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Originally published online as doi:10.1189/jlb.1005614 on February 24, 2006

Published online before print February 24, 2006
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(Journal of Leukocyte Biology. 2006;79:1043-1051.)
© 2006 by Society for Leukocyte Biology

Inhibition of platelet-activating factor biosynthesis by adenosine and histamine in human neutrophils: involvement of cPLA2{alpha} and reversal by lyso-PAF

Nicolas Flamand, Julie Lefebvre, Gabriel Lapointe, Serge Picard, Lise Lemieux, Sylvain G. Bourgoin and Pierre Borgeat1

Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ (CHUL), Faculté de Médecine, Université Laval, Québec, Canada

1Correspondence: Centre de Recherche en Rhumatologie et Immuologie, Centre de Recherche du CHUQ (CHUL), Office T1-49, 2705 boul. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: Pierre.Borgeat{at}crchul.ulaval.ca


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ABSTRACT
 
Leukotrienes (LT) and platelet-activating factor (PAF) are important lipid mediators of inflammation. We and others reported previously that autacoids such as adenosine, histamine, prostaglandin E2, and ß-adrenergic agents inhibit LT biosynthesis in activated human polymorphonuclear leukocytes (PMN). In this study, we demonstrate that CGS-21680 (a selective agonist of the adenosine A2A receptor) and histamine also potently inhibit PAF biosynthesis in agonist [formyl Met-Leu-Phe (fMLP)]- and thapsigargin-activated human PMN. The observed inhibitions of PAF biosynthesis were reversed effectively by exogenous 1-O-alkyl-lyso-sn-glyceryl-3-phosphocholine (lyso-PAF), suggesting that these effects of CGS-21680 and histamine implicate the blockade of cytosolic phospholipase A2{alpha} (cPLA2{alpha}) activity and lyso-PAF release and that the acetyl-coenzyme A/lyso-PAF acetyl transferase is not inhibited by the autacoids. Accordingly, the cPLA2{alpha} inhibitor pyrrophenone completely blocked PAF formation, and lyso-PAF similarly prevented this effect of pyrrophenone. The inhibitory effects of CGS-21680 and histamine on PAF biosynthesis were prevented by the protein kinase A inhibitor H-89, supporting roles for the Gs-coupled receptors A2A and H2, respectively, and cyclic adenosine monophosphate in the inhibitory mechanism. The fMLP-induced phosphorylations of p38 and extracellular signal-regulated kinase 1/2 were not altered significantly by the CGS-21680, indicating that inhibition of these kinases is not involved in the inhibitory effect of the adenosine A2A receptor ligand on LT and PAF biosynthesis. These data further emphasize the multiple and potent inhibitory effects of adenosine and histamine on leukocyte functions, in particular, on the biosynthesis of two classes of important lipid mediators and their putative regulatory roles in immune processes in health and diseases.

Key Words: lipid mediator • leukotriene • cAMP • leukocyte • pyrrophenone • arachidonic acid


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INTRODUCTION
 
Platelet-activating factor (PAF) exhibits numerous potent, biological properties, and its ubiquity and pleiotropicity support its importance in the regulation of various biological processes, in particular, in the immune response. Indeed, PAF shows several proinflammatory properties: It promotes platelet aggregation, production of reactive oxygen species (ROS), leukotriene (LT) biosynthesis, chemotaxis, vascular permeability, and expression of adhesion molecules [1 ].

PAF originates from membrane phospholipids; its biosynthesis involves the cytosolic phospholipase A2{alpha} (cPLA2{alpha}) and closely parallels arachidonic acid (AA) release and eicosanoid formation in many cell types, as demonstrated in studies involving cPLA2{alpha}-deficient mice [2 3 4 ]. The cPLA2{alpha} cleaves membrane phospholipids to release AA and lyso-phospholipids. The released AA is then transformed into prostaglandins (PG) by the cyclooxygenase pathways or to hydoxy-eicosatetraenoic acids, LT, and lipoxins by the 5-, 12-, and 15-lipoxygenases. The lyso-phospholipids released following the activation of human polymorphonuclear leukocytes (PMN) mainly consist of 1-acyl-2-lyso-glyceryl-3-phosphocholine, 1-O-alkenyl-2-lyso-glyceryl-3-phosphoethanolamine, and 1-O-alkyl-lyso-glyceryl-3-phosphocholine (lyso-PAF) [5 , 6 ], which is then acetylated by the acetyl-coenzyme A (CoA)/lyso-PAF acetyl transferase to generate PAF. In human PMN, the biosynthesis of PAF is triggered by stimuli such as opsonized zymosan or pharmacological agents such as the Ca2+ ionophore A23187 [6 7 8 ].

