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Originally published In Press as doi:10.1074/jbc.M410725200 on January 19, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14433-14442, April 15, 2005
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Prostaglandin E2 Induces Interleukin-8 Gene Transcription by Activating C/EBP Homologous Protein in Human T Lymphocytes*

Silvana Caristi, Giovanna Piraino, Maria Cucinotta, Andrea Valenti, Saverio Loddo, and Diana Teti{ddagger}

From the Department of Experimental Pathology and Microbiology, University of Messina, 98125 Messina, Italy

Received for publication, September 17, 2004 , and in revised form, January 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of prostaglandin E2 (PGE2) in regulating the synthesis of the pro-inflammatory chemokine inter-leukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE2 induced IL-8 mRNA transcription through prostaglandin E2 receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE2-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-{kappa}B-independent. Our data suggest that PGE2 acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin (PG)1 E2, a product of the cyclooxygenation of arachidonic acid, is a potent mediator of immune responses (1–3) and inflammation (4–6). PGE2 is thought to down-regulate cell-mediated immunity by inhibiting the functional activities of macrophages (7–10) and T cell proliferation and differentiation, expression of membrane receptors, secretion of diverse cytokines, and other effector functions in cellular immune reactions (11). On the other hand, it has been shown that PGE2 is also able to stimulate some cellular immune activities, such as the expression and activation of matrix metalloproteases in HSB.2 human leukemic T cells (12–13), the escape of some T cells from activation-induced apoptosis (14), and the macrophage production of interleukin (IL)-6 and IL-10 (15–17). Thus, it has been postulated that the heterogeneity of PGE2 immunoregulatory effects may depend on distinctive signaling mechanisms of different PGE2 receptors. PGE2 also displays a complex regulatory function in IL-8 gene expression depending on the concentrations employed and the cell specificity. IL-8 is a potent chemoattractant for neutrophils in local inflammatory sites and is produced by a wide variety of cells in response to different stimuli (18). At physiological as well as pathological concentrations up to 100 µM, PGE2 is able to up-regulate endogenous IL-8 expression in human intestinal epithelial cells (19–20) and to enhance IL-8 production in human synovial fibroblasts stimulated with IL-1{beta} (21). In contrast, PGE2 has no effect on neutrophil-derived IL-8 induced by lipopolysaccharide (22); down-regulates IL-8 in response to lipopolysaccharide in human alveolar macrophages and blood monocytes (23); and suppresses the production of chemokines, including IL-8, in human macrophages (24). The production of IL-8 in lymphocytes, viz. T cells, is crucial for the ability of the immune system to control infection and inflammation. However, despite the well known activity of PGE2 as a mediator of immune and phlogistic responses, the role of this molecule in T cell production of IL-8 has not been well documented. In this study, we observed that PGE2 significantly increased the amount of IL-8 by inducing gene transcription through a novel signaling mechanism mediated by C/EBP homologous protein (CHOP). We demonstrate that the signaling pathways triggered by EP1 and EP4 receptors and p38 MAPK are involved in this process. Therefore, we postulate that PGE2 may enhance the phlogistic response by inducing the release of the chemokine IL-8 from activated T lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—PGE2, PGF2{alpha}, and staurosporine were obtained from Sigma (Milan, Italy). Anti-human CD3 and anti-human CD28 monoclonal antibodies were purchased from Immunotech (Marseilles, France). SB 203580, PP2, caffeic acid phenethyl ester, H-89 dihydrochloride, bisindolylmaleimide I (GF 109203X), and LY 294002 were purchased from Calbiochem. PD98059 was obtained from New England Biolabs Inc. (Beverly, MA). SC-19220, AH 6809, sulprostone, butaprost, 17-phenyltrinor-PGE2 (PT-PGE2), 11-deoxy-PGE1, and PGI2 were obtained from Cayman Chemical (Ann Arbor, MI).

Cells and Culture Conditions—Peripheral T lymphocytes were isolated from human venous blood obtained from health adult volunteers as described previously (25). Briefly, diluted whole blood (100 ml) was layered in centrifuge tubes onto 120 ml of Histopaque-1077 gradient (Sigma). T lymphocytes were magnetically separated from B cells using colloidal paramagnetic microbeads conjugated to mouse anti-CD19 surface antigen monoclonal antibody expressed on B lineage cells using negative selection columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). T cell expression of activation markers such as CD25 and CD71 was tested using cytofluorometric analysis. Only CD25– and CD71– lymphocytes were processed. Jurkat T cells (clone E6.1) were obtained from the European Collection of Cell Cultures (Sigma) and maintained in a humidified atmosphere of 5% CO2 and 95% air (37 °C) in RPMI 1640 medium (Euroclone, Milan) supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin (Euroclone), and 20% heat-inactivated fetal calf serum (Euroclone). Cells were routinely tested for mycoplasma infection, and cultures were renewed from frozen stocks every 2 months.

Cytokine Production Assay—T cells were cultured in round-bottom 24-well plates (1 x 106 cells/well) in the presence of anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies (26). Cells were then immediately stimulated with PGE2, or PGI2, at different concentrations (stock solutions (100 mM) were prepared in ethanol and stored at –20 °C) and cultured for 24 h. In some experiments, cells were treated with various protein kinase inhibitors and EPR1/2 antagonists 1 h prior to the addition of PGE2. In other experiments, cells were treated with EPR1–4 agonists only and cultured for 24 h. After stimulation, supernatants were collected and analyzed for IL-8 using an enzyme-linked immunosorbent assay (ELISA) kit (BioSource International, Nivelles, Belgium) according to the manufacturer's instructions.

