PPARγ but not PPARα Ligands Are Potent Repressors of Major Histocompatibility Complex Class II Induction in Atheroma-Associated Cells
Peroxisome proliferator-activated receptors (PPARs) are essential in glucose and lipid metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as diabetes and dyslipidemia. Conversely, antidiabetic glitazones and hypolipidemic fibrate drugs, known as PPARγ and PPARα ligands, respectively, reduce the process of atherosclerotic lesion formation, which involves chronic immunoinflammatory processes. Major histocompatibility complex class II (MHC-II) molecules, expressed on the surface of specialized cells, are directly involved in the activation of T lymphocytes and in the control of the immune response. Interestingly, expression of MHC-II has recently been observed in atherosclerotic plaques, and it can be induced by the proinflammatory cytokine interferon-γ (IFN-γ) in vascular cells. To explore a possible role for PPAR ligands in the regulation of the immune response, we investigated whether PPAR activation affects MHC-II expression in atheroma-associated cells. In the present study, we demonstrate that PPARγ but not PPARα ligands act as inhibitors of IFN-γ–induced MHC-II expression and thus as repressors of MHC-II–mediated T-cell activation. All different types of PPARγ ligands tested inhibit MHC-II. This effect of PPARγ ligands is due to a specific inhibition of promoter IV of CIITA and does not concern constitutive expression of MHC-II. Thus, the beneficial effects of antidiabetic PPARγ activators on atherosclerotic plaque development may be partly explained by their repression of MHC-II expression and subsequent inhibition of T-lymphocyte activation.
- major histocompatibility complex class II
- peroxisome proliferator-activated receptors
- human endothelial cells
- human macrophages
- T-lymphocyte proliferation
Atherosclerosis and its clinical manifestations of heart attack, stroke, and peripheral vascular insufficiency continue to be the principal cause of death and disability in Western societies. Histological analysis of atherosclerotic plaques suggests that lesion development represents an inflammatory and proliferative response to lipid metabolic disturbances in regions of the vascular tree exposed to turbulent blood flow.1–3⇓⇓ Increasing evidence implicates the involvement of immune mechanisms in atherogenesis.4–6⇓⇓ Indeed, atherosclerotic plaques contain significant amounts of T lymphocytes, many of which are in an activated state.7–9⇓⇓ Furthermore, functional CD40 ligand (CD40L) is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages, and interruption of CD40-CD40L signaling limits atherosclerotic plaque development in mice.10–12⇓⇓ Finally, major histocompatibility complex class II (MHC-II) molecules are expressed in atherosclerotic lesions, not only on endothelial cells but also on macrophages and smooth muscle cells nearby activated T lymphocytes.13,14⇓ Taken together, these observations support the notion that atherosclerosis may be an immune-modulated disease.
MHC-II molecules play a critical role in the induction of immune responses by presenting peptides of foreign antigens to CD4+ T lymphocytes, which results in their activation and proliferation. A very tight regulation of MHC-II expression is thus crucial for the control of the immune response. Two main types of MHC-II expression can be distinguished, constitutive or inducible.15 MHC-II is constitutively expressed in only a very restricted number of cell types, specialized in antigen presentation, such as dendritic cells and B lymphocytes. MHC-II expression can be induced by interferon-γ (IFN-γ) in a large variety of other cell types, among which vascular endothelial cells and macrophages. Expression of MHC-II genes is regulated primarily at the level of transcription, and the class II transactivator CIITA has been found to be a master regulator in this process.16,17⇓ CIITA expression patterns correlate with that of MHC-II genes, such that it is constitutively expressed in MHC-II–positive professional antigen-presenting cells and that it is an obligatory mediator of IFN-γ–induced MHC-II expression.
Several regulatory pathways, including for example the transcription factor NF-κB, have been identified that control the expression of proinflammatory chemokines and adhesion molecules important in atherogenesis.1 Recent studies have described the expression of peroxisome proliferator-activated receptors (PPARs) in macrophage foam cells, endothelial cells, and smooth muscle cells of both human and murine atherosclerotic plaques.18–21⇓⇓⇓ PPARs are nuclear receptors that function as ligand-activated transcriptional regulators of genes controlling lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as diabetes and dyslipidemia.22–25⇓⇓⇓ Interestingly, antidiabetic glitazones and hypolipidemic fibrate drugs, known as PPARγ and PPARα ligands, respectively, reduce the progressive formation of atherosclerotic lesions in vivo,26–28⇓⇓ and PPARα deficiency reduced insulin resistance and atherosclerosis in mice.29 Knowing the involvement of immune mechanisms in the pathogenesis of atherosclerosis, we investigated the effects of PPAR activation on IFN-γ–inducible MHC-II expression in atheroma-associated cells.