Some of the molecular mechanisms implicated in the up-regulation of cPLA2{alpha} activity are well understood. Upon cell stimulation, a rise in intracellular [Ca2+] ([Ca2+]i) occurs, along with the activation of the mitogen-activated protein kinase (MAPK) pathway; cPLA2{alpha} binds Ca2+ at its N-terminal ß-barrel domain, similar to the C2 domain of protein kinase C (PKC). This binding of Ca2+ to the cPLA2{alpha} results in a dramatic (more than two orders of magnitude) increase in enzyme activity, as assessed by in vitro enzyme assay [9 ]. The cPLA2{alpha} can be serine-phosphorylated at four different sites (Ser-437, Ser-454, Ser-505, and Ser-727), and it has been demonstrated that the extracellular signal-regulated kinase (ERK) and p38 pathways are responsible for these phosphorylation events [10 ]. Although Ser-505 phosphorylation has clearly been shown to result in enhanced cPLA2{alpha} activity, the impact of other phosphorylation events on cPLA2{alpha} activity is not fully understood yet. For example, the inhibition of the ERK pathway by the MAPK kinase inhibitors PD 98,059 and U-0126 was recently shown to inhibit cPLA2{alpha} activity (AA release) in MDCK cells without affecting its translocation or phosphorylation on Ser-505 [11 ].

The elevation of intracellular cyclic adenosine monophosphate ([cAMP]i) represents an efficient, physiological mechanism of down-regulation of leukocyte functions. Of particular relevance to the present study, adenosine and histamine, two important regulatory autacoids present at inflammatory sites, have been shown to suppress PMN functional responses such as the production of ROS, adhesion, and chemotaxis, the release of lysosomial enzymes, and the biosynthesis of LT in activated PMN [12 , 13 ], acting through the Gs-coupled A2A and H2 receptors, cAMP and PKA. PAF biosynthesis is also down-regulated by elevated [cAMP]i. Phosphodiesterase inhibitors and ß-adrenergic agents, which cause accumulation of cAMP in leukocytes, have been shown to decrease the biosynthesis of PAF, mainly in A23187-activated cells [14 , 15 ]. The inhibition of LT and PAF biosynthesis by elevated [cAMP]icorrelates with an inhibition of AA release in PMN [12 , 13 , 15 ], pointing to a mechanism involving blockade of cPLA2{alpha} activity.

In this study, we demonstrate that histamine and CGS-21680 potently inhibit PAF biosynthesis in thapsigargin- and formyl Met-Leu-Phe (fMLP)-activated human PMN. The inhibition induced by these cAMP-elevating agents is reversed by the addition of lyso-PAF, indicating that the inhibitory effect on PAF biosynthesis is mediated by the blockade of cPLA2{alpha} activity. We also show that the autacoid-induced PAF inhibition does not involve inhibition of the acetyl-CoA/lyso-PAF acetyltransferase or MAPK (ERK and/or p38) activation. These data provide additional evidence in support of the important down-regulatory roles of adenosine and histamine on the inflammatory process.


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MATERIALS AND METHODS
 
Material
2H4-PAF [1-O-(7,7,8,8-2H4)hexadecyl-2-acetyl-glyceryl-3-phosphorylcholine] and PGE2 were purchased from Cayman Chemical (Ann Arbor, MI). Dimethyl sulfoxide (DMSO), A23187, AA, adenosine deaminase (ADA), cytochalasin B (CB), fMLP, PAF, lyso-PAF, PGB2, and 19-OH-PGB2 were obtained from Sigma Chemical Co. (St. Louis, MO). 1-O-Alkenyl-2-lyso-sn-glyceryl-3-phosphorylethanolamine and 1-oleoyl-2-lyso-sn-glyceryl-3-phosphorylethanolamine were purchased from Biotrend Chemicals (Destin, FL). Thapsigargin was from Research Biochemicals International (Natick, MA), and H-89 was obtained from Calbiochem (La Jolla, CA). CP 105,696, MK-0591, LY 293111, and BN 50739 were obtained from Pfizer Corp. (Groton, NJ), Merck Frosst (Montreal, Canada), Eli Lilly (Indianapolis, IN), and Institut Henri Beaufour (Paris, France), respectively. Pyrrophenone was a gift from Dr. Kaoru Seno, Shionogi and Co. Ltd. (Osaka, Japan). Ficoll-Paque and Trypan blue were purchased from Wisent Laboratories (St-Bruno, Québec, Canada). C18 solid-phase extraction (SPE) cartridges (60 mg, 3 ml capacity) and Immobilon-P polyvinylidene difluoride (PVDF) blotting membranes were obtained from Waters Corp. (Milford, MA), and Bond Elut SPE silica cartridges (100 mg, 2 ml) were purchased from Varian (Harbor City, CA). Phospho-ERK 1/2 rabbit polyclonal antibody and phospho-p38 mouse monoclonal antibody were purchased from Cell Signaling (Beverly, MA). The enhanced chemiluminescence (ECL) detection kit was from Perkin Elmer (Boston, MA).

Isolation of human PMN
PMN were obtained as described previously [13 ]. Briefly, venous blood was obtained from healthy donors and collected in 10 ml tubes containing 143 United States Pharmacopeia units heparin. The heparinized blood was centrifuged at room temperature for 20 min at 250 g. 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, and a hypotonic lysis was performed on the granulocyte cell pellet to remove the remaining erythrocytes. The granulocyte suspension contained mainly PMN (≥95%), and cell viability was always greater than 98%, as measured by trypan blue exclusion. PMN were finally resuspended in Hanks’ balanced saline solution (HBSS) containing 1.6 mM CaCl2 at 5 x 106 cells/ml.