RNA Isolation and Reverse Transcription-PCR—T cells were cultured in 35-mm well plates (5 x 106 cells/well) in the absence or presence of anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies, and cells were then immediately stimulated for the indicated times with PGE2 at final concentrations of 10 and 100 µM. Total RNA was extracted with TRIzol (Invitrogen, Milan) according to the manufacturer's instructions. For reverse transcription-PCR, 1 µg of total RNA was reverse-transcribed in a total volume of 20 µl with an IMProm-IITM reverse transcriptase kit (Promega, Milan) according to the manufacture's instructions. 20 µl of reverse transcription products were brought to a volume of 100 µl containing 2 mM MgCl2, 0.2 mM PCR nucleotide mixture, a 1 µM concentration of both the upstream and downstream PCR primers (Sigma), 5 units of Taq DNA polymerase (Transgenomic, Inc., Bergamo, Italy), and 10x PCR buffer (Transgenomic, Inc.). Two pairs of primers were used in this study. The primer sequences were as follows: IL-8, 5'-ATG ACT TCCAAG CTG GCC GTG GCT-3' (sense) and 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3' (antisense); and {beta}-actin, 5'-TGA CGG GGT CTA CCC ACA CTG TGC CCC ATC TA-3' (sense) and 5'-CTA GAA GCA TTG CGC TGG ACG ATG GAG GG-3' (antisense). Amplification was carried in a DNA thermal cycler (Applied Biosystems, Milan) after an initial denaturation at 94 °C for 4 min. This was followed by 37 cycles of PCR using the following temperature and time profile: denaturation at 94 °C for 3 min, primer annealing for 50 s at 58 °C for IL-8 and at 62 °C for {beta}-actin, primer extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. The PCR products were visualized by electrophoresis on 1% agarose gel in 1x buffer containing 89 mM Tris borate and 2 mM EDTA (pH 8.3) after staining with 0.5 µg/ml ethidium bromide. The UV light-illuminated gels were photographed, and the relative sum intensity was calculated by normalizing the sum intensity of the IL-8 product to the {beta}-actin mRNA control.

Protein Kinase C (PKC) Assay—T cells were grown in 10-cm dishes and, where indicated, transfected with PTGER1 small interfering RNA (siRNA). The cells were then incubated with the appropriate stimuli for 15 min and homogenized in 50 mM Tris-HCl (pH 7.5) containing 0.3% (w/v) {beta}-mercaptoethanol, 5 mM EDTA, 10 mM EGTA, 50 µg/ml phenylmethylsulfonyl fluoride, and 10 mM benzamidine. PKC activity in the lysates was determined using the protein kinase assay from Amersham Biosciences (Milan) according to the manufacturer's protocol.

Western Blot Analysis—Cells were cultured in 35-mm well plates at a concentration of 5 x 106 cells/well. After stimulation, cells were lysed at 4 °C in 150 µl of M-PER lysis buffer (Pierce), 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 mg/ml leupeptin. Cell lysates were centrifuged at 14,000 x g for 15 min at 4 °C, and the supernatants were collected. Protein concentrations of extracts were determined using a standard Bradford protein assay (Bio-Rad, Milan). Cell lysates were dissolved in 4x SDS-PAGE sample buffer containing 0.5 M Tris-HCl (pH 6.8) 10% SDS, 10% glycerol, 1% mercaptoethanol, and bromphenol blue and boiled for 3 min. After centrifugation at 14,000 x g for 3 min, cell lysates were analyzed on 9% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in Tris-buffered saline/Tween supplemented with 1% bovine serum albumin for 1 h. The blots were subsequently incubated with primary rabbit polyclonal antibodies (1:200 dilution) to the EP1 and EP4 receptors (Cayman Chemical) or primary phospho-specific rabbit IgG (1:500 dilution) recognizing p38 MAPK phosphorylated at Thr180 and Tyr182 or activating transcription factor-2 (ATF-2) phosphorylated at Thr71 (Cell Signaling Technology, Milan). Primary anti-{beta}-actin monoclonal antibody (1:1000 dilution; Sigma) was used to normalize protein loading to that of specific proteins in each lane. After overnight incubation at 4 °C, membranes were washed with Tris-buffered saline/Tween and incubated with horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Biosciences) or goat anti-mouse (Cell Signaling Technology) IgG as secondary antibody at a dilution of 1:10,000 for 1 h at room temperature. Immunoreactive bands were detected by autoradiography using the SuperSignal West Pico chemiluminescent substrate system (Pierce) according to the manufacturer's instructions.

Transient Transfection—Liposome-mediated transient gene transfer was carried out with DMRIE-C (Invitrogen) as recommended by manufacturer. Briefly, cells were seeded at 2 x 106 cells in 35-mm plates and transfected with a total of 2 µg of plasmid DNA using 6 µg of DMRIE-C/ml of RPMI 1640 medium without fetal calf serum. Transfected DNAs included 1 µg of pFR-Luc, a luciferase reporter gene including multimerized Gal4 upstream activating sequences upstream of the minimal promoter, the transactivator Gal-CHOP (50 ng; Stratagene, La Jolla, CA), pSV-nlsLacZ DNA, a {beta}-galactosidase expression vector (0.5 µg), and an empty plasmid DNA (pBSM) at a final concentration of 2 µg/plate. Jurkat cells (clone E6.1) were also transiently cotransfected with the pIL8-Luc plasmid (1 µg; kindly provided by Dr. Hector R. Wong, Cincinnati Children's Hospital Medical Center, Cincinnati, OH), pSV-nlsLacZ DNA, and an expression plasmid for CHOP (0.2 µg; kindly provided by Dr. Hidetoshi Hayashi, Nagoya City University, Nagoya, Japan) as described in the figure legends. In all experiments, transfections were stopped after 6 h by the addition of an equal volume of RPMI 1640 medium containing 20% fetal calf serum. 24 h after transfection, cells were treated with 10 or 100 µM PGE2. After an additional 6 h, cells were harvested, and protein extracts were prepared for the luciferase activity assay using luciferin (Promega) as the substrate. Luciferase activity was normalized to {beta}-galactosidase activity produced by cotransfected plasmid pnlsLacZ.

