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The Journal of Immunology, 2002, 169: 1228-1235.
Copyright © 2002 by The American Association of Immunologists

Dendritic Cell Immunogenicity Is Regulated by Peroxisome Proliferator-Activated Receptor {gamma}1

Alessio Nencioni, Frank Grünebach, Anke Zobywlaski, Claudio Denzlinger, Wolfram Brugger and Peter Brossart2

Department of Hematology, Oncology, and Immunology, University of Tübingen, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are the most potent APCs known that play a key role for the initiation of immune responses. Ag presentation to T lymphocytes is likely a constitutive function of DC that continues during the steady state. This raises the question of which mechanism(s) determines whether the final outcome of Ag presentation will be induction of immunity or of tolerance. In this regard, the mechanisms controlling DC immunogenicity still remain largely uncharacterized. In this paper we report that the nuclear receptor peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}), which has anti-inflammatory properties, redirects DC toward a less stimulatory mode. We show that activation of PPAR-{gamma} during DC differentiation profoundly affects the expression of costimulatory molecules and of the DC hallmarker CD1a. PPAR-{gamma} activation in DC resulted in a reduced capacity to activate lymphocyte proliferation and to prime Ag-specific CTL responses. This effect might depend on the decreased expression of costimulatory molecules and on the impaired cytokine secretion, but not on increased IL-10 production, because this was reduced by PPAR-{gamma} activators. Moreover, activation of PPAR-{gamma} in DC inhibited the expression of EBI1 ligand chemokine and CCR7, both playing a pivotal role for DC migration to the lymph nodes. These effects were accompanied by down-regulation of LPS-induced nuclear localized RelB protein, which was shown to be important for DC differentiation and function. Our results suggest a novel regulatory pathway for DC function that could contribute to the regulated balance between immunity induction and self-tolerance maintenance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The current model for the generation of Ag-specific immune responses predicts that specialized APCs, e.g., dendritic cells (DC),3 acquire Ags in the periphery and migrate to the afferent lymph nodes (1, 2, 3). In this work, DC present to T lymphocyte Ag-derived epitopes in the context of MHC class I and class II molecules. Cumulating evidence suggests that tissue-resident DC might acquire tissue Ags and migrate to the afferent lymph nodes even during the steady state (3), and it has been suggested that the constitutive presentation of self-Ags may contribute to maintain self-tolerance (3, 4, 5, 6, 7). Several factors have been proposed that could determine whether the immune system will be primed or tolerized against a defined Ag (reviewed in Ref. 3). These include the amount of Ag, the immunogenic effects of pathogen-derived products or danger signals capable of activating DC, and the existence of different subsets of DC, with different immunological properties. In this context, Albert et al. (7) have recently demonstrated that mature DC are required for both cross-priming and cross-tolerance, thus indicating that still-undefined signal(s) probably determine the final outcome.

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear transcription factors which, upon binding of the ligand, form heterodimers with the receptor for 9-cis retinoid acid, retinoid X receptor, and subsequently activate target gene transcription (8). So far, three isotypes for these receptors have been identified: {alpha}, {beta}, and {gamma}. PPAR-{alpha} expression can be detected in heart, liver, kidney, adipose tissue, and skeletal muscle. PPAR-{beta} expression appears to be less selective, as this receptor can be found in many tissues (9). PPAR-{gamma} is expressed at high levels in adipose tissue, where it exerts critical effects by promoting adipocyte differentiation and regulating tissue homeostasis (10, 11). Natural ligands of PPAR-{gamma} include the cyclopentenone metabolites of PGD2, PGJ2, and 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) (12, 13, 14), some unsaturated fatty acids (9, 10, 11, 12), and oxidized phospholipids (15). Furthermore, PPAR-{gamma} was recognized as the molecular target of the thiazolidinedione class of antidiabetic drugs (12, 16, 17), which are currently being used for the treatment of type II diabetes.

Detection of PPAR-{gamma} expression in hematopoietic cells (18) suggested a broader range of function for this receptor. It was demonstrated that some PPAR-{gamma} ligands, such as the cyclooxygenase-2-derived cyclopentenone PGs, possess strong anti-inflammatory properties and play a key role in the resolution of inflammation (19, 20). The cyclopentenone 15d-PGJ2 and other PPAR-{gamma} agonists prevent macrophage oxidative burst and lead to impaired cytokine production in monocytes and macrophages (21, 22, 23, 24). Ligand activation of PPAR-{gamma} induces caspase activation and apoptotic cell death in human activated macrophages (25). Moreover, consistent with their emerging anti-inflammatory properties, PPAR-{gamma} ligands inhibit colitis development in animal models of inflammatory bowel disease (26, 27).

