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Prostaglandin E₂ Inhibits the Phospholipase D Pathway Stimulated by Formyl-methionyl-leucyl-phenylalanine in Human Neutrophils. Involvement of EP₂ Receptors and Phosphatidylinositol 3-kinase

Centre de Recherche en Rhumatologie-Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Pavillon CHUL (C.B., N.T., S.L., S.S.) and Departments of Anatomy-Physiology (S.G.B.) and Medicine (P.H.N.), Université Laval, Québec, Canada

Received November 17, 2003; accepted April 27, 2004

	Abstract

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Prostaglandin E₂ (PGE₂), originally discovered as a pro-inflammatory mediator, also inhibits several chemoattractant-elicited neutrophil functions, including adhesion, secretion of cytotoxic enzymes, production of superoxide anions, and chemotaxis. In this study, we have examined the effects of PGE₂ and prostaglandin E (EP) receptor-selective agonists/antagonists on several steps of the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation pathway in human neutrophils to elucidate the PGE₂ inhibitory mechanism. PGE₂ and EP₂ receptor agonists inhibited the stimulation of the activity of PLD induced by fMLP in a concentration-dependent manner. The fMLP-stimulated translocation to membranes of protein kinase C , Rho, and Arf GTPases was diminished in the presence of PGE₂ or EP₂ agonists. Moreover, PGE₂ and EP₂ agonists decreased the activation of phosphatidylinositol 3-kinase (PI3K) and Tec kinases as well as the tyrosine phosphorylation of proteins stimulated by fMLP. These data provide strong evidence that 1) the inhibitory effects of PGE₂ on the fMLP-induced PLD activation pathway were mediated via EP₂ receptors and that 2) the suppression of PI3K activity was the crucial step in the EP₂-mediated inhibition of the fMLP-induced signaling cascade.

Besides their role in the regulation of PLD activity, the Rho GTPase family members (Rho, Rac, and Cdc42) also modulate multiple cellular processes by cycling between a GDP-bound inactive form and a GTP-bound form that directly activates several downstream targets. They are implicated in the regulation of the dynamics of the actin cytoskeleton, in changes in cell shape and motility (Hall, 1998), and they regulate various leukocyte functions including adhesion (Laudanna et al., 1996), phagocytosis (Chimini and Chavrier, 2000), and the migratory response to chemoattractants (Servant et al., 2000). In phagocytic cells, Rac also plays a major role in the production of superoxide anions by the NADPH oxidase complex (Babior, 1999). Rho GTPases are therefore critical regulators of the functional responsiveness in neutrophils.

Prostaglandins E (PGEs) are cyclooxygenase products that exert pleiotropic effects in a paracrine fashion. The physiological activities of PGEs are mediated via G-protein-linked seven transmembrane domain receptors that have been classified into four subtypes, EP₁ to EP_4, according to their structure and coupling to distinct signaling pathways (Breyer et al., 2001). EP₂ and EP₄ stimulate adenylyl-cyclase, whereas EP₁ triggers intracellular calcium release. The various EP₃ isoforms generated by alternative splicing are connected either to cAMP or to calcium metabolism. Pro-inflammatory functions have been attributed to PGEs by virtue of their vasodilator and nociceptive properties. However, PGEs also display inhibitory effects on most leukocyte functions and are considered modulators of inflammation rather than strictly pro-inflammatory mediators (Tilley et al., 2001). Most neutrophil functional responses, including adhesion to epithelial cells (Bloemen et al., 1997), motility and chemotaxis (Rivkin et al., 1975), production of superoxide anions (Fantone and Kinnes, 1983), and the release of cytotoxic enzymes and leukotriene B₄ (Ham et al., 1983; Hecker et al., 1990) are diminished by PGE₂ or PGE₁. Pharmacological studies have shown that the inhibitory effects of PGE₂ on fMLP-induced superoxide production, enzyme release, and chemotaxis are mediated via the EP₂ receptor (Armstrong, 1995; Talpain et al., 1995).

The effects of PGE₂ on signal transduction pathways induced by chemoattractants in neutrophils have only been incompletely characterized so far. PGE₂ has been shown to inhibit fMLP but not phorbol 12-myristate 13-acetate–induced PLD activation in human neutrophils (Agwu et al., 1991), but the mechanism involved has not been clarified. Therefore, we investigated further the effects of PGE₂ (and of specific EP receptor agonists) on the signaling cascade that leads to PLD activation upon fMLP stimulation. In this article, we report that PGE₂ inhibits the stimulation of the activity of PLD, the translocation of PKC, Arf, and Rho GTPases and Tec kinases to membranes, the accumulation of PtdIns(3,4,5)P₃ and the activity of PI3K induced by fMLP. These effects were mediated via EP₂ receptors. Our results suggest that suppression of fMLP-induced PI3K activity by PGE₂ plays a prominent role in the inhibition of downstream signaling events such as the activation of small GTPases and of PKC and Tec kinases, thereby resulting in decreased PLD activation.

	Materials and Methods

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Antibodies. The anti-PKC (P16520 [GenBank] ) and anti-Cdc42 (C70820 [GenBank] ) monoclonal Abs were purchased from BD Transduction Laboratories (Mississauga, ON, Canada). Anti-RhoA (SC-179), anti-Rac2 (SC-96), anti-Tec (SC-1109), and anti-Btk (SC-1107) polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-EP₂ receptor Ab and the EP₂ receptor blocking peptide were obtained from Cayman Chemical (Ann Arbor, MI). The polyclonal anti-Arf1 and anti-p110 antisera were raised in rabbits as described previously (Houle et al., 1999; Naccache et al., 2000). The anti-phosphotyrosine Ab (clone 4G10) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Secondary anti-mouse and anti-rabbit Abs were obtained from Amersham Biosciences (Baie d'Urfé, Québec, Canada) and the secondary anti-goat Ab from Santa Cruz Biotechnology.

