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The Journal of Immunology, 2007, 178, 6130 -6139
Copyright © 2007 by The American Association of Immunologists, Inc.
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*TRANS-RETINOIC ACID

9-cis-Retinoic Acid (9cRA), a Retinoid X Receptor (RXR) Ligand, Exerts Immunosuppressive Effects on Dendritic Cells by RXR-Dependent Activation: Inhibition of Peroxisome Proliferator-Activated Receptor {gamma} Blocks Some of the 9cRA Activities, and Precludes Them to Mature Phenotype Development1

Fernando Zapata-Gonzalez*, Félix Rueda§, Jordi Petriz{dagger}, Pere Domingo{ddagger}, Francesc Villarroya*, Africa de Madariaga* and Joan C. Domingo2,*

* Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain; {dagger} Cryopreservation Unit, Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; {ddagger} Infectious Diseases Unit, Internal Medicine Department, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Barcelona, Catalonia, Spain; and § Laboratory of Oncological Immunology, Department of Medical and Molecular Genetics, IDIBELL-Cancer Research Institute, Hospitalet del Llobregat, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
At nanomolar range, 9-cis-retinoic acid (9cRA) was able to interfere in the normal differentiation process from human monocyte to immature dendritic cell (DC) and produced a switch in mature DCs to a less stimulatory mode than untreated cells. 9cRA-treated mature DCs secreted high levels of IL-10 with an IL-12 reduced production. The phenotypic alterations unleashed by 9cRA were similar but not identical to other specific retinoid X receptor (RXR) agonists and to those already reported for rosiglitazone, a PPAR{gamma} activator, on DCs. The simultaneous addition of 9cRA and rosiglitazone on DCs displayed additive effects. Moreover, addition to cultures of GW9662, a specific inhibitor of PPAR{gamma}, or the RXR pan-antagonist HX603, blocked these changes. All these results suggest an activation of PPAR{gamma}-RXR and other RXR containing dimers by 9cRA in DCs. Finally, both GW9662 and HX603 by themselves altered the maturation process unleashed by TNF{alpha}, poly(I:C) or LPS on human DCs further suggesting that the heterodimer PPAR{gamma}-RXR must fulfill a significant role in the physiological maturation process of these cells in addition to the repressing effects reported till now for this nuclear receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 References
 
Immune responses form an extraordinarily sophisticated and potent system that must be instructed and regulated to avoid errors that could be fatal for the host (1, 2). The cells that accomplish this function are the APCs. Among them, dendritic cells (DCs),3 continuously produced from hemopoietic stem cells in the bone marrow (3, 4), display unique properties aimed at leading immunity against Ags that invade the organism (1, 5). They stand out because of their ability to induce primary immune responses and, as more evidence is recently arising, by their capacity to inhibit these immune responses depending on the circumstances as well (2, 6, 7, 8, 9). The mechanisms by which dendritic cells induce immunity or tolerance, following Ag presentation, are not clearly understood.

Retinoic acid and its derivatives, particularly 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA), are a group of structurally simple lipid molecules derived from vitamin A (retinol) that transactivate numerous genes and exert pleiotropic effects on cellular growth, differentiation and homeostasis both in vivo and in vitro in all vertebrates (10, 11, 12). Retinoids also exert wide effects on immune systems, and their effects on T cells (13, 14), monocytes (15, 16), and DCs have been widely demonstrated (17, 18). They modulate the expression of their target genes by binding to two classes of nuclear receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR). ATRA can only bind efficiently to RAR, but 9cRA is a ligand for both nuclear receptors RAR and RXR (19). 9cRA is present in the developing embryo and two 9-cis-retinol dehydrogenases that can mediate their synthesis in vivo have been identified (20, 21, 22).

RXR is located in the nucleus in the absence of ligand, and it forms homodimers or heterodimers with other members of the nuclear hormone receptor superfamily (19). Depending on the heterodimer, the RXR moiety remains sensitive to ligand activation (e.g., in peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})-RXR or liver X receptor (LXR)-RXR heterodimers) whereas the sensitivity of RXR to ligand activation as a part of RAR-RXR heterodimers depends on concurrent activation by RAR agonists. Other RXR heterodimers, such as RXR-vitamin D receptor, are nonpermissive for ligand-dependent activation of RXR (23).

One of the partners of the RXR in heterodimerization, which has been implicated in immune regulation, is PPAR{gamma}, a member of the PPARs. The PPARs belong to the steroid/thyroid/retinoid receptor superfamily and are nuclear receptors that control gene expression in multiple cell types (24). There are three different PPARs ({alpha}, beta, and {gamma}) with specific patterns of expression in different cell types. PPAR{gamma} is highly expressed in adipose tissue and is a molecular target for antidiabetic drugs such as thiazolinediones. Recently expression of PPAR{gamma} in the immune system has been reported where it fulfils different roles as apoptotic cell inducer following T lymphocyte activation (25, 26, 27), antiproliferative effects on B lymphocytes (28), inhibition of cytokine production in monocytes and macrophages (29, 30), and regulation of DC immunogenicity (31, 32, 33, 34). RXR homodimers can selectively bind to functional PPAR response elements and induce the transactivation of PPAR{gamma} genes (22). Retinoids perform different effects on DCs as well: they trigger apoptosis through a RAR-RXR pathway (17) or activate DC immunogenicity in the case of ATRA (18).

