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Laboratoire d’Immunologie, Unité Mixte de Recherche Centre National de la Recherche Scientifique, Université Victor Segalen, Bordeaux, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2 is thought to suppress cell-mediated immune
response while enhancing humoral immune responses (7, 8, 9).
In macrophages the down-regulation of cell-mediated immune responses by
PGE2 is suggested by a dramatic reduction in
LPS-mediated TNF-
and IL-12 production (10, 11).
PGE2 modulates a variety of physiological
processes, including APC function (12) and the production
of inflammatory cytokines (13). Previous studies reported
that PGE2 is a strong inhibitor of IL-12
production (11, 14), which implies a feedback mechanism at
the level of the APC (11). Both PGE2
and IL-12 are produced by monocytes, macrophages, and other APCs in
response to a variety of compounds, including bacterial products
(15), and have opposite effects on Th1 and Th2
responses.
PGE2 is a potent inducer of IL-10 (11, 14, 16), which is produced by a variety of cells including monocytes (17), and exerts suppressive effects on Th1 responses (18, 19, 20). Similar to PGE2, IL-10 inhibits both the ability of DC to produce IL-12 (21, 22) and their stimulatory capacity (12, 21, 23), inducing the development of a tolerogenic type of DC (23, 24). Because PGE2 triggers the synthesis of IL-10 that is known to inhibit important aspects of cell-mediated immunity (25), we investigated the hypothesis that PGE2 modulates the DC-mediated immune response via the induction of IL-10.
PGE2 is synthesized from arachidonic acid (AA), which can be rapidly liberated from membrane phospholipids by the action of phospholipases, in particular phospholipase A2 (PLA2) (26). The critical step in the formation of proinflammatory PGs is the oxygenation of free AA by cyclooxygenase (COX) enzymes (also named PGH synthase or PG endoperoxide synthase), (27). Two isoforms of COX exist. The type I enzyme (COX-1) is constitutively present in many cell types, whereas type II (COX-2) is usually absent under basal conditions but can be induced by several stimuli, such as cytokines and other factors (28). LPS is a strong inducer of COX-2, which is responsible for the elevated production of PGs during inflammatory processes (29, 30). LPS can also increase PLA2 phosphorylation and AA release (31). To study the respective role of each isoform of COX in PGE2 production and APC functions, we used drugs, such as indomethacin, a COX-1 preference inhibitor (32, 33); SC-560, a COX-1-selective inhibitor (34); and NS-398, a COX-2-selective inhibitor (35, 36).
Because IL-12 production by APC is central to the orchestration of both innate and acquired cell-mediated immune responses to many pathogens (37), in contrast to PGE2 and IL-10, we examined the modulation of IL-12 production by bone marrow-derived DC (BM-DC). We used LPS-mediated stimulation and COX-selective inhibitors to investigate mouse BM-DC phenotype, function, and cytokine production.
We demonstrate that although BM-DC express both isoforms of COX, production of PGE2 derives largely from COX-2 after LPS exposure. In agreement with other investigators (16), our study shows that COX-2-produced PGE2 is a strong inducer of IL-10 and plays a pivotal role in the reciprocal regulation of IL-10 and IL-12 balance. By contrast, leukotriene B4 (LTB4) and NO produced in response to LPS did not affect BM-DC cytokine production. In these studies we clearly demonstrate that the suppressive activity of PGE2 on DC-mediated responses is induced by the endogenously produced IL-10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complete medium was RPMI 1640 (Life Technologies, Paisley, U.K.)
supplemented with 10% heat-inactivated FCS (Dominique Dutscher,
Brumath, France), 1% streptomycin (Life Technologies; 1000 µg/ml), 2
mM L-glutamine (Sigma-Aldrich, St. Louis, MO), 50 µM 2-ME
(Sigma-Aldrich), and 2 mM sodium pyruvate (Life Technologies). Growth
factors used for the generation of DC were recombinant mouse GM-CSF (20
ng/ml), IL-4 (10 ng/ml; purchased from PeproTech, Rocky Hill, NJ), and
TNF- (10 ng/ml; Valbiotech, Paris, France).
