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Originally published online as doi:10.1189/jlb.1203627 on April 1, 2004

Published online before print April 1, 2004
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(Journal of Leukocyte Biology. 2004;76:227-236.)
© 2004 by Society for Leukocyte Biology

Prostaglandin E2 receptors EP2 and EP4 are up-regulated in peritoneal macrophages and joints of pristane-treated mice and modulate TNF-{alpha} and IL-6 production

Jun Akaogi*, Hidehiro Yamada{dagger}, Yoshiki Kuroda*, Dina C. Nacionales*, Westley H. Reeves*,{ddagger} and Minoru Satoh*,{ddagger},1

* Division of Rheumatology and Clinical Immunology, Department of Medicine, and
{ddagger} Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville; and
{dagger} Department of Internal Medicine, St. Marianna University, Kawasaki, Japan

1Correspondence: Division of Rheumatology and Clinical Immunology, University of Florida, P.O. Box 100221, Gainesville, FL 32610-0221. E-mail: satohm{at}medicine.ufl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin E2 (PGE2) can have pro- or anti-inflammatory effects, depending on engagement of different PGE2 receptor (EP) subtypes. The role of EPs in regulating autoimmune inflammation was studied in the murine arthritis/lupus model induced by pristane. Peritoneal macrophages were isolated (biomagnetic beads) from BALB/c, DBA/1, or C57BL/6 mice treated with pristane (intraperitoneally, 3 months earlier) or thioglycolate (3 days earlier) or with untreated controls. EPs, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells were cultured unstimulated or stimulated with lipopolysaccharide (LPS) or LPS + interferon-{gamma} in combination with EP subtype-specific agonists. Tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-6 production was tested by enzyme-linked immunosorbent assay (culture supernatant) and flow cytometry. TNF-{alpha} mRNA levels also were examined. High levels of EPs (EP4/2>EP1>EP3), iNOS, and COX-2 mRNA were expressed in peritoneal macrophages from pristane-treated but not untreated or thioglycolate-treated mice (RT-PCR). TNF-{alpha} production was inhibited 50–70% at 2–24 h by EP4/2 agonists, whereas IL-6 was enhanced up to ~220%. TNF-{alpha} inhibition is mediated partly via the protein kinase A pathway and partly via IL-6. Intracellular TNF-{alpha} staining was inhibited 20% by EP4/2 agonists. TNF-{alpha} mRNA levels were inhibited 50–70% at 2–24 h, indicating that TNF-{alpha} inhibition was partly at the level of transcription. EP1/3 agonists had little effect. Synovial cells from mice with pristane-induced arthritis (DBA/1) also expressed EP2/4, and the EP2/4 agonist inhibited TNF-{alpha} production. PGE2 can modulate inflammatory reactions via the EP2/4 receptor through its regulation of TNF-{alpha} and IL-6. Modification of EP signaling may be a new therapeutic strategy in inflammatory/autoimmune diseases.

Key Words: EP receptors • agonists • cytokines • pristane • arthritis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin E2 (PGE2) is an essential biological mediator synthesized via the cyclooxygenase (COX) pathway in a variety of cells triggered by physiological or pathological stimuli [1 ]. Whether PGE2 should be considered a pro- or an anti-inflammatory lipid has been controversial [2 3 4 ]. However, recent studies have revealed the presence of four subtypes of PGE2 receptor (EP), designated EP1, EP2, EP3, and EP4, and suggested that the effects of PGE2 in inflammatory responses vary, depending on which EP subtype is stimulated [1 , 5 ]. These receptors are coupled with different G proteins, each mediating unique intracellular signaling pathways. EP1 is coupled with the Gq protein, which signals through the phospholipase C (PLC) pathway, increasing intracellular Ca2+ concentration. EP2 and EP4 are coupled with the Gs protein and activate adenylate cyclase, increasing cyclic adenosine monophosphate (cAMP) levels and signaling through the protein kinase A (PKA) pathway. EP3 has three different isoforms in mice ({alpha}, ß, and {gamma}), products of alternate splicing of the same gene, coupled to different signaling systems. EP3-{alpha} and -ß decrease cAMP by inhibiting adenylate cyclase and increase intracellular Ca2+ concentration via Gi-mediated PLC activation, whereas EP3-{gamma} signaling increases cAMP via the Gs protein [1 ]. The EPs are distributed throughout the body and play an essential role in a variety of pharmacological and biochemical processes.

Proinflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-6, IL-1ß, and granulocyte macrophage-colony stimulating factor (GM-CSF) as well as PGE2, COX-2, and inducible nitric oxide synthase (iNOS) all are produced in inflammatory sites and are considered important participants in the pathophysiology of rheumatoid arthritis (RA) and other inflammatory diseases [4 , 6 ]. In particular, TNF-{alpha} has been shown to play a central role in RA and is an important therapeutic target [6 ]. IL-6 is induced together with the proinflammatory cytokines TNF-{alpha} and IL-1ß in many stress conditions and plays an essential role in the induction of acute-phase reactants [7 ]. Therefore, IL-6 also has been considered as a proinflammatory cytokine in RA. Biological therapy targeting IL-6 using anti-IL-6 antibodies or a soluble IL-6 receptor has been investigated in RA and other inflammatory diseases [8 ]. However, it has been shown that IL-6 can act as an anti-inflammatory regulator in vitro [9 ] and in vivo by controlling the level of TNF-{alpha} [7 ].

