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Originally published online as doi:10.1189/jlb.1002483 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;73:756-763.)
© 2003 by Society for Leukocyte Biology

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes

Hedi Harizi, Christophe Grosset and Norbert Gualde

Laboratoire d’Immunologie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5540, Université de Bordeaux 2, Cedex, France

Correspondence: Dr. Harizi, Laboratoire d’Immunologie, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5540, Université de Bordeaux 2, 33076 Bordeaux Cedex, France. E-mail: harizi33{at}yahoo.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have reported previously that PGE2 inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose-dependent manner, lipopolysaccharide (LPS) enhanced EP2R/EP4R but not EP1R/EP3R expressions. NS-398, a cyclooxygenase (COX)-2-selective inhibitor, suppressed LPS-enhanced EP2R/EP4R expression, suggesting that COX-2-issued prostaglandin E2 (PGE2) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP2R agonist, and PGE1 alcohol, an EP2R and EP2R/EP4R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin-10 production from DC. However, no effect was observed with sulprostone and 17-phenyl-{omega}-trinor-PGE2, selective agonists for EP1R and EP1R/EP3R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE2-induced, inhibitory effects. Taken together, our data demonstrate that EP2R/EP4R are efficient for mediating PGE2-induced modulation of DC functions.

Key Words: PGE2 • EP receptors • agonists • cytokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) and play a central role in immune-response development by their ability to present antigens and secrete bioactive molecules [1 ]. For many years, macrophages have been the focus of studies on the generation of arachidonic acid (AA)-derived mediators, and these cells were considered as the principal source of inflammatory mediators [2 , 3 ]. Considerable amounts of data have been accumulated implicating the ability of DC to produce AA metabolites, which modulate cytokine production and immune responses. Recently, we have reported that the in vitro-generated DC are an important source of AA products, in particular prostaglandin E2 (PGE2) [4 , 5 ].

Cyclooxygenase (COX) enzymes tightly controlled the biosynthesis of PGE2 in response to physiological and nonphysiological stimuli [6 , 7 ]. There are two isoforms of the COX enzyme: COX-1 and COX-2, produced from differentially regulated genes. COX-1 is constitutively expressed in most tissues [8 ], and COX-2 is undetectable in normal tissues or resting-immune cells, but its expression can be modulated by several stimuli, such as lipopolysaccharides (LPS) [9 , 10 ]. The effects of PGE2 are exerted by specific receptors on the plasma membrane of target cells [11 , 12 ]. Based on pharmacological and cDNA cloning studies, four subtypes of PGE receptors, designated E prostanoid receptors (EPRs), EP1–4R, have been identified and have been shown to differ in their signal transduction pathways [13 14 15 ]. The EP1R activates phospholipase C and phosphatidylinositol turnover and stimulates the release of intracellular calcium via a poorly characterized G protein-mediated mechanism [16 ]. The EP2R and EP4R are coupled to Gs and signal by stimulating adenylate cyclase, which increases the intracellular levels of cyclic adenosine monophosphate (cAMP) [17 , 18 ]. Signaling by the EP3R is more complex because of multiple EP3R isoforms generated by alternative splicing from a single EP3R gene. These EP3R isoforms are coupled to different signaling pathways including Gi, Gs, and calcium. The existence of this complex family of EPRs coupled to distinct intracellular signals provide a molecular basis for the diverse physiological and sometimes opposing actions of PGE2.