siRNA and Transfection—Double-stranded siRNA sequences targeting PTGER1 (GenBankTM/EBI accession number NM000955) and PTGER4 (accession number NM000958) mRNAs corresponding to the coding regions of residues 1009–1027 (5'-CCAGCTTGTCGGTATCATG-3') and 954–972 (5'-TCAACCATGCCTATTTCTA-3'), respectively, were obtained from Dharmacon (Lafayette, CO). A nonspecific duplex (5'-CAGUGGAGAUCAACGUGCAAGUU-3'; Dharmacon) was used as a control, which did not significantly affect PTGER1 and PTGER4 mRNA and protein levels relative to the untransfected controls. Concentrations of siRNA and time of incubation were tested. EP1 and EP4 receptor knockdown reached the maximum at a concentration of 100 nM and at 24–48 h post-transfection. After 72 h, there was a modest increase in EP1 and EP4 receptor levels compared with 48 h post-transfection. To assess gene silencing, the protein levels of the EP1 and EP4 receptors were determined by immunoblotting. Jurkat T cells were plated in 35-mm well plates in RPMI 1640 medium without fetal bovine serum and transfected with double-stranded siRNA using the DMRIE-C reagent according to the manufacturer's instructions.

24 h after the siRNA transfections, the cells were incubated in the absence or presence of anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies with PGE2 at final concentrations of 10 and 100 µM for an additional 24 h. All cells were harvested for total protein extraction, and supernatants were processed for IL-8 production by ELISA. In another set of experiments, transfection with the Gal-CHOP chimeric transcription factor was followed by siRNA (PTGER1 and PTGER4) transfection 4 h later.

Densitometry and Statistical Analysis—The relative intensities of protein and nucleic acid bands were analyzed using the Digital Sciences 1D program from Kodak Scientific Imaging Systems (New Haven, CT). Standard curves were run, and the data that were obtained were in the linear range of the curve. In addition, all values were normalized to their respective lane loading controls. Data are expressed as means ± S.E. of n determinations. Results were analyzed by two-tailed Student's t test. p values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-8 Protein Production after Stimulation with Prostaglandins—To determine whether PGE2 could enhance the synthesis of IL-8 protein, anti-CD3/CD28 antibody-stimulated T cells were cultured with different concentrations of PGE2, PGF2{alpha}, or PGI2, and IL-8 levels were measured in the supernatant by ELISA. Fig. 1 (A and B) shows that human peripheral T lymphocytes and Jurkat T cells produced significant elevated levels of IL-8 only when treated with PGE2 at 10 or 100 µM, with a higher significance at 100 µM. Therefore, stimulation with PGE2 leads to a dose-dependent release of IL-8 in T cells.



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  		FIG. 1.
PGE2 induces IL-8 protein synthesis in T cells. Peripheral (A) and Jurkat (B) T cells were stimulated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies and treated for 24 h with or without PGE2, PGF2{alpha}, or PGI2 (100 nM to 100 µM). IL-8 protein was measured in cell supernatants by ELISA. Data are depicted as means ± S.E. of five independent experiments. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells (based on two-tailed Student's t test).

 
PGE2 Induces IL-8 mRNA in Human T Cells—Reverse transcription-PCR analysis showed that stimulation of Jurkat cells with anti-CD3/CD28 antibody and subsequently with 10 or 100 µM PGE2 induced expression of IL-8 mRNA at 4 h, reaching a maximum at 5 h (Fig. 2A and B, respectively). The increase in IL-8 mRNA was not observed at 8 h after PGE2 treatment. The values of IL-8 mRNA were 2–4-fold over the basal expression of untreated cells when 10 µM PGE2 was employed, whereas they were 3–5-fold higher in cells treated with 100 µM PGE2. These results suggest that PGE2 increases IL-8 production at the transcriptional level in a time- and dose-dependent fashion.



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  		FIG. 2.
PGE2 induces IL-8 mRNA in Jurkat T cells. Jurkat T cells stimulated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies were incubated for 0, 1, 2, 4, 5, 6, and 8 h with 10 (A) or 100 (B) µM PGE2, respectively. IL-8 and {beta}-actin (as an internal control) mRNAs were measured by reverse transcription-PCR. The expected product sizes of IL-8 and {beta}-actin are 289 and 391 bp, respectively. The relative densities were calculated by dividing the density of the IL-8 band by the density of the {beta}-actin band at the same time point. Data are depicted as means ± S.E. of five independent experiments. **, p < 0.01 versus control untreated cells (based on two-tailed Student's t test).

 
PGE2 Stimulates IL-8 Production via the EP1 and EP4 Receptors—To determine the EP receptors involved in IL-8 production by PGE2, cells were stimulated with the EP1 agonist PT-PGE2 (27), the EP2 receptor agonist butaprost (27), the EP3 receptor agonist sulprostone (27), and the EP4 agonist 11-deoxy-PGE1 (28). PT-PGE2 and 11-deoxy-PGE1 induced a significant increase in IL-8 release, whereas butaprost and sulprostone were not able to induce chemokine synthesis (Fig. 3A). In parallel experiments, both the selective EP1 antagonist SC-19220 (28) and the EP1/2 antagonist AH 6809 (29) significantly inhibited the production of IL-8 induced by either 10 or 100 µM PGE2 (Fig. 3B). The effect of AH 6809 was most probably due to inhibition of EP1, but not EP2, since butaprost had no effect on IL-8. To further verify whether the effects of PGE2 on IL-8 production are mediated through the EP1 and EP4 receptors, we performed post-transcriptional gene silencing experiments for both receptors using synthetic siRNAs and evaluated IL-8 production. Under optimal conditions (100 nM siRNA, 24 h post-transfection), we achieved 70 and 85% knockdown of the EP1 and EP4 receptors at the protein level, respectively (Fig. 3, C and D, upper). The EP1 and EP4 receptor silencing by specific siRNAs reduced the IL-8 production in PGE2-treated cells by ~35 and 45%, respectively (Fig. 3, C and D, lower). In contrast, the nonspecific control duplex did not affect IL-8 synthesis by PGE2. Therefore, these data indicate that the pro-inflammatory action of PGE2 is exerted via the EP1 and EP4 receptors.