Interestingly, PPAR-{gamma} was recently shown to inhibit lymphocyte activation and to favor lymphocyte apoptotic cell death (28, 29, 30). PPAR-{gamma} expression could also be detected both in mouse and human DC (31, 32). In this context, it was suggested that PPAR-{gamma} activation might contribute to redirect Th2 immune responses due to a down-regulated IL-12 secretion and to a selective inhibition of Th1 lymphocyte-recruiting chemokines in DC (32). These findings suggest that PPAR-{gamma} might also play a role in the regulation of responses mediated by adaptive immunity effectors.

In the present work, we have explored the effect of PPAR-{gamma} on the differentiation of human monocytes into DC. We show that activation of PPAR-{gamma} affects DC properties and reverts them to a less stimulatory mode, possibly via inhibition of RelB, an NF-{kappa}B family member playing an important role in DC development and function.


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

The medium used for cell cultures was RPMI 1640 supplemented with 10% inactivated FCS, 50 nM 2-ME, and antibiotics, all purchased from Life Technologies (Grand Island, NY). The cell line Croft is an EBV-immortalized HLA-A2+ B cell line kindly donated by O. J. Finn (University of Pittsburgh, Pittsburgh, PA). GM-CSF (Leucomax) was from Novartis (Basel, Switzerland). LPS was obtained from (Sigma-Aldrich, Deisendorf, Germany). IL-2, IL-4, IL-12, and TNF-{alpha} were purchased from R&D Systems (Wiesbaden, Germany). Troglitazone (TGZ) and BRL49653 were kindly donated by Sankyo (Tokyo, Japan) and GlaxoSmithKline, respectively. 15d-PGJ2 was from Biomol (Plymouth Meeting, PA).

DC generation

DC were generated from peripheral blood adhering monocytes as described previously (33). In brief, PBMC were isolated by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation of heparinized blood from buffy coat preparations of healthy volunteers. Cells were seeded (1 x 107 cells/well) into six-well plates (Costar, Cambridge, MA) in medium. After 2 h of incubation at 37°C, nonadhering cells were removed and adherent monocytes were cultured in medium supplemented with GM-CSF (100 ng/ml) and IL-4 (1000 U/ml). Differentiating DC were fed with cytokines every 2–3 days. In some experiments DC were further induced to mature by adding LPS (100 ng/ml) at day 6 of culture. TGZ, BRL49653, or 15d-PGJ2 were added to the culture medium starting from the first day together with GM-CSF and IL-4. DC were enumerated by flow cytometry as lineage (CD14, CD3, CD19) negative and HLA-DR bright, and purity was confirmed by morphology. Furthermore, analysis of the expression of the DC markers CD1a and CD83 was performed.

Immunostaining

Cells were stained using FITC- or PE-conjugated mouse mAbs against CD14, CD80, and CD54 (BD Biosciences, Heidelberg, Germany); CD36, CD40, and CD86 (all purchased from BD PharMingen, Hamburg, Germany); CD1a (DAKO, Hamburg, Germany); CD83 (Immunotech, Marseille, France); and mouse IgG isotype control. All flow cytometry analysis were performed on a FACSCalibur (BD Biosciences).

MLR assay

A total of 105 responding cells from allogeneic PBMC were cultured in 96-well flat-bottom microplates (Nunc, Roskilde, Denmark) with various numbers of stimulator cells. Thymidine incorporation was measured on day 5 by a 16-h pulse with [3H]thymidine (0.5 µCi/well; Amersham Life Science, Little Chalfont, U.K.).

Induction of Ag-specific CTL response using the HLA-A2-restricted peptide E75 from Her-2/neu

The induction of Her-2/neu-specific CTL was performed as described (33). The Her-2/neu-derived peptides E75 (369–377: KIGSFLAFL) and GP-2 (654–662: IISAVVGIL) were synthesized using standard F-moc chemistry on a peptide synthesizer (432A; Applied Biosystems, Weiterstadt, Germany) and analyzed by reversed-phase HPLC and mass spectrometry. For CTL induction, 5 x 105 DC were pulsed with 50 µg/ml E75 peptide for 2 h, washed, and incubated with 3 x 106 autologous PBMC with or without addition of IL-12 (10 ng/ml). After 7 days of culture, cells were restimulated with autologous peptide-pulsed PBMC, and 1 ng/ml IL-2 was added on days 1, 3, and 5. The cytolytic activity of induced CTL was analyzed on day 5 after the last restimulation in a standard 51Cr-labeled release assay.

CTL assay

The standard 51Cr-labeled release assay was performed as described (33). Target cells (Croft cells) were pulsed with 50 µg/ml peptide for 2 h and labeled with 51Cr for 1 h at 37°C. Cells (104) were transferred to a well of a round-bottom 96-well plate. Varying numbers of CTLs were added to give a final volume of 200 µl and were incubated for 4 h at 37°C. At the end of the assay, supernatants (50 µl/well) were harvested and counted in a beta-plate counter. The percentage of specific lysis was calculated as follows: 100 x (experimental release - spontaneous release/maximal release - spontaneous release). Spontaneous and maximal releases were determined in the presence of either medium or 1% Triton X-100, respectively.