Materials and Methods
Human recombinant IFN-γ was obtained from Endogen (Woburn, Mass), 15d-PGJ2 from Calbiochem (La Jolla, Calif), ETYA from Sigma (St. Louis, Mo), and ciglitazone and WY14643 from Biomol (Plymouth Meeting, Pa) BRL49653 was a gift from SmithKline Beecham (Philadelphia, Pa), and troglitazone was a gift from Park Davis Pharmaceuticals (Morris Plains, NJ). Mouse anti-human MHC-II and MHC-I fluorescein isothiocyanate-conjugated (FITC) and unconjugated monoclonal antibodies were purchased from PharMingen (San Diego, Calif).
Cell Isolation and Culture
Human vascular endothelial cells (ECs) were isolated from saphenous veins by collagenase treatment (Worthington Biochemicals, Freehold, NJ) and cultured in dishes coated with gelatin (Difco, Liverpool, England) as described elsewhere.10 Cells were maintained in medium 199 (M199; BioWhittaker, Wokingham, England) supplemented with 100 U/mL penicillin/streptomycin (BioWhittaker), 5% FCS (Gibco, Basel, Switzerland), 100 μg/mL heparin (Sigma), and 50 μg/mL endothelial cell growth factor (Pel-Freez Biological, Rogers, Ark). Culture media and FCS contained <40 pg lipopolysaccharide/mL as determined by chromogenic Limulus amoebocyte-assay analysis (QLC-1000; BioWhittaker). Endothelial cells were >99% CD31 positive as characterized by flow cytometry and were used at passages 2 to 4 for all experiments.
Monocytes and T lymphocytes were isolated from freshly prepared human peripheral blood mononuclear cells obtained from leukopacs of healthy donors following Ficoll-Hypaque gradient and subsequent differential adherence to plastic culture flasks (90 minutes, 37°C). They were cultured in RPMI 1640 medium (BioWhittaker) containing 10% FCS. After 10 days of culturing, macrophages (Mφ) derived from monocytes were >98% CD64 positive as determined by flow cytometry.
The human monocytic ThP1 cell line obtained from American Type Culture Collection (Manassas, Va) was grown in RPMI 1640 medium containing 10% FCS.
Total RNA was prepared with Tri reagent (MRC Inc, Cincinnati, Ohio) according to the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed and amplified using a one-step RT-PCR kit (Qiagen AG, Basel, Switzerland). For the amplification of PPARγ cDNA, two oligonucleotide primers amplifying a 473-bp fragment were used30: sense primer 5′-TCTCTCCGTAATG-GAAGACC-3′ and antisense primer 5′-GCATTATGAGCA-TCCCCAC-3′. For the amplification of PPARα cDNA, two oligonucleotide primers amplifying a 276-bp fragment were used31: sense primer 5′-AGATTTCGCAATCCATCGGC-3′ and antisense primer 5′-GCGTGGACTCCGTAATGATA-3′. The polymerase chain reaction was carried out in a 1:1 mixture of standard buffer and Q solution with 0.3 μmol/L of each primer (Microsynth, Balgach, Switzerland) and 2 μL of enzyme mix for 30 cycles. For relative quantitative analyses, 1 μL of 18S PCR primer pair and 4 μL of 18S PCR Competimers (QuantumRNA, Ambion, Austin, Tex) were included per reaction resulting in a 489-bp product. Polymerase chain products (12 μL/50 μL) were analyzed on a 2% agarose gel.
Cells were incubated with FITC-conjugated specific antibody (60 minutes, 4°C) and analyzed in a Becton Dickinson FACScan flow cytometer (Franklin Lanes, NJ) as described.10 At least 50 000 viable cells were analyzed per condition. Data were analyzed using CELLQUEST software (Becton Dickinson).
Cells grown on coverslips were fixed for 5 minutes with methanol at −20°C. The coverslips were rinsed and incubated successively with 0.2% Triton X-100 in PBS for 1 hour, 0.5 mol/L NH4Cl in PBS for 15 minutes, and PBS supplemented with 2% BSA (Sigma) for another 30 minutes. Cells were then incubated overnight with primary antibody (1:200) in 10% normal goat serum (Sigma)/PBS. After rinsing, the coverslips were incubated with secondary antibodies FITC-conjugated (1:1000) for 4 hours. All steps were performed at room temperature and in between incubation steps cells were rinsed with PBS. Cells were counterstained with 0.03% Evans Blue in PBS. Coverslips were mounted on Vectashield slides (Vector Laboratories, Burlingame, Calif). Cells were examined using a Zeiss Axiophot microscope equipped with appropriate filters. Specificity of the immunolabeling was checked for by replacing the primary antibody with PBS.