COXs and lipoxygenase inhibitors
Inhibitors used in our experiments were indomethacin, a COX-1 preference inhibitor; nor-dyhydroguarinic acid (NDGA), a 5-lipoxygenase (5-LO) inhibitor (Sigma-Aldrich); NS-398, a COX-2-specific inhibitor; and SC-560, a COX-1 selective inhibitor (Cayman Chemicals, Ann Arbor, MI). These drugs were dissolved in absolute ethanol (Merck, Darmstadt, Germany) and stored as stock solutions of 1 mM at -20°C. The required dilutions were prepared immediately before use, and equivalent quantities of ethanol were added to the cultures to serve as controls for these agents.
DC generation
DC were generated in vitro from mouse bone marrow as we previously reported (12, 38, 39). At the end of the culture period DC were positively purified using anti-CD11c (N418) microbeads and a MACS system column (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified BM-DC (routinely >98% CD11c+) were used for phenotypic, molecular, and functional analyses.
FACS analysis
Cells were subjected to analysis with a three-color FACSCalibur
(BD Biosciences, Mountain View, CA) using the following mouse Abs:
anti-CD11c-PE, anti-CD40-FITC, anti-CD80-PE,
anti-CD86-PE, and anti-I-Ad-PE (BD
PharMingen Europe, Pont de Claix, France), and data were collected for
1 x 104 cells. The primary Ab was directed
toward a panel of cell surface markers and compared with their
appropriate isotype-matched controls: hamster IgG1
-PE, rat hamster
IgM-FITC, hamster IgG-PE, rat IgG2ak-PE, and mouse IgG2bk-PE,
respectively (BD PharMingen, San Diego, CA). Cells showing a typical
dendritic morphology were always >95%, and the DC marker CD11c was
always present on >98% of the cells. Less than 1% of cells expressed
the CD14 marker. Expression of these markers was also analyzed by FACS
on DCs matured for 48 h in the presence of COX inhibitors.
Preparation of DC cytoplasmic extracts and Western blot analysis
BM-DC obtained at 98% purity from bone marrow cells in vitro
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 DTT, 0.5% Nonidet P-40, 8 µg/ml aprotinin, 8 µg/ml
leupeptin, and 10 µg/ml PMSF. Nuclei were removed by centrifugation
(1250 x g) at 4°C for 5 min. The bicinchoninic acid
protein assay reagent (Pierce, Rockford, IL) was used for analyzing the
protein concentration. Cytoplasmic extracts (15 µg protein/lane) were
resolved on 7% SDS-polyacrylamide gels, and Western blotting analysis
was performed using an ECL kit (Amersham, Little Chalfont, U.K.). The
blots were probed with specific Abs directed against COX-1 (1/1,000
dilution; Cayman Chemicals), COX-2 (1/500 dilution; BD Transduction
Laboratories, Lexington, KY), cytosolic PLA2
(cPLA2; 1/1,000; Santa Cruz Biotechnology, Santa
Cruz, CA), or -tubulin (2/10,000; Sigma-Aldrich). The blots were
subsequently incubated with the secondary Ab (peroxidase-labeled
anti-mouse Ab; 2/1,000 dilution; Amersham) and analyzed by an
Amersham ECL and Kodak XAR-5 film (Eastman Kodak, Rochester,
NY).
Quantification of released AA
Samples (DC and LPS-stimulated DC) with 10 million cells (pellet) were used for the fatty acid analysis; hepatodecanoyl acid (C17; 10 mg/sample) was added as an internal standard. Lipids were extracted according to the method of Folch et al. (40). The organic phase was evaporated to dryness under nitrogen, and transesterification was conducted with 2.5% H2SO4 in methanol for 1 h at 80°C. Fatty acid methyl esters were then extracted with 1 vol hexane/H2O (1/1, v/v) and analyzed in Hewlett-Packard 5890 series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a Carbowax capillary column and a flame ionization detector. Fatty acid methyl esters were identified by comparison of relative retention times with standards and were quantified by comparison with the internal standard.