Cytokine determination

Cytokine concentrations in supernatants from DC cultures were measured with commercially available two-site sandwich ELISAs from R&D Systems (IL-15) or Immunotech Diagnostics (Hamburg, Germany; IL-12, IL-4, IL-10, IL-6, IFN-{gamma}, and TNF-{alpha}), according to the manufacturer’s instructions. DC were incubated at 1 x 106/well in 2 ml medium and stimulated with different cytokine combinations. Supernatants were harvested after 24 h and stored at -70°C until use for cytokine determination.

RT-PCR

RT-PCR was performed with some modifications as previously described (33, 34, 35). Total RNA was isolated from cell lysates using the High Pure RNA Isolation kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. This protocol includes a DNase incubation that digests contaminating DNA. For standardization of the various PCR experiments 250 ng of total RNA were subjected to a 20-µl cDNA synthesis reaction (First Strand cDNA Synthesis kit for RT-PCR; Roche Diagnostics). Oligo(dT) was used as primer. A total of 2 µl of cDNA were used for PCR amplification. To control the integrity of the RNA and the efficiency of the cDNA synthesis, 1 µl of cDNA was amplified by an intron-spanning primer pair for the {beta}2-microglobulin ({beta}2m) gene. PCR temperature profiles and primer sequences were described elsewhere (31), except for {beta}2m, IL-12 p40, and PPAR-{gamma}. Primers were as follows: {beta}2m, 5'-GATGCTGCTTACATGTCTCGA-3' and 5'-GGGTTTCATCCATCCGACAT-3'; IL-12 p40, 5'-GAGAAATGGTGGTCCTCACCTGTG-3' and 5'-GAGTGTAGCAGCTCCGCACGTC-3'; PPAR-{gamma}, 5'-CAGAAATGACCATGGTTGACAC-3' and 5'-ATCCTTCACAAGCATGAACTCC-3'. PCR temperature profiles were as follows: 5-min pretreatment at 94°C and 22 cycles at 94°C for 15 s, and annealing at 55°C for 30 s and 72°C for 30 s for the {beta}2m cDNA; 5-min pretreatment at 94°C, 35 cycles at 94°C for 15 s, and annealing at 60°C for 30 s and 72°C for 30 s for the IL-12 p40 and PPAR-{gamma} cDNA. A total of 10 µl of the RT-PCR were electrophoresed on a 3% agarose gel and stained with ethidium bromide for visualization under UV light.

Preparation of nuclear extracts

Nuclear extracts were prepared from DC as described previously (31). Briefly, cell pellets were washed in 1 ml of ice-cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM PMSF, and 1 mM DTT) and incubated for 10 min on ice in 1 ml buffer A plus 0.4% Igepal CA-630 (Sigma, Munich, Germany). Cell membranes thus obtained were centrifuged at 750 x g for 5 min. Pellets were resuspended in 200 µl buffer B (20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, and 1 mM DTT) and nuclei were mechanically lysed for 2 h at 4°C. Cell debris were pelleted for 15 min at 7500 x g, and supernatant was recovered and stored at -70°C until use. Proteinase inhibitors (aprotinin and leupeptin; Sigma) were added to buffers just before use.

PAGE and Western blotting for detection of RelB protein

Protein concentration of nuclear extracts were determined using a bicinchoninic acid assay (Pierce, Rockford, IL). Twenty micrograms of total protein were separated on a 12% polyacrylic amide gel, blotted on a polyvinylidene difluoride membrane, and probed with a polyclonal rat RelB Ab C-19 (Santa Cruz Biotechnology, Santa Cruz, CA); bands were visualized by ECL staining (Amersham Pharmacia, Freiburg, Germany).

Western blotting for detection of PPAR-{gamma} expression

Cell pellets were boiled with 5x Laemmli buffer for 5 min and 7.5% SDS-PAGE was performed. Thereafter, proteins were transferred to nitrocellulose by electroblotting. The nitrocellulose membranes were incubated with the first Ab (anti-PPAR-{gamma}; WAK-Chemie Medical, Bad Homburg, Germany; kindly provided by J. Auwerx, Institut Pasteur, Paris, France) overnight at 4°C. Membranes were washed before incubating with HRP-conjugated secondary Ab for 1 h at room temperature. Bands were visualized by ECL staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR-{gamma} is expressed in circulating human monocytes and in human DC at each stage of maturation

To determine whether PPAR-{gamma} may play a role in DC differentiation and function, we first analyzed its expression at mRNA and protein levels (Fig. 1Go) in peripheral blood adherent monocytes and in different DC populations. In agreement with a previous report (36), peripheral blood monocytes were found to express low levels of PPAR-{gamma}. DC generated from adherent monocytes cultured in the presence of GM-CSF and IL-4 for 5–7 days were also found to be positive for PPAR-{gamma}, and the expression of this receptor was not affected by the addition of known maturation stimuli, including LPS. PPAR-{gamma} levels of expression were also not influenced by the administration of the PPAR-{gamma} ligands TGZ, BRL49653, or 15d-PGJ2, concomitantly with GM-CSF and IL-4 during DC development.