Reagents. Dextran T-500 was purchased from Pharmacia Biotech (Dorval, PQ, Canada) and Ficoll-Paque from Wisent (St-Bruno, PQ, Canada). Adenosine deaminase (ADA) was purchased from Roche Diagnostics (Laval, PQ, Canada), and di-isopropylfluorophosphate (DFP) from Serva (Heidelberg, Germany). fMLP and cytochalasin B (CB) were obtained from Sigma-Aldrich Canada (Oakville, ON, Canada). 1-O-[³H]alkyl-2-lyso-phosphatidylcholine was obtained from Amersham Biosciences (Baie d'Urfé, PQ, Canada). PGE₂, the butaprost acid-form CAY 10399, 11-deoxy PGE₁, sulprostone, and AH 23848 were purchased from Cayman Chemical (Ann Arbor, MI). [³²P]Orthophosphate (1000 Ci/mmol) and [-³²P]ATP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Fura-2/AM was obtained from Molecular Probes (Eugene, OR).

Isolation of Human Neutrophils. Venous blood was collected from healthy adult volunteers in isocitrate anticoagulant solution. Neutrophils were separated as described previously (Marcil et al., 1999). In brief, whole blood was centrifuged at 180g for 10 min and the resulting platelet rich plasma was discarded. Leukocytes were obtained after erythrocytes sedimentation in 2% Dextran T-500. Mononuclear cells were removed by centrifugation on Ficoll-Paque cushions and contaminating erythrocytes in the neutrophil pellets were removed by a 20-s hypotonic lysis in water. Neutrophils were resuspended in Hanks' balanced salt solution (HBSS), pH 7.4, containing 1.6 mM Ca²⁺ but no Mg². The cells were sterilely purified to avoid bacterial contamination of the neutrophil suspensions.

PLD Measurements. Neutrophils were labeled with 1-O-[³H]alkyl-2-lyso-phosphatidylcholine (2 µCi/10⁷ cells) for 90 min as described previously (Marcil et al., 1999). The cells were washed and resuspended at 10⁷ cells/ml in HBSS. Cell suspensions (0.5 ml) were warmed at 37°C for 5 min and then pretreated for 5 min with 10 µM CB and 0.1 U/ml ADA to eliminate endogenous adenosine and in the presence or the absence of the indicated concentrations of agonists or antagonists. Neutrophils were stimulated with 100 nM fMLP for 10 min in the presence of 1% ethanol to monitor the formation of radiolabeled phosphatidylethanol (PEt) catalyzed by PLD. Incubations were stopped by adding 1.8 ml of chloroform/methanol/HCl (50:100:1, v/v/v) and unlabeled PEt as a standard. Lipids were extracted and the levels of [³H]PEt were quantified as described previously (Marcil et al., 1999).

Measurement of Cytoplasmic Free Calcium Concentrations. Neutrophils (10⁷ cells/ml) were incubated at 37°C for 30 min with 1 µM Fura-2/AM. The cells were washed and resuspended at 5 x 10⁶ cells/ml in HBSS with 1.6 mM Ca²⁺. Neutrophils were transferred to the thermostated cuvette compartment of a spectrofluorimeter (SLM 8000C) and preincubated 5 min at 37°C with 0.1 U/ml ADA in the presence of various EP receptor agonists/antagonists or the equal volume of dimethyl sulfoxide. Cells were stimulated with 100 nM fMLP at the time indicated by the arrow. The fluorescence of the cells was monitored at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The internal calcium concentrations were calculated using the following formula: 224[(y – F_min)(F_max – y)^–1] in which y represents the fluorescence of the samples. F_max was obtained by disrupting the cells with 1% Triton X-100 and F_min was obtained by adding 5 mM of both EGTA and NaOH.

Manganese Influx Measurement. Neutrophils were loaded with Fura-2/AM, washed and resuspended in HBSS without calcium and magnesium just prior incubation with ADA and PGE₂ or EP receptor agonists for 5 min at 37°C in a thermostated cuvette as described above. The wavelength used for these experiments were 360 and 505 nm for excitation and emission, respectively. After 10 s, 200 µM MnCl₂ was added to the cell suspensions that were then stimulated by 100 nM fMLP for 70 s.

Translocation Assays. Neutrophils (4 x 10⁷ cells/ml) were treated with 1 mM DFP for 10 min at room temperature. The cell suspensions were centrifuged once and the cells were resuspended in HBSS at 10⁷ cells/ml. The cells were warmed for 5 min at 37°C and treated 5 min with 10 µM CB, 0.1 U/ml ADA and PGE₂ or EP receptor agonists or an equal volume of diluent (dimethyl sulfoxide) as a control. Neutrophils were stimulated with 100 nM fMLP at 37°C. Incubations were stopped by diluting the cells 5-fold with ice-cold HBSS, and total membrane proteins were collected as described previously (Marcil et al., 1999). Protein samples (10–20 µg) were resolved on a 7.5 to 20% gradient SDS-polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Western blots were performed using anti-Arf1 (1/2500), anti-RhoA (1/1000), anti-PKC (1/1000), anti-Rac2 (1/200) and anti-Cdc42 (1/250), anti-p110 (1/1000), anti-Tec (1/1000) or anti-Btk (1/1000) Abs. Proteins were revealed with HRP-conjugated secondary anti-mouse or anti-rabbit Ab (1/20,000) or anti-goat Ab (1/15,000) and the Renaissance detection system (PerkinElmer Life and Analytical Sciences).