A previous study has reported that the major cellular constituents of the immune system express EPR isoforms [19 ]. Recently, it has been reported that PGE2 inhibits T cell responses via EP2R and macrophage functions via EP2/EP4Rs [20 ]. DC are not refractory to the effects of PGE2. In fact, we have demonstrated that exogenously added or endogenously released PGE2 acts on the DC themselves, by the induction of endogenous interleukin (IL)-10, which suppresses IL-12 production and alters antigen presentation by inhibiting major histocompatibility complex (MHC) class II protein expression [21 ]. Other investigators have reported that some cAMP-elevating agents, such as PGE2, inhibited DC function via an IL-10-dependent mechanism [22 ]. Although the inhibitory actions of PGE2 were clearly established on DC, and many studies examining EPR expression in macrophages, B and T lymphocytes, have been performed, nothing is known about the precise EPR subtypes involved in the inhibitory effects of PGE2 on DC. Thus, in the present report, we designed experiments to investigate the profile of EPR expression and to identify the role for each EPR in DC function. We examined the effects of EPR-selective agonists and dibutyryl cAMP (dbcAMP) on cytokine production, MHC class II molecule expression, and APC function of DC.

Western blot analyses demonstrate that resting DC express all EPRs. Flow cytometry analyses show that EPRs are expressed at the DC surface. In a dose-dependent manner, LPS up-regulates EP2R and EP4R expressions without any effect on EP1R and EP3R levels. The enhancement of EP2R/EP4R expression seems to be closely connected with COX-2 induction by LPS, as the COX-2-selective inhibitor NS-398 prevented the up-regulation of EP2R and EP4R expression in response to LPS. Treatment of cells with dbcAMP, an analog of cAMP, mimics PGE2-induced inhibition of DC functions, indicating that PGE2 acts on DC via Gs-coupled EP2R and/or EP4R subtypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media, reagents, and cell cultures
Complete medium (CM) was RPMI 1640 (Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Dominique Dutscher, Brumath, France), 1% streptomycin (Life Technologies; 1000 µg/ml), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), 50 µM 2ß-mercaptoethanol (Sigma-Aldrich), and 2 mM sodium pyruvate (Life Technologies).

DC were prepared from mice bone marrow cells, as we have previously reported [4 ]. Briefly, bone marrow cells isolated from BALB/c mice (Iffa Credo, Lyon, France) were cultured in CM in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF; 20 ng/ml) and IL-4 (10 ng/ml; PeproTech, Rocky Hill, NJ) for 6 days. DC were positively purified (routinely >98% CD11c+) using anti-CD11c (N418) MicroBeads and a magnetic cell separation system column (Miltenyi Biotec, Bergisch Gladbach, Germany).

CD4+ T lymphocytes prepared from a C57Bl/6 mouse and used as responder cells in an allogeneic mixed lymphocyte reaction (MLR) were obtained as previously reported [23 ]. Briefly, female C57Bl/6 8-week-old mice were killed by cervical dislocation, and spleen cells were isolated, separated by flotation on Ficoll (Sigma-Aldrich), and were allowed to stick on plastic Petri dishes (Costar, Dominique Dutscher SA) for 4 h in CM at 37°C in a regular incubator. At the end of the incubation period, nonadherent cells were gently removed with a pastor pipette, washed twice, and kept in CM; adherent cells were discarded. Nonadherent mononuclear cells obtained contained less than 0.5% CD14+ cells and more than 80% CD4+ T lymphocytes.

Preparation of EPR agonists
Sulprostone (Sul), a selective agonist for EP1R; Butaprost (But), a selective agonist for EP2R; 17-phenyl-{omega}-trinor PGE2 (17-Ph), a selective agonist for EP1R and EP3R; and PGE1 alcohol (PGE1 alc), a selective agonist for EP2R and EP4R, were purchased from Cayman Chemical (Ann Arbor, MI). Desiccated agonists were reconstituted in absolute ethanol (Merck, Darmstadt, Germany) and stored at –20°C, according to the manufacturer’s instructions. The required dilutions were prepared immediately before use, and equivalent quantities of ethanol were added to cultures to serve as controls for these synthetic molecules.