In the present study, the role of EPs in regulation of TNF-{alpha} and IL-6 has been studied using inflammatory macrophages from mice with chronic arthritis and lupus induced by pristane [10 11 12 ], a model closely resembling the chronic destructive arthritis in human RA, with future application of EP biomodulating therapy of RA in mind. Among EP subtypes, EP2 and EP4 were up-regulated in peritoneal macrophages as well as in arthritic synovial tissue. The data indicate that PGE2 can modulate macrophage inflammatory reactions via EP2 or EP4 receptor through regulation of TNF-{alpha} at mRNA and protein level. EP4/EP2 agonists enhanced IL-6 production while inhibiting TNF-{alpha} production. Inhibition of TNF-{alpha} appeared to be mediated partially by direct EP4/EP2 signaling and in part via inhibitory effects on IL-6, as a PKA inhibitor and IL-6 neutralization had partial inhibitory effects. Modification of EP receptor signaling may be a new therapeutic strategy in inflammatory/autoimmune diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Four-week-old female C57BL/6, BALB/c, DBA/1 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in a virus-free animal facility in barrier cages. At 3 months of age, mice received a single intraperitoneal (i.p.) injection of pristane (0.5 ml, 99% pure, Sigma Chemical Co., St. Louis, MO) [11 , 12 ]. Another group of age-matched mice received an i.p. injection of 0.5 ml thioglycolate broth 3 days before harvesting cells. Age-matched, untreated mice were used as control.

EP agonists
EP-selective agonists, 17-phenyl trinor PGE2 (EP1/3), butaprost (EP2), sulprostone (EP3/1), and misoprostol (EP4/2/3), were purchased from Cayman Chemical Co. (Ann Arbor, MI). The properties of EP-selective agonists were described previously [1 ].

Preparation of cells
Peritoneal cells were harvested by lavaging the peritoneal cavity of 6-month-old mice (3 months after injection of pristane or untreated controls) with 5 ml phosphate-buffered saline (PBS) containing 1 U/ml heparin. Red blood cells were lysed with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 2 mM EDTA). Peritoneal CD11b (+) macrophages were positively selected using biomagnetic beads (Miltenyi Biotech, Auburn, CA) following the manufacturer’s instructions after removing CD11c (+) dendritic cells and CD19 (+) B cells. Cells were usually >95% positive for CD11b and negative for Ly-6C (Gr-1) by flow cytometry.

Cell culture
Peritoneal CD11b (+) macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 10 mM HEPES, glutamine, and penicillin/streptmycin (complete DMEM) [13 ] in 24-well culture dishes (5x105 cells /ml). Cells were cultured unstimulated or with lipopolysaccharide (LPS; Salmonella Minnesota, Sigma Chemical Co.; 100 ng/ml, 10 µg/ml) or the combination of LPS (100 ng/ml) and interferon-{gamma} (IFN-{gamma}; 5 ng/ml, BD Bioscience, East Meadow, CA). EP-selective agonists were added at 1 µM in ethanol. Controls received ethanol alone. Culture supernatants were harvested at 0, 2, 4, 8, 12, and 24 h and were stored at –80°C until analysis. RNA was extracted from cells in culture using TRIzol (Invitrogen, Carlsbad, CA). In other experiments, anti-IL-6-neutralizing antibodies (1 µg/ml, BD Bioscience) or the cell-permeable inhibitor of PKA, Rp-cAMP (adenosine 3',5'-cyclic phosphorothiolate-Rp, 20 µM, Calbiochem, San Diego, CA), were added to culture.

Preparation of synovial tissue
Synovial tissue was microsurgically harvested from arthritic knee joints of DBA/1 mice with pristane-induced arthritis 6 months after treatment. Tissue was crushed into a cell strainer (70 µm nylon, Falcon, Franklin Lakes, NJ), and then single-cell suspensions of cells were prepared lysing erythrocytes. Cells from four mice were pooled and cultured in 24-well cell-culture plates (106 cells/well/1 ml in complete DMEM) without stimulation or in the presence of LPS (100 ng/ml) and IFN-{gamma} (5 ng/ml, BD Bioscience). Samples were harvested after 24 h culture at 37°C with a 5% CO2 atmosphere and were frozen at –80°C until analysis.

Detection of mRNA for iNOS, COX-2, and EPs by reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from whole peritoneal cells, purified cell fractions (magnetic beads), and synovial tissue using TRIzol. RNA was precipitated with isopropanol, washed with 75% (v/v) ethanol, and treated with DNase I (Invitrogen) to remove genomic DNA. cDNA was synthesized with RT (Super Script II RT, Invitrogen), and 1 µl was added to the following PCR amplification mixture: 5 µl 10x PCR buffer (contains 15 mM MgCl2), 1 µl 10 mM deoxyribonucleotide triphosphate, 0.5 µl HotStar Taq polymerase (5 U/µl; Qiagen, Valencia, CA), and sense and antisense primers (0.2 µmol each) in a final volume of 50 µl. Amplification was performed for 15 min at 95°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min in a thermal cycler (GeneAmp 9600-R, Perkin-Elmer, Wellesley, MA). The primers were as described previously for EP receptors [5 ], iNOS [14 ], and COX-2 [15 ]: EP1 (sense 5'-TTA ACC TGA GCC TAG CGG ATG-3' and antisense 5'-CGC TGA GCG TAT TGC ACA CTA-3'); EP2 (sense 5'-GTG GCC CTG GCT CCC GAA AGT C-3' and antisense 5'-GGC AAG GAG CAT ATG GCG AAG GTG-3'); EP3 (sense 5'-TGA CCT TTG CCT GCA ACC TG-3', EP3-{alpha} antisense 5'-AGC TGG AAG CAT AGT TGG TG-3', EP3-ß antisense 5'-GAC CCA GGG AAA CAG GTA CT-3', EP3-{gamma} antisense 5'-AGA CAA TGA GAT GGC CTG CC-3'); EP4 (sense 5'-AGT AGC TAA AGG GGG AAT CTT-3' and antisense 5'-AAC ACT TTG GCC TGA ACT TGT-3'); COX-2 (sense 5'-ACT CAC TCA GTT TGT TGA GTC ATT C-3' and antisense 5'-TTT GAT TAG TAC TGT AGG GTT AAT G-3'); iNOS (sense 5'-ACA GGG AAG TCT GAA GCA CTA G-3' and antisense 5'-CAT GCA AGG AAG GGA ACT CTT C-3'). ß-Actin (sense 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3' and antisense 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3') was used as positive control, and no RT mRNA samples served as negative controls. The amplified products were run on 1.5–2% agarose gels and stained with ethidium bromide.