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  		FIG. 3.
PGE2 induces IL-8 protein synthesis via the EP1 and EP4 receptors. A, Jurkat T cells activated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies were treated with PGE2 (10 or 100 µM) or EP1–4 receptor agonists (10 µM). B, stimulated Jurkat T cells were treated with PGE2 (10 or 100 µM) in the absence or presence of EP1 (10 µM SC-19220) or EP1/2 (10 µM AH 6809) receptor antagonists for 24 h. After 24 of incubation with EP1–4 receptor agonists and antagonists, IL-8 protein was measured in cell supernatants by ELISA. Data are depicted as means ± S.E. of five independent experiments. C, shown is a representative immunoblot of the EP1 receptor in Jurkat T cells transfected with either control (C) or EP1 receptor (EP1R) siRNA (100 nM), activated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies, and treated with 100 µM PGE2 (upper) and IL-8 protein production in activated cells transfected with control or EP1 receptor siRNA and exposed to 10 and 100 µM PGE2 (lower). D, shown is a representative immunoblot of the EP4 receptor in Jurkat T cells transfected with either control or EP4 receptor (EP4R) siRNA (100 nM), activated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies, and treated with 100 µM PGE2 (upper) and IL-8 protein production in activated cells transfected with control or EP4 receptor siRNA and exposed to 10 and 100 Mµ PGE2 (lower). {beta}-Actin was used to control for lane loading. After 48 h of incubation with EP1 and EP4 receptor siRNAs, IL-8 protein was measured in cell supernatants by ELISA. Data are depicted as means ± S.E. of three independent experiments. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells; {circ}{circ}, p < 0.01 versus 10 µM PGE2-treated cells; ++, p < 0.01 versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 
Inhibition of PKC, Src Family Kinases, and Phosphatidylinositol 3-Kinase (PI3K), but Not Protein Kinase A (PKA), Blocks the PGE2-induced Synthesis of IL-8—The EP1 receptor induces changes in intracellular calcium levels that are believed to be mediated either by the phospholipase C signaling pathway, most likely via G{alpha}q, or via direct activation of the calcium channel (28). The signal transduction pathway activated by EP1 is represented mainly by PKC (30, 31) and Src family kinases (32). To further determine the signaling mechanisms of PGE2, cells were pretreated with the PKC inhibitors bisindolylmaleimide I, staurosporine, and H-89 or with the specific Src tyrosine kinase inhibitor PP2, and complete dose responses were evaluated (data not shown). At the concentrations reported in Fig. 4A, all compounds were able to completely suppress IL-8 synthesis induced by 10 or 100 µM PGE2.



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  		FIG. 4.
PGE2-induced IL-8 protein synthesis is mediated by the activation of PKC, Src family kinases, and PI3K. A, Jurkat T cells stimulated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies were treated with PGE2 (10 or 100 µM) in the absence or presence of the PKC inhibitors bisindolylmaleimideI(BIM; 10 µM), staurosporine (ST; 100 nM), and H-89 (5 µM) and the Src family kinase inhibitor PP2 (10 µM). B, stimulated T cells were treated with PGE2 (10 or 100 µM) in the absence or presence of the PI3K inhibitor LY 294002 (LY; 20 µM) and the PKA inhibitor H-89 (50 nM). After 24 h of incubation, IL-8 protein was measured in cell supernatants by ELISA. Data are depicted as means ± S.E. of five separate experiments. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells; {circ}{circ}, p < 0.01 versus 10 µM PGE2-treated cells; ++, p < 0.01 versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 
It has been shown that the EP4 receptor primarily utilizes a PI3K-dependent pathway and is less efficiently coupled to adenylyl cyclase and/or has additional pathways not involving cAMP/PKA signaling (33). Thus, we examined the role of these signaling pathways in the enhancement of IL-8 release by PGE2 using LY 294002, an inhibitor of PI3K, at a concentration of 20 µM, as resulted from a dose-response curve (data not shown), and H-89 in a nanomolar range, which inhibits PKA only (34). As shown in Fig. 4B, LY 294002 inhibited IL-8 production by 40%, whereas H-89 treatment did not interfere with the increase in IL-8 synthesis induced by the two concentrations of PGE2 employed. These results suggest that PKC, Src family kinases, and PI3K, but not PKA, participate in IL-8 synthesis.

PGE2 Induces PKC Activation in T Cells—To support the evidence of the involvement of PKC in PGE2 induction of IL-8 synthesis, a PKC activity assay was performed as described under "Materials and Methods." Fig. 5 shows that PGE2 (10 or 100 µM) induced a significant increase in basal PKC activity. The same effect was observed in anti-CD3/CD28 antibody-stimulated T cells (data not shown). Pretreatment with the PKC inhibitors completely suppressed the PGE2-induced PKC activity enhancement. To examine whether the modulation of PKC is mediated through the EP1 receptor, the cells were treated with EP1 agonist (PT-PGE2) or PGE2 and EP1 antagonist (SC-19220). PT-PGE2 caused a significant increase in PKC, whereas SC-19220 completely reverted the stimulatory effect of PGE2 on PKC activity. Similar results were obtained in cells transfected with PTGER1 siRNA and treated with PGE2 (data not shown).