Fluorescein-activated cell sorter (FACS) analysis
Purified DC were treated with 1 µM PGE2 or various agonists for 18 h. The designated treatments had no effect on the cell viability quantified by the trypan blue test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide essay. At the end of the incubation period, cells were harvested, washed with phosphate-buffered saline (PBS; BioWhittaker, Verviers, Belgium), and subjected to analysis by FACS (Becton Dickinson FACSort flow cytometer, Pont de claix, France) using the following murine mouse antibodies (Ab): anti-CD11c-phycoerythrin (PE) and anti-I-Ad-PE (PharMingen Europe, Becton Dickinson). Data were collected on 1 x 104 cells. The primary Ab were directed toward a panel of cell-surface markers and compared with the appropriate isotype-matched control: hamster immunoglobulin G (IgG)1{lambda} PE and mouse IgG2bk-PE, respectively (PharMingen). Flow cytometry analysis was also used to monitor the level of DC surface expression of EPRs before and after LPS stimulation. For that purpose, cell were stained with Ab against EPRs for 30 min, washed with PBS, and incubated with anti-rabbit IgG–PE (PharMingen). Results were compared with specific isotypes. The median fluorescence intensity (MFI) of isotype-matched controls was subtracted to get the values reported in the figures.

Preparation of DC cytoplasmic extracts and Western blot analyses
Cytoplasmic lysates were prepared, as we have described previously [21 ]. Briefly, following the designated treatments, the in vitro-generated DC, obtained at 98% purity, were washed twice with PBS and lysed in ice-cold lysis buffer containing 10 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, 2 mM dithiothreitol, 0.5% Nonidet P-40, 8 µg/ml leupetin, and 10 µg/ml phenylmethylsulfonyl fluoride. Nuclei were then removed by centrifugation at 1250 g at 4°C for 5 min. The bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford IL) was used for analyzing the protein concentration. Cytoplasmic extracts (15 µg protein/lane) were resolved on 8% sodium dodecyl sulfate-polyacrylamide gels and analyzed by Western blotting using an enhanced chemiluminescence (ECL) kit (Amersham, Little Chalfont, UK). The blots were probed with specific Ab directed against human EP1R, EP2R, EP3R, and EP4R (Cayman Chemical; 1/5000 dilution) or {alpha}-tubulin (Sigma-Aldrich; 2/10,000). Protein bands were detected with the ECL Western blotting analysis system from Amersham.

Effect of dbcAMP and assessment of cytokine production by DC
DC were harvested on day 6 of culture, washed with PBS, and placed in fresh CM at a concentration of 1 x 106 cell/ml in a six-well plate. The DC were then treated for 18 h with 1 µM PGE2 or various agonists for EPRs. As the binding of PGE2 on EP2R and/or EP4R is known to enhance the production of cAMP, the effects dbcAMP (N6, O2'-dibutyryl adenosine-3',5'-cyclic monophosphate; Sigma-Aldrich), an analog of cAMP, were tested on cytokine release. For that purpose, cells were incubated in CM with graded concentrations of dbcAMP for 18 h. Cell-free supernatants were collected from DC cultures, and cytokine (IL-6 and IL-10) production was measured by commercial enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN). The detection limits were 3.1 pg/ml for IL-6 and 4 pg/ml for IL-10.

MLR
To more precisely identify the EPR isoforms that regulate T cell proliferation in allogeneic MLR, DC were treated with 1 µM PGE2 or EPR agonists for 18 h at 37°C. After extensive washing with PBS, DC were counted and incubated with 10 µM mitomycin C (Sigma-Aldrich) for 35 min at 37°C and then washed four times with PBS. C57Bl/6 CD4+ T lymphocytes used as responder cells were seeded (2x105/ml) into a 96-well flat-bottom plate (Costar, Dominique Dutscher SA) together with mitomycin C-treated (10 mg/ml, 35 min, 37°C; Sigma-Aldrich) DC (2x104/ml). Controls included DC treated with mitomycin without lymphocytes and lymphocytes cultured alone. All experiments were set up in triplicate. After 4 days of incubation at 37°C in 5% CO2, cell cultures were pulsed with 1 µCi/well [3H] methyl-thymidine (specific activity, 2 Ci/mmole; Amersham Pharmacia Biotech, Buckinghamshire, UK) for 6 h. The plates were harvested onto glass-fiber filters with an IH-110 harvester (Inotech, Dottikon, Switzerland) and filter-counted for 5 min in a 1450 millicroplate counter (Wallac, Turku, Finland).