Effects of LPS on PGE2 production
Immature BM-DC were seeded at 1 x 106 cells/ml into six-well plates (Costar, Cambridge, MA) for an additional 48 h at 37°C in complete medium with increasing concentrations of LPS (Escherichia coli, serotype E26:B6; Sigma-Aldrich). At the end of the culture period some cultures were supplemented with AA (Calbiochem, Meudon, France) at a final concentration of 1 µM for 30 min. Supernatants were collected by centrifugation at 2500 x g for 15 min, and PGE2 production was measured by ELISA (Cayman Chemicals). The detection limit was 15 pg/ml.
Effects of selective COX inhibitors on eicosanoid production
To address the relative contribution of each isoform of COX to PGE2 production, 1 x 106 immature DC/ml were cultured for 48 h in complete medium with and without LPS (1 µg/ml) and graded concentrations of a selective COX-1 inhibitor, SC-560, or a selective COX-2 inhibitor, NS-398, without exogenous AA. To study the effect of endogenous PGE2 on LTB4 production, parallel cultures were treated with both COX selective inhibitors (4 µM indomethacin, 20 nM SC-560, and 10 µM NS-398) or NDGA (1 µM). Controls included DC stimulated with LPS without inhibitors. PGE2 and LTB4 production was measured by ELISA in the presence and the absence of exogenous AA in all supernatants. The detection limit for LTB4 was 4 pg/ml.
Nitrite assay
The release of NO was measured in the absence of exogenous AA by the assessment of NO2-, a stable metabolite, determined by colorimetric reaction (41). After cell stimulations, 50 µl culture supernatant was transferred in triplicate into 96-well plates, to which 50 µl 0.1% (v/v) N-1-naphthylethylene diamine HCl in distilled H2O and 50 µl 1% (v/v) sulfanilic acid in 5% (v/v) phosphoric acid were added. Results were determined with a microplate reader at 540 nm. Standard curves were generated with dilutions of NaNO2 (0–100 µM). The detection limit was 0.1 mM/106 cells.
IL-10 and IL-12p70 measurement
Cytokines were measured by ELISA (R&D Systems, Abingdon, U.K.) in the supernatants of BM-DC matured in the presence of PGE2 or LPS with and without COX inhibitors. Controls included cells matured in complete medium alone. IL-12p70 and IL-10 were also measured in supernatant from cells treated with graded concentrations of LTB4 or with NDGA after LPS stimulation. To investigate whether PGE2-induced IL-10 mediated inhibition of IL-12 production, LPS-treated DC cultures were treated with increasing concentrations of anti-IL-10 (purified rat anti-mouse IL-10 mAb, clone JES5-16E3; BD PharMingen) or an isotype control (purified Rat IgG2b; BD PharMingen). The effects of NO on IL-12 production by LPS-stimulated cells were also tested by the use of 0.5 mM NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase, or 0.25 mM S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a donor of NO. The detection limits were 4 pg/ml for IL-10 and 2.5 pg/ml for IL-12p70.
Effects of exogenous IL-10 on IL-12p70 production
To evaluate the effects of exogenous IL-10 on IL-12 production, LPS-treated DC were exposed to complete medium containing 10 µM NS-398 with increasing concentrations of exogenous IL-10 (mouse rIL-10; PeproTech). Controls included LPS-stimulated DC treated with NS-398 alone. After 48 h supernatants were collected for the assessment of IL-12p70 production. The percentages of suppression of IL-12-p70 production were calculated and compared with control values.