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  		FIG. 5.
PGE2 induces the activation of PKC in Jurkat T cells. Jurkat T cells were incubated with PGE2 (10 or 100 µM) for 15 min in the absence or presence of the PKC inhibitors bisindolylmaleimide I (BIM; 10 µM), staurosporine (ST; 100 nM), and H-89 (5 µM) and the EP1 antagonist SC-19220 (10 µM) or treated with the EP1 agonist PT-PGE2 (10 µM). PKC activity in cell lysates was determined by PKC activity assay as described under "Materials and Methods." Data are depicted as means ± S.E. of five independent experiments. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells; {circ}{circ}, p < 0.01 versus 10 µM PGE2-treated cells, ++, p < 0.01 versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 
Induction of IL-8 Synthesis by PGE2 Is Not Mediated by NF-{kappa}B—IL-8 is an important NF-{kappa}B-regulated gene (35). To verify whether NF-{kappa}B activation is essential in the production of PGE2-induced IL-8 in activated human T cells, Jurkat T cells were treated with the NF-{kappa}B inhibitor caffeic acid phenethyl ester (1–20 µM) 1 h prior to stimulation with PGE2 (10 or 100 µM). None of the caffeic acid phenethyl ester concentrations employed was able to modify the effects of PGE2 on IL-8 production (data not shown), thus suggesting that the induction of IL-8 by PGE2 in human T cells is NF-{kappa}B-independent.

IL-8 Induction by PGE2 in Human T Cells Occurs via p38 MAPK—The mechanism of IL-8 induction has been reported to be mediated through the p38 MAPK pathway (36, 37). To verify that PGE2 enhances IL-8 synthesis from stimulated T cells via this MAPK pathway, the p38 MAPK inhibitor SB 203580 (38) was added to cultures 1 h before treatment with PGE2 (10 or 100 µM). Fig. 6 shows that this inhibitor significantly reduced PGE2-induced IL-8 production in T cells in a dose-dependent manner.



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  		FIG. 6.
IL-8 protein synthesis induced by PGE2 is mediated by the activation of p38 MAPK. Jurkat T cells stimulated with anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies were pretreated with the specific p38 MAPK inhibitor SB 203580 (SB) 1 h prior to incubation with PGE2 (10 or 100 µM) for 24 h. IL-8 protein was measured in cell supernatants by ELISA. Data are depicted as means ± S.E. of five independent experiments. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells; {circ} and {circ}{circ}, p < 0.05 and 0.01, respectively, versus 10 µM PGE2-treated cells; + and ++, p < 0.05 and 0.01, respectively, versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 
PGE2 Activates p38 MAPK in T Cells—As inhibition of p38 MAPK prevented PGE2-induced IL-8 synthesis, we investigated the effect of PGE2 on the phosphorylation of p38 MAPK. After treating cells with PGE2 for different times (15–480 min), total cell proteins were extracted and subjected to Western blotting with an antibody specific for the phosphorylated form of p38 MAPK. As shown in Fig. 7A, the phosphorylation of p38 MAPK occurred upon treatment of cells with either 10 or 100 µM PGE2 after 60 min and persisted for up to 360 min. The phosphorylation of p38 MAPK induced by PGE2 resulted in the activation of the kinase since ATF-2, a substrate of p38 MAPK, was also phosphorylated with the same pattern. The specific inhibitor SB 203580 was able to prevent the phosphorylation as well as the activation of p38 MAPK induced by PGE2 (Fig. 7B).



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  		FIG. 7.
PGE2 induces the activation of p38 MAPK in Jurkat T cells. A, Jurkat T cells were treated with PGE2 (10 or 100 µM) for the indicated time periods. Total cell lysates of T cells were loaded onto gels (60 µg of protein/lane) and subjected to SDS-PAGE and immunoblotting using polyclonal antibodies that recognize the phosphorylated forms of p38 MAPK and ATF-2. B, Jurkat T cells were pretreated for 1 h with the p38 MAPK inhibitor SB 203580 (SB; 20 µM) and subsequently incubated with PGE2 (10 or 100 µM). Total cell lysates were loaded onto gels (60 µg of protein/lane) and subjected to SDS-PAGE and immunoblotting using rabbit polyclonal antibodies specific for the phosphorylated forms of p38 MAPK and ATF-2. Shown are representative immunoblots of phospho-p38 and phospho-ATF-2. The densitometry values are depicted as means ± S.E. of five independent experiments. All densitometry values were normalized to the endogenous {beta}-actin protein. * and **, p < 0.05 and 0.01, respectively, versus control untreated cells; $$, p < 0.01 versus PGE2-treated cells (based on two-tailed Student's t test).

 
Activation of p38 MAPK by PGE2 Is Not Dependent on the PKC, Src Tyrosine Kinase, PI3K, and PKA Pathways—To investigate whether PKC, Src family kinases, PI3K, and PKA are involved in the phosphorylation of p38 MAPK induced by PGE2, Jurkat T cells were treated with the specific PKC inhibitor bisindolylmaleimide I, the specific Src tyrosine kinase inhibitor PP2, the PI3K/Akt inhibitor LY 294002, or the PKA inhibitor H-89 1 h prior to treatment with PGE2 and examined for the activated form of p38 MAPK by Western blot analysis. Treatment with bisindolylmaleimide I, PP2, LY 294002, or H-89 for 1 h did not affect the phosphorylation of p38 MAPK induced by PGE2 (Fig. 8). Similar results were obtained after treatment with 10 µM PGE2 (data not shown).