Statistical analyses
Results are expressed as mean ± SEM. Statistical analysis was performed using the Student’s t-test. P < 0.01 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DC generated in vitro from mice bone marrow express EP1, EP2, EP3, and EP4Rs
Before this investigation, nothing was known regarding the EPR expression in DC. Western blot and FACS analyses were performed to examine the profile of EPR expression in DC generated in vitro from mice bone marrow cells in the presence of GM-CSF and IL-4. Western blot analyses demonstrate that resting DC express EP1, EP2, EP3, and EP4R proteins (Fig. 1A ). It has been previously demonstrated that LPS modulate the expression of EPRs in B cells [24 , 25 ]. To examine whether similar effects are found in DC, EPR expression was analyzed in DC stimulated with increasing concentrations of LPS for 18 h. We observed that in a dose-dependent manner, LPS up-regulated EP2R and EP4R but not EP1R and EP3R protein levels in DC (Fig. 1A) . As LPS is also known to enhance COX-2 protein expression and PGE2 production, as we have previously shown [21 ], we examined the effects of COX-2-produced PGE2 on the expression of EPRs. Treatment of cells with increasing concentrations of NS-398, a COX-2 selective inhibitor, in the presence of 1 µg/ml LPS dose-dependently inhibited the expression of EP2R and EP4R (Fig. 1B) . These results suggest that PGE2 produced from DC via LPS-induced COX-2 acts on DC themselves, by increasing EP2R and EP4R levels without any effect on EP1R and EP3R. The same results were observed with exogenous PGE2 (data not shown). As Western blotting of DC cell lysates does not allow a discrimination between EPRs in the cytoplasma and receptors displayed on the surface of DC, flow cytometry analysis was used to monitor the levels of cell-surface expression of EPRs before and after LPS stimulation. Results obtained in Figure 2A show that EPRs are mainly expressed at the DC surface, and LPS enhanced EP2R and EP4R expressions without any effect on EP1R and EP3R levels. We also found that NS-398 inhibited the LPS-induced EP2R and EP4R expression (Fig. 2B) . A dose of 5 µg/ml NS-398 caused a 55% and 47% decrease in EP2R and EP4R expression, respectively (Fig. 2C) . Taken together, our data indicate that exogenous- or LPS-induced COX-2-produced PGE2 differentially modulated EPR expression in DC. In addition to its inhibition of COX-2 enzyme activity, NS-398 may act by modulating the expression of EPRs, suggesting that nonsteroidal anti-inflammatory drugs (NSAIDs) may have another regulatory action in terms of cellular responsiveness to PGE2.



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  		Figure 1. Western blot analysis of EPR expressions in DC. Purified DC (1x106/ml) were incubated for 18 h in the presence of a graded concentration of LPS (A) or COX-2-selective inhibitor NS-398 (B) with 1 µg/ml LPS. Cytoplasmic extracts were prepared and analyzed as described in Materials and Methods by Western blot analysis. One of five representative Western blots is shown.

 


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  		Figure 2. DC-surface expression of EPRs. The levels of EPR expression on DC surface were examined by flow cytometry before and after LPS stimulation. Cells (1x106/ml) were treated with 1 µg/ml LPS in the absence (A) and the presence of 5 µM NS-398 (B). After an 18-h incubation, cell-surface expression of each EPR was analyzed as described in Materials and Methods. Representative histograms for the expression of each EPR are shown from one of three separate experiments, and results are expressed as the MFI (C).