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  		FIGURE 1. PPAR-{gamma} expression in human monocytes and monocyte-derived DC. A, Adherent monocytes were cultured in different conditions and were further analyzed by RT-PCR for PPAR-{gamma} expression. Lane 1, immediate monocyte harvest and RNA extraction; lane 2, GM-CSF and IL-4 for 6 days; lane 3, GM-CSF and IL-4 for 6 days, LPS at day 6, harvest at day 7; lane 4, GM-CSF, IL-4, and TGZ (10-5 M) for 6 days; lane 5, GM-CSF, IL-4, and BRL49653 (3 x 10-5 M) for 6 days; lane 6, GM-CSF, IL-4, and 15d-PGJ2 (10-5 M) for 6 days. As a positive internal control, RT-PCR amplification for {beta}2m RNA was performed. B, Protein lysates were prepared from 106 adherent monocytes harvested after 2 h of adherence or were stimulated as indicated in A. PPAR-{gamma} expression was detected by immunoblotting.

 
Activation of PPAR-{gamma} pathway skews DC differentiation

In the first series of experiments we explored whether PPAR-{gamma} ligation would have an effect on the normal differentiation of monocytes into DC by monitoring the acquisition of a DC morphology and phenotype. Besides the two synthetic PPAR-{gamma} agonists thiazolidinediones TGZ and BRL49653, we used the natural PG 15d-PGJ2. When cultured in the presence of GM-CSF and IL-4 for 5–7 days, adherent peripheral blood monocytes differentiated into large, round, loosely adherent cells showing the typical cell protrusions in the form of veils or dendrites (data not shown). Addition of PPAR-{gamma} agonists during DC differentiation did not substantially affect the morphological development of DC (data not shown) and led to a significant up-regulation of the target gene CD36 in comparison with the untreated control DC, thus indicating that the PPAR-{gamma} pathway can be activated in DC (Fig. 2GoA). Phenotypic analysis of GM-CSF/IL-4-genarated DC demonstrated loss of CD14 expression and acquisition of a DC phenotype characterized by CD1a and HLA-DR expression; the two costimulatory molecules B7.1 and B7.2 (CD80 and CD86, respectively) were still found to be expressed at low levels. Addition of the PPAR-{gamma} agonists TGZ, BRL49653, or 15d-PGJ2, together with GM-CSF and IL-4 from the first day of culture, skewed DC toward the acquisition of an unusual phenotype, characterized by reduction of CD1a and CD80 expression and selective CD86 up-regulation. CD14 down-regulation as well as CD54 (data not shown) expression were not significantly affected (Fig. 2GoB), whereas HLA-DR was up-regulated and CD40 was slightly reduced.



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  		FIGURE 2. PPAR-{gamma} activators skew the acquisition of a normal DC phenotype. Human peripheral blood adhering monocytes were incubated for 6 days in the presence of GM-CSF and IL-4 with or without TGZ (10-5 M), BRL49653 (3 x 10-5 M), or 15d-PGJ2 (10-5 M). On day 6 cells were washed and analyzed by FACS for CD36 expression (A) and for the expression of DC markers (B). The surface expression level is indicated in B as mean fluorescence intensity.

 
In this context, it has recently been demonstrated that IL-4 leads to up-regulated PPAR-{gamma} expression in macrophages and to production of endogenous PPAR-{gamma} ligands (37). However, the effects on DC phenotype observed in the presence of TGZ, BRL49653, or 15d-PGJ2 were not mimicked by the sole addition of increasing doses of IL-4 together with GM-CSF to the cultures of differentiating DC (data not shown), suggesting that IL-4-dependent production of PPAR-{gamma} ligands might not be significant in DC.

The observed effect of PPAR-{gamma} agonists was found to be concentration dependent for all three PPAR-{gamma} ligands, as monitored by evaluating CD1a (Fig. 3Go), CD86, and CD80 (data not shown) expression. According to their reported affinities for PPAR-{gamma}, BRL49653 and TGZ were the most potent modulators of DC differentiation, because they had a significant activity at a concentration as low as 10-8 M, whereas the weakest ligand, 15d-PGJ2, significantly interfered with the acquisition of a normal DC phenotype only upon higher concentrations. Addition of LPS, TNF-{alpha}, or soluble CD40 ligand (CD40L) to the cultures of differentiating DC was not able to revert the effect of PPAR-{gamma} activation on CD1a, CD86, and CD80 expression (data not shown). Importantly, PPAR-{gamma} is known to promote cell death in numerous cell types, including macrophages and lymphocytes (25, 30). However, when checking the viability of the examined cell populations by propidium iodide cell staining, we did not detect any increase in the rate of dead cells following exposure to PPAR-{gamma} agonists (data not shown).