We report here for the first time that 9cRA, a RXR ligand, exerts intense effects on dendritic cell morphology, phenotype, cytokine secretion profile, and lymphoproliferative induction. These effects are similar but not identical to those previously published for PPAR{gamma} activators (31, 32, 33, 34), and were also observed in treatments with other RXR agonists, suggesting that 9cRA could act through PPAR{gamma}-RXR heterodimer and possibly RXR homodimers. We also report that PPAR{gamma}-RXR must be necessary for the normal development of DC maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 References
 
Cells and reagents

The medium used for cell cultures was RPMI 1640 supplemented with 10% inactivated FCS purchased from Invitrogen Life Technologies and antibiotics obtained from Biological Industries. 9cRA and GW9662 were purchased from Sigma-Aldrich. Rosiglitazone (ROSI) was purchased from GlaxoSmithKline. HX630 and HX603 were provided by Dr. H. Kagechika (University of Tokyo, Japan), CH55 was purchased from Wako Pure Chemicals Industries, and 24,25-epoxycholesterol was obtained from Biomol International.

Cell isolation and generation of DCs

Human monocytes were purified from PBMC isolated from fresh buffy coats (provided by the Center for Transfusion and Tissue Bank in Barcelona) using a Ficoll-Paque gradient (Amershan Bioscience). After that, cells were washed and centrifuged twice at 1500 rpm to avoid platelets.

Monocytes were obtained by 20-min plastic adherence of 20 x 106 PBMC/ml to a gelatin-coated flask. Nonadherent PBMC were then removed and used as an autologous lymphocyte source. The adherent cells (70–80% monocytes) were detached by addition of 10 mM EDTA for 30 min. Then they were seeded (1 x 106 cells/well) into 6-well plates (Costar). Lymphocytes were resuspended in 10% DMSO human autologous serum and frozen until use.

A negative selection purification method using a monocyte negative isolation Kit II according to the instructions of the manufacturer was also used (Dynal Biotech ASA). In brief, cells were resuspended in 5 x 107 mononuclear cells/ml in 0.1% BSA, added blocking reagent and Ab mixture and incubated for 10 min at 4–8°C. Later on, the cells were washed and centrifuged for 8 min at 500 x g. Washed beads were added to the cells and incubated for 15 min at 4–8°C. Then they were placed in a Dynal magnetic particle concentrator, and supernatant (with monocytes) was transferred to a fresh tube. After magnetic retention of the dynabead-attached cells, the supernatant fraction contained >85% CD14+ cells.

Monocytes were then differentiated to immature DCs by adding IL-4 (750 U/ml; Sigma-Aldrich) and GM-CSF (100 ng/ml; Leucomax 400; Novartis) in RPMI 1640 medium. Cultures were fed with cytokines every 2–3 days. On day 6, DC showed phenotypic characteristics (CD14neg, CD83low, CD1ahigh, HLA-DRhigh, CD80low, CD86low) of immature DCs. When mature DCs were needed, LPS (500 ng/ml, Sigma Aldrich; EC 055:B5), TNF-{alpha} (Sigma-Aldrich) or poly(I:C) (Sigma-Aldrich) were added on day 6, and DCs were cultured during for 2 additional days.

DC treatments

Once the monocytes were purified and before the addition of DC-differentiating cytokines, ROSI, 9cRA, RXR agonist HX630, RAR agonist CH55, or LXR agonist 24,25-epoxycholesterol were added at the indicated concentrations. When necessary, 10 µM PPAR{gamma} antagonist GW9662 or 1 µM RXR pan-antagonist HX603 were added to cultures and incubated for 90 min at 37°C 5% CO2 before any other treatment. GW9662 addition was repeated on day 3 and only, in LPS maturation, on day 6.

Flow cytometry

For analysis of DC phenotype, DCs were collected and mAbs directly conjugated to FITC or PE were subsequently used. The FITC-conjugated mouse mAbs were anti-CD1a, anti-CD83, anti-HLA-DR and anti-CD36 (BD Pharmingen). The PE-conjugated mouse mAbs were anti-CD14, anti-CD80, anti-TLR4, and anti-CD86 (BD Pharmingen). Data for at least 5 x 103 DCs region/sample were acquired and analyzed on a FACScan flow cytometer using CellQuest (BD Biosciences), FlowJo (Tree Star), Modfit (Verity Software), median fluorescence intensity (MFI) software (from Eric Martz Formula www.umass.edu/microbio/mfiFormula ), and WinMDI software.