 
PGE2 inhibits MHC class II protein expression through an EP2R/EP4R-dependent mechanism
PGE2 acts by binding and interacting with one of four receptor subtypes designated EP1–EP4Rs [12 ]. The expression of MHC class II molecules is essential for DC to act as potent APC during the induction of immune responses. Others and we have shown that PGE2 inhibits MHC class II expression in DC [4 ] and in macrophages [26 ] and limits their ability to act as APC by stimulating T cell proliferation. To determine the subtype of EPRs involved in PGE2-suppressive effects on MHC class II molecules, we have used specific agonists for each EPR subtype. As shown in Figure 3 , treatment of cells with 1 µM exogenous PGE2, But, a selective agonist for EP2R, or PGE1 alc, a selective agonist for EP2/EP4Rs, caused a significant decrease in the expression of MHC class II molecules (P<0.01 when compared with control cells). However, selective agonist for EP1 and/or EP3Rs had no effects. The expression of CD11c antigens was also analyzed in the same conditions. Results obtained in Figure 3 demonstrate that PGE2 failed to affect the expression of a CD11c marker in DC. Taken together, these results suggest the involvement of EP2 and EP4Rs in the inhibition of MHC class II expression by PGE2.



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  		Figure 3. PGE2 down-regulates MHC class II molecule expression in DC via EP1 and EP2Rs. DC (1x106/ml) were incubated for 18 h in the presence of PGE2 (1 µM) or various EPR agonists used at 1 µM. These synthetic products did not affect the cell viability. At the end of the incubation period, cells were washed, stained with Ab to MHC class molecules (I-Ad), or CD11c-conjugated to fluorochromes listed in Materials and Methods, and FACS analysis was performed. Results for five representative experiments are expressed as the MFI. *, P< 0.01, when compared with control cells.

 
EPRs that modulate cytokine production by DC
DC shape cellular immune responses in their remarkable and unique capacity as professional APC and by producing a variety of cytokines that can influence the functions of antigen-specific T cells [1 ]. It is established that eicosanoids, in particular PGs and leukotrienes (LTs), can influence immune response by modulating cytokine release from immune cells. For example, PGE2 plays important roles in modulating IL-10 and IL-12 balance in DC [21 ] and can modulate the production of IL-6 in vivo [27 ]. Other investigators have shown that LTB4, a 5-lipoxygenase (5-LO) metabolite, which can be produced by DC [5 ], stimulates IL-6 release from human monocytes [28 ]. When we examined the effects of these eicosanoids on DC, we found that treatment of cells with graded concentrations of PGE2 stimulates, in dose-dependent manner, the production of IL-10 but not IL-6 (Fig. 4A ). However, LTB4 dose-dependently stimulates the release of endogenous IL-6 from DC without any effect on IL-10 (Fig. 4B) . These results suggest that eicosanoids differentially modulate cytokine production from DC. Next we examined the relative contribution of each EPR subtypes in the stimulation of IL-10 biosynthesis by PGE2. For that purpose, cells were stimulated with EPR-selective agonists (used at 1 µM). We observed that treatment of DC with But or PGE1 alc mimics the inhibitory effect of PGE2 by significantly increasing the production of endogenous IL-10 from DC (Fig. 4C) . However, EP1R agonists (Sul) and EP1/EP3R-selective agonist (17-Ph) had no effects on DC-IL-10 release (Fig. 4C) . Taken together, our findings strongly suggest the involvement of EP2 and/or EP4Rs in PGE2-induced modulation of IL-10 production from the in vitro bone marrow-derived DC.



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  		Figure 4. PGE2 and LTB4 differentially modulate cytokine release from DC and involvement of EP2/EP4R in the induction of IL-10 by PGE2. DC (1x106/ml) were incubated for 18 h in the presence of increasing concentrations of PGE2 (A) or LTB4 (B). Parallel DC cultures were incubated for 18 h in the presence of PGE2 (1 µM) or various EPR agonists used at 1 µM (C). At the end of the incubation period, all cell-free supernatants were harvested, and ELISA assessed cytokine production. Cytokine levels are expressed as pg/ml or percentage of control (untreated cells). Data are the mean ± SEM of five independent experiments.