Phosphatidylinositol 3,4,5-trisphosphate Formation. Neutrophils (5 x 10⁷ cells/ml) were incubated with 0.5 mCi/ml of [³²P] orthophosphate for 1 h at 37°C in a labeling buffer (137 mM NaCl, 5 mM KCl, 10 mM glucose, 20 mM HEPES, pH 7.4). Unincorporated radioactivity was discarded and the cells were washed twice in HBSS. The cells were resuspended in HBSS and preincubated with PGE₂/EP agonists or diluent in the presence of ADA 0.1 U/ml for 5 min at 37°C. The cells were stimulated with 100 nM fMLP for 30 sec and reactions were stopped by addition of 400 µl of chloroformmethanol (1:1, v/v). The samples were processed as described previously (Gilbert et al., 2003) and the final products visualized by exposure of X-ray films at –80°C or with a bio-imaging analyzer (Fujifilm BAS-1800).

Measurements of PI3K Activity. Cell suspensions (4 x 10⁷ cells/ml) were preincubated for 5 min at 37°C with 10^–5M PGE₂ (or an equal volume of dimethyl sulfoxide) and 0.1 U/ml ADA. Cells were stimulated with 100 nM fMLP and the reaction was stopped by addition of an equal volume ice-cold buffer I (phosphate-buffered saline containing 1 mM CaCl₂, 1 mM MgCl₂, 100 µM Na₃VO₄). Samples were processed as already described for the p110 immunoprecipitation and the PI3K assays (Naccache et al., 2000).

Tyrosine Phosphorylation Patterns. Neutrophils (2 x 10⁷ cells/ml) were preincubated at room temperature with 1 mM DFP, with or without the indicated concentrations of PGE₂ or EP agonists for 10 min at 37°C before stimulation with 100 nM fMLP for the indicated times. The reactions were stopped by transferring 100 µlof the cell suspensions to an equal volume of boiling 2x Laemmli sample buffer (SB) (1x is 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 5% -mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.025% bromphenol blue) and boiled for 7 min. The samples were then subjected to 7.5 to 20% gradient SDS-polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidene difluoride membranes. Immunoblotting was performed using the monoclonal anti-phosphotyrosine Ab 4G10 (1/4000) and revealed as described previously (Gilbert et al., 2003).

Statistical Analysis. The data were analyzed using the one-sample Student's t test for the PLD and translocation assays data or the Student paired t test (two-tailed) for the PI3K assay data. The levels of significance (*, p < 0.05; **, p < 0.01) were determined between the treated samples and the appropriate controls.

	Results

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fMLP-Induced PLD Activity Is Inhibited by PGE₂ via EP₂ Receptor. As is the case for other cAMP elevating substances (adenosine, forskolin, adrenergic ₂-agonists), PGE₂ has also been shown to inhibit fMLP-induced PLD activity in the presence of phosphodiesterase inhibitors (3-isobutyl 1-methylxanthine or theophylline) (Agwu et al., 1991). We have first investigated the effect of PGE₂ on PLD activity in our experimental conditions (i.e., in the absence of phosphodiesterase inhibitor and in the presence of ADA to eliminate the inhibitory effect of endogenous adenosine that could interfere with that of PGE₂) (Thibault et al., 2000). As shown in Fig. 1A, PGE₂ inhibited [³H]PEt formation in a concentration-dependent manner. The amounts of [³H]PEt formed were 25.89 ± 5.01% (p = 0.0045), 25.44 ± 5.74% (p = 0.0059), 35.27 ± 6.83% (p = 0.011), and 43.3 ± 5.21% (p = 0.0083) of the fMLP-stimulated control in the presence of 10^–5, 10^–6, 10^–7, and 10^–8 M PGE_2, respectively. To determine which EP receptor subtype mediated the inhibition of PLD activity by PGE₂,[³H]PEt formation was assessed in the presence of EP receptor selective agonists and antagonists, all of which are PGE₂ analogs. We tested the effects of one EP₂ receptor-selective agonist (CAY 10399), one EP₂/EP₄ agonist (11-deoxy-PGE₁), one EP₁/EP₃ agonist, sulprostone, and one selective EP₄ receptor antagonist (AH 23848) on PLD activity. fMLP-induced [³H]PEt formation was significantly reduced to 25.67 ± 2.37 (p = 0.01), 40.89 ± 5.8 (p = 0.0095), 65.3 ± 3.68 (p = 0.011), and 88.24 ± 2.15 (p = 0.032) of its level in untreated controls in the presence of 10^–5, 10^–6, 10^–7, and 10^–8 M 11-deoxy PGE₁, respectively. In the presence of 10^–5, 10^–6, and 10^–7 M CAY 10399, [³H]PEt formation was also reduced to 33.5 ± 4.25 (p = 0.004), 41.8 ± 3.9 (p = 0.004), and 58.75 ± 4,45 (p = 0.011) of its level in the untreated controls, respectively. The selective EP₁/EP₃ receptor agonist sulprostone had no effect on the fMLP-induced PLD activity (Fig. 1A). The selective EP₄ receptor antagonist AH 23848 did not affect the inhibitory effect of PGE₂ on the stimulation of the activity of PLD by fMLP (Fig. 1B), indicating that PGE₂ did not exert its inhibitory effect via the EP₄ receptor. Because the EP₁/EP₃ agonist sulprostone did not affect fMLP-stimulated PLD activity and the EP₄ antagonist did not modify the PGE₂-mediated inhibition of PLD activity, these data suggested that EP₂ receptors mediated the inhibitory effects of PGE_2.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Effect of PGE2 and EP receptor agonists/antagonists on fMLP-induced PLD activity. A and B, neutrophils (107 cells/ml) were preincubated with the indicated concentrations of PGE2 or/and the EP agonists/antagonists for 5 min at 37°C in the presence of 0.1 U/ml ADA and 10 µM CB. Cells were stimulated with fMLP in the presence of 1% ethanol for 10 min. The amounts of [3H]PEt formed were evaluated as described under Materials and Methods. The levels of [3H]PEt formed are expressed as the percentage of the fMLP-stimulated control. The data are from three different experiments performed in duplicate and are expressed as the means ± S.E.M. C, presence of EP2 receptors on neutrophil membrane. Membranes were prepared as described under Materials and Methods and were analyzed by immunoblotting with the anti-EP2 receptor Ab alone (lane 1) or preneutralized with the immunizing peptide (lane 2). The immunoblot shown is representative of three different experiments.