Carboxyfluorescein diacetate succinimidyl ester staining

To determine proliferation, lymphocytes obtained as described were thawed and quickly washed with PBS to eliminate DMSO. Cells were incubated for 15 min at 4–8°C with DNase and MgCl2 to avoid DNA debris. Then they were incubated with 2.5 µM carboxyfluorescein diacetate succinimidyl ester (Molecular Probes) for 10 min in a rocker at 100 rpm. After this, the same volume of FCS was added, allowed to rest for 3 min and, washed twice with PBS/10% FCS and once with PBS alone.

Dendritic cells autologous stimulation of lymphocytes and MLR

Primary DCs were pulsed with 1 µg/ml, 100 ng/ml, or 10 ng/ml SEB Ag (staphylococcal enterotoxin B; Sigma-Aldrich) for 2 h at 37°C, in a humid atmosphere of 5% CO2 before the coincubation with lymphocytes. Afterward, SEB-pulsed DCs and nonpulsed DCs for MLR were irradiated with a gamma radiation source (25 Gy) to prevent non-CFSE lymphocyte proliferation. Stimulation was conducted in 96-well culture plates in 200 µl of RPMI 1640 containing 10% inactivated FCS and antibiotics. DCs were mixed with 1 x 105 autologous CFSE-labeled lymphocytes at a ratio DC/lymphocyte 1:10 for SEB-pulsed DCs and 40:100 for MLR. After 96 h of proliferation the cells were collected and analyzed in a FACScan flow cytometer using CellQuest (BD Biosciences), FlowJo proliferation platform (Tree Star), Modfit (Verity software) and WinMDI 2.8 (Joseph Trotter). A proliferation index (a measure of the median number of divisions for each lymphocyte in the population) was obtained.

Chemotaxis assay

Five x 105 cells were seeded into a Transwell chamber (5 µm; Millipore) in a 12-well plate and migration to MIP3beta (10 ng/ml; PeproTech) was analyzed after 1 h by counting the migrated cells by gating DCs in a FACSCalibur cytometer for 4 min.

Analysis of endocytic capacity

For the analysis of endocytic activity, 2 x 105 cells were incubated with FITC-dextran (Sigma-Aldrich) for 30 min at 37°C. As a control, 2 x 105 cells were precooled to 4°C before the incubation with dextran at 4°C for 30 min. The cells were washed four times and analyzed on a FACSCalibur cytometer.

Measurement of cytokine secretion

On day 7, LPS was added to the DC cultures and 24 h later supernatants were harvested. Also, supernatants from 96-h proliferating lymphocytes were harvested. Samples were frozen at –80°C until use. The human IL-10 and IL-12 levels were determined in DC supernatants using the human IL-12 and IL-10 BD OptEIA ELISA set according to the instructions of the manufacturer (BD Bioscience). The human IFN-{gamma} levels were determined in lymphocyte supernatants using the IFN-{gamma} ELISA Ready-SET-GO from eBioscience (Bionova) according to the instructions of the manufacturer.

Statistics

Statistical analysis was performed using a SPSS 12.0 statistical package. The nonparametric Wilcoxon signed rank test was used for comparisons between control and experimental groups (35). The significance level was placed at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 References
 
9cRA affects normal differentiation from monocyte to immature DC

DCs express retinoid receptors that trigger apoptosis (17), enhance dendritic immunogenicity (17, 18), or fulfil a role in the differentiation process from monocyte to DC (15). Our results indicate that 9cRA, a RXR and RAR nuclear receptor ligand, at higher concentrations than 100 nM trigger apoptosis in immature DCs as already reported (17). At 1 µM 9cRA, >50% of immature DCs died by the 9th day of culture whereas LPS-matured DCs remained alive. However, at concentrations of 100 nM or less, this apoptotic effect was minimized and other effects arose.

To examine these effects, we studied the differentiation process from monocyte to immature DC in the presence or absence of 9cRA or the specific RXR agonist HX630 (Fig. 1A and Table I). In these conditions, untreated monocytes differentiated to normal immature DCs that displayed low CD86 and CD80, no CD83 or CD14, and were CD36+, HLA-DR+ and CD1a2+. However, these agonists impacted the phenotype of immature DCs, increasing the maturation markers CD86, HLA-DR and CD83, and decreasing CD1a (completely blocked), CD80 and CD36 whereas still remaining CD14–.


Figure 1
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  		FIGURE 1. A, Phenotypic changes in the differentiation process from monocyte to immature DC in the presence of low concentrations of 9cRA (10 nM), ROSI (1 µM), and HX630 (1 µM) with or without GW9662 (10 µM), a PPAR{gamma} antagonist, and HX603 (1 µM), a RXR antagonist. B, Same studies but in the presence of the RAR agonist CH55 and of the LXR agonist 24,25-epoxycholesterol (eCH). Results are the MFI means from the analysis by flow cytometry of DCs obtained from at least six different buffy coats. Results from immature DCs were collected on day 6 of culture of adherent monocytes with GM-CSF and IL-4.