Stimulation of PAF and LT biosynthesis
In experiments where PMN were stimulated with fMLP, prewarmed PMN suspensions (37°C, 5x106 cells/ml in HBSS containing 1.6 mM CaCl2) were preincubated 30 min with 1.5 nM tumor necrosis factor {alpha} (TNF-{alpha}), 700 pM granulocyte macrophage-colony stimulating factor (GM-CSF), and 10 µM CB for priming of PAF and LT biosynthesis and then stimulated with 300 nM fMLP for 5 min unless stated otherwise (in figure legends). Exposure of human PMN freshly isolated from blood to GM-CSF/TNF/CB strongly enhances their functional responses to a second stimuli (such as fMLP or PAF) [16 17 18 ] and was used in the present study to enhance PAF and LT biosynthesis induced by fMLP. All incubations were carried out using 1 ml PMN suspension per condition. In experiments involving stimulation with thapsigargin, unprimed, prewarmed PMN suspensions (as above) were stimulated with 100 nM thapsigargin for 10 min unless stated otherwise (in figure legends). In all experimental settings, 0.3 U/ml ADA was added to incubation media 10 min prior to stimulation to eliminate the inhibitory constraint exerted by endogenous adenosine [19 ].

Analysis of PAF and lyso-PAF
For the determination of PAF and lyso-PAF, cell incubations were stopped by the addition of 1 vol cold (4°C) ethanol (EtOH) containing 5 ng 2H4-PAF as internal standard and stored overnight at –20°C. The denatured samples were then centrifuged (600 g, 20 min at room temperature); PAF and lyso-PAF were recovered from the supernatants and analyzed as described previously [20 ] with minor modifications. Briefly, the samples were loaded on a C18 SPE cartridge, which was successively washed with 4 ml H2O and 2 ml EtOH/H2O (50/50, v/v). PAF and lyso-PAF were then eluted from the C18 cartridge with 2 ml EtOH/H2O (98/2, v/v), and this eluate was loaded directly onto an EtOH-conditioned silica SPE cartridge, which was then washed with 2 ml EtOH, and PAF and lyso-PAF were eluted from the silica cartridge with 1.1 ml MeCN/H2O (60/40, v/v). Samples were evaporated to dryness under reduced pressure in a Speed-Vac evaporator, and the residues were solubilized in 50 µl high-pressure liquid chromatography (HPLC) mobile phase [hexane/isopropanol/20 mM aqueous ammonium acetate, 3/4/0.7 (v/v/v)]. Analysis of PAF was then performed by liquid chromatography/mass spectrometry (LC-MS)/MS in a negative ion mode by the measurement of the PAF:2H4-PAF ratio [(m/z 508->59)/(m/z 512->59)]. The levels of lyso-PAF were determined by the measurement of the lyso-PAF:2H4-PAF ratio [(m/z 466->377)/(m/z 512->59)]. Quantitation was achieved using standard curves generated by analysis (ratio determination) of solutions containing increasing amounts of PAF or lyso-PAF and a fixed amount of 2H4-PAF.

Analysis of LT
For the determination of 5-lipoxygenase (5-LO) products, cell incubations were stopped by the addition of 0.5 vol cold (4°C) stop solution (MeOH/MeCN, 1/1, v/v) containing 12.5 ng each PGB2 and 19-OH-PGB2 as internal standards. The denatured samples were centrifuged (600 g, 10 min), and the supernatants were analyzed by reverse phase-HPLC using an on-line extraction procedure as described previously [21 ]. The sum of LTB4, 20-COOH-LTB4, 20-OH-LTB4, 6(E)-LTB4, and 6(E)-12-epi-LTB4 was compiled and is referred to as LT.

Analysis of MAPK phosphorylation
PMN suspensions (1 ml/condition) were incubated and centrifuged (see Fig. 6 legend); the pellets were resuspended in 600 µl cold (4°C) Nonidet P-40 (NP-40) lysis buffer [0.1% NP-40, 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The cell lysates were vortexed for 15 s, immediately solubilized by addition of 150 µl electrophoresis sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, 10% glycerol, 0.01% bromophenol blue, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF], boiled for 10 min, and then analyzed by SDS-polyacrylamide gel electrophoresis, as described by Laemmli [22 ] on 10% acrylamide gels. Proteins were then transferred at 0.5 A for 3 h at 4°C onto an Immobilon-P PVDF blotting membrane. Transfer efficiency as well as loading were visualized by Ponceau Red staining. For the determination of phospho-p38 and phospho-ERK, the membranes were soaked for 30 min at 25°C in Tris-buffered saline (25 mM Tris-HCl, pH 7.6, 0.2 M NaCl, 0.15% Tween 20) containing 5% dried milk (w/v), blotted with the primary antibody, and revealed by chemiluminescence using a horseradish peroxidase-coupled antibody and the ECL detection kit. The densitometric analyses of the bands were performed using the software Scion Image for Windows 4.0.3.2. In the case of phospho-ERK, the densitometric analyses were performed on both bands.


