (A), 0.023 ± 0.009% (B), and 0.053 ± 0.01% (C), and those formed in response to AA and LTB4 (control 100%) were 0.28 ± 0.1% and 0.68 ± 0.13% (A), 0.45 ± 0.14% and 0.70 ± 0.23% (B), and 0.24 ± 0.06 and 0.61 ± 0.11% (C) of total radioactivity, respectively. *, P < 0.05 using the Student’s paired t-test.

 
Importance of the 5-LO pathway in stimulus-induced PLD activity in human PMN
Pharmacological agents such as calcium ionophores (ionomycin, A23187) and the Ca2+-ATPase blocker thapsigargin or particulate agonists such as MSU are frequently used to investigate the regulation of AA metabolism in human PMN. They are indeed very potent inducers of LT biosynthesis [15 ]. fMLP also stimulates LT biosynthesis (although to a lesser extent) in human PMN when cells are pre-exposed to priming agents such as TNF-{alpha}, GM-CSF, or lipopolysaccharide [24 , 27 , 28 ]. As AA-induced PLD activity is dependent on de novo LTB4 biosynthesis in human PMN (Fig. 2) , we investigated herein the putative role of LTB4 in PLD activation in human PMN exposed to other stimuli. As shown in Figure 3 , a suboptimal concentration of fMLP (1 nM) and thapsigargin (100 nM) induced significant PLD activity in human PMN. Neither the BLTR1 antagonist CP 105,696 (Fig. 3A) nor the LTA4 hydrolase inhibitor SC57461A (not shown) affected the fMLP-induced PLD activity.



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  		Figure 3. Effect of 5-LO pathway inhibitors and antagonist on fMLP- and thapsigargin-induced PLD activity. Human PMN were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine and suspended in HBSS (8x106 cells/ml). Cell suspensions were prewarmed at 37°C for 5 min and incubated with CB (10 µM) and ADA (1 U/ml) for an additional 5 min. Cells were then stimulated for 10 min with ethanol (1% final concentration) and (A) 1 nM fMLP or (B and C) 100 nM thapsigargin. CP 105,696 (10 nM) and SC57461A (1µM) were always added 5 min before stimulation with fMLP or thapsigargin. Incubations were stopped with the addition of 3.6 vol cold (4°C) stop solution containing unlabeled PEt as TLC standard. [3H]PEt was recovered as described in Materials and Methods, and radioactivity content was analyzed. Data are the mean ± SEM of at least three separate experiments, each performed in duplicates. *, P < 0.05 using the Student’s paired t-test.

 
To properly evaluate whether fMLP and PAF-stimulated PLD activity is dependent on BLTR1 occupation under proinflammatory conditions where LTB4 formation is greatly enhanced, human PMN were primed with GM-CSF and TNF-{alpha} before stimulation, and the activation of PLD and the formation of 5-LO products were evaluated. In these experiments, the final concentrations of 5-LO products in cell suspensions were estimated to be 50.4 ± 20 nM and 42.4 ± 9 nM in response to stimulation with fMLP and PAF, respectively. As shown in Figure 4A , the addition of the BLTR1 antagonist CP 105,696 or of the LTA4 hydrolase inhibitor SC57461A resulted in a partial inhibition of PAF and fMLP-induced LT synthesis (30–60% inhibition). Furthermore, the synthesis of 5-LO products was totally abrogated by the LT biosynthesis inhibitor MK-0591 (Fig. 4A) . In contrast, CP 105,696, SC57461A, and MK-0591 were without significant effect on fMLP- and PAF-stimulated PLD activity (Fig. 4B) . Similarly, CP 105,696 or SC57461A did not significantly alter PLD activity in unprimed PMN stimulated with MSU and A23187 (data not shown). Conversely, the results do not totally exclude a minor role for LTB4 in fMLP- and PAF-stimulated PLD activity. However, the contribution of LTB4 could not be detected, as it is a weak agonist for the activation of PLD in PMN. Indeed, the levels of [3H]PEt formed in response to stimulation with 10–100 nM LTB4 were 10–15% of those induced by supraoptimal concentrations of fMLP or PAF (300 nM) in primed and unprimed PMN. Conversely, CP 105,696 and SC57461A (Fig. 3B and 3C , respectively) completely inhibited the activation of PLD induced by thapsigargin.