 

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  		Table I. Impact of 9cRA concentrations on immature and mature DC phenotypea

 
To further study whether PPAR{gamma}-RXR or RXR-RXR were involved in the mechanism induced by the 9cRA and RXR ligands on DCs, the PPAR{gamma}-specific inhibitor, GW9662 or the RXR pan-antagonist HX603 were tested. GW9662 inhibits PPAR{gamma} activation covalently and selectively with regard to PPAR{alpha} and beta, blocks heterodimer formation (36) and has been extensively used to determine PPAR{gamma} inhibition effects in different kinds of cells (36, 37, 38, 39, 40, 41, 42, 43, 44). In the presence of GW9662, 9cRA activation would be expected to be blunted if the pathway is PPAR{gamma}-RXR dependent. HX603 should be able to impair 9cRA actions both when acting via PPAR{gamma}-RXR heterodimer and other RXR-containing dimers.

As can be observed in Fig. 1, the addition of GW9662 or HX603 effectively blocked 9cRA effects on the expression of CD86, CD80, and CD36 (p < 0.05) in immature dendritic cells, whereas the down-regulation effect on CD1a expression was partially inhibited (p < 0.05). This blocking effect was stronger for HX603 suggesting that both homo- and heterodimers could be involved in the process. Finally, HX603 up-regulated CD80 expression (p < 0.05) with regard to controls unlike GW9662 which reduced it (p < 0.05).

The results obtained for 9cRA were extraordinarily similar to those already reported by Nencioni et al. (33) obtained when DCs were treated with the PPAR{gamma} agonist ROSI. To identify in our system whether the activities mediated via RXR were the same as those induced through PPAR{gamma}, we repeated the experiments with ROSI instead of 9cRA (Table II). As it can be observed, immature DCs treated with 9cRA or ROSI behave in a similar manner except for the increments in HLA-DR and CD83 depicted in 9cRA but not in ROSI-treated DCs. RXR agonist HX630 showed the same effects performed by 9cRA and PPAR{gamma} agonists (Fig. 1A).


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  		Table II. Impact of rosiglitazone concentrations on immature and mature DC phenotypea

 
These phenotypical changes were accompanied by morphological variations in which DC treated with 1, 10, or 100 nM 9cRA were morphologically long shaped resembling mature ones. These morphological changes were reproduced with the RXR agonist HX630 but not with the PPAR{gamma} agonist ROSI at these levels. Morphological changes were inhibited by both the PPAR{gamma} antagonist GW9662 and the RXR antagonist HX603 (data not shown).

9cRA modulates endocytosis capacity, CCR7 expression, and MIP3beta chemotaxis in DCs

As the changes induced by 9cRA suggested a more mature differentiated phenotype of the immature DCs, the expression of the chemokine receptor CCR7 (usually up-regulated in mature DCs) and the chemotactic capacity to its ligand, MIP3beta of the 9cRA treated immature and mature DCs was assayed. As can be observed in Fig. 2A, 9cRA induced a slight increase in the expression of CCR7 in immature DCs, whereas in mature DCs both 9cRA and ROSI reduced the expression in a statistically significant manner (p < 0.05). This process was unaffected by GW9662 (data not shown). The migratory capacity to MIP3beta displayed a correlation with CCR7 expression in both immature and mature DCs (Fig. 2B).


Figure 2
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  		FIGURE 2. A, Expression of CCR7 on immature and mature DCs treated with 9cRA (10 nM) or ROSI (1 µM). B, Chemotaxis induced by MIP3beta on immature and mature DCs. C, FITC-dextran uptake by 9cRA (10 nM), ROSI (1 µM), or GW9662 (10 µM) treated immature DCs. Mean of 14 different assays. Results are expressed as mean of MFIs of the different flow cytometry assays. Statistical analysis was Wilcoxon signed rank test, *, p < 0.05 (different from untreated).

 
We also tested the FITC-dextran endocytic capacity of treated immature DCs. As shown in Fig. 2C, treatments with 9cRA and ROSI yielded variations that were not significant, whereas GW9662 induced the most striking effects (p < 0.05).

Maturation process in DCs is also altered by 9cRA

LPS-matured DCs displayed a phenotype characterized by CD862+, HLA-DR2+, CD83+, CD80+, CD1a+, with CD36 expressed at very low levels (Fig. 3A and Table I). Surprisingly, as opposed to the effects displayed in some markers on immature DCs, when DCs were matured with LPS in the presence of 9cRA, all studied costimulation and Ag presentation markers were down-regulated in a concentration-dependent manner with respect to the untreated DCs except by the slight up-regulation of CD36 (p < 0.05). These effects were not dependent on the expression of TLR4, which was unaffected (data not shown). Furthermore, similar results to those obtained for LPS maturation were found using other maturation stimuli as poly(I:C) and TNF-{alpha} except in the case of CD86 in TNF-{alpha} maturation, which was slightly up-regulated (Fig. 3B).