 
The cAMP analog, dbcAMP, mimics PGE2-induced effects on DC
Previous investigations examining signaling pathways activated by EP2R and EP4R in diverse cell types reveal that EP2R and EP4R elevate cAMP via activating a G{alpha}s protein and adenylate cyclase [17 , 18 ], and agents that elevate cAMP mimic the effect of PGE2. To confirm the data implying EP2R and EP4R in mediating the effects of PGE2 on IL-10 production, DC were treated with graded concentrations of dbcAMP, an analog of cAMP, for 18 h. As shown in Figure 5A , dbcAMP failed to affect the release of endogenous IL-6 from DC. In contrast, it dose-dependently stimulates the production of endogenous IL-10, indicating that G{alpha}s protein-coupled EP2 and/or EP4Rs are involved in the induction of endogenous IL-10 by PGE2. We also examined the effect of dbcAMP on the expression of MHC class II and CD11c antigen. Results obtained in Figure 5B , which reflect the MFI, indicated that dbcAMP mimics PGE2-induced inhibition of MHC class II molecule expression and confirmed no effect on CD11c antigens. Together, our results demonstrate that G{alpha}s protein-coupled EP2 and/or EP4Rs mediate the inhibitory actions of PGE2 on DC functions.



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  		Figure 5. dbcAMP mimics the effects of PGE2 on DC (1x106/ml), which were incubated for 18 h in the presence of increasing concentrations of dbcAMP, an analog of cAMP. Cytokine production (pg/ml±SEM;A) and MHC class II expression (MFI; B) were analyzed as described in Materials and Methods. Data are representative of five separate experiments. *, P< 0.01, compared with respective controls.

 
Role of EPRs in MLR
Next, we examined the capacity of PGE2 and EPR agonist-treated DC to induce the proliferation of allogeneic T cells. We observed that 17-Ph (EP2/EP4R agonist) mimics PGE2-induced inhibition of T cell proliferation in allogeneic MLR (Fig. 6 ). Significant inhibition (*, P<0.01) was also observed when cells were treated with But (EP2R-selective agonist) or PGE1 alc (EP2/EP4R-selective analog). However, DC treated with EP1 and EP3R agonists induced a significant proliferation of allogeneic T cells, suggesting that EP2 and EP4Rs are involved in inhibiting proliferation of T cells in MLR.