RNAse Protection Assay
Total RNA was prepared with Tri reagent (MRC Inc) according to the manufacturer’s instructions. RNAse protection assays with 15 μg of RNA per reaction were carried out as described previously32 using human probes for MHC-II (DR-α), CIITA, and GAPDH as a control for RNA loading. Signal quantitation was determined using a PhosphorImager analysis system (Molecular Dynamics, Sunnyvale, CA). Levels of DR-α and CIITA RNA in any given sample were normalized to the GAPDH signal for that sample.
Reporter Gene Assay
A CIITA promoter IV-reporter gene plasmid was created by subcloning the CIITA 5′-flanking region, ie, −461(KpnI)/+75 fragment of exon 1 type IV, upstream of the firefly luciferase gene of plasmid pGL3-basic (Promega, Madison, Wis). Primary human vascular ECs (0.5×106) were transiently transfected using 7.2 μL FuGENE transfection reagent (Roche, Indianapolis, Ind), 0.3 μg pGL3/461, and 25 ng pRL (Promega) in 150 μL of M199. Eight hours later, cells were rinsed and cultured for an additional 12 hours under the respective stimulating conditions. Reporter gene expression was measured using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions.
Mixed Lymphocyte-Like Reaction (MLR)
Human ECs were cultured on 96-well plates, pretreated with the respective stimuli, and irradiated. Purified allogenic human T lymphocytes (1×105) were then added to the wells containing the adherent cells and cultured for 5 days at 37°C without stimuli. The proliferative response was determined by the amount of [3H]thymidine (10 μCi; 25 mCi/mmol; Hartmann Analytic, Braunschweig, Germany) incorporated after an additional 14 hours of culture. Assays were performed in duplicate.
Results and Discussion
To explore possible interfaces between immune mechanisms and atherogenesis, we analyzed the effects of PPAR ligands on various features of the control of MHC-II expression in atheroma-associated cells and of subsequent T-lymphocyte activation. Vascular ECs and monocyte-macrophages (Mφ), both of human origin, are known to express PPARα and PPARγ.19,30⇓ To establish the expression of PPARγ and PPARα in the primary human saphenous vein ECs used in our experiments, RT-PCR was performed. Indeed, ECs originating from different donors all contained mRNA for both PPARs (Figure 1A). Moreover, expression levels of these mRNAs were not affected by either IFN-γ or PPARγ ligands (Figure 1B). Experiments were performed to measure surface expression of MHC-II by flow cytometry (Figures 2a through 2f) and by immunofluorescence (Figures 2g through 2l). As shown in Figure 2a, human ECs did not express MHC-II under resting conditions whereas treatment with IFN-γ induced expression of this molecule. The natural PPARγ ligand 15d-PGJ2 as well as three different synthetic PPARγ ligands (BRL49653, troglitazone, and ciglitazone) effectively repressed this induction of MHC-II by IFN-γ (Figure 2a) in a dose-dependent manner (Figure 2b), whereas two PPARα ligands (ETYA and WY14643) showed almost no effect (Figure 2c). Similar flow cytometry results were obtained in a macrophage cell line (ThP1, Figure 2f) and in primary human Mφ using immunochemical fluorescent labeling (Figures 2g through 2l). The concentration range at which the PPAR ligands were used in the present study is comparable to the ones previously reported19–21,30⇓⇓⇓ and did not affect cell viability or protein synthesis of human ECs and Mφ (data not shown). Treatment with PPAR ligands alone had no effect on MHC-II expression (data not shown). As shown in Figure 2d, ECs expressed MHC-I under resting conditions, and IFN-γ treatment further induced expression of this molecule. However, neither PPARγ (Figure 2d) nor PPARα (Figure 2e) ligands inhibited MHC-I expression. Taken together, these findings point to specific effects of PPARγ ligands on the MHC-II gene activation pathway.