Figure 6
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  		Figure 6. Effect of CGS-21680 on ERK and p38 phosphorylation in cytokine-primed, fMLP-activated PMN. Prewarmed human PMN suspensions (37°C, 107 cells/ml) were primed (or not) with 700 pM GM-CSF, 1.5 nM TNF-{alpha}, and 10 µM CB for 30 min and then stimulated with 300 nM fMLP for 2 or 5 min. Incubations were stopped by the addition of 2 vol cold (4°C) incubation buffer, and samples were immediately centrifuged (525 g, 90 s, 4°C). Supernatants were collected and processed for analysis of LT content as described in Materials and Methods. The cell pellets were disrupted in lysis buffer and analyzed for phospho-ERK and phospho-p38 as described in Materials and Methods. The Western blots shown are representative of three experiments. LT biosynthesis and densitometry data are the mean (±SD) of three separate experiments. N.D., Not determined.


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RESULTS
 
Kinetics of biosynthesis and localization of PAF in activated PMN
As preliminary experiments to the present studies, we determined the kinetics of PAF generation in PMN stimulated with thapsigargin and fMLP. As shown in Figure 1A , the biosynthesis of PAF is observed in unprimed, thapsigargin-activated PMN and in GM-CSF/TNF-{alpha}/CB-treated, fMLP-activated PMN; however, the kinetics of PAF accumulation was clearly different in the two experimental conditions. In fMLP-activated PMN, the biosynthesis of PAF occurs rapidly (within 5 min), and PAF level remains elevated for 15 min and then decreases over time. In sharp contrast, the biosynthesis of PAF in thapsigargin-activated human PMN increases for up to 45 min. fMLP did not trigger measurable PAF formation in unprimed PMN (data not shown), as assessed by LC-MS/MS analysis.


Figure 1
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  		Figure 1. Kinetics of biosynthesis and cellular localization of PAF in PMN. (A) Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were stimulated as described in Materials and Methods with 300 nM fMLP or 100 nM thapsigargin for the indicated times. Incubations were stopped by the addition of 1 vol cold EtOH containing 5 ng 2H4-PAF. (B) PMN were incubated as described above at the indicated cell concentrations and stimulated with 100 nM thapsigargin. For measurement of PAF in cell suspensions, incubations were stopped by the addition of 1 vol EtOH containing 5 ng 2H4-PAF. For measurement of PAF in cell pellets or supernatants, incubations were stopped by quickly centrifuging the cell suspensions (525 g, 90 s at 4°C). The supernatants were then collected, and the cell pellets were resuspended in 1 ml incubation buffer. EtOH (1 vol), containing 5 ng 2H4-PAF, was then added to both fractions. The denatured samples (A and B) were then centrifuged, and PAF was recovered and analyzed by LC-MS/MS as described in Materials and Methods. Data represent the mean (±SEM) of three different experiments, each performed in duplicate.

It has been reported previously that newly biosynthesized PAF can be found cell-associated or in the extracellular milieu, depending on the cell type and/or the stimulatory conditions [1 ]. We therefore carried out preliminary studies to assess PAF localization in thapsigargin-activated PMN. Figure 1B shows that all PAF was cell-associated; this cell association of PAF was still observed 60 min after the stimulation with thapsigargin. Figure 1B also shows that PAF formation did not vary with PMN concentration over the range of 5–20 x 106 cells/ml. In the next series of experiments, PAF biosynthesis was measured at 5 and 10 min incubation times with fMLP and thapsigargin, respectively, from EtOH-extracted whole cell suspensions.

Effect of CGS-21680 and histamine on PAF biosynthesis in PMN
We then evaluated the putative, inhibitory effect of histamine and the adenosine A2A receptor agonist CGS-21680 on PAF biosynthesis in human PMN; other agents that elevate the [cAMP]i were tested in parallel for comparison purposes. As shown in Figure 2 , all the autacoids tested abolished the biosynthesis of PAF in activated human PMN. The inhibitions observed were nearly complete, and similar inhibitory concentration of 50% (IC50) was observed for CGS-21680, PGE2, and the ß-adrenergic agonist isoproterenol (IC50 ~2 and 30 nM for thapsigargin- and fMLP-activated PMN, respectively). Histamine also inhibited PAF biosynthesis in thapsigargin- and fMLP-activated PMN with IC50of 30 nM and 10 µM, respectively.


Figure 2
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  		Figure 2. Inhibitory effect of histamine, CGS-21680, isoproterenol, and PGE2 on PAF biosynthesis in thapsigargin- and fMLP-activated PMN. Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were stimulated as described in Materials and Methods for 10 min with (A) 100 nM thapsigargin or (B) 300 nM fMLP in the presence of histamine, CGS-21680, PGE2, or isoproterenol at the indicated concentrations. The cAMP-elevating agents were added 10 min before the addition of fMLP or thapsigargin. All incubations were stopped by the addition of 1 vol EtOH containing 5 ng 2H4-PAF. The denatured samples were then centrifuged, and PAF was recovered and analyzed by LC-MS/MS as described in Materials and Methods. Data represent the mean (±SEM) of three different experiments, each performed in duplicate.