The presence of EP₂ receptors on neutrophil membranes was assessed next by immunoblotting of the membrane fractions with an anti-EP₂ antibody. We observed a band at 53 kDa that was no longer detected when the antibody was preneutralized with the immunizing peptide (Fig. 1C).

PGE₂ Inhibition of Ca ²⁺ Influx Is Mediated via EP₂ Receptor. fMLP induces a rapid increase in free cytoplasmic calcium resulting from the intracellular release of Ca²⁺ stores mediated by phospholipase C-released inositol 3,4,5-trisphosphate followed by a sustained Ca²⁺ influx from the extracellular medium. fMLP-induced PLD activity has been shown to be dependent on this rise in Ca²⁺ (Exton, 1999) as well as on extracellular calcium (Pai et al., 1988). First, we examined the effect of PGE₂ on calcium mobilization in fMLP-stimulated human neutrophils. As already described, fMLP induced a rapid and transient increase in cytoplasmic free calcium as monitored using the fluorescent probe Fura-2. PGE₂ had no effect on the magnitude of the rapid rise in free cytoplasmic calcium but reduced the duration of the calcium spike suggesting that the contribution of calcium influx to the mobilization of calcium could be partially decreased by PGE₂ (Fig. 2A). We next assessed whether the effects of PGE₂ on calcium mobilization were also mediated via EP₂ receptors. The EP₂ receptor-selective agonist CAY 10399 reduced the duration of the calcium spike in a manner akin to PGE₂, (Fig. 2B), whereas the EP₄ receptor antagonist AH 23848 did not modify the reduction of the mobilization of cytosolic calcium induced by PGE₂ (Fig. 2C). Furthermore, the EP₁/EP₃ receptor agonist sulprostone influenced neither the magnitude nor the duration of the elevation of cytoplasmic calcium elicited by fMLP (Fig. 2D).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effects of PGE2 and of EP agonists/antagonists on cytoplasmic free calcium concentration. A–D, neutrophils (5 x 106 cells/ml) were preincubated for 5 min at 37°C with PGE2 or EP agonists/antagonists in the presence of 0.1 U/ml ADA. Cells were stimulated with fMLP at the time indicated by the arrow and cytosolic calcium concentrations were monitored as described under Materials and Methods. E, effect of PGE2 and CAY 10399 on fMLP-stimulated Mn2+ influx. Neutrophils resuspended in HBSS with no Ca2+ were preincubated 5 min at 37°C with PGE2 or EP agonists/antagonists in the presence of 0.1 U/ml ADA. 200 µM MnCl2 and fMLP were added at the times indicated by the arrows. The results are representative of three separate experiments.

To confirm that PGE₂ and EP₂ agonists acted on the influx of calcium, we monitored the quenching of intracellular Fura-2 fluorescence that results from the entry of Mn²⁺ through divalent cation channels (Fig. 2E). This experiment showed that a pretreatment of neutrophils with PGE₂ or the EP₂ agonist CAY 10399 partially reduced the fMLP-induced rate of quenching of Fura-2, indicating that the stimulation of the EP₂ pathway exerted an inhibitory effect on the fMLP-stimulated calcium influx.

Effect of PGE₂ on Recruitment of PKC, Arf, and Rho GTPases to Membranes. The cytosolic PLD activation factor PKC and the small GTPases Arf1 and RhoA migrate to membranes, where they mediate the activation of PLD by fMLP (Houle et al., 1995). To investigate the mechanism implicated in the inhibition of fMLP-induced PLD activity by PGE₂, we assessed whether PGE₂ interfered with the translocation to membranes of the PLD1 activation factors. As shown in Fig. 3, 10^–5 M PGE₂ alone had no noticeable effect on the basal membrane levels of PKC, Arf1, and RhoA. In comparison, PGE₂ pretreatment before fMLP stimulation resulted in a significant and dose-dependent inhibition of the translocation of PKC, Arf1, and RhoA to the membranes (compared with the fMLP-stimulated control). The recruitment to membranes of the two other Rho GTPase family members Rac2 and Cdc42 was also decreased by PGE₂ pretreatment.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Effect of PGE2 on the fMLP-induced translocation of PKC, Arf1, and Rho GTPases to the membranes. Neutrophils (107 cells/ml) were preincubated with the indicated concentrations of PGE2 for 5 min at 37°C, in the presence of 0.1 U/ml ADA and 10 µM CB. Cell suspension were stimulated with fMLP for 2 min and the incubations were stopped as described under Materials and Methods. The samples were analyzed for PKC, Arf1, RhoA, Rac2, and Cdc42 by immunoblotting. A, the immunoblots shown are representative of at least three independent experiments. B, the data of densitometric analyses are expressed as the percentage of fMLP-stimulated control and are the means ± S.D. from at least three experiments. The numbers identify the corresponding lanes of the immunoblots.