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  		Figure 6. EP2R and EP4R are involved the modulation of T cell proliferation in allogeneic MLR. C57BL/6 T lymphocytes (2x105) were incubated with DC (5x104), treated as described in Materials and Methods. Controls (<500 counts per minute, histograms not shown) included DC treated with mitomycin without lymphocytes and lymphocytes cultured alone. Data are the mean ± SD of triplicate culture from five representative experiments. *, P< 0.01, when compared with control cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PGE2, an AA-derived metabolite, modulates several immune and inflammatory processes, such as cytokine production [29 ], Ab formation [30 ], phagocytosis, and cell multiplication. Several lines of evidence suggest that PGE2 exerts anti-inflammatory effects and induces immune suppression by decreasing T cell proliferation, IL-2 production [31 ], and IL-2 receptor expression [32 ]. In this way, PGE2 blunts the proliferation of T lymphocytes, a crucial step in the expansion of T cell clones [33 ]. It also inhibits the secretion of interferon-{gamma} [34 ]. Thus, PGE2 inhibits the production of T helper cell type 1 (Th1) cytokines, switching the immune response toward a Th2-type cytokine profile. Many new and interesting data have emerged concerning the role of PGE2 in cells of the immune system, in particular APC. These data suggest that PGE2 is a lipid mediator with a complex biology in the immune response. PGE2 has stimulatory and inhibitory effects on the activation of DC, depending on the site of encounter. In peripheral tissues, PGE2 seems to have a stimulatory effect on DC, inducing their activation and migration [35 , 36 ]. Once the cells have migrated to lymphoid organs, PGE2 assumes a suppressive action, inhibiting the maturation of DC and their ability to present antigen [4 ]. In murine bone marrow-derived DC, we demonstrated that PGE2 down-regulated IL-12 production [21 ] and altered antigen presentation by inhibiting MHC class II protein expressions [5 ]. Other investigators have reported that PGE2 acts as enhancer of DC differentiation and affects the Th-driving ability of DC. For example, the addition of PGE2 in combination with proinflammatory cytokines, such as IL-1ß + tumor necrosis factor {alpha} (TNF-{alpha}) ± IL-6, promotes human DC maturation [37 , 38 ] and enhances the expression of CCR7 required in DC migration in response to CCL19 or CCL21. When considering the effects of PGE2 on DC, it is important to note that mice and human DC are not necessarily equivalent between species. Besides the problem of species, there are some sensitivity differences to the suppressive effect of PGE2 among mouse strains [39 ]. In addition, Luft et al. [35 ] have reported some differences between tow subsets of human DC, as peripheral blood DC, in contrast to monocyte-derived DC, were not as dependent on the presence of PGE2 for acquisition of migratory functions. It is also important to note that without the standardization of APC culture conditions, the results from different studies cannot be directly compared.

In addition to PGE2, PGD2, another COX metabolite produced, for example, in the epidermis, plays an important role in modulating APC function. As shown herein for PGE2 and its inhibitory effects, PGD2 and its adenylate cyclase-coupled PGD2 receptor have a similar, suppressive effect on Langerhans cell (LC) function, as PGD2 not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity response after challenge [40 ].

The actions of PGE2 require the expression of EPRs, which bind PGE2 and mediate its complex and sometimes opposite effects. Before this investigation, nothing was known regarding which subtypes of EPRs are expressed and involved in regulating DC functions. The data reported herein are the first to describe coexpression of all EPRs in DC generated in vitro from mice bone marrow. In addition, we show that PGE2 suppresses DC functions through an EP2/EP4R-dependent mechanism. These results are in agreement with a recent report [20 ] in which authors systematically deleted the four EPRs in mice and showed their nonredundant functions in dampening immune responses. They demonstrated that T cells lacking EP1 or EP3R remained sensitive to PGE2, whereas EP2R-deficient T cells were resistant to PGE2 inhibition. They also found that only EP4R-deficient macrophages showed defects in antigen presentation and cytokine production. In contrast, we demonstrate that EP2R and EP4R play important roles in modulating cytokine production and antigen presentation by DC. One possible explanation is that DC and macrophages are not necessarily equivalent in terms of EPR profile expression and cellular responsiveness.

As LPS is known to stimulate PGE2 production via the COX-2 enzyme in monocytes [41 ] and modulate the expression of EPR in the other cells such as macrophages [42 ], the effects of LPS on EPR expression were examined. In a dose-dependent manner, LPS up-regulate EP2R and EP4R expressions, without any effect on EP1R and EP3R. We have reported recently that LPS strongly increased the expression of the COX-2 enzyme, which resulted in the production of high levels of PGE2 [21 ]. In a recent study, it has been shown that COX-2 inhibition by NS-398, a COX-2-selective inhibitor, modulates EPR expression in mouse M-1 cells [43 ]. In agreement with other investigators [42 ], we show that following treatment of DC with NS-398, EP2R and EP4R, but not EP1R and EP3R, expression was found to decrease. These results suggest links between COX-2 and EP2R/EP4R expressions and indicate that endogenous PGE2 produced via LPS-induced COX-2 acts on DC by increasing EP2 and EP4R expressions. Although the main mechanism of action of NSAIDs such as NS-398 is to inhibit COX activity, which in turn leads to decreases in PGE2 production, there may be another regulatory level in terms of the cellular responsiveness to PGE2. It is accepted that the effects of PGE2 are mediated via G protein-coupled plasma membrane receptors [44 ]. However, recent data implied that PGE2 might act intracellularly. In fact, previous studies have reported the presence of nuclear EPRs [45 , 46 ]. In the present study, although flow cytometry analyses show that EPR are mainly expressed at the DC surface, we cannot exclude the presence of cytoplasmic or nuclear EPRs on DC. It would be of interest in future work to study the regulatory effect of COX-2 inhibitors on nuclear PGE2 signaling mechanisms and the regulation of gene transcription by NSAIDs.

