* 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
d’Anatomie-Physiologie et de
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
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Key Words: phospholipase A2 • 5-lipoxygenase • chemotaxis • inflammation • LTB4 receptors • adenosine deaminase • adenosine receptors • CGS-15943 • CGS-21680 • chlorostyryl caffeine
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
(TNF-) 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- 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).
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-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.
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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.
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, 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.
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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- 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.
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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.
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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.
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 
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- 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- [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.
Received July 25, 2002; revised December 18, 2002; accepted December 23, 2002.
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C. Kim and M. C. Dinauer Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid J. Leukoc. Biol., January 1, 2006; 79(1): 223 - 234. [Abstract] [Full Text] [PDF]
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