Quantification TNF-{alpha} mRNA
TNF-{alpha} mRNA in peritoneal macrophages from pristane-treated mice was examined using the Quantikine mRNA kit (R&D Systems, Minneapolis, MN) following the manufacturer’s instruction.

Cytokine and cAMP enzyme-linked immunosorbent assay (ELISA)
TNF-{alpha} and IL-6 in culture supernatants were measured by sandwich ELISA using antibody pairs and cytokine standards from BD Bioscience [16 ]. OD405 was converted to concentration based on standard curves produced by recombinant cytokines using a four-parameter logistic equation (Softmax Pro 4.3 software, Molecular Devices, Sunnyvale, CA) [16 ]. cAMP production in culture supernatant was measured using cAMP enzyme immunoassay kit (Cayman Chemical Co.) following the manufacturer’s protocol.

Intracellular TNF-{alpha} staining
Peritoneal cells (106/ml) were cultured unstimulated or stimulated with LPS (10 µg/ml) with GolgiStop (monensin) and GolgiPlug (brefeldin A, BD Bioscience) for 6 h. EP-selective agonists (1 µM in ethanol) or ethanol alone also were added. After staining for cell-surface fluorescein isothiocyanate (FITC) conjugated anti-CD11b, cells were fixed with 1% paraformaldehyde in PBS and permeabilized with 0.1% saponin in PBS, 0.5% bovine serum albumin, for 10 min. Intracellular TNF-{alpha} was then stained using allophycocyanin (APC)-conjugated rat anti-mouse TNF-{alpha} monoclonal antibodies (mAb; BD Bioscience). Samples were examined by flow cytometry (FACSCaliber, Becton Dickinson, San Jose, CA), and data were analyzed using Flow Jo 4.2 software (Tree Star, Stanford, CA). Staining was compared with appropriate isotype controls.

Statistical analysis
Frequencies were compared by Fisher’s exact test. Levels of cytokines were compared using Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EP, COX-2, and iNOS mRNA expression in peritoneal cells from untreated, pristane-treated, or thioglycolate-treated mice
Peritoneal cells were harvested from 6-month-old BALB/cJ mice, treated with pristane (3 months earlier) or thioglycolate (3 days earlier) or untreated. EP, COX-2, and iNOS mRNA expression by RT-PCR is shown (Fig. 1 ). Peritoneal cells from pristane-treated mice expressed high levels of EP2, EP4, COX-2, and iNOS and low levels of EP1 and EP3. In contrast, cells from untreated mice did not express these molecules. Cells from some thioglycolate-treated mice expressed low levels of EP2, EP4, and COX-2. Similar results were obtained in C57BL/6 and DBA/1 strains (not shown).



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  		Figure 1. EP, COX-2, and iNOS mRNA expression in peritoneal cells from untreated, pristane-treated, or thioglycolate-treated mice. Peritoneal cells were harvested from BALB/c mice treated with pristane (3 months earlier) or thioglycolate (3 days earlier) or untreated. EP, COX-2, and iNOS mRNA expression by RT-PCR is shown. Representative data of four mice/group from experiments using six to 18 mice/group are shown. TG, Thioglycolate

 
As immune responses can differ depending on the genetic background, mice that are prone to T helper cell type 1 (Th1; C57BL/6) or Th2 (BALB/cJ) responses [17 ] as well as mice susceptible to pristane-induced arthritis (DBA/1 and BALB/cJ; C57BL/6 is resistant) [10 ] were examined in this study. The frequencies of detectable EP, iNOS, and COX-2 mRNA by RT-PCR in peritoneal cells from three strains of mice, comparing pristane-treated versus untreated, are summarized (Table 1 ). Peritoneal cells from nearly all pristane-treated mice expressed all EPs, iNOS, and COX-2 (pristane vs. control C57BL/6, P<0.01; BALB/cJ and DBA/1, P<0.05, for all molecules; Fisher’s exact test). In contrast, expression of these molecules was absent among untreated mice except for EP2 and EP4, which were expressed in some B6 mice. These results suggest that regardless of the background of the mice, pristane treatment induces expression of EPs and proinflammatory enzymes iNOS and COX-2. The expression of these molecule in pristane-treated mice was much higher at 3 months after treatment than that at 6 months (not shown), suggesting that expression was the highest at the time of maximal cytokine production (IL-6, IL-12, TNF-{alpha}) [18 ] and in parallel, with the onset of autoantibody production and chronic destructive arthritis [11 , 12 ].