Preparation of C57BL/6 lymphocytes and allogeneic MLR
Spleen C57BL/6 lymphocytes used for allogeneic MLR as responder cells were obtained as previously reported (42). Briefly, 8-wk-old female C57BL/6 (H-2Db) mice (Iffa Credo, Lyon, France) were killed by cervical dislocation. Splenocytes were isolated and separated by flotation on Ficoll (Sigma-Aldrich). Mononuclear splenocytes were allowed to stick to plastic petri dishes (Costar) for 4 h in RPMI 1640 complete medium at 37°C in a regular incubator. Then nonadherent cells were gently removed, washed twice, and kept in RPMI 1640 complete medium, and adherent cells were discarded. Nonadherent cells contained <0.5% CD14+ cells and at least 80% CD4+ lymphocytes. The allostimulatory capacity of DCs was tested in MLR. DCs were stimulated for 48 h with 1 µM exogenous PGE2 or 1 µg/ml LPS in the presence and the absence of COX inhibitors. After extensive washing with PBS (BioWhittaker, Walkersville, MD), DC were counted and incubated with 10 µg/ml (final concentration) mitomycin C (Sigma-Aldrich) for 35 min at 37°C in humidified atmosphere containing 5% CO2, then washed four times with PBS. To test the effects of PGE2 and IL-10 on the cell stimulatory function of DC in allogeneic MLR, T lymphocytes (2 x 105/ml) were seeded into 96-well flat-bottom plates (Costar) together with 5 x 104/ml mitomycin C-treated DC. Controls included DC treated with mitomycin without lymphocytes and lymphocytes cultured alone. Some LPS-treated DC were incubated with T cells in the presence of anti-IL-10 (100 ng/ml). Plates were incubated for 4 days at 37°C in 5% CO2. All experiments were performed in triplicate. On day 4 of the MLR, cell cultures were pulsed with 1 µCi/well [methyl-3H]thymidine (sp. act., 2 Ci/mmol; Amersham) for 6 h. The plates were harvested onto glass-fiber filters with an IH-10 harvester (Ionetech, Dottikon, Switzerland), and the filters were counted for 1 min in a 1450 Millicroplate counter (Wallac, Turku, Finland).
Statistical analysis
The results are expressed as the mean ± SEM. Analysis of data was performed using Student’s t test; p < 0.01 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AA release and the generation of lipid mediators require the
expression and the activation of PLA2. Western
blot analyses show that although cPLA2 is
constitutively expressed in BM-DC, it is also inducible by LPS. Fig. 1
A shows that LPS
dose-dependently up-regulates cPLA2 expression.
In terms of AA release, LPS-treated BM-DC released substantial amounts
of AA (2.73 µg/106 cells) compared with
unstimulated cells (Fig. 1, B and C).
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Western blot analyses were performed to evaluate COX protein
expression in mature BM-DC. Fig. 2A shows that resting BM-DC
constitutively express COX-1 but not COX-2. COX-2 protein was
detectable after LPS exposure, and its expression was dose-dependently
up-regulated by LPS. No changes were observed in COX-1 protein
expression under the same conditions. When we assessed eicosanoid
production from BM-DC, we found that PGE2 was the
predominant PG produced by BM-DC, while production of other COX-issued
prostanoids was quite weak. As shown in Table I, BM-DC are able to produce
PGE2 in the absence and the presence of exogenous
AA. These results show that the addition of exogenous AA is not
required for the generation of measurable amounts of
PGE2. Treatment of BM-DC with 1 µg/ml LPS
resulted in the production of high quantities of
PGE2 (>3 ng/ml/1 x
106 cells; Table I), and LPS enhances
PGE2 production in dose-dependent fashion (Fig. 2B). When we assessed COX-1- and COX-2-mediated
PGE2 production, COX-2 appeared to be the
predominant isoform of COX involved in the production of endogenous
PGE2 by LPS-stimulated DCs, because NS-398
dose-dependently decreased PGE2 production (Table II). A dose of 10 µM NS-398 caused an
81% decrease in PGE2 production
(p < 0.01; n = 4). The lack of
effect of SC-560, a COX-1-selective inhibitor, suggests that COX-1 did
not contribute significantly to the total pool of
PGE2 in LPS-stimulated cells. However, in resting
BM-DC, SC-560 decreased PGE2 production in
dose-dependent fashion (Table II). Indomethacin
(IC50 (COX-1), 1.67 µM) used at 1 and 4 µM,
which inhibited only COX-1, did not affect the higher production of
PGE2 following LPS exposure (Table I). Added
together, COX-1 and COX-2 inhibitors caused >94% inhibition in
PGE2 production (Table I).