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  		FIGURE 3. Concentration-dependent effect of PPAR-{gamma} agonists on DC surface marker expression. Adhering monocytes were incubated for 6 days in medium containing GM-CSF, IL-4, and concentrations of TGZ, BRL49653, or 15d-PGJ2 ranging from 0 to 10-5 M; cells were harvested at day 6 and analyzed by FACS. CD1a expression level is indicated as mean fluorescence intensity.

 
PPAR-{gamma} inhibits DC maturation

To further evaluate the degree of responsiveness to a standard activation stimulus, DC generated in the presence of PPAR-{gamma} agonists were exposed to LPS at day 6 of culture and examined for the acquisition of a mature phenotype 24 h later. Upon activation with LPS, DC generated from peripheral blood monocytes with GM-CSF and IL-4 reverted to an adherent, long-shaped morphology (data not shown), and, as detected by flow cytometric analysis, they acquired high levels of HLA-DR and CD83 expression and up-regulation of CD40, CD80, and CD86. Cells differentiated in the presence of PPAR-{gamma} ligands demonstrated the morphological changes described for the normal control DC when analyzed by light microscopy (data not shown). However, as shown with TGZ, immunostaining of these cells revealed an unbalanced response to LPS stimulation (Fig. 4Go): while displaying a normal HLA-DR up-regulation, DC were found to express lower levels of CD1a, CD83, CD80, and CD40. Again, CD86 up-regulation prevailed.



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  		FIGURE 4. PPAR-{gamma} activation primes an unbalanced response of DC to LPS. DC were generated by culturing adherent monocytes in GM-CSF and IL-4 with or without TGZ (10-5 M) for 6 days. At day 6, DC were stimulated with LPS and, after 24 h, they were harvested and analyzed by FACS.

 
PPAR-{gamma} ligation skews DC cytokine and chemokine expression patterns

When evaluating whether the effects of PPAR-{gamma} agonists on DC phenotype were associated to a skewed cytokine secretion, we found that exposure of differentiating DC to TGZ, BRL49653, or 15d-PGJ2 led to a poorer IL-6 and TNF-{alpha} secretion (Fig. 5GoA). Moreover, PPAR-{gamma} agonists were found to inhibit IL-10 secretion (Table IGo). Activation of immature DC differentiated in the absence of PPAR-{gamma} ligands with LPS at day 6 of culture determined a severalfold increase in IL-6, IL-10, and TNF-{alpha} secretion and primed DC to produce IL-15 and IL-12. Exposure of DC differentiated in the presence of TGZ, BRL49653, or 15-PGJ2 to LPS resulted in a reduced production of IL-10, IL-15, and IL-12 in comparison with the control (Fig. 5GoB). Inhibition of cytokine secretion correlated with the concentration of PPAR-{gamma} agonist, as shown in Fig. 5GoC for IL-12 down-regulation via TGZ. RT-PCR analysis also revealed a concentration-dependent inhibition for IL-12 p40 expression at the mRNA level in DC exposed to TGZ during differentiation (Fig. 6Go).



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  		FIGURE 5. PPAR-{gamma} activation during DC differentiation affects DC cytokine secretion. A, Peripheral blood adhering monocytes were induced to differentiate into DC by a 6-day culture in the presence of GM-CSF (GM) and IL-4 with or without PPAR-{gamma} agonists including TGZ (10-5 M), BRL49653 (3 x 10-5 M), or 15d-PGJ2 (10-5 M). Cells were harvested at day 6 and cultured at 106/well in 2 ml medium. After 24 h supernatants were harvested and IL-6 and TNF-{alpha} concentrations were determined using a commercially available ELISA. B, DC were generated from peripheral blood monocytes by a 6-day culture in GM-CSF and IL-4, with or without PPAR-{gamma} activators; cells were harvested at day 6, plated at 106/well in 2 ml medium, and stimulated with LPS. Supernatants were harvested after 24 h and IL-15 and IL-12 concentrations were determined by commercially available ELISAs. n.d., Not detectable. C, DC generated in the presence of different concentrations of TGZ were harvested at day 6 of culture, plated at 106/well, and stimulated by LPS. Supernatants were harvested after 24 h and IL-12 production was determined by ELISA.