Figure 3
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  		FIGURE 3. A, Phenotypic changes in the differentiation process from monocyte to mature DCs in presence of 9cRA (10 nM), ROSI (1 µM), and HX630 (1 µM) with or without GW9662 (10 µM) and HX603 (1 µM). B, Same studies but in function of the maturation stimuli used: LPS, TNF-{alpha} or poly(I:C). C, Same studies but in the presence of the RAR agonist CH55 or the LXR agonist 24,25-epoxycholesterol (eCH) on LPS-matured DCs. On 6th day LPS (or where indicated other maturation stimuli) was added to the culture, and on day 8, mature DCs from at least 6 different buffy coats were collected and analyzed. GW9662 (10 µM) was added on days 1, 3, and 6 (the last time was 90 min before maturation stimuli) except in GW9662 2T in which the addition of GW9662 was only on the 1st and 3rd day.

 
GW9662 and HX603, independently of the maturation stimulus used, were able to block the changes displayed by 9cRA (Fig. 3A). CD86 in 9cRA-treated DCs did not increment its expression in presence of HX603 or GW9662 (p < 0.05). The same happened with CD83, HLA-DR, and CD80 (p < 0.05), but surprisingly, CD36, a hallmark of PPAR{gamma} activation, was up-regulated by both antagonists (p < 0.05). In the case of CD1a, the presence of GW9662 or HX603 partially blocked the inhibition induced by both 9cRA and ROSI during the differentiation and maturation process (p < 0.05).

GW9662 and HX603 produced effects on mature DCs by themselves as well. In untreated DCs, they retained immature phenotypes by blocking important hallmarks of the maturation process, suggesting that activation of PPAR{gamma}-RXR could be important for DC maturation in its natural physiological process (Fig. 3A). In this sense, CD86 and CD83 were completely down-regulated whereas CD80 down-regulation was only partial (p < 0.05). Both compounds up-regulated CD36 expression (p < 0.05) but have no effect on CD1a expression in LPS-mature DCs. At least GW9662 could act independent of the maturation stimuli, but blocking the maturation with LPS needed three additions of GW9662 (the third, 90 min before LPS addition) whereas poly(I:C) and TNF-{alpha} only needed two (the 1st and 3rd day of differentiation) (Fig. 3B).

According to these results it seems that PPAR{gamma}-RXR nuclear receptor could be acting not only to modulate the immune response but also on the normal development of the maturation process in dendritic cells.

9cRA and ROSI when combined perform additive effects in DCs

The results obtained with 9cRA-treated DCs, similar to those with PPAR{gamma} ligands, point toward an action of 9cRA through the RXR monomer in the PPAR{gamma}-RXR heterodimer. In this case, the simultaneous activation of each partner could yield complementary effects. To check this hypothesis, DCs were treated with low concentrations of 9cRA (1 nM) and ROSI (100 nM), which themselves alone showed similar slight effects on DCs. When both compounds were added together to the DC cultures, then additive effects arose (Fig. 4). For immature DCs (Fig. 4A), CD86, HLA-DR, and CD36 displayed additive effects with the combination treatment (p < 0.05). CD1a and CD80 were highly down-regulated (p < 0.05) whereas when cultured with ROSI or 9cRA alone, the effects were partial.


Figure 4
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  		FIGURE 4. Phenotypic additive effects obtained by combining ROSI and 9cRA on DCs. MFI mean from eight healthy individuals. DCs were incubated with 1 nM 9cRA and/or 100 nM ROSI. These compounds were present in culture medium from the 1st day of transformation from monocyte to DC. In A, immature DCs, at day 6, were collected and analyzed with a FACScan (BD Biosciences) for each studied marker. In B the same, but with 2 additional days of culture with LPS for mature DCs. Statistical analysis was Wilcoxon signed rank test, *, p < 0.05 (different from untreated).

 
In mature DCs, the behavior of the markers was additive as well (Fig. 4B). CD86 down-regulated its expression in the presence of the 9cRA-ROSI combination, even though the monocomponent treatment alone displayed no effect. CD83, HLA-DR, and CD80 were also inhibited when both compounds were present at the same time. CD1a was more deeply affected, because both compounds individually had already strong inhibitory activity on its expression. These effects were dose-dependent for both mature and immature DCs (data not shown).