PGE₂ Decreases PKC, Arf, and Rho GTPases Recruitment to Membranes through EP₂ Receptor. We next assessed whether EP₂ agonists affected the membrane translocation of PKC and small GTPases. As was the case with PGE₂, the EP₂-selective agonist CAY 10399 alone did not affect the basal level of PKC, Arf, and Rho GTPases associated with membranes, but preincubation of cells with CAY 10399 before fMLP stimulation significantly decreased the amounts of membrane-translocated PKC, Arf, and Rho GTPases in a concentration-dependent manner (Fig. 4). These data have been buttressed by the use of another selective EP₂ agonist: the 19(R)-hydroxy-PGE₂, which showed effects similar to those of CAY 10399 (data not shown), indicating that the inhibition of the translocation of PLD cofactors by PGE₂ was mediated via the EP₂ receptors.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of CAY 10399, an EP2 agonist, on the fMLP-induced translocation of PKC, Arf1 and Rho GTPases to the membranes. Neutrophils (107 cells/ml) were preincubated with the indicated concentrations of CAY 10399 for 5 min at 37°C, in the presence of 0.1 U/ml ADA and 10 µM CB. Cell suspensions were stimulated with fMLP 100 nM for 2 min, and the reactions were stopped as described under Materials and Methods. The samples were analyzed for PKC, Arf1, RhoA, Rac2, and Cdc42 by immunoblotting. A, the immunoblots shown are representative of at least three independent experiments. B, the data of densitometric analyses are expressed as the percentage of fMLP-stimulated control and are the means ± S.D. from at least three experiments. The numbers identify the corresponding lanes of the immunoblots.

Effect of PGE₂ on PtdIns(3,4,5)P₃ Formation and PI3K Activity. PLD1 activation is also regulated by phosphoinositides and pharmacological inhibitors of PI3K strongly inhibit PLD activity induced by chemoattractants (Reinhold et al., 1990). Moreover, PI3K exerts a regulatory role over small GTPase activation, especially on Rac2 and Cdc42 (Benard et al., 1999). Thus, we tested the effects of PGE₂ on fMLP-induced PtdIns(3,4,5)P₃ formation in human neutrophils. As shown in Fig. 5A, PGE₂ (10^–5 to 10^–8 M) decreased the amounts of PtdIns(3,4,5)P₃ formed after fMLP stimulation. The amounts of PtdIns(3,4,5)P₃ formed in response to fMLP were also monitored in the presence of CAY 10399. As shown in Fig. 5B, CAY 10399 (10^–5 and 10^–6 M) also inhibited fMLP-stimulated PtdIns(3,4,5)P₃ formation, indicating that PGE₂ exerted its inhibitory effect on PtdIns(3,4,5)P₃ formation via the EP₂ receptors. Decreased accumulation of PtdIns(3,4,5)P₃ could be caused by decreased transformation of phosphatidylinositol 4,5-bisphosphate into PtdIns(3,4,5)P₃ or to accelerated dephosphorylation of PtdIns(3,4,5)P₃. To distinguish between these possibilities, we monitored the activity of p110 induced by fMLP in the presence of PGE₂, for p110 is the major PI3K isoform activated by chemoattractants in neutrophils (Hirsch et al., 2000; Naccache et al., 2000). In brief, PI3K was immunoprecipitated using an anti-p110 Ab at different times after fMLP stimulation, and PI3K activity was tested using an exogenous substrate (PtdIns). The results of these experiments (Fig. 6A) indicated that fMLP-induced p110 activity was maximal at 15 to 30 s, and this peak of p110 activity was totally abolished by PGE₂ (p = 0.0264 at 15 s and p = 0.0089 at 30 s). The activation of p110 by chemotactic factors is characterized by its rapid recruitment to membranes, and we have therefore examined the effects of PGE₂ and EP₂ agonists on this translocation step. PGE₂ and CAY 10399 alone had no effect on the amounts of membrane-associated p110 in unstimulated cells but reduced the levels of membrane-associated p110 to that of the unstimulated controls in fMLP-stimulated neutrophils (Fig. 6B). Thus, the inhibition of the fMLP-stimulated accumulation PtdIns(3,4,5)P₃ by PGE₂ seems to be the direct consequence of the inhibition of p110 activity.

View larger version (67K): [in this window] [in a new window]   Fig. 5. Effect of PGE2 and CAY 10399 on the fMLP-induced accumulation of PtdIns(3,4,5)P3. [32P]-labeled neutrophils (5 x 107 cells/ml) were preincubated 5 min at 37°C with the indicated concentrations of PGE2 (A) or CAY 10399 (B) in the presence of 0.1 U/ml ADA. Cells were stimulated with fMLP for 30 s at 37°C and the amounts of PtdIns(3,4,5)P3 were measured as described under Materials and Methods. The autoradiograms shown are representative of three independent experiments. C, the data of the densitometric analyses are expressed as the percentage of the fMLP-stimulated control and are the means ± S.D. from three experiments. The numbers identify the corresponding lanes of the autoradiograms.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Effect of PGE2 on fMLP-induced p110 activity and translocation to membranes. A, neutrophils (107 cells/ml) were preincubated 5 min at 37°C with 10–5 M PGE2 in the presence of 0.1 U/ml ADA. Cells were stimulated with fMLP for the indicated times, lysed, and the immunoprecipitated p110 was assayed for lipid kinase phosphorylation as described under Materials and Methods. The results are from four independent experiments and are expressed as percentage of the unstimulated sample at t = 0. B, neutrophils (107 cells/ml) were preincubated with PGE2 or CAY 10399 at the indicated concentrations for 5 min at 37°C, in the presence of 0.1 U/ml ADA and 10 µM CB. Cell suspensions were stimulated with fMLP for 30 s and the reactions were stopped as described under Materials and Methods. The samples were analyzed for p110 by immunoblotting. The immunoblot shown is representative of three independent experiments. C, the data of densitometric analyses are expressed as the percentage of fMLP-stimulated control and are the means ± S.D. from at least three experiments. The numbers identify the corresponding lanes of the immunoblots.