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  		FIG. 8.
Effects of PKC, Src family kinases, PI3K, p38 MAPK, or PKA inhibitors on p38 phosphorylation. Jurkat T cells were pretreated for 1 h with the PKC inhibitor bisindolylmaleimide I (BIM; 10 µM), the Src family kinase inhibitor PP2 (10 µM), the PI3K inhibitor LY 294002 (LY; 20 µM), or the PKA inhibitor H-89 (50 nM) and subsequently incubated with PGE2 (100 µM). Total cell lysates were loaded onto gels (60 µg of protein/lane) and subjected to SDS-PAGE and immunoblotting using rabbit polyclonal antibody specific for the phosphorylated form of p38 MAPK. Shown are representative immunoblots of phospho-p38. The densitometry values are expressed as means ± S.E. of PGE2-treated cells exposed to the inhibitors at different times in five different experiments. All densitometry values were normalized to the endogenous {beta}-actin protein. **, p < 0.01 versus untreated cells (based on two-tailed Student's t test).

 
PGE2-induced IL-8 Synthesis Is Regulated by the Transcription Factor CHOP—To determine whether PGE2 regulates IL-8 gene expression, experiments were performed by transfecting resting and activated Jurkat T cells with the wild-type IL-8 promoter, and after treatment with PGE2, luciferase activity in cell lysates was measured. Fig. 9 shows that PGE2 slightly activated the IL-8 promoter in resting T lymphocytes, whereas it was able to further increase anti-CD3/CD28 antibody-induced gene expression. The cotransfection of CHOP up-regulated the luciferase response with respect to anti-CD3/CD28 antibody-stimulated cells. Treatment of cotransfected stimulated cells with PGE2 further increased the luciferase activity, indicating that PGE2 induces transcription of the IL-8 gene by activating the transactivation of CHOP. A very high significance was obtained with either 10 or 100 µM PGE2.



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  		FIG. 9.
PGE2-induced IL-8 synthesis is regulated by the transcription factor CHOP. Jurkat T cells were transfected with the wild-type IL-8 promoter (pIL8-Luc reporter). After transfection, Jurkat T cells were treated with or without anti-CD3 (1 µg/ml) plus anti-CD28 (250 ng/ml) antibodies in the absence or presence of PGE2 (10 and 100 µM). Where indicated, Jurkat T cells were cotransfected with CHOP expression plasmid. Data are means ± S.E. of five independent experiments and are expressed as -fold luciferase activation. {beta}-Galactosidase levels were determined for transfection efficiency. **, p < 0.01 versus untreated cells; ++, p < 0.01 versus anti-CD3/CD28 antibody-stimulated cells; {circ}{circ}, p < 0.01 versus anti-CD3/CD28 antibody-stimulated and cotransfected cells (based on two-tailed Student's t test).

 
PGE2 Induces the Transactivation Potential of CHOP—Since we demonstrated that the induction of IL-8 synthesis by PGE2 occurs through p38 MAPK activation, we investigated whether PGE2 induces the potential transactivation of CHOP, the main substrate of p38 MAPK. Jurkat T cells were transiently cotransfected with the p38 MAPK-responsive Gal-CHOP chimeric transcription factor, which consists of the DNA-binding domain of yeast Gal4 and the transactivation domain of CHOP, together with a luciferase reporter plasmid (pFR-Luc) and a bacterial {beta}-galactosidase expression vector (pSV-nlsLacZ). Cells were then treated with 10 or 100 µM PGE2 at different time intervals. Control cells were treated with ethanol only. As shown in Fig. 10, the luciferase activity of Gal-CHOP exhibited a 3- or 4-fold increase over the base-line levels of ethanol-treated cells at 6 h after treatment with 10 or 100 µM PGE2, respectively. A less but also significant increase occurred at 4 h with either 10 or 100 µM PGE2. These effects required the CHOP transactivation domain since treatment with PGE2 failed to induce reporter expression in cells transfected with a plasmid encoding the Gal4 DNA-binding domain only (residues 1–147) and lacking the transactivation domain of CHOP (data not shown).



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  		FIG. 10.
Effects of PGE2 on CHOP activation in Jurkat T cells. Jurkat T cells were transfected with a Gal-TATA-luciferase reporter gene; expression vectors encoding Gal-CHOP or, where indicated, the Gal4 DNA-binding domain (GAL); and a bacterial {beta}-galactosidase expression plasmid. After transfection, the cell cultures were treated with or without PGE2 (10 or 100 µM) or ethanol at different time intervals. Data are means ± S.E. of five independent experiments and are expressed as -fold luciferase activation. {beta}-Galactosidase levels were determined for transfection efficiency. * and **, p < 0.05 and 0.01, respectively, versus ethanol-treated cells (based on two-tailed Student's t test).

 
PGE2 Activates the Transcriptional Activity of CHOP via the EP1 and EP4 Receptors—Since our data demonstrated an involvement of the EP1 and EP4 receptors in IL-8 production, we investigated whether these receptors are also involved in CHOP activation. The EP1 agonist PT-PGE2 and the EP4 agonist 11-deoxy-PGE1 significantly increased the transcriptional activity of CHOP at a level 2–2.5-fold higher than in ethanol-treated cells. In contrast, the EP2 agonist butaprost and the EP3 agonist sulprostone did not induce the activation of CHOP-mediated transcription (Fig. 11A). Cotransfection experiments with Gal-CHOP and PTGER1 and PTGER4 siRNAs confirmed the exclusive EP1 and EP4 receptor involvement in the PGE2-induced activation of CHOP (Fig. 11B).