The agonists of other nuclear receptors act in a similar but not identical manner as 9cRA

Additionally, as other nuclear receptors are known to heterodimerize with RXR and in the case of RAR can be itself activated by 9cRA, the effects that other agonists of nuclear receptors could perform on DCs were tested—receptors like 24,25-epoxycholesterol (for LXR) and CH55 (for RAR). We found that all of them were able to up-regulate CD86 expression in immature DCs and down-regulate CD80, CD36, and CD1a (Fig. 1B). When the DCs were matured with LPS in presence of RAR and LXR agonists, CD1a, CD80, and CD86 were down-regulated in a dose-dependent way (p < 0.05) (Fig. 3C). CD36, unlike 9cRA and ROSI treatments, was down-regulated by these nuclear receptor agonists (p < 0.05).

9cRA modulates stimulation of autologous lymphocytes by DC and MLR which correlates with IFN-{gamma} secretion

As the 9cRA-treated mature DCs displayed a lower expression of costimulatory and Ag presentation molecules, the DC lymphocyte stimulating capacity could be altered. Therefore lymphoproliferation triggered by these DCs was investigated. CFSE-labeled lymphocytes were cocultured with SEB Ag pulsed and irradiated DCs, which had been maturated in the presence of different concentrations of 9cRA and/or ROSI (Fig. 5A and Table III). The lymphoproliferative stimulation capacity of 9cRA-treated DCs decreased in a concentration-dependent manner. ROSI could also inhibit autologous lymphocyte proliferation stimulated by DCs and its combination with 9cRA slightly increased the effects at least with SEB 100 ng/ml. MLR was also inhibited by 9cRA (Table III). All the inhibitory effects on lymphoproliferation were in correlation with a down-regulation in the IFN-{gamma} secretion of the lymphocyte cultures (Fig. 5).


Figure 5
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  		FIGURE 5. A, Histograms of a lymphoproliferation triggered by 100 ng/ml SEB pulsed and irradiated DCs representative from five healthy individuals. All the histograms have been compared with control (white histogram), and the gray histograms are: 10 µM GW9662-treated DCs (I), 10 µM GW9662 plus 10 nM 9cRA (II), 10 µM GW9662 plus 1 µM ROSI (III), 1 µM ROSI (IV), 1 nM 9cRA (V), 10 nM 9cRA (VI), 100 nM 9cRA (VII), 10 nM 9cRA and 1 µM ROSI (VIII). The ratio DC/lymphocyte was 1:10 beginning with 100,000 lymphocytes. B, Secretion of IFN-{gamma} by lymphoproliferations stimulated by 100 ng/ml SEB pulsed and irradiated DCs with different treatments. Statistical analysis was Wilcoxon signed rank test, *, p < 0.05 (different from untreated).

 

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  		Table III. Impact of 9cRA, rosiglitazone, and GW9662 in SEB and MLR DC lymphoproliferationsa

 
The altered expression of the surface costimulation molecules CD83, CD80, and CD86 obtained with GW9662-treated DCs (Figs. 1A and 3A), could reasonably suggest an inhibitory role on the DCs ability to activate lymphoproliferation. Surprisingly, when GW9662 was present in the differentiation process from monocyte to DC, the SEB lymphoproliferation and IFN-{gamma} secretion levels were similar to or even higher than those of the untreated DCs (Table III, Fig. 5) independently of the presence of 9cRA or ROSI. Unlike the results with SEB-induced lymphoproliferations, MLR induction was effectively down-regulated by GW9662 (Table III).

Cytokine pattern expression is altered by 9cRa

Due to phenotypic changes, further characterization of the 9cRA-treated mature DCs was performed. Therefore, the cytokine secretion pattern on 9cRA-treated mature DCs was studied. IL-12 and IL-10 were evaluated in supernatants of DCs cultured for 24 h with LPS (Fig. 6). We found that in 9cRA-treated DCs the secretion of IL-12p70 was diminished in a dose-dependent manner (70–80%) (p < 0.05), while IL-10 secretion did not show significant variations with respect to untreated mature DCs. The behavior observed with ROSI-treated DCs was similar to that described earlier (33) (Fig. 6).


Figure 6
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  		FIGURE 6. ELISA displaying IL-12 and IL-10 secretion at different 9cRA and ROSI concentrations in the presence or absence of the PPAR{gamma} antagonist GW9662. GW9662 2T means addition of GW9662 on the 1st and 3rd day. GW9662 3T means addition of GW9662 on days 1, 3, and 6 (last time 90 min before maturation stimuli). All the compounds were present in the culture medium from the 1st day of transformation from monocyte to DC. At day 6, LPS was added to the medium, and 24 h later supernatants were collected and congealed at –80°C until evaluation through ELISA. Statistical analysis was Wilcoxon signed rank test, *, p < 0.05 (different from untreated).