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). An
80% inhibition of LTB4 synthesis was observed in
the presence of 1 µM NDGA. In addition, production of
LTB4 was 2-fold higher when both COX pathways
were blocked (Table I), with or without addition of exogenous AA. These
results suggest that COX metabolites may inhibit
LTB4 synthesis. Compared with control cells,
addition of exogenous AA to cells did not significantly modify BM-DC
eicosanoid production, suggesting that PGE2, the
major PG formed, did not inhibit the production of
LTB4 by limiting substrate availability in BM-DC.
In addition, we found that exogenous PGE2
inhibited the production of LTB4 without any
change in 5-LO expression (H. Harizi and N. Gualde, manuscript in
preparation). It should be interesting to investigate the
interaction between COXs and 5-LO pathways that might regulate tissue
homeostasis and contribute to the modulated production of
eicosanoids. LPS-stimulated BM-DC produce high level of NO
Among the other soluble mediators induced by LPS is NO. Compared
with control cells, treatment of BM-DC with 1 µg/ml LPS resulted in
the production of >50 µM NO (Table III). A 90% inhibition of NO production
was observed after addition of the NO synthase inhibitor,
L-NMMA. When we examined the effect of
PGE2 on NO production, we found that exogenous
PGE2 at 1 µM did not affect the production of
NO by either BM-DC or T cells (Table III). In addition, COX-2-selective
inhibitors did not modify the production of NO, suggesting that
COX-2-produced PGE2 did not affect the endogenous
production of NO.
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BM-DC express relevant surface Ags such as CD11c, CD40, CD80,
CD86, and MHC class II (Fig. 3). To
examine whether endogenous PGE2 affects the BM-DC
phenotype, we analyzed surface marker expression of BM-DC matured in
the presence of inhibitors. We have recently shown that exogenous
PGE2 reduces MHC class II expression in BM-DC
(12). In this study we observed that COX inhibitors did
not modify the expression of CD11c and CD40 markers, but the COX-2
inhibitor NS-398 up-regulated the expression of the MHC class II, and
costimulatory molecules (Fig. 3). We also found that indomethacin,
SC-560 (a COX-1 inhibitor), and NDGA did not affect the expression of
any marker (data not shown).
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In our study supernatants from various culture conditions (see
Materials and Methods) were assayed by ELISA to
determine the production of cytokines in the absence and the presence
of LPS, COX inhibitors, or exogenous PGE2. As
shown in Fig. 4, which summarizes the
average of six experiments, BM-DC did not spontaneously produce a
significant quantity of IL-10. However, they secreted >1.5
ng/ml/106 cells of IL-12p70. Addition of 1 µM
exogenous PGE2 enhanced the generation of IL-10
and simultaneously reduced the production of IL-12p70 (-95%). LPS
alone (1 µg/ml) induced a similar effect as exogenous
PGE2 (Fig. 4). In fact, the effects of the PG
were mimicked by LPS (which stimulates COX-2) and reversed by the
addition of 10 µM NS-398, a COX-2-specific inhibitor, which
up-regulates IL-12p70 and down-regulates IL-10 production. These
results suggest that COX-2-mediated PGE2 plays a
central role in the reciprocal regulation of IL-10 and IL-12, because
addition of indomethacin or SC-560, a COX-1 selective inhibitor, did
not significantly affect the production of IL-12p70 after LPS exposure
(Fig. 4). When we analyzed the effects of LTB4 on
BM-DC cytokine production, we found that treatment of BM-DC with
increasing concentrations of exogenous LTB4 did
not modify the production of either IL-10 or IL-12. Compared with
controls (LPS alone and LPS plus NS-398), the combination of LPS and
NDGA (which caused 80% inhibition of endogenous
LTB4 production) produced no significant decrease
in cytokine release (Fig. 5). These
results suggest that LTB4 had no effect on
cytokine production from BM-DC.