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  		Table 1. Frequency of EP Receptor Subtypes mRNA in Peritoneal Cells by RT-PCR

 
Some previous studies used thioglycolate-elicited or resident macrophages to examine the biological function of EPs [19 , 20 ]; however, little EPs are expressed in these cells (Fig. 1 and Table 1 ). RAW 264.7 and other macrophage-like tumor cell lines used in other studies [21 ] express high levels of EPs (see Fig. 2 ); however, their behavior may or may not reflect a physiological response of inflammatory macrophages. EPs are induced by inflammatory stimuli, and the macrophages at inflammatory site express high levels of EPs and iNOS, arginases, and other enzymes involved in inflammation. The role of EPs in regulation of inflammation should be better understood by examining inflammatory macrophages expressing EPs. Because of this reason, macrophages from pristane-induced arthritis/lupus model mice were used in the present study.



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  		Figure 2. EP, COX-2, and iNOS mRNA expression in peritoneal macrophages from pristane-treated versus untreated mice. Peritoneal cells were harvested from C57BL/6, BALB/c, or DBA/1 mice treated with pristane (3 months earlier) or untreated. EP, COX-2, and iNOS mRNA expression by RT-PCR in CD11b (+) peritoneal macrophages (biomagnetic beads) and the macrophage cell line RAW264.7 is shown. Representative data from four independent experiments are shown. (–), untreated; P, pristane-treated.

 
Expression of the EPs, COX-2, and iNOS in peritoneal macrophages
Macrophages play a central role in peritoneal inflammation in pristane-treated mice, producing IL-6, TNF-{alpha}, IL-12, and other cytokines [16 ] as well as PGE2 [22 ]. Therefore, EP expression of peritoneal CD11b (+) macrophages was examined (Fig. 2) . In all three strains of mice (B6, BALB/c, and DBA/1), peritoneal macrophages expressed high levels of EP2 and EP4 mRNAs and lower levels of EP1 and EP3, indicating that the EP expression pattern is similar in various strains. EP2 and EP4 were induced prominently in pristane-treated mice. COX-2 and iNOS mRNA was also detected in pristane-elicited peritoneal macrophages from all three strains of mice, in contrast to their absence in untreated mice. These results indicate that induction of EPs is associated with the expression of proinflammatory enzymes such as iNOS and COX-2.

Regulation of cytokine production via EPs
To determine the role of EPs in regulating inflammation, the effects of EP-selective agonists on TNF-{alpha} and IL-6 production were studied. CD11b (+) peritoneal macrophages from pristane-treated BALB/c mice were cultured with or without LPS or with the combination of LPS and IFN-{gamma} for 24 h (Fig. 3 ). EP-selective agonists or ethanol (control) were added at 1 µM. In preliminary experiments, the EP agonists and PGE2 all inhibited cytokine production in a dose-dependent manner between 10 µM and 0.1 nM (not shown), as reported earlier [23 ]. At a concentration of 0.01%, the diluent (ethanol) had no effect on cytokine production (not shown). The EP4/2/3 agonist, misoprostol, inhibited TNF-{alpha} production substantially in all three conditions (Fig. 3A) . In particular, misoprostol showed ~70% inhibition of TNF-{alpha} production by LPS + IFN-{gamma}-stimulated cells. The EP2 agonist, butaprost, also significantly inhibited TNF-{alpha} production by LPS (33%) or LPS + IFN-{gamma}-stimulated cells (64%). Inhibition by EP1/3 agonist, 17-phenyl-trinor PGE2, or EP3/1 agonist, sulprostone was much less dramatic than that seen with EP4/EP2 agonists (Fig. 3A) . The weak inhibition of TNF-{alpha} with 17-phenyl-trinor PGE2 and sulprostone may be explained by their interaction with EP3-{gamma}, which shares the Gs-PKA pathway with EP4/2. These results suggest that the binding of PGE2 to EP2 and EP4 may have significant, anti-inflammatory effects mediated by an inhibition of TNF-{alpha} production by macrophages. In contrast, butaprost and misoprostol dramatically enhanced IL-6 production (Fig. 3B) . IL-6 production was increased as much as ~120% by EP2 or EP4 agonists. The effect of misoprostol (EP4/2/3) was most clearly pronounced in LPS-stimulated cells, whereas butaprost also increased IL-6 production most clearly in LPS plus IFN-{gamma}-stimulated cells. In striking contrast, the EP1/3 agonist had little effect on IL-6 production in all three conditions. Similarly, the EP3/1 agonist had little effect in unstimulated or LPS-stimulated cells. The EP3/1 agonist sulprostone also moderately increased IL-6 production in LPS + IFN-{gamma}-stimulated cells. These results suggest that the effects of EP agonists are selective, as different EP agonists had distinctive effects on TNF-{alpha} and IL-6 production. Also, EP4 and EP2 and EP1 and EP3 agonists, respectively, appeared to have similar effects, probably reflecting the shared G proteins and signaling pathway.