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  		Figure 4. Inhibition of LT synthesis does not inhibit fMLP- and PAF-induced PLD activation. PMN (5x106/ml) were primed with 1.5 nM TNF-{alpha} and 700 pM GM-CSF and were stimulated with 300 nM fMLP or PAF in the presence of 1 U/ml ADA and 10 µM CB as described in Materials and Methods. (A) Supernatants were analyzed by RP-HPLC to determine LT synthesis. The data are the mean ± SEM of two separate experiments, each performed in triplicate. (B) The formation of [3H]PEt by labeled PMN was monitored in parallel. The data are the mean ± SEM of two separate experiments, each performed in duplicate.

 
The release of AA from membrane phospholipids is an important step in LT biosynthesis [15 ]. Figure 5 shows that the addition of thapsigargin to human PMN stimulated the release of AA. A lag of ~2 min was observed before a measurable release of AA could be detected. The presence of 1 U/ml ADA to eliminate extracellular adenosine was required to achieve an experimental condition in which the release of AA can be evaluated. As previously observed for LT synthesis [23 ], the addition of the adenosine A2AR agonist CGS-21680 caused a total inhibition of thapsigargin-induced AA release (Fig. 5) . To confirm that the activation of PLD is a result of the sequential release of AA from membrane phospholipids and the formation of 5-LO products in thapsigargin-stimulated PMN, we monitored the effect of the cPLA2 inhibitor pyrrophenone [29 ]. The addition of 1 µM pyrrophenone to PMN effectively inhibited the stimulation of PLD activity induced by thapsigargin (69.3±4.9%) but not fMLP (11.1±0.7%). These results demonstrate that the fMLP-stimulated PLD activity does not require LTB4, and the PLD activity induced by thapsigargin is totally dependent on the sequential release of AA from cellular phospholipids by a cPLA2 and the de novo LTB4 biosynthesis.



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  		Figure 5. Time dependence of AA release in thapsigargin-activated neutrophils. Human PMN (5x106/ml) were treated with 1 U/ml ADA and simultaneous exposed to 0.1 µM CGS-21680 or the diluent (Me2SO) 5 min before stimulation with 100 nM thapsigargin for the indicated times. AA was isolated from the cell suspensions by RP-HLPC and analyzed by liquid chromatography–mass spectrometry. The data shown are from single incubations from one experiment representative of three others.

 
AA induces the translocation of the small GTPases RhoA and ARF1 to membrane fractions
Of the PLD isoforms characterized so far (PLD1, its spliced variant PLD1b, and PLD2), only the expression of PLD1a protein has been documented in human PMN [21 ]. PLD1 has a low basal activity but is stimulated several times in a guanosine 5'-triphosphate (GTP)-dependent manner by ARF and the GTP-binding proteins of the Rho family [30 ]. fMLP-stimulated PLD activity in human PMN is associated with an increase in the translocation of ARF1 and RhoA to cellular membranes [19 ]. It was previously demonstrated that the kinetics of the synthesis of LTB4 in PMN exposed to AA in the presence of ADA was maximal within 2 min [18 ]. Therefore, we investigated if AA-induced PLD activity also implicates an increase in the translocation of ARF1 and RhoA to cellular membranes. The data illustrated in Figure 6A and 6B , demonstrate that stimulation of PMN with AA (2.5 µM) and LTB4 (100 nM) increased the amounts of ARF1 and RhoA associated with cellular membranes and that AA-induced translocation events are time-dependent and maximal at ~2.5 min. There was a lag period before a significant translocation of small GTPases could be detected, and maximal recruitment varied between 1 and 5 min, depending on PMN preparation. The translocation of ARF1 and RhoA induced by AA was blocked by the LTA4 hydrolase inhibitor SC57461A and the BLTR1 antagonist CP 105,696 (Fig. 7. A and 7B ). These results provide evidence that the cellular events associated with the induction of PLD activity by fMLP in human PMN are also observed in AA-stimulated PMN and that the biotransformation of AA to LTB4 by the 5-LO is required to stimulate the translocation of ARF1 and RhoA.



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  		Figure 6. Effect of AA and LTB4 on the translocation of RhoA and ARF1 to the membranes. PMN suspensions (4x107 cells/ml) were treated with 1.1 mM DFP (30 min, 24°C), prewarmed at 37°C for 5 min, and incubated with CB (10 µM) and ADA (1 U/ml) for an additional 5 min before stimulation with LTB4 (100 nM) or AA (2.5 µM) for the indicated times. Incubations were stopped with the addition of 5 vol cold (4°C) incubation buffer, and all cell suspensions were immediately centrifuged. Cell pellets were processed as described in Materials and Methods, and the membrane fractions were analyzed for their content in (A) RhoA and (B) ARF1. Histogram values (densitometric analysis) are the mean ± SEM of at least three separate experiments. * P < 0.05 using the Student’s paired t-test. NS,.