Effect of PGE₂ on the fMLP-Induced Pattern of Tyrosine Phosphorylation. Tyrosine kinases are well known regulators of PLD activity induced by various agonists and have been shown to act upstream of fMLP-induced recruitment of cytosolic PLD cofactors to membranes (Houle et al., 1999). We have therefore sought evidence for an effect of PGE₂ on the tyrosine phosphorylation of proteins stimulated by fMLP. The pattern of tyrosine phosphorylation induced by fMLP was examined in the presence or absence of PGE₂ or the EP₂ agonist CAY 10399. As shown in Fig. 7A, incubation of neutrophils with 10^–5 M PGE₂ or CAY 10399 before fMLP stimulation decreased the intensity and the duration of phosphorylation of the band at 120 kDa. For a few donors, the tyrosine phosphorylation of the 42- to 44-kDa bands corresponding to the mitogen-activated protein kinases as evidenced by reprobing the membranes with an anti-phospho extracellular signal-regulated kinase 1/2 Ab (data not shown) was also reduced by PGE₂ (middle) but not by CAY 10399 (right). Taken together, these data indicate that PGE₂ reduced the activation of some still unidentified tyrosine kinase(s) via EP₂ receptor.

View larger version (80K): [in this window] [in a new window]   Fig. 7. Effect of PGE2 and CAY 10399 on the global profile of tyrosine phosphorylation and on the translocation of Tec kinases induced by fMLP. A, neutrophils (2 x 107/ml) were preincubated with 10–5 M PGE2 or 10–5 M CAY 10399 in the presence of 0.1 U/ml ADA at 37°C for 10 min. The assay was performed as described under Materials and Methods. The results shown are representative of at least three independent experiments. B, neutrophils (107 cells/ml) were preincubated with the indicated concentrations of PGE2 or CAY 10399 for 5 min at 37°C in the presence of 0.1 U/ml ADA and 10 µM CB. Cell suspensions were stimulated with fMLP for 30 sec and the reactions were stopped as described under Materials and Methods. The samples were analyzed for Btk and Tec by immunoblotting. The immunoblots shown are representative of three independent experiments. C, the data of densitometric analyses are expressed as the percentage of fMLP-stimulated control and are the means ± S.D. from at least three experiments. The numbers identify the corresponding lanes of the immunoblots.

Several members of the Tec family kinases (Tec, Btk, and Bmx) have been shown to be involved in response to chemoattractants in human neutrophils (Lachance et al., 2002). The activation of Tec kinases is dependent on PI3K and characterized by a rapid translocation to the PtdIns(3,4,5)P₃-enriched membranes. We thus examined the effect of PGE₂ and EP₂ agonist on this index of activation of Tec kinases. The data shown in Fig. 7B indicate that PGE₂ as well as CAY 10399 (10^–5-10^–7 M) decreased the amounts of Tec and Btk translocated to membranes in fMLP-stimulated cells.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have investigated the mechanisms underlying the inhibition of PLD activity by PGE_2. We demonstrated that PGE₂ inhibited several critical events stimulated by fMLP, including the influx of calcium, translocation of PKC and small GTPases, the activation of PI3K and tyrosine kinases, and the stimulation of PLD activity as well. Using various agonists and antagonists of the EP receptors, we demonstrated that EP₂ but not EP₁, EP₃, or EP₄ receptors mediate the inhibitory effect of PGE₂ on the fMLP-induced PLD activation pathway. Taken together, our results are consistent with previous pharmacological studies attributing a major role for EP₂ receptors in PGE₂-induced inhibition of neutrophil functional responses (Armstrong, 1995; Talpain et al., 1995).

The mechanism underlying the PGE₂-induced inhibition of the stimulation of PLD activity by fMLP has not yet been clarified. The fact that PGE₂ inhibited fMLP-but not phorbol 12-myristate 13-acetate–induced PLD activity suggested that PGE₂ did not exert its inhibitory effect directly on PLD but rather at a site nearer to the fMLP receptors (Agwu et al., 1991). This hypothesis led us to examine the effect of PGE₂ on the signaling steps located upstream of PLD in the fMLP-stimulated PLD activation pathway. We confirmed that PGE₂ did not influence the mobilization of Ca²⁺ from intracellular stores but decreased the influx of Ca²⁺ from the extracellular medium (Ahmed et al., 1995). The inhibitory mechanism of PGE₂ on influx has not yet been elucidated, but several hypotheses can be advanced. An up-regulating role for PI3K in Ca²⁺ influx has been reported in chemokine-stimulated cells (Kansra et al., 2001), but this does not seem to be the case in our model, because we and others (Ahmed et al., 1995) have found that wortmannin, a potent PI3K inhibitor, does not decrease fMLP-stimulated calcium influx in neutrophils; on the contrary, it slightly augments this influx (P. H. Naccache, unpublished results). Therefore, the inhibition of PI3K by PGE₂ cannot explain the reduced calcium influx observed in our experimental model. Because PGE₂ has no impact on the release of Ca²⁺ from intracellular stores, the capacitive calcium entry is probably not affected by PGE₂, and a relevant hypothesis is that PGE₂ rather inhibits the opening of a receptor-operated calcium channel (Ahmed et al., 1995). Another possibility is that, as shown previously for adenosine and other cAMP elevating agents, PGE₂ could promote an accelerated clearance of cytosolic calcium by up-regulating the endo-membrane Ca²⁺-ATPase that recaptures cytosolic calcium (Theron et al., 2002). However, this hypothesis would account in part for the decreased mobilization of calcium but not for the inhibition of Mn²⁺ influx observed in the presence of PGE₂. Because the influx of Ca²⁺ has been shown to increase PLD activity stimulated by fMLP (Pai et al., 1988), the reduction of this influx of Ca²⁺ by PGE₂ could possibly down-regulate fMLP-induced PLD activity, although this effect of PGE₂ is probably of minor influence on PLD activation because other factors than calcium directly regulate PLD.