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  		Figure 3. Effects of EP agonists on TNF-{alpha} and IL-6 production by peritoneal macrophages. CD11b (+) peritoneal macrophages (biomagnetic beads) from pristane-treated BALB/c mice were cultured (5x105/ml) unstimulated or stimulated with LPS (100 ng/ml) or with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml). EP subtype-selective agonists also were added (1 µM). TNF-{alpha} and IL-6 in culture supernatant were measured by ELISA 24 h later. Mean ± SEM of triplicates is shown. (A) Inhibition of macrophage TNF-{alpha} production by EP agonists. Effect of EP agonists on TNF-{alpha} in culture supernatant is shown as percent inhibition compared with levels in control (ethanol-treated cultures). Actual levels in ethanol-treated cultures: unstimulated (unTX), 138.5 ± 2.1 pg/ml; LPS, 1401.7 ± 134.3 pg/ml; LPS + IFN-{gamma}, 11,503.7 ± 1373.6 pg/ml. (B) Enhancement of macrophage IL-6 production by EP agonists. Effects of EP agonists are shown as percent enhancement of IL-6 production versus control. Levels in control: unstimulated (unTx), 11.0 ± 0.1; LPS, 426.9 ± 27.0; LPS + IFN-{gamma}, 700.9 ± 26.6 pg/ml; *, P < 0.05; **, P < 0.01; ***, P < 0.005, by Student’s t-test. Representative data from four independent experiments performed in triplicates are shown.

 
Time-course of effects of EP agonists on TNF-{alpha} and IL-6 production
Although EP4 and EP2 agonists appeared to have the most significant effects on TNF-{alpha} production, it is possible that some reagents have different effects at earlier time-points, or their effect is short-lived. Therefore, the time-course of TNF-{alpha} and IL-6 production by macrophages treated with EP agonists was examined (Fig. 4 ). Throughout a 24-h period, the effects of the four EP agonists on TNF-{alpha} were very consistent, suggesting that the onset of inhibition is rapid (<2 h) and long-lived (Fig. 4A) . Butaprost and misoprostol inhibited TNF-{alpha} production by 50–70% from 2–24 h. The effect of EP agonists on IL-6 was more variable (Fig. 4B) . Misoprostol induced enhanced IL-6 production rapidly and dramatically (up to ~220% of the control, peaking at 8 h). Although butaprost and misoprostol had similar effects on TNF-{alpha} (Fig. 4A) , the enhancement of IL-6 production by butaprost was delayed compared with that of misoprostol, becoming apparent only after 12 h. Thus, although the EP2 and EP4 signaling pathways are similar, the effects of butaprost and misoprostol on IL-6 induction differed. The rapid increase of IL-6 peaked at 8 h by EP4 agonist may reflect down-regulation of IL-6 receptor [24 ] and short-term agonist-induced desensitization reported in EP4 but not in EP2 [25 ]. These results were reproducible in several experiments using whole peritoneal cells or purified macrophages, as well as the RAW264.7 cells (data not shown), suggesting that the effects of EP agonists on TNF-{alpha} and IL-6 production are common effects with biological significance.



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  		Figure 4. Time-course of effects of EP agonists on TNF-{alpha} and IL-6 production by peritoneal macrophages, which from pristane-treated mice, were cultured with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml) and EP agonists (1 µM). TNF-{alpha} and IL-6 levels in culture supernatants were measured by ELISA (2, 4, 8, 12, and 24 h). (A) Time-course of inhibition of TNF-{alpha} production by EP agonists. The effect of EP agonists is shown as a percentage of inhibition of TNF-{alpha} production compared with control (ethanol). The data reflect production between time 0 and each time-point, as the culture medium was not replaced. TNF-{alpha} inhibition = (control TNF-{alpha}–TNF-{alpha} with EP agonist)/control TNF-{alpha}. Time 0: 26.5 ± 2.0, 2 h; 374.9 ± 7.2, 4 h; 972 ± 51.8, 8 h; 1372.7 ± 148.4, 12 h; 6101.0 ± 145.0, 24 h; 11,503 ± 1373.6 pg/ml. (B) Time-course of effects of EP agonists on enhancement of IL-6 production. Enhancement of IL-6 production in the presence of EP agonists compared with control (ethanol) is shown (control=0%). IL-6 enhancement = (IL-6 levels with EP agonist–control IL-6)/control IL-6. Time 0: <1, 2 h; 2.7 ± 2.2, 4 h; 80.9 ± 4.0, 8 h; 120.4 ± 3.2, 12 h; 188.6 ± 8.8, 24 h; 700.9 ± 26.6 pg/ml. Representative data from three independent experiments performed in triplicates are shown.

 
Effects of anti-IL-6-neutralizing antibodies or PKA inhibitor on TNF-{alpha} inhibition and IL-6 enhancement by EP agonists
EP agonists induced IL-6, which can inhibit TNF-{alpha} production under certain conditions, and TNF-{alpha} production was inhibited. IL-10 was not produced in any experiments. Whether the inhibition of TNF-{alpha} was a result of the inhibitory effect of IL-6 or to a direct effect of EP signaling was studied by adding IL-6-neutralizing antibodies (Fig. 5A ) or the PKA inhibitor Rp-cAMP (Fig. 5B) to this system. Addition of anti-IL-6-neutralizing antibodies enhanced TNF-{alpha} production by ~30%, consistent with the inhibitory effects of IL-6 on TNF-{alpha} production (Fig. 5A) . TNF-{alpha} production was inhibited ~50% by butaprost (EP2, Fig. 5A , left) or misoprostol (EP4/2/3, Fig. 5A , right), consistent with other experiments. However, the inhibition of TNF-{alpha} by EP2/4 agonists was reversed ~50% less by adding anti-IL-6-neutralizing antibodies (Fig 5A) , indicating that the effects of EP2/4 agonists were partially via production of IL-6. Isotype-control antibodies had no significant effect (not shown). To further confirm that the TNF-{alpha} inhibition is mediated by direct signaling via EP2/EP4 and cAMP/PKA-dependent mechanisms, the PKA inhibitor (Rp-cAMP) was added to this system (Fig. 5B) . Addition of Rp-cAMP enhanced TNF-{alpha} production consistent with the role of the PKA pathway in regulation of TNF-{alpha}. The TNF-{alpha} inhibition by EP2 or EP4 agonist was inhibited ~50% by addition of Rp-cAMP, suggesting that direct regulation via EP2/4 was involved. Thus, the TNF-{alpha} inhibition appears to be mediated partially by the increased IL-6 production, resulting from signaling through EP receptors and partially by direct regulation through EPs. In contrast, Rp-cAMP had little effect on IL-6 induction by EP2/4 agonists (Fig. 5C) , suggesting that the PKA pathway does not mediate IL-6 induction.