 


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  		Figure 7. Effect of the BLTR1 antagonist CP 105,696 and of the LTA4 hydrolase inhibitor SC57461A on the translocation of RhoA and ARF1 in AA-stimulated PMN. PMN suspensions (4x107 cells/ml) were treated with 1.1 mM DFP (30 min, 24°C), prewarmed at 37°C for 5 min, and incubated with CB (10 µM) and ADA (1 U/ml) for an additional 5 min before stimulation with LTB4 (100 nM) or AA (2.5 µM) for 2.5 min. Incubations were stopped with the addition of 5 vol cold (4°C) incubation buffer, and all cell suspensions were immediately centrifuged. Cell pellets were processed as described in Materials and Methods, and the membrane fractions were analyzed for their content in (A) RhoA and (B) ARF1. Where indicated, CP 105,696 (CP; 100 nM) and SC57461A (SC; 1 µM) were added 5 min before cell stimulation. The immunoblots shown are from one experiment representative of three others. NS, not stimulated.

 
Adenosine exerts an inhibitory constraint on AA- and LTB4-induced PLD activation
We previously reported that adenosine released by isolated PMNs into the extracellular milieu exerts, through A2AR, an inhibitory constraint on agonist- and AA-induced LT biosynthesis [17 , 18 ] and fMLP-induced PLD activation [19 ]. Thus, in all experiments described above, cell suspensions were routinely incubated in the presence of ADA to eliminate adenosine from the PMN environment, and a series of experiments was subsequently undertaken to define the impact of endogenous adenosine on AA- and LTB4-induced PLD activation in PMN. As shown in Figure 8 , incubation of PMN suspensions in the presence of ADA increased the stimulation of PLD by AA (Fig. 8A and 8C) or LTB4 (Fig. 8B and 8D) in comparison with similar incubations in the absence of ADA. A2AR are more effective in blocking the conversion of AA to LTB4 [18 ] than in inhibiting activation of PLD by LTB4. Accordingly, addition of increasing concentrations of the selective A2AR agonist CGS-21680 to these PMN suspensions inhibited the PLD activity induced by AA and to a lesser extent, LTB4 (Fig. 8A and 8B) . To further investigate the inhibitory effect of endogenous adenosine on PLD activity, PMN suspensions were incubated in the absence of ADA (endogenous adenosine levels allowed to build up in PMN suspensions) and were stimulated with AA or LTB4 in the presence of increasing concentrations of CGS-15943, an adenosine receptor antagonist, or CSC, a selective A2AR antagonist. Both pharmacological agents caused a concentration-dependent increase of PLD activity induced by AA (Fig. 8C and 8E) , which is fully consistent with the inhibitory effect of endogenous adenosine on AA-induced LT biosynthesis previously reported [18 ]. The stimulatory effect of the two A2AR antagonists was less important in LTB4- (Fig. 8D and 8F) than in AA-activated PMN.



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  		Figure 8. Effect of ADA, A2AR agonist, and antagonists on AA- and LTB4-induced PLD activity. PMN were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine and suspended in HBSS (8x106 cells/ml). Cell suspensions were prewarmed at 37°C for 5 min and incubated with CB (10 µM) for an additional 5 min. ADA (1 U/ml; A and B) or vehicle (C–F) was added simultaneously with CB. PMN were then stimulated for 10 min in the presence of 1% ethanol and 2.5 µM AA (A, C, E) or 100 nM LTB4 (B, D, F). In all experimental settings, CGS-21680, CGS-15943, and CSC were added (at the indicated concentrations) 5 min before stimulation with AA or LTB4. PLD activity was monitored as described in Materials and Methods. Data are the mean ± SEM of at least three separate experiments, each performed in duplicates. The basal levels of PEt formed were 0.052 ± 0.01% (A), 0.045 ± 0.005% (B), 0.017 ± 0.004% (C), 0.017 ± 0.004% (D), 0.076 ± 0.011% (E), and 0.071 ± 0.01% (F) of total radioactivity and were not significantly altered by the highest concentrations of CGS-21680, CGS-15943, and CSC. *, P < 0.05 using the Student’s paired t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AA is a 20 carbon tetra-unsaturated fatty acid present in the form of esters, mainly as phospholipids, in biological membranes; AA is also present (in free form) in micromolar concentration in human plasma. AA stimulates numerous biological responses by itself, including the activation of the NADPH oxidase [4 ]. AA is the immediate precursor of numerous biologically active lipids, such as prostaglandins, thromboxanes, LTs, and lipoxin. In activated PMN, the predominant metabolic pathway of AA is the 5-LO pathway, and LTB4 is the major 5-LO metabolite of AA [12 ]. LTB4 is a potent activator of PMN and is considered a lipid mediator of inflammation and host defense. The de novo LTB4 biosynthesis by blood PMN regulates their transmigration elicited by a wide variety of proinflammatory mediators [31 ]. LTB4, by activating and recruiting leukocytes at inflammatory sites, is also important for the clearance of particles including microbes [32 ]. Accordingly, 5-LO knockout mice show increased susceptibility to infection [33 ]. Conversely, overproduction of LTB4 is observed in various inflammatory diseases such as rheumatoid arthritis and ulcerative colitis arthritis [34 , 35 ].