Involvement of PKA in CGS-21680- and histamine-mediated inhibition of PAF biosynthesis in PMN
The inhibitory effects of autacoids and other cAMP-elevating agents on LT biosynthesis in PMN were previously shown to involve PKA as shown by reversal of the inhibition by PKA inhibitors such as H-89 and KT-5720 [13 , 23 ]. Similar experiments were conducted in this study to assess the role of PKA in the CGS-21680- and histamine-mediated inhibition of PAF biosynthesis in activated PMN. As shown in Figure 3 , the inhibition of PAF biosynthesis mediated by autacoids (or by pharmacological ligands of autacoid receptors) was prevented when PMN were incubated in the presence of the PKA inhibitor H-89, indicating that the observed inhibition was also a PKA-mediated phenomenon.


Figure 3
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  		Figure 3. Reversal by the PKA inhibitor H-89 of the inhibitory effect of histamine, CGS-21680, isoproterenol, and PGE2 on thapsigargin-induced PAF biosynthesis. Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were stimulated for 10 min with 100 nM thapsigargin (TG) in the presence of 10 nM CGS-21680, PGE2, isoproterenol, and 300 nM histamine in the presence and absence of 10 µM H-89, and H-89 and the autacoids were added 20 and 10 min, respectively, before the addition of thapsigargin. All incubations were stopped by the addition of 1 vol EtOH containing 5 ng 2H4-PAF. The denatured samples were then centrifuged, and PAF was recovered and analyzed by LC-MS/MS as described in Materials and Methods. Data represent the mean (±SEM) of three different experiments, each performed in duplicate.

Involvement of cPLA2{alpha} in the CGS-21680- and histamine-mediated inhibition of PAF biosynthesis in PMN
We previously demonstrated that the adenosine- and histamine-mediated inhibition of LT biosynthesis correlated with an inhibition of AA release and thus, likely involved inhibition of cPLA2{alpha} activity [12 , 13 ]. Considering that cPLA2{alpha} is involved in eicosanoid and PAF biosynthesis in PMN, experiments were conducted to assess a similar role of this enzyme in CGS-21680- and histamine-induced inhibition of PAF formation. Figure 4A shows that exogenous lyso-PAF fully restored the biosynthesis of PAF in CGS-21680-treated PMN activated with fMLP in a concentration-dependent manner. In these experiments, addition of 1-acyl-2-lyso-sn-glyceryl-3-phosphocholine and 1-oleoyl- or 1-O-alkenyl-2-lyso-sn-glyceryl-3-phosphoethanolamine did not elicit nor restore PAF biosynthesis (data not shown), ruling out an unspecific effect of lyso-phospholipids on PAF formation. It is noteworthy that these data do not support the involvement of a CoA-independent transacylase-dependent PAF biosynthesis in fMLP-stimulated PMN, as observed previously in permeabilized PMN [24 ] and in intact PMN [25 ] incubated with 1-O-alkenyl-2-lyso-sn-glyceryl-3-phosphoethanolamine. As shown in Figure 4B , exogenous lyso-PAF results in the formation of significant quantities of PAF in resting PMN (unprimed, unstimulated), and histamine does not inhibit this lyso-PAF-induced PAF biosynthesis; similar results were obtained on lyso-PAF-induced PAF biosynthesis in unstimulated but GM-CSF/TNF-{alpha}/CB-treated PMN (data not shown). It is interesting that the stimulatory effect of lyso-PAF on PAF biosynthesis was observed at lower concentrations of lyso-PAF in GM-CSF/TNF-{alpha}/CB-primed, fMLP-activated PMN than in the unprimed, unstimulated PMN.


Figure 4
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  		Figure 4. Reversal of the CGS-21680-mediated inhibition of PAF biosynthesis by lyso-PAF and effect of histamine on lyso-PAF-induced PAF biosynthesis. (A) Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were incubated for 10 min with or without 10 µM CGS-21680 and then treated simultaneously with increasing concentrations of lyso-PAF and 300 nM fMLP for 10 min. (B) Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were preincubated in the presence or absence of 1 µM histamine and then stimulated with increasing concentrations of lyso-PAF for 10 min. Incubations were stopped by the addition of 1 vol EtOH containing 5 ng 2H4-PAF. The denatured samples were then centrifuged, and PAF was recovered and analyzed by LC-MS/MS as described in Materials and Methods. Data represent the mean (±SEM) of three different experiments, each performed in duplicate.

In separate experiments, we investigated the effect of the potent and specific cPLA2{alpha} inhibitor pyrrophenone [26 ] on PAF biosynthesis in thapsigargin-stimulated PMN. Figure 5A shows that the cPLA2{alpha} inhibitor concentration-dependently blocked PAF formation with an IC50 of ~20 nM and that the inhibition curves for PAF and lyso-PAF were perfectly superposed, as expected. Moreover, the inhibition of PAF formation was reversed efficiently by exogenous lyso-PAF (Fig. 5B) , as observed for the adenosine-mediated inhibition of PAF biosynthesis (Fig. 4A) .