 
The effects of GW9662 on the cytokine pattern expression of DCs were also studied. Addition of GW9662 during the differentiation process from monocytes to immature DCs but not before LPS addition, made DCs to inhibit IL-10 secretion whereas IL-12 remained at untreated DCs secretion levels (p < 0.05) (Fig. 6). However, with a third GW9662 addition before LPS stimulus (GW9662 3T), neither IL-12 nor IL-10 cytokine secretion could be detected (p < 0.05) suggesting that GW9662 is able to completely block the cytokine secretion process. Moreover, in the presence of GW9662, the ROSI- or 9cRA-treated DCs displayed the same cytokine profile as GW9662-treated DCs indicating that the 9cRA and ROSI effects on cytokine secretion were also blocked.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 References
 
Retinoids have recently been the subject of an exhaustive study as their roles in differentiation, development, and homeostasis in many different cell types are elucidated (10). 9cRA stands out from other retinoids because of its ability to activate RXR in addition to RAR nuclear receptors. In DCs, retinoids act by triggering the expression of retinoid receptors along their differentiation from monocytes (15, 16, 17), have been used to obtain DCs in absence of IL-4 (18), induce DC apoptosis through a RAR-RXR pathway at high concentrations (17) and, through a crosstalk with inflammatory cytokines, trigger membrane MHC II and costimulatory molecule expression enhancing Ag-specific T cell response through a RXR-dependent, RAR-independent pathway (17).

In this report, we focused on the possibility that low concentrations of 9cRA could exert an effect in DC differentiation from monocyte. As RXR Kd for 9cRA is between 5 and 15 nM (45) we expected to find other effects at a wide range of noncytotoxic 9cRA concentrations below 100 nM. We duly found that 9cRA on immature DCs altered costimulation and Ag presentation markers (Fig. 1 and Table I), triggered a more mature morphology (data not shown) and, slightly increased their chemotactic capacity to MIP3beta (Fig. 2B).

Unexpectedly, in the presence of several maturation stimuli, IL-12 secretion, costimulation and Ag presentation markers were significantly down-regulated whereas CD36 remained up-regulated and IL-10 was secreted at similar levels to untreated DCs. All the changes in the phenotype of 9cRA-treated DCs were consistent with the reduction in their lymphoproliferative capacity and the diminished IFN-{gamma} secretion by lymphocytes (Fig. 5).

In this way, the down-regulation of HLA-DR in mature DCs reduces their immunogenicity. The same behavior was observed for CD83, CD80, and CD86, which are essential for both the T lymphocyte proliferation and the maturation process in DCs (46, 47, 48, 49) and CD1a, which is implicated in the activation of a particular subpopulation of T cells able to produce Th1 cytokines. The up-regulation of CD36, which is under the transcriptional control of PPAR{gamma} (50, 51), also suggests a decreased stimulatory capacity for DCs as this receptor is involved in the apoptotic cell capture, a process that usually avoids the activation of the immune response. It is known that Leishmania donovani and Plasmodium falciparum, use the strategy of modulating CD1 and CD36, respectively, to escape from the immunological response induced by DCs (50, 52). DCs exposed to P. falciparum through the CD36 internalization of infected erythrocytes from malaria patients were not able to mature in vitro when LPS or TNF-{alpha} stimuli were added. MHC-II and costimulatory molecule expression, as well as IL-12 secretion were inhibited. However, IL-10 secretion was incremented (50). Our results, that complement and confirm those obtained by Geissman et al. (17), point toward a bimodal effect of 9cRA in DCs, which would be dependent on the maturation stage.

9cRA is a ligand for RXR, and the PPAR{gamma}-RXR and LXR-RXR heterodimers are know to retain ligand-dependent activation of RXR together with specific PPAR{gamma} or LXR activation (10, 24, 53, 54, 55, 56). It is possible that 9cRA act in DCs via the RXR moiety of PPAR{gamma}-RXR, or LXR-RXR heterodimers (57, 58, 59) or even by activation of RXR homodimers (which have recently been described to be able to bind to functional PPAR response elements) (22, 60). Eventually, because 9cRA is a potent ligand for RAR, activation through this pathway is likely as well.

Our studies found important similarities in the action of the nuclear receptors RAR, LXR, RXR, and PPAR{gamma} (Figs. 1B and 3B), which could suggest the existence of a common pathway of action in DCs. These facts are in agreement with a widespread point of view that there is a close relation among all these receptors, suggesting a complex and tight regulation dependent on secondary messengers and cofactors. This regulation has already been described in macrophages and Wnt signaling in other cell types (61, 62) but, to our knowledge, not until now in DCs. In this sense, there would be a nuclear receptor modulation of the DC response depending on the external signaling or the extracellular medium composition, together with an intrinsic stage of the cells.

Increasing evidence suggests that PPAR{gamma} may be an important regulator of many immunogenic functions in monocytes, macrophages, lymphocytes, and DCs (25, 26, 27, 29, 30, 31, 32, 33, 34, 63, 64, 65, 66). It has been proposed that PPAR{gamma} activators have the ability to switch DC toward a less stimulatory mode of Th2 (32) or, even more, to an induction of anergy/tolerance in T lymphocytes (33). The results depicted here for 9cRA-treated DCs are extraordinarily similar to those already reported for PPAR{gamma} activators in DCs (31, 32, 33, 34) and lymphocytes (42) (see Table II, Figs. 5 and 6).