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We studied the production of IL-12-p70 by BM-DC treated with
exogenous IL-10. As shown in Fig. 6A, addition of exogenous
recombinant mouse IL-10 to DC in the presence of NS-398, a
COX-2-selective inhibitor, dose-dependently decreased the production of
IL-12p70. A dose of 2 ng/ml IL-10 caused a 90% decrease in IL-12
release (p < 0.01; n = 6). In
view of the potential of PGE2 to stimulate IL-10
production, we investigated whether IL-10 acted as an intermediate in
the suppression of IL-12p70 by PGE2. For that
purpose we used an anti-IL-10 neutralizing mAb. As shown in Fig. 6B, anti-IL-10 dose-dependently neutralizes endogenous
IL-10, and a dose of 100 ng/ml resulted in the neutralization of >95%
of the endogenously induced IL-10 and a >88% increase in IL-12p70
production, whereas the isotype-matched control mAb had no effect (Fig. 7). IL-12p70 production increased as the
concentration of IL-10 neutralizing mAb increased (data not shown) and
was maximal at a concentration exceeding 100 ng/ml. These results
suggest that the endogenously produced IL-10 was a potent inhibitor of
IL-12p70 production. Our results also show that treatment of DC with
NS-398 enhances the synthesis of IL-12p70 (Fig. 7), as in
anti-IL-10-treated DC. However, SC-560 and indomethacin had no
effect on IL-12 (Fig. 4), suggesting that COX-2, but not COX-1, was
involved in the modulation of IL-12 production by DC.
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As shown in Fig. 7, LPS- and PGE2-treated DC
produce low levels of IL-12p70 compared with control cells
(p < 0.01). In addition, the inhibitory effect
of PGE2 (either exogenous or LPS-induced) was
prevented by anti-IL-10. Our results confirm the involvement of
IL-10 in IL-12 suppression by PGE2. We also found
that addition of L-NMMA, an inhibitor of NO
synthase, or SNAP, an NO donor, did not affect the suppression of IL-12
production by PGE2 or LPS in BM-DC (Fig. 7),
suggesting that endogenous NO does not mediate the suppressive effect
of PGE2. Taken together with the influence of
exogenous IL-10 (Fig. 6A), we demonstrate that COX-2
PGE2-induced IL-10 suppresses the production of
IL-12p70 in BM-DC.
BM-DC stimulatory capacity
We therefore tested the capacity of DC treated with COX
inhibitors, exogenous PGE2, or anti-IL-10 to
induce the proliferation of allogeneic T cells. We observed that the
proliferative response of T lymphocytes was significantly diminished
(50% inhibition) when DC were exposed to 1 µM exogenous
PGE2 (Fig. 8;
p < 0.01 compared with unstimulated cells). Compared
with control cells (LPS alone), LPS-stimulated DC treated with NS-398
or anti-IL-10 induced a significantly higher proliferation of
allogeneic T cells (Fig. 8; p < 0.01), while thymidine
uptake by the allo-stimulated lymphocytes was not increased by
indomethacin alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because we found no significant differences in the profile of the relative amounts of eicosanoids (PGE2 and LTB4) in the presence and the absence of exogenous AA, and addition of exogenous AA is not required for the formation of measurable amounts of PGE2, we performed all experiments in the absence of exogenous AA. Selective COX-1 or COX-2 inhibitors decreased PGE2 production from endogenous AA.