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  		Figure 5. Effects of IL-6-neutralizing antibodies or PKA inhibitor (Rp-cAMP) on TNF-{alpha} inhibition and IL-6 induction by the EP agonist. Peritoneal macrophages from pristane-treated mice were cultured for 24 h with LPS + IFN-{gamma}. (A) Effect of IL-6-neutralizing antibodies. Percent production (LPS+IFN-{gamma} stimulation, 100%, 3334.1±112.0 pg/ml) of TNF-{alpha} by cells treated with EP agonist, IL-6-neutralizing antibodies (clone MP5-20F3, 1 µg/ml, anti-IL-6), or with EP agonist + IL-6-neutralizing antibodies. (B) Effect of Rp-cAMP (PKA inhibitor). Percent TNF-{alpha} production (LPS+IFN-{gamma} stimulation 100%, 3334.1±112.0 pg/ml) by cells treated with EP agonist Rp-cAMP (20 µM) or EP agonist + Rp-cAMP, compared with LPS + IFN-{gamma} alone. (C) Effect of Rp-cAMP on IL-6 induction by EP agonists. Percent IL-6 production (LPS+IFN-{gamma} stimulation=100%, 325.6±3.5 pg/ml) by cells treated with EP agonist, Rp-cAMP (20 µM), or EP agonist + Rp-cAMP (20 µM). Representative data from three independent experiments. Mean + SEM of triplicates is shown.

 
Effects of EP agonists on expression of TNF-{alpha} mRNA
Although post-translational regulation plays a critical role in TNF-{alpha} release [6 ], signaling through EPs also could affect transcription of TNF-{alpha}. Therefore, the effect of EP agonists on TNF-{alpha} mRNA was examined (Fig. 6A ). TNF-{alpha} mRNA rapidly increased upon LPS + IFN-{gamma} stimulation, reaching a maximum after 12 h. EP2/EP4 agonists reduced TNF-{alpha} mRNA as early as 2 h and by up to 50–70% at later time-points (Fig. 6A) . These data clearly indicate that inhibition of TNF-{alpha} by EP agonists also has affects at the mRNA level. In parallel to the inhibition of TNF-{alpha} mRNA, levels of cAMP were increased after 2–24 h in culture with EP agonists, particularly with EP2/EP4 agonists (butaprost, misoprostol). Representative data after 2 h are shown (Fig. 6B) . These results support the involvement of the cAMP/PKA pathway in TNF-{alpha} inhibition and are consistent with the effects of Rp-cAMP shown in Figure 5 .



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  		Figure 6. EP2 or EP4 agonists also inhibit TNF-{alpha} at mRNA levels. (A) TNF-{alpha} mRNA levels. Peritoneal macrophages from pristane-treated mice were cultured with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml) and EP agonists (1 µM). Time-course (2, 4, 8, 12, and 24 h) of TNF-{alpha} mRNA was examined by the Quantikine mRNA kit (R&D Systems). (B) Effects of EP agonists on cAMP levels, which in culture supernatant 2 h after culture with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml) and EP agonists (1 µM), were measured by ELISA. Effects of EP agonists on cAMP levels are shown as percent changes compared with the level in control (ethanol alone, cAMP level, 4.82±0.34 pmol/ml). Representative data from three independent experiments performed in triplicates are shown.

 
Intracellular TNF-{alpha} staining
The effect of EP agonists on TNF-{alpha} protein levels was examined by intracellular staining of peritoneal macrophages forTNF-{alpha} (Fig. 7 ). TNF-{alpha}-producing cells increased from 2.8% (unstimulated) to 72% after LPS stimulation. EP2/EP4 agonists significantly decreased the number of TNF-{alpha}-positive cells (53% by misoprostol and 55% by butaprost) and shifted the distribution of TNF-{alpha} staining toward left. These results indicate that the EP agonists also influence the levels of TNF-{alpha} protein (increased translation or decreased turnover).



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  		Figure 7. Intracellular TNF-{alpha} staining by flow cytometry. Peritoneal macrophages from pristane-treated mice were cultured with LPS (10 µg/ml) and EP agonists for 6 h. TNF-{alpha} production was evaluated by intracellular staining (flow cytometry). The number of TNF-{alpha}-positive cells (rat mAb clone MP6-XT22) increased from 2.8 to 72% following LPS stimulation (unstimulated vs. LPS control panels). Representative data from five independent experiments are shown.

 
EP, COX-2, and iNOS mRNA expression in arthritic synovial tissue from pristane-treated mice
EP, COX-2, and iNOS mRNA expression by RT-PCR in synovial tissue from arthritic knee joints of DBA/1 mice, 6 months after pristane treatment, is shown (Fig. 8 ). Synovial tissue from pristane-induced arthritis expressed EP2, EP4, and iNOS in a pattern similar to the expression in peritoneal macrophages (Figs. 1 and 2) . COX-2 expression was very weak.