In this study, we characterized the stimulatory effect of AA on PLD activity in human PMN. Our data demonstrated that AA does activate PLD activity in PMN in a dose-dependent manner, and several lines of evidence support the necessary involvement of LTB4 in this stimulatory effect of AA. First, considering that incubation of PMN with AA results in significant LTB4 biosynthesis [18 ] and that LTB4 directly activates PLD, it seems likely that endogenous LTB4 could contribute, at least in part, to the observed effect of AA on PLD activity. Second and most importantly, the stimulatory effect of AA on PLD activity in human PMN was dose-dependently inhibited by three pharmacological agents acting on distinct elements of the 5-LO pathway: the highly selective LTB4 antagonist CP 105,696, the LTA4 hydrolase inhibitor SC57461A, and the LT biosynthesis inhibitor MK-0591. Of these three pharmacological agents, only the LTB4 antagonist inhibited LTB4-induced PLD activity, demonstrating that the inhibitory effect of SC57461A and MK-0591 on AA-induced PLD activation is not the result of an unspecific effect of the drugs on PLD activity or activation mechanisms. In addition, it seems relevant to point out that our previous report that AA-induced LTB4 biosynthesis is strongly inhibited by the A2AR agonist CGS-21680 [18 ] is fully consistent with a role of LTB4 in AA-induced PLD activation, as this process was also shown herein to be highly sensitive to inhibition by A2AR engagement. Finally, EPA, a penta-unsaturated fatty acid closely related to AA (contains one additional double bond at {Delta}17) and a good substrate for the 5-LO, did not activate PLD activity; this intriguing lack of effect of EPA on PLD activity is likely the consequence of the fact that the 5-LO metabolite of EPA, LTB5, is 25 times less potent than LTB4 in activating LTB4 receptors in PMN [36 ]. Taken together, these data conclusively demonstrated that LTB4 is an obligatory mediator of the stimulatory effect of AA on PLD activity in PMN.

To explore the possible involvement of LTB4 in PLD activation by other agonists in PMN, we assessed the effect of the BLTR1 antagonist CP 105,696 on fMLP-induced PLD activity. The results obtained clearly demonstrated that LTB4 is not a necessary mediator of the stimulatory effect of fMLP on PLD activity. It is likely that fMLP, which shares with LTB4 several signal transduction mechanisms, triggers, by itself, the signaling pathway(s) implicated in PLD activation in PMN, in which case, the autocrine effects of endogenous LTB4 may be redundant and unnecessary; this also likely accounts for the noninvolvement of LTB4 in MSU- and A23187-induced PLD activity. In contrast, the potent, stimulatory effect of thapsigargin on PLD activity in PMN was strongly inhibited by the LTA4 hydrolase inhibitor SC57461A and the LTB4 antagonist CP 105,696, demonstrating that thapsigargin-induced PLD activity in PMN is strongly dependent on an autocrine stimulatory loop of endogenous LTB4. This is in agreement with previous reports that several thapsigargin-induced responses in PMN, such as Ca2+ mobilization and AA release, are dependent on an autocrine effect of endogenous LTB4 [18 ].