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  		FIG. 11.
PGE2 induces CHOP activation in Jurkat T cells via the EP1 and EP4 receptors. A, Jurkat T cells were transfected with a Gal-TATA-luciferase reporter gene; expression vectors encoding Gal-CHOP or, where indicated, the Gal4 DNA-binding domain (GAL); and a bacterial {beta}-galactosidase expression plasmid. After transfection, Jurkat T cells were treated with the specific EP1 agonist PT-PGE2 (10 µM), the EP2 agonist butaprost (10 µM), the EP3 agonist sulprostone (10 µM), the EP4 agonist 11-deoxy-PGE1 (10 µM), or ethanol. B, 4 h after transfection, Jurkat T cells were cotransfected with or without specific EP1, EP4, or control (C) siRNAs (100 nM) for 24 h and then with 10 or 100 µM PGE2 for 6 h. Data are means ± S.E. of five independent experiments and are expressed as -fold luciferase activation. {beta}-Galactosidase levels were determined for transfection efficiency. **, p < 0.01 versus ethanol-treated cells; {circ}{circ}, p < 0.01 versus 10 µM PGE2-treated cells; ++, p < 0.01 versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 
PKC, Src Family Kinases, PI3K, and p38 MAPK, but Not PKA, Are Involved in the PGE2-induced Transcriptional Activation of CHOP—To investigate the kinase pathways involved in the PGE2-induced transcriptional activation of CHOP, transfection experiments were performed with inhibitors of PKC, Src tyrosine kinases, PI3K, p38 MAPK, and cAMP/PKA. Fig. 12 shows that the Gal-CHOP activity stimulated by 10 or 100 µM PGE2 was completely blocked by both staurosporine and bisindolylmaleimide I. A significant inhibition of CHOP activation was also caused by treatment with PP2, LY 294002, and SB 203580. H-89, at a nanomolar concentration inhibiting PKA (34), did not interfere with the transcriptional activation of CHOP induced by PGE2.



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  		FIG. 12.
PGE2-induced transcriptional activation of CHOP in Jurkat T cells is mediated by PKC, Src family kinases, PI3K, and p38 MAPK. Jurkat T cells were transfected with a Gal-TATA-luciferase reporter gene; expression vectors encoding Gal-CHOP or, where indicated, the Gal4 DNA-binding domain (GAL); and a bacterial {beta}-galactosidase expression plasmid. After transfection, Jurkat T cells were treated with or without PGE2 (10 or 100 µM) in the absence or presence of the PKC inhibitors bisindolylmaleimide I (BIM; 10 µM) and staurosporine (ST; 100 nM), the Src family kinase inhibitor PP2 (10 µM), the PI3K inhibitor LY 294002 (LY; 20 µM), the p38 MAPK inhibitor SB 203580 (SB; 20 µM), and the PKA inhibitor H-89 (50 nM). Data are means ± S.E. of five independent experiments and are expressed as -fold luciferase activation. {beta}-Galactosidase levels were determined for transfection efficiency. **, p < 0.01 versus ethanol-treated cells; {circ} and {circ}{circ}, p < 0.05 and 0.01, respectively; versus 10 µM PGE2-treated cells; + and ++, p < 0.05 and 0.01, respectively, versus 100 µM PGE2-treated cells (based on two-tailed Student's t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that PGE2 at physiological as well as pathological concentrations, usually reached in several neoplastic, phlogistic, and immunological processes (5, 6, 20, 39), induced IL-8 production in peripheral as well as Jurkat T cells. These results are of particular interest since the other PGs employed (PGF2{alpha} and PGI2) were ineffective. PGE2 is ubiquitously synthesized and reaches high levels in inflammatory processes, characterized by infiltrates of neutrophils, macrophages, lymphocytes, and plasma cells (40), following the induced expression of cyclooxygenase-2. Evidence has been reported that 15-deoxy-{Delta}12,14-PGJ2 (an active metabolite of PGD2) increases the synthesis of IL-8 in peripheral human T lymphocytes and several human T cell lines (26). In contrast, PGE2 was tested only on CCRF-CEM cells (acute human lymphoblastic leukemia) and was ineffective also at the concentrations we showed to be able to induce IL-8 synthesis by peripheral and Jurkat T cells. The results obtained by us therefore highlight a new important role for PGE2 in regulating IL-8 production by T lymphocytes, confirming the pro-inflammatory activity of this prostaglandin.

A variety of transcription factors such as NF-{kappa}B, NF-IL6, activator protein-1, and octamer-1 have been shown to regulate IL-8 gene transcription (41–44). To our knowledge, the data reported here describe for the first time that CHOP functions as a transcription factor that regulates IL-8 mRNA transcription. In fact, a regulatory role of CHOP has been shown only in IL-6 gene transcription in human melanoma cells (45, 46). Although inflammatory stimuli are known to activate IL-8 gene transcription through NF-{kappa}B, our study shows that PGE2 induces IL-8 synthesis through an NF-{kappa}B-independent pathway.

Using agonists and antagonists of different EP receptor subtypes and specific PTGER1 and PTGER4 siRNAs, we have shown that the EP1 and EP4 receptors, but not the EP2 and EP3 receptors, are involved in PGE2-induced IL-8 synthesis and CHOP activation in T cells. Under optimal conditions (100 nM siRNA, 24 or 48 h post-transfection), we achieved an average of 80% knockdown of the EP1 and EP4 receptors at the protein level. Recent data suggest that a complete knockdown is hard to be reached in Jurkat T cells (maximum of 70–80%) because the silencing is less effective in these cells than in others (47). We demonstrated that EP1 triggering by PGE2 leads to the activation of PKC, most likely via G{alpha}q (48, 49), which then increases IL-8 gene transcription. Although it was previously believed that the interaction of PGE2 with the EP4 receptor transiently increases intracellular cAMP and activates PKA, which then phosphorylates downstream effector proteins such as CREB (50). A recent report (34) indicates that EP4 binding induces mainly the activation of PI3K. Our results with the PKA and PI3K inhibitors clearly show that PKA is not involved in PGE2-induced CHOP activation and IL-8 synthesis and that PI3K participates in augmenting the transactivation activity of CHOP in the IL-8 promoter. Fiebich et al. (29) hypothesized that the activation of PKC by PGE2 may be mediated by a possible EP4-like receptor in astroglial cells. Our data rule out the involvement of an EP4-like receptor and lead to the hypothesis that PGE2 induces IL-8 synthesis in T lymphocytes through the canonic EP4 receptor and EP1-mediated PKC and Src kinase activation.