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  		Figure 8. Eps, COX-2, and iNOS mRNA expression in arthritic synovial tissue from pristane-treated mice. Synovial tissue was harvested from arthritic knee joints from DBA/1 mice, 6 months after pristane treatment. EP, COX-2, and iNOS mRNA expression by RT-PCR is shown. Data were reproducible in three independent experiments.

 
Effects of EP agonists on TNF-{alpha} and IL-6 production by synovial cells from mice with pristane-induced arthritis
Synovial cells from knee joints of DBA/1 mice, 6 months after pristane treatment, were cultured with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml) stimulation with EP subtype-selective agonists butaprost, misoprostol, or ethanol alone (control; Fig. 9 ). Cells from synovial tissues produced high levels of TNF-{alpha} and IL-6. As in peritoneal macrophages, EP2 agonist butaprost and EP4/2/3 agonist misoprostol inhibited TNF-{alpha} production while enhancing IL-6 production. These results indicate that synovial inflammatory tissue has expression of EP2, EP4, and proinflammatory enzyme in a pattern identical to peritoneal inflammatory macrophages from pristane-treated mice and that their TNF-{alpha} and IL-6 production can be modified via EP2/4 signaling similar to inflammatory macrophages in peritoneal cavity from these mice.



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  		Figure 9. Effects of EP agonists on TNF-{alpha} and IL-6 production by synovial tissue from mice with pristane-induced arthritis. Synovial tissue was harvested from arthritic knee joints from DBA/1 mice 6 months after pristane treatment. Cells were cultured (5x105/ml) with LPS (100 ng/ml) + IFN-{gamma} (5 ng/ml) stimulation. EP subtype-selective agonists also were added (1 µM). TNF-{alpha} and IL-6 in culture supernatant were measured by ELISA 24 h later. Mean ± SEM of triplicates is shown. Ethanol (control). Essentially the same data were obtained from two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PGE2, synthesized through the COX pathway and triggered by proinflammatory stimuli such as cytokines, is a key mediator of inflammation [1 , 26 ]. However, whether PGE2 is a pro- or anti-inflammatory lipid is controversial. Some studies showed that neutralization of PGE2 suppresses inflammation [3 ], whereas others reported that PGE2 inhibits the proinflammatory cytokine TNF-{alpha} [2 ]. Four subtypes of PGE2 receptors have been identified (EP1, -2, -3, and -4), each mediating unique signals through coupled G proteins [1 , 26 ]. Recent studies using EP subtype knockout mice, selective agonists, or antagonists have shown that the effects of PGE2 on inflammation vary depending on which EPs are involved [5 , 27 28 29 ]. Most studies suggest that EP4 and EP2 are anti-inflammatory in vitro [23 , 25 ] and in vivo [28 ], although EP4 may be proinflammatory in collagen-induced arthritis (CIA) [27 ] and certain allergic diseases [29 ]. The inhibition of TNF-{alpha} production by misoprostol or butaprost shown here and in other studies [5 , 25 ] clearly indicates that PGE2 cannot be simply classified as a proinflammatory lipid.

It appears that there are several factors affecting whether PGE2 is pro- or anti-inflammatory. First, the expression of EPs on target cells is critical, as stimulation through different EP signaling has distinctive effects on inflammatory responses. Second, the role of cytokines in the pathogenesis of the particular type of inflammation is critical. The inflammatory process is heterogeneous, and the same cytokine can have opposite effects depending on the type of inflammatory response. For example, IL-6 also can be pro- or anti-inflammatory (see below) [7 , 9 ]. In addition, the stage of inflammation when PGE2 interacts with EPs may be very important. A number of mediators once considered to be purely proinflammatory, such as IFN-{gamma}, iNOS [30 ], COX-2 [31 ], and nuclear factor-{kappa}B [32 ], can be pro- or anti-inflammatory depending on stage of inflammation. It is likely that PGE2–EP interactions at different stages of inflammation can have different outcomes on the expression of these molecules.

In the peritoneum of pristane-treated mice, EPs (especially EP2 and EP4), COX-2, and iNOS are produced (Figs. 1 and 2) along with TNF-{alpha}, IL-6, IL-12 [13 , 16 ], and PGE2 [22 ]. EP4/2 agonists specifically inhibited TNF-{alpha} production in culture, consistent with observations in other systems [23 , 33 ]. Experiments using synovial cells from arthritic joints showed the same results. Furthermore, the present data clearly showed that the EP agonists affect the level of TNF-{alpha} mRNA (Fig. 6) and TNF-{alpha} protein (Fig. 7) as well as secretion into culture supernatant (Figs. 3A and 4A) . EP4/2 agonists also enhanced IL-6 production, as reported previously [34 , 35 ]. In contrast to the clear reversal of EP4/2-mediated TNF-{alpha} inhibition, the PKA inhibitor Rp-cAMP did not affect IL-6 induction (Fig. 5C) , suggesting that different signaling pathways mediated TNF-{alpha} inhibition and IL-6 induction [35 ]. EP4/EP2-mediated IL-6 induction was PKA-dependent in an early human T cell line [34 ], whereas PKC and p38 mitogen-activated protein kinase (MAPK) dependence was reported in human astroglioma cells [35 ]. EPs may regulate IL-6 differentially depending on the mode of stimulation, species, cell types, and other factors. The different effects of LPS versus LPS + IFN-{gamma} (Fig. 2) may reflect EP2 and EP4 induction by LPS versus EP4 induction and EP2 inhibition by LPS + IFN-{gamma} [36 ]. In addition to the PKA pathway shared by EP2 and EP4 signaling [1 ], EP4 signaling may stimulate the phosphatidylinositol-3 kinase pathway [37 ], PKA-independent p38 MAPK, or the PKC pathway [35 ].