Figure 5
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  		Figure 5. Inhibitory effect of pyrrophenone on PAF (and lyso-PAF) formation in thapsigargin-stimulated PMN and reversal by lyso-PAF. Prewarmed PMN suspensions (37°C, 5x106 cells/ml) were treated with the indicated concentrations of pyrrophenone for 10 min and then stimulated for 10 min with 100 nM thapsigargin (A) or treated (or not) with 100 nM pyrrophenone for 10 min and simultaneously stimulated for 10 min with 100 nM thapsigargin and the indicated concentrations of lyso-PAF (B). Incubations were stopped by the addition of 1 vol EtOH containing 2H4-PAF. The denatured samples were then centrifuged, and PAF was recovered and analyzed by LC-MS/MS, as described in Materials and Methods. Data represent the mean (±SEM) of three different experiments, each performed in duplicate.

Effect of CGS-21680 on ERK and p38 phosphorylation
It was proposed previously that the inhibitory effects of elevated [cAMP]i may be the consequence of MAPK inhibition [27 ]. As cPLA2{alpha} is phosphorylated by ERK and p38 at Ser-505, which results in a significant increase in enzyme activity [10 ], we analyzed the putative inhibitory effect of CGS-21680 on ERK and p38 phosphorylation in activated PMN. In these experiments, cPLA2{alpha} activity was assessed by measurement of LT in cell supernatants, as the cell pellets were used for protein analysis. Figure 6 shows that CGS-21680 clearly inhibited the fMLP-induced LT biosynthesis, as shown previously [12 ], but only caused a slight inhibition of ERK and p38 phosphorylation at 5 min, when most of PAF biosynthesis has already occurred (see Fig. 1 ), suggesting that the mechanism by which CGS-21680 blocks lipid mediator biosynthesis does not involve ERK and/or p38 inhibition. This result is in complete agreement with our previous observation that cPLA2{alpha} phosphorylation at Ser-505 (visualized by a decreased electrophoretic motility) in GM-CSF/TNF-{alpha}/CB-primed, fMLP-activated PMN is not inhibited by histamine and the adenosine A2A receptor agonist CGS-21680 [13 ].

Distinct mechanisms in thapsigargin- and agonist-induced PAF biosynthesis
Comparison of IC50in Figure 2A and 2B , reveals that thapsigargin-induced PAF biosynthesis is much more sensitive (one to three orders of magnitude) to inhibition by cAMP-elevating autacoids than is fMLP-stimulated PAF biosynthesis, clearly indicating the involvement of distinct mechanisms in the regulation of PAF formation under the two stimulatory conditions. Accordingly, Figure 7 shows that PAF biosynthesis is completely inhibited in thapsigargin-activated PMN pretreated with 100 nM LTB4 receptor 1 (BLT1) antagonists CP 105,696 and LY 293111 or the 5-LO-activating protein antagonist MK-0591 but not by a PAF receptor antagonist (BN 50739), demonstrating an obligatory role for LTB4 in this process, as previously reported for thapsigargin-induced PLA2 activity and AA release in PMN [6 ]. In sharp contrast, fMLP-induced PAF biosynthesis in primed PMN was only slightly reduced by a BLT1 antagonist, confirming the involvement of different regulatory mechanisms, which might account for the striking difference observed in the susceptibility of PAF biosynthesis to inhibition by the autacoids (see Discussion).


Figure 7
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  		Figure 7. Effects of LT (and PAF) antagonists and inhibitors on PAF biosynthesis in thapsigargin- and fMLP-activated PMN. Prewarmed human PMN suspensions (37°C, 5x106 cells/ml) were primed and stimulated for 10 min with 300 nM fMLP or preincubated 30 min at 37°C and directly stimulated with 100 nM thapsigargin for 10 min. PMN suspensions were treated with 100 nM of the indicated antagonists or inhibitor 5 min prior to stimulation with fMLP or thapsigargin. Incubations were stopped, and PAF analyses were carried out as described in Figure 5 legend. Data represent the mean (±SEM) of triplicate incubations from one experiment representative of three.


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DISCUSSION
 
The use of pharmacological agents and cPLA2{alpha}-deficient mice has revealed that the cPLA2{alpha} is the enzyme responsible for the release of AA and lyso-PAF, the respective precursors of LT and PAF in activated PMN [13 , 28 , 29 ]. LT and PAF are implicated in the recruitment and activation of human PMN and other leukocytes and are important regulators of inflammation, as demonstrated with cPLA2{alpha}-deficient mice, which showed sharply decreased inflammatory responses [2 3 4 ]. Numerous studies have shown that physiological stimuli and pharmacological agents (such as the ionophore A23187), which activate PMN, also stimulate PAF and LT biosynthesis. In contrast, few studies have addressed the inhibitory mechanisms of lipid mediator biosynthesis, and to date, the only known physiological processes by which PAF biosynthesis is down-regulated in intact cells are the elevation of [cAMP]i, for instance, by ligation of a Gs-coupled receptor such as PGE2 receptor (EP2) or the ß-adrenergic receptor [14 , 15 ] and the inhibition of p38, which results in the inhibition of the acetyl-CoA/lyso-PAF-acetyltransferase [8 , 30 ]. We have in the past years demonstrated that adenosine, a ubiquitous autacoid with potent inhibitory effects on PMN functions, and more recently, histamine, an autacoid present at inflammatory sites, which also inhibit PMN functional responses, efficiently suppress AA release and LT biosynthesis in a cAMP-dependent manner [12 , 13 , 19 ]. In the present study, we demonstrate that histamine and CGS-21680 (an adenosine A2A receptor agonist) are also potent inhibitors of PAF biosynthesis. It is important that part of the present data has been obtained using agonist (fMLP)-activated PMN, in support of the physiological relevance of the inhibitory effects reported herein.