As additive or synergistic effects on glucose, lipid metabolism and gene activation have been described for RXR and PPAR{gamma} ligands (67, 68, 69, 70, 71, 72, 73), experiments of coincubation of 9cRA with ROSI were performed (Fig. 4). At concentrations in which each compound alone had little effect on DCs, the combination of both compounds displayed potent effects on the studied DCs markers, suggesting that 9cRA could be acting through RXR and PPAR{gamma} heterodimers.

Finally, the PPAR{gamma}-specific inhibitor GW9662 and the RXR pan-antagonist HX603 were used to test whether 9cRA could be acting through RXR and PPAR{gamma} heterodimers. Both GW9662 and HX603 inhibited most phenotypic alterations induced not only by ROSI, but also those by 9cRA whatever maturation stimuli was used (Figs. 1 and 3), suggesting that the 9cRA mechanism of action should involve the PPAR{gamma}-RXR heterodimer and/or others RXR-containing dimer pathways.

Unexpectedly, when GW9662 or HX603 were tested alone, DCs displayed a significant different phenotype from the untreated DCs. GW9662- or HX603-treated DCs maturated with LPS, poly(I:C), or TNF-{alpha}, showed no CD86 and a lower expression of CD83, HLA-DR, and CD80 compared with controls. CD36 was strongly up-regulated and CD1a down-regulation was partially blocked. This phenotype was similar to that of immature DCs except for CD80, which was expressed at higher levels in GW9662- and HX603-treated DCs. These effects were reproduced with LPS, TNF-{alpha} or poly(I:C) stimuli but only LPS maturation required a third addition of GW9662 to block costimulation markers up-regulation. Additionally, with two additions of GW9662, DCs secreted normal levels of IL-12, but an IL-10 production deeply inhibited, independently of the presence of 9cRA or ROSI in the culture, but with a third addition 90 min before LPS, there was no cytokine secretion (Fig. 6). This would suggest an effect of the LPS-TLR4 pathway on PPAR{gamma} expression or activation which has been reported in other cell types included monocytes (74, 75). In this sense, a third addition of GW9662 before LPS would be necessary to avoid the production and activation of PPAR{gamma} de novo in DCs, which—in accordance with our results—would be enough to unleash IL-12 secretion but not enough to repress this process or to increment IL-10 secretion in sufficient levels. Further supporting this hypothesis are the results obtained in the lymphoproliferations induced with GW9662-treated DCs. GW9662 is able to induce more potent lymphocyte activations than even the controls when SEB is used and whose role could be through activation and up-regulation of TLR4 (76, 77) and where an inhibition of lymphoproliferation is obtained with the same DCs in MLR where TLRs would not play any role (Table III). Once again this surprising effect of GW9662 suggests that it is likely that PPAR{gamma} plays an unforeseen physiological role in these processes.

From these results, it could be speculated that, as has already been suggested in lymphocytes (42), PPAR{gamma}-RXR heterodimer should play some kind of necessary role in the DC maturation process, which could be controlled by endogenous PPAR{gamma} ligands. Futhermore, these results show the pivotal role of RXR activation via PPAR{gamma}-RXR or other ligand-dependent RXR hetero- or homodimers in the control of DC regulation.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 References
 
The results presented here show that treatment with PPAR{gamma} and RXR inhibitors prevents maturation of DCs in a normal way despite the maturation stimuli used, suggesting that PPAR{gamma}-RXR could be involved in the DC physiological maturation process. Moreover, 9cRA interferes in the differentiation process from monocyte to DC, leading DCs to a less stimulatory mode, which is accompanied by an impaired IL-12 and a normal IL-10 secretion. These effects were similar to those reported for PPAR{gamma} activators, which have been confirmed in our work except for IL-10 secretion. Finally, although these results suggest a 9cRA mechanism through PPAR{gamma}-RXR and/or other RXR homodimer and heterodimer activation, further investigation to elucidate its molecular mechanisms of action is required.


   Acknowledgments
 
We thank Dr. Andrew Bantly for excellent technical assistance in CFSE proliferation cytometry and Dr. Anabel Fernández-Valledor for assistance in the LXR tests.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo FISS01/0880. F. Z.-G. was the recipient of a fellowship from the University of Barcelona. Back

2 Address correspondence and reprint requests to Dr. Joan Domingo, Department of Biochemistry and Molecular Biology, University of Barcelona, Avenida Diagonal 645 Edificio Nuevo, Planta-1, 08028 Barcelona, Spain. E-mail address: jcdomingo{at}ub.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; 9cRA, 9-cis-retinoid acid; ATRA, all-trans-retinoid acid; RAR, retinoid acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; SEB, staphylococcal enterotoxin B; ROSI, rosiglitazone; MFI, median fluorescence intensity. Back

Received for publication November 16, 2005. Accepted for publication March 9, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
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