Although the enhancement of IL-6 production following misoprostol treatment (Fig. 3B) seems paradoxical in view of the proposed anti-inflammatory effect of EP4/2, IL-6 can mediate not only proinflammatory but also anti-inflammatory signals [7 , 9 ]. The regulation of IL-6 and TNF-{alpha} is complex. TNF-{alpha} is a potent inducer of IL-6, whereas IL-6 can down-regulate TNF-{alpha} expression in vitro and in vivo [9 , 38 ]. The induction of EP2/EP4 by IL-6 [39 ] also may contribute to inhibition of TNF-{alpha} by EP4/EP2 signaling. In the present study, the PKA inhibitor Rp-cAMP and IL-6-neutralizing antibodies partially reversed the inhibition of TNF-{alpha} production by the EP4/2/3 or EP2 agonist (Fig. 5) . Thus, TNF-{alpha} inhibition via EP2/EP4 is mediated partially by direct signaling through EP2/EP4 and partly via IL-6 receptor signaling as a result of enhanced IL-6 production. Pristane activates macrophages by unknown mechanisms, inducing TNF-{alpha}, IL-6, PGE2, iNOS, COX-2, and EP expression. Signaling through EP2/EP4 in inflammatory macrophages may be a key part of the homeostatic mechanism limiting the inflammatory response to pristane.

RA is a systemic autoimmune disease characterized by chronic destructive synovitis. Locally produced proinflammatory cytokines such as TNF-{alpha}, IL-1ß, GM-CSF, and IL-6, as well as PGE2, are important participants in the pathophysiology of the disease. In particular, TNF-{alpha} is believed to play a central role in the pathogenesis and has become a major therapeutic target in RA [6 ]. However, although some patients respond dramatically to anti-TNF-{alpha} therapy [6 ], others benefit from anti-IL-6 therapy [8 ]. Heterogeneity also is apparent in animal models of chronic arthritis. Mice with pristane-induced arthritis produce a large amount of TNF-{alpha}, IL-6, and IL-12 [16 ], and anti-TNF-{alpha} therapy is effective [40 ]. Although there are some conflicting reports, EP2 and EP4 appear to be the subtypes predominantly expressed in synovial cells in human RA [41 ] as well as in animal models [42 ]. Our data in the pristane-induced arthritis model are consistent with other studies highlighting the anti-inflammatory role of EP2 and EP4 signaling in chronic arthritis [42 ] but seem at odds with the proinflammatory role of EP4 in the CIA model induced by a cocktail of anticollagen type II mAb [27 ]. CIA can be inhibited by IL-6 gene deletion [43 ] or anti-IL-6 receptor therapy [44 ]. Elevated levels of IL-6 have been emphasized in several studies, suggesting that IL-6 is a key player in CIA. As EP4/2 stimulation inhibits TNF-{alpha} but enhances IL-6 production, EP4/2 agonists may be beneficial for TNF-{alpha}-mediated inflammatory disease, not for IL-6-dependent disease such as CIA [27 ]. The ineffectiveness of misoprostol in CIA [33 ] is consistent with this interpretation.

Although several studies have emphasized the anti-inflammatory role of EP4 signaling based on decreased TNF-{alpha} production, a recent study showed that EP4 stimulation can mediate allergic diseases [29 ]. The development of antinuclear antibodies and lupus-like autoimmunity in human patients treated with anti-TNF-{alpha} therapy also has been well described [6 ]. These observations suggest that altering the cytokine balance may have unanticipated effects, including the induction of autoimmune or allergic diseases. This is a potential concern for the therapeutic use of EP agonists as well.

Beneficial effects of PGE2 in chronic arthritis have been described [45 ], and anti-PGE2-neutralizing antibodies [3 ] or COX inhibitors [46 ] paradoxically inhibit inflammation. With the recent progress in characterizing distinct biological functions of EP subtypes in inflammatory regulation, we have started to understand the complex role of PGE2 and its receptors in regulation of chronic inflammation. Although the evidence for EP4/EP2-mediated TNF-{alpha} suppression based on the reagents currently available is quite solid, some reagents are not monospecific. More specific EP agonists and antagonists, currently under development [28 ], would help to further define the mechanisms in the future. Another limitation of in vitro study using agonists is the potential difference of in vitro and in vivo effects as a result of metabolism of agonists and interaction of different type of cells. Heterogeneity of the inflammatory process, depending on the model or disease, stage or timing, or cells involved, as discussed above, may also make the interpretation more complicated.

The present data support the hypothesis that the release of PGE2 and induction of EPs in inflammatory sites represent a negative-feedback mechanism that limits the inflammatory response. In addition to the anti-inflammatory effects, EP4 activation may have a beneficial effect on bone mineralization, which may enhance its attractiveness as a potential therapy for RA [47 ]. With the availability of increasingly selective EP agonists, there is reason for cautious optimism that these agents may prove valuable additions to the therapeutic armamentarium for RA and other inflammatory diseases.


   ACKNOWLEDGEMENTS
 
NIH Grants R21-AR 056001, R01-AR44731, and AI44074 supported this work. This material is the result of work supported with resources and the use of facilities at the Malcom Randall VA Medical Center (Gainesville, FL). We thank Ms. Minna Honkanen-Scott for technical assistance.

Received December 10, 2003; revised February 26, 2004; accepted March 1, 2004.


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 DISCUSSION
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