 

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  		Table I. Impact of PPAR-{gamma} activation on IL-10 secretion by DC1

 


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  		FIGURE 6. PPAR-{gamma} activation modulates IL-12 p40, MCP-2, ELC, and CCR7 expression in differentiating DC. RNA was extracted from DC generated from adhering monocytes by exposure to GM-CSF and IL-4 in the presence of different concentrations of the PPAR-{gamma} activator TGZ. IL-12 p40, MCP-2, ELC, and CCR7 expression were subsequently analyzed by RT-PCR.

 
We also examined by RT-PCR whether activators of PPAR-{gamma} would have an effect on the chemokine profile expressed by DC. Interestingly, we found that, while not affecting the expression of macrophage-inflammatory protein-1{alpha} and DC-CK-1 (data not shown), activation of PPAR-{gamma} during DC differentiation down-regulated monocyte chemoattractant protein-2 (MCP-2), EBI1 ligand chemokine (ELC), and CCR7 in a concentration-dependent fashion (Fig. 6Go).

PPAR-{gamma} modulates the stimulation of allogeneic lymphocytes and Ag-specific CTL by DC

We further analyzed the ability of DC generated in vitro in the presence of PPAR-{gamma} agonists to activate lymphocyte responses. When blood monocytes were exposed to TGZ, BRL49653, or 15d-PGJ2 concomitantly with GM-CSF and IL-4 during their differentiation, DC had an impaired capacity to stimulate allogeneic T lymphocytes, as evaluated in standard MLR (Fig. 7GoA). As shown with TGZ, the effect of PPAR-{gamma} activation on DC stimulatory capacity was found to be concentration dependent, with a significant inhibition still being detectable at a TGZ concentration of 10-7 M (Fig. 7GoB). The addition of LPS (Fig. 7GoC) or soluble CD40L plus IFN-{gamma} (data not shown) at day 6 of culture could not restore the stimulatory capacity of DC differentiated in the presence of PPAR-{gamma} activators to levels comparable with the untreated control. Because activation of PPAR-{gamma} down-regulates the production of IL-12 by DC and this cytokine is known to play a relevant role in the induction of cell-mediated immune responses (1, 2), we evaluated the effect of IL-12 supplementation on DC stimulatory capacity. As shown in Fig. 7GoC, IL-12 addition failed to restore DC capacity to the control level, suggesting that other factors beside IL-12 shortening, such as the skewed expression of costimulatory molecules and/or the down-regulated production of cytokines and chemokines, may play a decisive role in the inhibitory effects of PPAR-{gamma} on DC function.



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  		FIGURE 7. PPAR-{gamma} impairs DC stimulatory capacity in MLR. A, Adhering peripheral blood monocytes were cultured for 6 days with GM-CSF and IL-4; GM-CSF, IL-4, and TGZ (10-5 M); GM-CSF, IL-4, and BRL49653 (3 x 10-5 M); or GM-CSF, IL-4, and 15d-PGJ2 (10-5 M). Cells were harvested at day 6 and used to stimulate 105 allogeneic PBMC at a stimulator:responder (S:R) ratio of 1:100. Thymidine incorporation was measured on day 5. The assay was conducted in triplicate and results show the mean cpm and SD of triplicates. B, Monocytes were incubated in GM-CSF and IL-4 with or without different concentrations of TGZ. Cells were harvested at day 6 and incubated with 105 responder allogeneic PBMC at a S:R ratio of 1:10. Thymidine incorporation was measured at day 5. C, Monocytes were cultured for 6 days in the presence of GM-CSF and IL-4 with or without TGZ (10-5 M); on day 6 cells were stimulated with LPS or left unstimulated. Cells were used 24 h later as stimulators in MLR (S:R ratio of 1:10) with (filled bars) or without (open bars) addition of IL-12. Thymidine incorporation was measured on day 5.

 
Flow cytometric analysis of PBMC stimulated in MLR by allogeneic DC demonstrated the expansion of a blast population expressing the activation markers CD25 and HLA-DR on both CD4+ and CD8+ cells (Fig. 8GoA). In contrast, stimulation of differentiating DC by TGZ impeded activation of allogeneic lymphocytes (Fig. 8GoB). Moreover, reduced levels of IFN-{gamma} were detected in the MLR cultures where DC generated in the presence of TGZ were used as stimulators (Fig. 8GoC), whereas no IL-4 or IL-10 production could be detected in the supernatants (data not shown).



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  		FIGURE 8. PPAR-{gamma} activation in DC prevents stimulation of lymphocytes. Adherent PBMC were cultured for 6 days in the presence of GM-CSF and IL-4 with (B) or without (A) TGZ (10-5 M) and further activated by LPS at day 6 of culture. Cells were harvested at day 7, irradiated, and plated at 5 x 105/well in 24-well plates together with 106 allogeneic PBMC. Five days later cells were harvested and analyzed by FACS. C, IFN-{gamma} concentration in the culture supernatants was determined by a commercially available ELISA.