Our study demonstrates that EP2R and EP4R play a central role in modulating DC functions by PGE2 and confirms the fact that EP2R and EP4R have generally been associated with immunological modulation. For instance, B cell differentiation to IgE-secreting plasma cells [30 ] as well as IL-8 production by human colonic epithelial cells [47 ] and TNF-{alpha} inhibition in human blood monocytes [48 ] were reported to be mediated by PGE2 via EP2R and EP4R subtypes. The function of EP1R and EP3R in DC is unknown. One possibility is that these receptors may be required for counteracting a PGE2-induced effect mediated by EP2R and EP4R and may represent a possible mean by which EP2R/EP4R expression returns to basal levels. This hypothesis is conceivable, in particular for EP3ß, as EP3ßRs dampen cAMP elevation via the action of inhibitory G protein on adenylate cyclase [49 ]. In addition to the hypothetic anti-EP2R/EP4R-regulatory functions, EP1R and EP3R may mediate other PGE2-induced effects, which have yet to be identified in DC.

IL-10 is an important cytokine induced by lipid mediators, such as platelet-activating factor or PGE2, and plays a central role in systemic immune suppression [50 ]. Others and we [21 , 22 ] have reported that PGE2-induced IL-10 plays an important role in regulating DC functions. Treatment of DC with PGE2 or dbcAMP, an analog of cAMP, resulted in high production of endogenous IL-10 but not of IL-6. In agreement with published data on IL-6 release from LTB4-stimulated monocytes [28 ], we demonstrate that treatment of DC with LTB4 dose-dependently enhanced the release of endogenous IL-6, without any effect on IL-10. These results suggest that eicosanoids, such as PGE2 and LTB4, differentially modulate the ability of DC to secrete bioactive cytokines. Collectively, our data show that PGE2 binds EP2R and/or EP4R of DC, which activate G{alpha}s protein, leading to an increase in cAMP that is required for enhancing the release of endogenous IL-10. This immunosuppressive cytokine plays a pivotal role in inhibiting the production of cytokine such as IL-12p70 [21 ] and the expression of MHC class II molecules in DC, limiting their ability to act as professional APC. It can also suppress the production of lipid mediators such as LTB4 by inhibiting the expression of 5-LO-activating protein, which is involved in the optimal activity of the 5-LO enzyme [51 ]. The induction of endogenous IL-10 by cAMP-elevating substances such as PGE2 was also observed in monocytic cells [52 ].

In summary, our study contributes to the sorting out of the complex biology of PGE2 in the immune system. We clearly demonstrate that the inhibitory effects of PGE2 on murine DC are mediated via EP2R/EP4R. Thus, EP2R and EP4R could represent promising targets to modulate DC-mediated immune responses. Further studies with the EPR knockout mice and different subsets of human DC are necessary to clarify the role of PGE2 in the generation of DC for therapeutic purposes in patients.


   ACKNOWLEDGEMENTS
 
This work was supported by la Ligue Régionale Contre le Cancer Comité Départemental des Charentes et Comité Départemental de Gironde.

Received October 10, 2002; revised January 25, 2003; accepted February 6, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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