The regulatory mechanisms implicated in cPLA2{alpha} activation, lyso-PAF release, and biosynthesis of PAF involve the sequential increase in [Ca2+]i followed by the translocation of cPLA2{alpha} to the peri-nuclear membranes where it hydrolyzes AA-containing phospholipids, mainly 1-O-alkyl-2-arachidonoyl-phosphatidylcholine. Following this phospholipid hydrolysis, the released AA is transformed by the lipoxygenases and/or the cyclooxygenases, and the released lyso-phospholipids are reacylated by transacylases or acetylated by the acetyl-CoA/lyso-PAF acetyltransferase to generate PAF. The availability of lyso-PAF limits PAF biosynthesis, which is triggered following PMN (and cPLA2{alpha}) activation or by the direct addition of lyso-PAF to PMN suspensions. In this study, the biosynthesis of PAF induced by exogenous lyso-PAF was not inhibited by histamine in unstimulated PMN nor by CGS-21680 in fMLP-activated PMN. Moreover, the phosphorylation of p38, which activates the acetyl-CoA/lyso-PAF acetyltransferase [30 ], was not altered significantly by CGS-21680. These results strongly suggest that histamine and A2A receptor agonist do not alter the activity of the acetyl-CoA/lyso-PAF acetyltransferase and that the mechanism responsible for the inhibition of PAF biosynthesis is located upstream of this biosynthetic step. It is interesting that we observed that there was a differential effect of exogenous lyso-PAF on PAF biosynthesis, and whether PMN were activated or not, more lyso-PAF was required to induce PAF formation in resting PMN. The activation of p38 and consequently of the lyso-PAF acetyltransferase by cytokines and fMLP (see Fig. 6 ) may account for this difference.

In agreement with an inhibitory mechanism of histamine and CGS-21680 on PAF biosynthesis located upstream of the lyso-PAF acetyltransferase, the present study points to an involvement of the cPLA2{alpha} and PKA, as shown by the complete reversal of inhibition by exogenous lyso-PAF and the PKA inhibitor H-89. These data are in complete agreement with previous studies involving the cPLA2{alpha} in the inhibitory mechanism of adenosine, histamine, and other cAMP-elevating agents on LT biosynthesis [12 , 13 , 19 ]. We previously demonstrated that although cAMP-elevating agents inhibit Ca2+ influx in agonist-activated PMN, this was not the mechanism by which cAMP inhibits fMLP-induced LT biosynthesis [12 , 13 ]. In addition, the cPLA2{alpha} phosphorylation at Ser-505 and the phosphorylation of p38 and ERK are only slightly inhibited by cAMP-elevating agents (ref. [13 ]; Fig. 6 ). Moreover, inhibition of cPLA2{alpha} translocation does not account for the observed, profound inhibitions of PAF and LT formation (≥90%), as in several experiments, inhibition of translocation was highly variable (often not observed) and thus poorly correlated with inhibition of PAF and/or LT biosynthesis. However, we have clearly shown herein and in previous studies that PKA inhibitors efficiently prevent the suppression of PAF and LT biosynthesis by histamine and adenosine, which strongly supports that a PKA-dependent mechanism must be implicated in the inhibition of cPLA2{alpha} activity in activated PMN treated with cAMP-elevating agents. The inhibition of tyrosine kinases by cAMP upstream of p38 and ERK [27 ] is not compatible with the present data; we rather demonstrated that cAMP-elevating agents only slightly alter the phosphorylation of the p38 and ERK 1/2 in fMLP-activated PMN at 5 min when LT and PAF biosynthesis are nearly complete (>90%). The amino acid sequence of cPLA2{alpha} shows three putative PKA-phosphorylation sites at residues 57–60 (RKRT), 281–284 (KKKS), and 282–285 (KKSS). An inhibitory, PKA-mediated phosphorylation of cPLA2{alpha} may therefore be involved in the cAMP-mediated inhibition of cPLA2{alpha}. Such an inhibitory phosphorylation event has in fact been reported before [31 ] but has yet to be characterized.

Peptide fragments from the N terminus of annexin-1 have been shown to interact with and inhibit the cPLA2{alpha} [32 , 33 ]. Accordingly, U937 cells lacking annexin-1 showed an increased cPLA2{alpha} activity [34 ], supporting that annexin-1 may play a role in the regulation of cPLA2{alpha} activity. Moreover, cAMP and the cAMP-responsive transcription factor cAMP response element-binding protein have been implicated in the up-regulation of annexin-1 [35 ]. However, the interaction of annexin-1 with cPLA2{alpha} led to a decreased cPLA2{alpha} phosphorylation at Ser-505 and translocation to the peri-nuclear membranes [36 , 37 ], two events that are not altered by histamine in human PMN [13 ]. Therefore, a role of annexin-1 in the cAMP-mediated inhibition of LT and PAF biosynthesis appears unlikely.