 
For the induction of a primary CTL response, DC generated form adherent HLA-A2-positive PBMC with or without addition of TGZ were pulsed with the synthetic HLA-A2-restricted peptide E75 from Her-2/neu and used as APC to stimulate autologous PBMC. As shown in Fig. 9Go, CTL lines obtained after two weekly restimulations showed high peptide-specific killing that was slightly enhanced by IL-12 addition during CTL induction. Conversely, DC generated in the presence of TGZ failed to activate Ag-specific CTL and, again, IL-12 supplementation did not significantly antagonize this effect.



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  		FIGURE 9. Induction of CTL responses by peptide-pulsed DC. Adherent PBMC from a HLA-A2-positive donor were cultured with GM-CSF and IL-4 with or without TGZ (10-5 M) for 6 days and activated at day 6 by stimulation with LPS. The in vitro generated cell populations were harvested at day 7, pulsed with the synthetic E75 peptide from Her-2/neu, and used as APC to induce a CTL response in vitro with (filled symbols) or without (open symbols) addition of IL-12. The cytotoxic activity of induced CTL was determined after two restimulations in a standard 51Cr assay using Croft cells (HLA-A2 positive) as target pulsed for 2 h with 50 µg of the cognate peptide E75 (squares) or irrelevant GP-2 peptide.

 
PPAR-{gamma} down-regulates nuclear localized RelB transcription factor

It has recently been demonstrated that members of the NF-{kappa}B family of transcription factors are important in DC differentiation and function (38, 39, 40, 41). We analyzed the expression of the NF-{kappa}B family member RelB in DC pretreated with the PPAR-{gamma} agonist TGZ upon stimulation with LPS. As shown in Fig. 10Go, expression of nuclear localized RelB protein was found to be reduced depending on the concentration of PPAR-{gamma} ligand, thus suggesting that PPAR-{gamma} inhibitory effects on DC might be due to blocking of RelB signaling.



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  		FIGURE 10. PPAR-{gamma} down-regulates nuclear localized RelB protein in DC. DC generated in the presence of different concentrations of TGZ were stimulated at day 6 of culture with LPS. Twenty-four hours later nuclear extracts were isolated from the cells and nuclear localized RelB protein was detected by PAGE and Western blotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tissue-resident DC originate from bone marrow-derived circulating precursors, including CD14+ monocytes, which home in peripheral tissues and can further develop into Ag-capturing DC (1, 2). The nature and the respective roles of factors regulating DC differentiation and acquisition of fully immunostimulatory capacity are still subjects of intensive investigation. GM-CSF and IL-4 are regarded as the most important cytokines driving circulating progenitors toward the acquisition of DC properties (42). However, several other signals seem to play an important role in this process. These include IL-10 (31, 43) and IL-6 (44), both known as inhibitors of DC differentiation, TNF-{alpha} (45), and T cell-derived signals like CD40 ligation (46), which, on the contrary, promote DC development and enhance their immunogenicity.

In this study, we show that activation of the nuclear transcription factor PPAR-{gamma} affects the differentiation of monocytes into DC. We found that PPAR-{gamma} ligands administered during GM-CSF/IL-4-induced differentiation of peripheral blood monocytes into DC, while normally permitting CD14 down-regulation, almost completely inhibited the expression of the DC hallmarker CD1a. Moreover, consistent with the results of Gosset et al. (32), we detected a selective up-regulation of the costimulatory molecule B7.2 (CD86), which was paralleled by B7.1 (CD80) down-regulation. In addition, we found that PPAR-{gamma} agonists also impaired CD83 and CD40 expression on DC, both effects being enhanced upon DC activation by LPS or CD40L plus IFN-{gamma} (data not shown). Several PPAR-{gamma} agonists, including TGZ, BRL49653, and the cyclopentenone 15d-PGJ2, that were used in our experiments have been reported to act also via PPAR-{gamma}-independent mechanisms (47, 48). However, such effects were described for very high concentrations of these agonists, thus making it extremely unlikely that the effects we describe in DC, which are reproduced upon concentrations of PPAR-{gamma} agonists in the nanomolar range, are mediated independently of PPAR-{gamma}.