We observed that PGE₂ and EP₂ agonists significantly decreased the translocation of PKC, Arf1 and RhoA to fMLP-stimulated neutrophil membranes. These PLD activation cofactors act synergistically to enhance PLD activity (Exton, 1999) and their simultaneous inhibition by PGE₂ provides a mechanistic explanation for the inhibitory effect of the EP₂ signaling pathway on fMLP-induced PLD activity. These results are in accordance with and further support a previous study from our laboratory indicating that adenosine inhibited the fMLP-induced PLD pathway via the A_2A receptor by diminishing membrane recruitment of PKC, Arf1, and RhoA (Thibault et al., 2000). The translocation of two other Rho GTPase family members, Rac2 and Cdc42, are involved in several functions inhibited by PGE₂ is also reduced by PGE₂ via the EP₂ receptors.

The concomitant inhibition of Rho and Arf GTPases suggested that PGE₂ exerts an inhibitory effect upstream of both small GTPase families. Although the intermediary signaling steps linking membrane bound receptors to small GTPase activation are not yet fully understood, PI3K and tyrosine kinases seem to play key roles in membrane recruitment and activation of small GTPases (Benard et al., 1999).

We report here that PGE₂ significantly reduced the amounts of PtdIns(3,4,5)P₃ formed in response to fMLP via EP₂ receptor. Moreover, we observed an almost complete inhibition of p110 kinase recruitment and activation by PGE₂. These results strongly suggest that PGE₂ inhibited the chemoattractant-induced activation of p110 rather than stimulating an accelerated rate of dephosphorylation of PtdIns(3,4,5)P₃. It is therefore unlikely that the phosphoinositide-specific phosphatases PTEN or SHIP are involved in the inhibitory effect of PGE₂ on PtdIns(3,4,5)P₃ accumulation. This inhibitory effect of PGE₂ on p110 adds a new element to the mechanism involved in EP₂-mediated inhibition of the fMLP-induced PLD signaling pathway.

The activation of small GTPases is tightly controlled by the opposite actions of guanine nucleotide exchange factors (GEFs) and guanine nucleotide activating proteins that stimulate GTP hydrolysis. GEFs for the Rho and Arf family GTPases contain a PH domain known to bind phosphoinositol lipids, thereby targeting GEF to PtdIns(3,4,5)P₃-enriched membrane regions. Increasing evidence indicates that Rho GTPases are indirectly regulated by PI3K via specific GEFs (Welch et al., 2003). Of particular relevance to our work is the observation that the Rho GEF Vav stimulated by the fMLP receptor mediates the activation of Rac by PI3K. The relationship between Arf GEFs and PI3K in vivo is more controversial, and reports differ according to the GEF and the cellular type studied. Nevertheless, the PH domain of cytohesin-1, the main Arf-GEF expressed in human neutrophils, binds to PtdIns(3,4,5)P₃ (Corvera and Czech, 1998), and we observed that Arf translocation to membranes is down-regulated by wortmannin (N. Thibault, unpublished results). These lines of experimental evidence linking PI3K to GEF activation in various cellular systems support our hypothesis that the inhibition of p110 by PGE₂ decreases the translocation of Arf and Rho GTPases.

The inhibition of PKC translocation by PGE₂ may at first seem intriguing because PGE₂ neither alters the first peak of cytosolic calcium released from intracellular pools by fMLP-induced phospholipase C activation nor effectively inhibits diacylglycerol formation in fMLP-stimulated neutrophils (Takenawa et al., 1986). The reduced calcium influx could account in part for the inhibitory effect of PGE₂ on PKC activation, but PKC inhibition is more likely to be attributable to the decreased activity of PI3K because PKC has been shown to be located downstream of PI3K in the fMLP signaling pathway (Vlahos et al., 1995).

PLD and small GTPase activation is also regulated by tyrosine phosphorylation events. There is no evidence for a direct tyrosine phosphorylation of PLD after fMLP stimulation in neutrophils (Marcil et al., 1999), but the translocation of PLD cofactors (PKC, Arf, and RhoA) as well as Rac2 and Cdc42 activation has been shown to occur downstream of tyrosine phosphorylation events in fMLP-stimulated HL-60 cells, providing a functional link between tyrosine kinases and PLD activation (Benard et al., 1999; Houle et al., 1999). PGE₂ (or CAY 10399) reduced the fMLP-stimulated tyrosine phosphorylation of a set of substrates around 120 kDa by as-yet-unidentified kinases. This suggests that tyrosine kinase(s) could be another target for PGE_2. Because the profile of tyrosine phosphorylation cannot provide precise information on the putative kinases involved, we further investigated the effect of PGE₂ on Tec kinases that have been shown to be activated by fMLP (Lachance et al., 2002). PGE₂ significantly decreased Tec and Btk translocation to membranes, an effect that can be viewed as a consequence of PI3K inhibition because the recruitment and the activation of these PH-domain containing kinases are dependent on PI3K. This latter result is to be related to a previous study that established a functional link between Btk (and possibly other Tec kinases) and the activation of Rho, Arf GTPases and PLD in human neutrophils (Gilbert et al., 2003) and further support the involvement of PI3K as a crucial target of the EP₂ inhibitory pathway.