Several studies have shown that the activation of signal transduction pathways by AA, including Ca2+ mobilization, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases [10 , 11 , 37 ], is dependent on receptors that are coupled to PT-sensitive G-proteins. Previous studies have established that most of the signals induced by LTB4 receptors (BLTR1 and BLTR2) are mediated by Gi-like G-proteins, as they are blocked by a pretreatment with PT [14 ]. Whereas the expression of BLTR2 has been reported in most human tissues, BLTR1 is exclusively expressed in leukocytes [14 ]. Accordingly, in PMN, the stimulatory effects of AA, thapsigargin, and LTB4 on PLD activity were dependent on the activation of the BLTR1, as evidenced by the blockade of PLD activation by CP 105,696, a selective BLTR1 antagonist [13 ].

Adenosine is a potent, endogenous, anti-inflammatory agent. Several studies have established that A2AR decreases the recruitment of leukocytes [38 39 40 ] and the production of proinflammatory cytokines such as interleukin (IL)-12 or increases IL-10 secretion [41 ]. It is interesting that in A2AR(-/-) animals the absence of A2AR-mediated suppressive signals on inflammatory processes results in elevated and prolonged production of proinflammatory cytokines and may explain the increased tissue damage and animal death [42 ]. We previously reported that adenosine released in the extracellular milieu by isolated PMN exerts an inhibitory constraint on agonist-, thapsigargin- and AA-induced LT biosynthesis [15 , 18 ] and fMLP-induced PLD activation [19 ]. In the present study, we found that the occupancy of A2AR by endogenous adenosine exerts a suppressive effect on AA- and LTB4-induced PLD activation and that this effect could be countered by ADA or the A2AR antagonists CSC and CGS-15943. Although the inhibitory effect of A2AR engagement on AA-induced PLD activity is the consequence of the efficient blockade of AA-induced LTB4 formation [15 , 18 ], our observations that A2AR engagement also blocks LTB4 (and fMLP-)-induced PLD activation clearly indicate a second mechanism by which A2AR agonists can regulate PLD activity; this other mechanism, which likely implicates cyclic adenosine monophosphate-regulated events [22 , 43 ], is currently under investigation in our laboratory.

Two mammalian PLDs, PLD1 and PLD2, have been cloned [44 ]. PLD1 is strongly activated by ARFs, RhoA, and PKC-{alpha} in vitro, as opposed to PLD2, which shows high basal activity and is weakly stimulated by these cofactors [30 ]. The PLD enzyme from human PMN is PLD1a, as demonstrated by immunoblot analysis [45 ] and by its activation by RhoA, ARF1, and PKC-{alpha} [30 ]. These cytosolic proteins are recruited to PMN membranes in response to fMLP stimulation and induce PLD activation [19 ]. We report here that ARF1 and RhoA are recruited to membranes in response to LTB4 and AA and that these effects of AA implicate its transformation into LTB4. The translocation of small GTPases induced by AA reached a plateau by 2.5 min, and it preceded the synthesis of LTB4 that was maximal within 2 min [18 ]. The data also demonstrate a role for BLTR1 in the stimulatory effects of AA and LTB4 in ARF1 and RhoA translocation.

In PLB-985 cells, ARF6 but not ARF1 regulates fMLP-mediated activation of PLD [46 ]. It is possible that ARF6 also contributes to AA-induced PLD activation in PMN. In this study, we have routinely measured the translocation of ARF1, as it is six- to sevenfold more abundant than ARF6, which is barely detectable in PMN membranes.

In conclusion, we demonstrate that AA and thapsigargin are efficient stimulators of PLD activity in human PMN. Furthermore, we show an absolute requirement for LTB4 in the activation of PLD by AA and thapsigargin and that LTB4 acts through BLTR1 and induces the translocation of the small GTPases ARF1 and RhoA to PMN membranes. We also demonstrated that adenosine inhibits AA-induced PLD activation, presumably by blocking AA-mediated LTB4 synthesis and by suppressing signaling through BLTR1. These findings, by indicating that adenosine present at inflammatory sites may control PLD activity and BLTR1 signaling, provide additional evidence for a role of adenosine in the down-regulation and/or resolution of inflammation and support the use of pharmacological agents capable of increasing adenosine levels at inflammatory sites (such as adenosine kinase inhibitors) or capable of selectively activating A2AR as novel classes of anti-inflammatory agents.


   ACKNOWLEDGEMENTS
 
This work was supported by a Senior Scholarship from the Arthritis Society of Canada to S. G. B. and from grants from the Canadian Institutes for Health Research (CIHR) to P. B., P. H. N., and S. G. B. S. G. is the recipient a postdoctoral fellowship and N. F., of a studentship from the CIHR.

Received July 25, 2002; revised December 18, 2002; accepted December 23, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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