PGE2 is accepted as a potent modulator of APC function and plays a prominent role in regulating Th1- and Th2-type responses (10, 13). We have recently reported that in our BM-DC model exogenous PGE2 alters Ag presentation by inhibiting the expression of MHC class II protein (12). These data are quite similar to those published by Snyder et al. (46), who reported that PGE2 diminished the expression of both MHC molecules and the accessory effect of macrophages. With regard to the effects of endogenous PGE2 on DC, we analyzed the phenotype and function of COX inhibitor-treated DC. For this purpose the in vitro-generated BM-DC were matured in the presence of COX-selective inhibitors and examined for their 1) expression of relevant surface markers, 2) production of IL-10 and IL-12, and 3) APC stimulatory function. To investigate whether endogenous PGE2 affects the maturation of murine DC, we analyzed the expression of CD11c, MHC class II, CD40, CD80, and CD86 in DC matured in the presence of inhibitors. We found that indomethacin, SC-560, and NDGA did not affect the expression of these markers (data not shown). However, although NS-398 did not modify CD11c and CD40 expression, it up-regulated the expression of CD80, CD86, and the MHC class II molecules. These data suggest that the predominant effects of PGE2 appear to be mediated through the COX-2 pathway, which affects the expression of molecules involved in the APC function of DC. Our data are not in agreement with a report by Whittaker et al. (47), who reported that blocking COX-2 in monocyte-derived DC had no effect on CD40, CD80, CD86, and HLA-DR expression. However, the expression of CD83 appears to be regulated by COX-2 produced prostanoids. One can explain the discrepancy between the two reports because Whittaker et al. (47) adopted a quite different model from the one we used.
The ability of mature DC to act as a potent APC is also related to
their production of cytokines, especially IL-12 (48, 50),
which is a major Th1-driving cytokine, promoting cell-mediated immunity
(51, 52). Resting BM-DC, which express only COX-1 and
produce low levels of PGE2, produce IL-12p70, but
not IL-10. However exogenous PGE2- or
LPS-mediated induction of COX-2 resulted in high levels of IL-10 and a
potent suppression of IL-12 production. This is in agreement with
earlier reports showing that IL-12 production by human monocytes and DC
is suppressed after LPS exposure (53, 54). The synthesis
of IL-12 is regulated by a positive feedback mechanism mediated by Th1
cells through IFN-
or by negative feedback through Th2 cells
secreting IL-10 (55). IL-10 is known to be a potent
suppressor of IL-12 gene expression at the transcriptional level
(56). In accordance with other investigators (22, 23), we found that addition of IL-10 to DC profoundly inhibited
the production of IL-12p70.
Confirming a recent study (57), we found that LPS-treated DC secreted high levels of IL-10, which appears to be an important regulator of DC biology and function. In contrast to Whittaker et al. (47), who showed that prostanoids did not modulate IL-10 production by human monocyte-derived DC, we demonstrate that COX-2-mediated PGE2 enhances the production of IL-10, and that COX-2-produced PGE2 might be a key point in the reciprocal regulation of IL-10 and IL-12 production, because it enhances IL-10 and spontaneously inhibits IL-12 production in LPS-treated BM-DC. This is in accordance with published data (14, 16).
Our data demonstrate that the inhibitory effect of PGE2 on IL-12 production was completely reversed by an anti-IL-10 mAb. Based on the influence of exogenous IL-10, PGE2, and NS-398 on BM-DC, it is likely that PGE2, IL-10, and IL-12, produced by murine DC might interact, hence regulating APC function and therefore the immune response. Together with the results obtained with anti-IL-10, we conclude that PGE2 inhibits IL-12 production in an IL-10-dependent mechanism. Our finding is not in agreement with studies using human macrophages or DC; when considering the effects of IL-10 on DC phenotype and function, one should keep in mind that mouse and human DC populations are obviously not the same. Our results could be related to the high sensitivity of that lineage to the suppressive effect of PGE2, as was reported by Kuroda et al. (58). However, in the data published by Kuroda, the distinctions between lineages exist only in terms of sensitivity, but there were no differences in terms of general orientation of the effects of PGE2 on the immune response among the different mice.
The biological actions of PGE2, including its effects on immunity, are mediated by G protein-coupled receptors, designated E prostanoid receptors (59). The existence of this complex family of E prostanoid receptors coupled to distinct intracellular signals provides a molecular basis for the diverse physiological actions of PGE2. It is clear that the reports on PGE2 effects on DC are sometimes confusing (12, 47). That could be related to the heterogeneity of the APC, the species used for the experiments, the signaling pathways affected in the maturation of the DC, etc.