We have shown that PGE2 activates CHOP and IL-8 gene transcription not only through the activation of PKC and Src family tyrosine kinases triggered by EP1 and PI3K/Akt triggered by EP4, but also through the activation of p38 MAPK. Interestingly, p38 MAPK led to the activation of CHOP independently of the other kinases since the PKC inhibitors employed did not block the phosphorylation of p38 MAPK induced by PGE2, even though they were able to completely suppress CHOP activation and IL-8 production. In agreement with our results, the activation of p38 MAPK by PGE2 has been reported to be independent of the activation of PKC in the enhancement of IL-6 synthesis in astrocytes (29). Since, in other cell systems, the activation of PKC by stimuli other than PGE2 is able to activate p38 MAPK, it may be hypothesized that not only the cell type, but also the stimulus employed has a pivotal role in regulating the activity of PKC. In fact, in the human lung adenocarcinoma cell line H441, hepatocyte growth factor, but not phorbol 12-myristate 13-acetate, simultaneously activates PKC and p42/44 and p38 MAPKs through parallel and nonintersecting pathways (51). Recently, mechanisms linking G protein-coupled receptors to the MAPK cascade have been investigated in different cell types (51), but few data are available regarding the role of tyrosine kinases in activating the p38 MAPK pathway. In human embryonic kidney 293 cells and in neutrophils, the activation of p38 MAPK by G{alpha}q/11 is mediated by the Src family kinase-dependent pathway (52, 53). Our studies employing a protein tyrosine kinase inhibitor (PP2) showed that the PGE2-induced activation of p38 MAPK is independent of Src tyrosine kinases, which are instead involved in CHOP activation and IL-8 production. Therefore, similar to PKC, PGE2-activated Src tyrosine kinases may also exert a different role in the activation of p38 MAPK, which may depend on cell specificity. However, additional experiments are needed to clarify the signaling pathway that links the EP receptors and the phosphorylation and thus the activation of p38 MAPK. Nevertheless, the involvement of PKC and Src tyrosine kinases appears to be necessary in CHOP activation and IL-8 synthesis since their inhibition completely blocked the effects of PGE2. In contrast, the role of p38 MAPK and PI3K appears to be important but not essential in that the p38 MAPK inhibitor SB 203580 and the PI3K inhibitor LY 294002 were able to inhibit only partially the PGE2-induced transcriptional activity of CHOP and the synthesis of IL-8. Since none of the kinases activated by EP1 and EP4 binding to PGE2 induced the phosphorylation of p38 MAPK, it may be hypothesized that these two receptors are not involved in p38 MAPK activation. Therefore, we propose that, in stimulated human T cells, PGE2 induces IL-8 mRNA transcription by the activation of several signal transduction pathways, including the p38 MAPK, EPR1-triggered PKC and Src family tyrosine kinase, and EPR4-triggered PI3K pathways (Fig. 13).



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  		FIG. 13.
Schematic drawing of the possible signal transduction cascades in PGE2-induced IL-8 synthesis by T cells. Pro-inflammatory stimuli increase the levels of PGE2, which induces IL-8 transcription through Src family kinases. PKC is activated via the EP1 receptor, and PI3K is activated via the EP4 receptor and p38 MAPK. These pathways are independently involved in the phosphorylation and activation of CHOP, which transactivates the IL-8 promoter and induces IL-8 protein synthesis. IL-8 release induced by PGE2 is independent of the activation of PKA and the EP2 and EP3 receptor-dependent signaling mechanisms. TNF-alpha, tumor necrosis factor-{alpha}; LPS, lipopolysaccharide.

 
In conclusion, the findings described in this study show that PGE2 induces activated human T lymphocytes to produce the chemokine IL-8. This IL-8 production is through the PKC, Src, and p38 MAPK pathways and is NF-{kappa}B independent. Our results indicate that PGE2 plays a key role in enhancing and sustaining inflammation via stimulation of IL-8 synthesis by activated T cells. Therefore, PGE2 has profoundly different effects on the production of this chemokine that are dependent on the cell type and environment. The characterization of the pathways stimulated in T cells by cyclooxygenase-2-dependent PGE2 may have practical clinical impact in addition to pathophysiological significance.


   FOOTNOTES
 
* This work was supported by the Progetto di Ricerca di Ateneo, University of Messina (to D. T.), and by the School of Specialization in Clinical Pathology, University of Messina. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Experimental Pathology and Microbiology, Torre Biologica (IV p.), Policlinico Universitario, Via Consolare Valeria 1, Gazzi, 98125 Messina, Italy. Tel.: 39-90-221-3340; Fax: 39-90-221-3341; E-mail: dteti{at}unime.it.

1 The abbreviations used are: PG, prostaglandin; IL, interleukin; C/EBP, CAAT/enhancer-binding protein; CHOP, C/EBP homologous protein; MAPK, mitogen-activated protein kinase; PT-PGE2, 17-phenyltrinorprostaglandin E2; ELISA, enzyme-linked immunosorbent assay; PKC, protein kinase C; siRNA, small interfering RNA; ATF-2, activating transcription factor-2; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; CREB, cAMP-responsive element-binding protein; EPR, prostaglandin E2 receptor. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Giancarlo Vecchio (University Federico II, Naples, Italy) for critically reading the manuscript and Drs. Hidetoshi Hayashi and Hector R. Wong for providing expression plasmids.



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