Ceramide-1-phosphate has recently been described as a potent activator of cPLA2{alpha} in A549 cells, probably acting by increasing enzyme interaction with membranes [38 , 39 ]. Thus, ceramide-1-phosphate is possibly involved in the regulation of LT and PAF biosynthesis in PMN, although the role of this endogenous mediator and its regulation by cAMP remain to be characterized in PMN.

Phosphatidylinositol-4,5-bisphosphate (PIP2) represents another putative, regulatory factor of cPLA2{alpha} activity [40 ]. Indeed, the activity of cPLA2{alpha} was strikingly enhanced (~60 fold) in an in vitro cPLA2{alpha} assay when 4 mol% PIP2 was added to phosphatidylcholine micelles. Cell stimulation leads to PLC activation and the breakdown of PIP2 into diacylglycerol and inositol-1,4,5-trisphosphate. This breakdown of PIP2 is followed by a resynthesis of PIP2 within 60–120 s, which has been shown to be inhibited by PGE2 in fMLP-activated PMN [41 ], probably through a cAMP-dependent inhibition of phophatidylinositol kinases. Such a mechanism could be implicated in the decreased cPLA2{alpha} activity observed when activated PMN are treated with cAMP-elevating agents.

In an attempt to explore the mechanism underlying the striking differences observed in the sensitivity of thapsigargin-activated PMN as opposed to fMLP-activated PMN to the inhibitory effect of autacoids, we compared the effects of BLT1 antagonists on PAF biosynthesis under both stimulatory conditions. It was indeed shown previously that LTB4, through an autocrine-stimulatory loop, has a significant impact on the activation of the cPLA2{alpha} pathway in human PMN [42 43 44 ], whereas it plays an essential role in thapsigargin-induced AA release in human PMN [45 ]. It is interesting that in the present study, thapsigargin-induced PAF formation was totally blocked by BLT1 antagonists (and the LT biosynthesis inhibitor MK-0591), in agreement with the suggestion that LTB4 exerted its up-regulatory effect on thapsigargin-induced AA release by acting at the level of PLA2 activation [45 ]. In sharp contrast, fMLP-induced PAF biosynthesis was only slightly inhibited by the BLT1 antagonist CP 105,696 compared with thapsigargin-activated PMN. These data clearly demonstrated that distinct mechanisms are involved in the regulation of PAF biosynthesis in thapsigargin- and fMLP-activated PMN, and it seems reasonable to speculate that the LTB4-mediated autocrine stimulatory loop may be responsible for the differences observed in the sensitivity to inhibition of PAF formation by the autacoids. In support of this hypothesis, we have reported previously that AA-induced LT biosynthesis in PMN, which also involves an obligatory LTB4-mediated autocrine stimulatory loop, is similarly much more sensitive (≥tenfold) to inhibition by the A2A receptor agonist CGS-21680 than fMLP-induced LT biosynthesis [1 , 12 ].

Preliminary data suggest that in unprimed PMN stimulated with thapsigargin, the newly biosynthesized LTB4 is essential for Ca2+ mobilization, ERK activation, and LT biosynthesis (data not shown), as shown herein for PAF biosynthesis (Fig. 7) . In contrast, LTB4 alone does not induce detectable levels of PAF biosynthesis in unprimed PMN (data not shown), indicating that other signals are necessary for PMN activation by thapsigargin. The crucial importance of LTB4 in thapsigargin-activated PMN suggests that the mechanism of activation of cPLA2{alpha} in this experimental condition involves the activation of a Gi-coupled receptor and its downstream signal transduction. This would explain why PMN activation by fMLP (which also signals through a Gi-coupled receptor) does not require LTB4. The activation of unprimed PMN with thapsigargin typically leads to a LT biosynthesis, which is twice that observed in GM-CSF/TNF-{alpha}/CB-treated, fMLP-activated PMN (~100 vs. 50 pmol/106 cells [13 ]). Additional studies are in progress to clarify the mechanisms involved in thapsigargin and cytokine-primed, fMLP-activated PMN for LT and PAF biosynthesis.

In conclusion, histamine and adenosine have the ability to inhibit the biosynthesis of LT and PAF in activated human PMN; this inhibitory effect is mediated by PKA and involves a down-regulation of cPLA2{alpha} activity. The present data suggest that the increased inflammatory responses observed in A2A, EP2, or H2 receptor-deficient mice [46 47 48 49 ] (three receptors positively coupled to the adenylate cyclase) may be, at least in part, the consequence of the loss of the inhibitory constraint exerted by adenosine, PGE2, and histamine on LT and PAF biosynthesis in PMN, other leukocytes, and endothelial cells and strongly support that these autacoids are implicated in the down-regulation and possibly the resolution of inflammation and other immune processes.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Canadian Arthritis Network (CAN), and The Arthritis Society of Canada (TAS). N. F. was the recipient of a doctoral award from the CIHR. J. L. is the recipient of a doctoral award from the CAN. N. F. and J. L. contributed equally to this study.

Received October 28, 2005; revised December 22, 2005; accepted January 11, 2006.


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