Among the panel of AA metabolites produced by BM-DC, we noticed that
LTB4 is released, but in a lesser quantity
compared with PGE2. Because it was reported
before that LTB4 up-regulates IL-1, IL-2, and
IFN- production and enhances NK cell activity (45),
effects quite opposite to whhat is usually known concerning the
immunomodulation induced by PGE2, we investigated
the effects of LTB4 on BM-DC phenotype and
functions. We found that neither the exogenously added nor the
endogenously produced LTB4 affected the
production of cytokines by BM-DC.
It is well known that LPS activates macrophages to release proinflammatory mediators. Among the other soluble mediators stimulated by LPS is NO. In murine macrophages NO regulates IL-12 gene expression (61). Using L-NMMA, an NO synthase inhibitor, or SNAP, a donor of NO, we were able to show that in our experimental setting NO produced by DC did not affect the production of IL-12. This is in accordance with published data (53).
Next, we examined the ability of COX inhibitor-treated BM-DC to stimulate T cells in allogeneic MLR. In contrast to Kalinski et al. (62), we found that blocking COX-2 with NS-398 enhances the stimulatory potential of BM-DC compared with the control. Because it is reported that the stimulatory capacity of APC is inhibited by IL-10 (19, 25), and that COX-2-mediated PGE2 up-regulates the production of IL-10 by DC, we performed allogenic MLRs in the presence of anti-IL-10. We found that the neutralization of the endogenously produced IL-10 markedly increased the stimulatory potential of BM-DC. These results suggest that PGE2 inhibits the APC function of DC via IL-10. This inhibitory influence of IL-10 may be due to the down-regulation of MHC class II and costimulatory molecules as was previously reported (20, 63, 64).
In the present paper we provide evidence that PGE2 enhances IL-10, which down-regulates IL-12p70 production, and DC stimulatory capacity. Our study confirms that inflammatory effects of PGE2 on DC functions are mediated by an autocrine feedback mechanism involving endogenous IL-10 and expands the concept that molecules produced during inflammation may influence IL-12 production by LPS-stimulated APC, which are highly susceptible to the induction of LPS tolerance induced by endogenous PGE2 from the COX-2 pathway. It should be also underlined that our assessment of eicosanoids was limited to PGE2 and LTB4. One cannot be sure that blocking COX-2 by NS-398 inhibits only PGE2 synthesis and does not weakly affect other PG production. However, our former assessment using gas chromatography and mass spectrometry showed that immune cells, among them APC, produce mostly PGE2 and that synthesis of other COX-issued eicosanoids (PGs, prostacyclin, thromboxane) is quite weak (65).
PGE2 belongs to a family of short-lived chemical paracrine messengers that has been studied over the past 20 years for its immunomodulatory properties (66). However, the precise role of this eicosanoid has remained more or less unknown. One can expect that, by reaching an upstream step in the immune response, it may be easier to better evaluate the different roles of PGE2 for suppression and/or organization of the direction of the immune response (as, for instance, Th1 vs Th2 or vice versa). Because DC, which were originally identified by Steinman (reviewed in Ref. 1), represent the pacemakers of the immune response, one could accept that PGE2, which contributes widely to that major function of the professional APC, might be considered a pacemaker mediator of the immune response.
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2 Address correspondence and reprint requests to Dr. Norbert Gualde, Laboratoire d’Immunologie, Unité Mixte de Recherche Centre National de la Recherche Scientifique, Université Victor Segalen, Bordeaux 2, 33076 Bordeaux, France. E-mailaddress: norbert.gualde{at}umr5540.u-bordeaux2.fr
3 Abbreviations used in this paper: DC, dendritic cell; AA, arachidonic acid; BM-DC, bone marrow-derived DC; COX, cyclooxygenase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; L-NMMA, NG-monomethyl-L-arginine; 5-LO, 5-lipoxygenase; LTB4, leukotriene B4; NDGA, nor-dyhydroguarinic acid; SNAP, S-nitroso-N-acetyl-D,L-penicillamine.
Received for publication July 31, 2001. Accepted for publication January 2, 2002.
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