The mechanism underlying the inhibition of PI3K by PGE₂ remains to be further investigated. PI3K is composed of a p110 catalytic subunit tightly bound to a p101 regulatory unit, and upon fMLP-receptor stimulation, this cytosolic complex is activated by binding to the G units of heterotrimeric G protein. The noncatalytic p101 subunit is thought to act as an adaptor that mediates the recruitment of the enzymatic complex to the membrane where G directly activates the catalytic p110 subunit (Brock et al., 2003). Because PGE₂ inhibits the translocation of the p110, we can suggest that PGE₂ prevents the binding of the enzymatic complex to G by targeting a site either on the p101/p110 complex or on G and that the decreased amount of p101/p110 recruited explains the suppression of the stimulation of p110 activity by the EP₂ pathway.

The signaling events linking the EP₂ receptor to its fMLP-stimulated targets (possibly PI3K) also remain to be characterized. The EP₂ receptor is coupled to a G_s protein that increases cAMP levels by stimulating adenylate cyclase (Breyer et al., 2001). Although several suppressive effects of PGE₂ on neutrophil functions have been paralleled by an increase in cAMP (Rivkin et al., 1975; Talpain et al., 1995; Bloemen et al., 1997), others (e.g., chemotaxis and aggregation triggered by fMLP) have not (Armstrong, 1995), suggesting that EP₂ could transmit signals through an alternative and still unknown pathway. Nevertheless, the fact that superoxide production stimulated by fMLP was inhibited by PGE₂ in a cAMP/cAMP-dependent protein kinase-dependent manner (Armstrong, 1995) would be in favor for a role of this pathway in the inhibition of PLD activity. A more detailed study of the involvement of cAMP-dependent protein kinase in the inhibition of the fMLP signaling cascades, more particularly in the inhibition of PI3K, is presently underway.

The present study also provides new insights into the signaling mechanisms underlying the EP₂ mediated inhibition of the respiratory burst, chemotaxis, and secretion elicited by chemoattractants in human neutrophils. The assembly and activation of the NADPH complex is tightly regulated at multiple levels and has been shown to require 1) the phosphorylation of several components by PKC, 2) the presence of PLD-derived PA implicated in phosphorylation processes, 3) the translocation of Rac2 and, finally, 4) the activation of PI3K, mainly p110 (Vlahos et al., 1995; Babior, 1999; Hirsch et al., 2000). The EP₂-mediated inhibitory effects on fMLP-induced PI3K and PLD activities as well as on the translocation of Rac2 and PKC can account for the strong reduction of the respiratory burst by PGE₂. PI3K and Rho GTPases also play essential roles in the polarization of the cell that initiates the chemotactic response (Servant et al., 2000), and their inhibition by the EP₂ pathway explains, at least in part, the impairment of chemotaxis by PGE₂. Exocytosis and degranulation processes are regulated by Arf and PLD in neutrophils (Cockcroft, 2001), and their inhibition by the EP₂ pathway might also account for the inhibition of the fMLP-stimulated release of cytotoxic enzymes by PGE₂.

In conclusion, the present study shows that PGE₂ inhibits the chemoattractant-stimulated PLD pathway by binding to the EP₂ receptor in human neutrophils. Moreover, we provide evidence that this inhibitory pathway decreases p110 activation by fMLP and, as a result activation of Tec kinases, small GT-Pases and PLD. The results of this investigation provide new insights into the signaling mechanisms implicated in the down-regulation by PGE₂ of the inflammatory response to chemoattractants.

	Acknowledgements

 
We thank Guillaume Paré and Danielle Harbour for their expert help in the measurement of Mn²⁺ influx and PLD activity, respectively.

	Footnotes

 
This work was supported in part by grants from the Canadian Institutes of Health Research. S.G.B. is the recipient of a Research Scientist Award from the Arthritis Society of Canada. P.H.N. is the recipient of the Canada Research Chair on the Molecular Physio-pathology of the human neutrophil.

ABBREVIATIONS: PLD, phospholipase D; PA, phosphatidic acid; PKC, protein kinase C; Arf, ADP-ribosylation factor; PI3K, phosphatidylinositol 3-kinase; PG, prostaglandin; EP receptor, E prostaglandin receptor; PtdIns(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; fMLP, formyl-methionyl-leucyl-phenylalanine; Ab, antibody; ADA, adenosine deaminase; DFP, di-isopropylfluorophosphate; PH, pleckstrin homology; CB, cytochalasin B; AM, acetoxymethyl ester; CAY 10399, 9-oxo-11, 16S-dihydroxy-17-cyclobutyl-prosta-5Z,13E-dien-1-oic acid; AH23848, [1(Z),2,5]-(±)-7-[5-[[(1,1'-biphenyl)-4-yl]methoxyl]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid; HBSS, Hanks' balanced saline solution; PEt, phosphatidylethanol; PtdIns, phosphatidylinositol; GEF, guanine nucleotide exchange factors.

Address correspondence to: Dr. Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie-Immunologie, Room T1-49, 2705 Boul Laurier, Ste Foy, Québec, Canada, G1V 4G2. E-mail: sylvain.bourgoin{at}crchul.ulaval.ca

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