In their report, Gosset et al. (32) speculated that activation of PPAR-{gamma} in DC may switch them toward a type 2 stimulatory mode due to inhibition of IL-12 secretion and down-regulation of chemokines capable to recruit Th1 lymphocytes. Our data indicate a more profound effect of PPAR-{gamma} activation on the DC system: PPAR-{gamma} agonists not only impeded the acquisition of a normal DC phenotype and down-regulated DC production of TNF-{alpha}, IL-6, IL-15, and IL-12 but also severely blunted DC capacity to activate lymphocyte proliferation in MLR and impeded the induction of Ag-specific T cell responses by DC. Moreover, while leading to a reduced production of IFN-{gamma}, lymphocyte stimulation by DC generated in the presence of PPAR-{gamma} agonists failed to prime the secretion of Th2 cytokines like IL-4 and IL-10. Therefore, we suggest that PPAR-{gamma} activation in DC may result in induction of anergy/tolerance in T lymphocytes instead of committing them toward a type 2 cytokine-secreting mode. In contrast, it is possible that the inhibition of DC function we observed depended on the sustained activation of PPAR-{gamma}, as it was realized in our experiments, whereas shorter activation periods like those used by Gosset et al. (32) probably result in milder immunological effects. The effects of PPAR-{gamma} on DC stimulatory capacity were not due to increased IL-10 secretion, the latter being reduced by PPAR-{gamma}. Moreover, the sole supplementation of IL-12 could not restore DC stimulatory capacity in MLR and upon induction of CTL, indicating that other factors, such as the skewed expression of the surface costimulation molecules CD40, CD80, and CD86, reasonably could play a decisive role in the inhibitory effects of PPAR-{gamma}. Finally, while not affecting expression of chemokines expressed by DC at the immature state, such as macrophage-inflammatory protein-1{alpha} and DC-CK-1, PPAR-{gamma} agonists selectively down-regulated ELC and the corresponding receptor, CCR7. These are normally up-regulated during DC maturation and have a crucial importance for DC migration from inflamed tissues to the afferent lymph nodes (49, 50).

RelB is a member of the NF-{kappa}B family of transcription factors that has been implicated in the differentiation of monocytes into DC and in DC maturation (38, 39, 40, 41, 42). Because, upon DC activation by LPS, we detected a down-regulation of nuclear localized RelB in DC pretreated with PPAR-{gamma} agonists, it seems likely that the effects of PPAR-{gamma} on DC are mediated, at least in part, via inhibition of RelB signaling and that other molecules might be involved in the effects mediated by PPAR-{gamma}. A link between PPAR-{gamma} and NF-{kappa}B has already been established (25, 27), and Chung et al. (51) recently demonstrated that PPAR-{gamma} can inhibit the NF-{kappa}B pathway by directly binding the NF-{kappa}B components p50 and p65. However, to the best of our knowledge, this is the first report indicating RelB p68 as a target of PPAR-{gamma} signaling.

Taken together, the effects of PPAR-{gamma} activation on differentiating DC delineate a novel mechanism for the regulation of DC immunogenicity and propose a pathway for the resolution of immune responses arising during an inflammatory process: TNF-{alpha}, LPS, IL-1, and PGE2 released during inflammation promote activation and migration of tissue-resident DC to promptly stimulate an Ag-specific immune response (1, 2). In contrast, at late stages of an inflammatory event, the inducible cyclooxygenase-2 probably redirects PG synthesis toward PGD2 and its cyclopentenone metabolites, whereas PGE2 levels may be strongly reduced (19, 20). At this time point, cyclopentenone PGs could contribute to avoid sustained immune stimulation by acting at the APC level, because PPAR-{gamma} activation in DC may switch them toward a less stimulatory mode with down-regulated cytokine secretion and impaired ELC and CCR7 expression. Detection of micromolar concentrations of PGD2 in rat tissue homogenates including spleen, intestine, bone marrow, and lung in the absence of inflammation (52) and the identification of APC including DC as one major source of endogenous PGD2 (53) raise the possibility that this pathway of regulation is active also under steady state conditions and contributes to modulate the acquisition of fully immunostimulatory capacity by DC. Remarkably, some thiazolidinediones currently represent a therapeutic option for patients with type 2 diabetes mellitus. Because these drugs reduce DC immunogenicity, administration to diabetic patients could potentially worsen immunodepression.


   Acknowledgments
 
We thank Dr. M. Kellerer for performing Western blot analysis of PPAR-{gamma} expression. We also thank S. Kurtz and S. Stephan for the excellent technical assistance. We thank Prof. H.-U. Häring for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a grant from Deutsche Forschungsgemeinschaft (SFB510, Projekt B7). A.N. acknowledges a fellowship from the University of Genoa (Genoa, Italy). Back

2 Address correspondence and reprint requests to Dr. Peter Brossart, Department of Hematology, Oncology, and Immunology, University of Tübingen, Otfried Müller Strasse 10, D-72076 Tübingen, Germany. E-mail address: peter.brossart{at}med.uni-tuebingen.de Back

3 Abbreviations used in this paper: DC, dendritic cell; 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; CD40L, CD40 ligand; PPAR, peroxisome proliferator-activated receptor; {beta}2m, {beta}2-microglobulin; TGZ, troglitazone; MCP-2, monocyte chemoattractant protein-2; ELC, EBI1 ligand chemokine; S:R, stimulator:responder. Back

Received for publication November 27, 2001. Accepted for publication May 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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