Regulation of expression of MHC-II genes is highly complex, and its precise control directly influences T-lymphocyte activation and thus the immune response. The elucidation of a molecular defect responsible for bare lymphocyte syndrome, a rare hereditary disease of MHC-II regulation, has contributed to our current understanding of the complex regulation of these genes.15 Analysis revealed that patients with similar symptoms of severe primary immunodeficiency could be affected genetically in one of four distinct trans-acting regulatory factors that are essential for MHC-II gene transcription. Of these factors, RFX5, RFX-AP, and RFX-ANK15,30⇓ are ubiquitously expressed, whereas the expression of CIITA, which controls MHC-II expression, is tightly regulated.16,17⇓ To determine at which level PPAR ligands exert their inhibitory action on IFN-γ–induced MHC-II expression, we measured the expression of mRNA for MHC-II (DRα) and the class II transactivator CIITA. As expected, human ECs did not express DRα and CIITA mRNA under resting conditions, but both mRNAs were induced on stimulation with IFN-γ (Figure 3). All four different PPARγ ligands tested repressed this induction of DRα and CIITA mRNA by IFN-γ (Figure 3) in a dose-dependent manner (Figure 4), whereas two different PPARα ligands had no significant effect (Figure 3). It is notable that the potency of PPARγ ligands as MHC-II repressors vary widely according to the different PPARγ ligands used in these experiments. Of the forms tested, the most powerful MHC-II repressor is the natural PPARγ ligand 15d-PGJ2. The better efficacy of this molecule to repress the induction of MHC-II might result from additional effects of 15d-PGJ2 on other signaling pathways, as recently described for human chondrocytes,33 as well as for antiinflammatory effects of PPARγ in macrophages.34,35⇓ To test for 15d-PGJ2 effect through PPARγ, we performed experiments using the specific PPARγ inhibitor prostaglandin F2α (PGF2α).36 As shown in Figure 5, the effect of 15d-PGJ2 on MHC-II expression was largely reduced when PGF2α was added to the culture conditions. Thus, these findings suggest that the effects of 15d-PGJ2 on MHC-II is mainly achieved via PPARγ-dependent pathways.
Expression of CIITA is controlled by several alternative promoters, operating under distinct physiological conditions.37 CIITA promoter I controls constitutive expression in dendritic cells, promoter III controls constitutive expression in B lymphocytes, whereas CIITA promoter IV is specifically responsible for the IFN-γ–inducible expression of CIITA and thus of MHC-II. Constitutive expression of MHC-II in dendritic cells and B lymphocytes was not affected by PPAR ligands (data not shown). To test the activity of CIITA promoter IV under various conditions, we tested a construct in which firefly luciferase was placed under the control of this promoter. After transient cotransfection of this promoter-reporter gene construct and a reference plasmid into human ECs, cells were stimulated and expression levels analyzed. As shown in Figure 6A, IFN-γ treatment increases the expression of firefly luciferase by ≈3-fold. The four different PPARγ ligands tested effectively repressed IFN-γ–induced expression to baseline levels, whereas PPARα ligands had almost no effect on IFN-γ–induced CIITA promoter IV activity. In addition, the effects of the PPARγ ligands on MHC-II and CIITA promoter IV activity appeared dose-dependent (Figure 6B). Altogether, these results point to specific actions of PPARγ ligands on the inducible promoter IV of the CIITA gene. It has been shown that three trans-acting factors, ie, Stat-1, USF-1, and IRF-1, are required and essential for activation of CIITA promoter IV by IFN-γ.15,32,37⇓⇓ The effect of PPAR ligands on gene expression is known to result from the formation of a heterodimer between PPAR and RXRα (9-cis-retinoic acid receptor), a protein complex that interacts with a peroxisome proliferator responsive element (PPRE) in the target gene.38 DNA sequence analysis did not reveal any known PPRE in CIITA promoter IV, suggesting that PPARγ ligands may exert their actions via trans-acting factors. We are currently investigating whether PPARγ ligands influence the availability and/or DNA binding capacity of the three trans-acting factors Stat-1, USF-1, and IRF-1 that bind to CIITA promoter IV.
MHC-II molecules play a critical role in the induction of immune responses by presenting peptides of foreign antigens to CD4+ T lymphocytes, which results in their activation and proliferation. They also contribute to the activation of T lymphocyte by alloreactivity. We investigated the functional consequences of PPARγ-induced repression of MHC-II expression in mixed lymphocyte-like reactions. Allogenic T lymphocytes were incubated with human ECs pretreated with IFN-γ alone or with IFN-γ and the PPARγ ligand 15d-PGJ2. IFN-γ–dependent T-lymphocyte proliferation could be blocked by anti–MHC-II monoclonal antibodies (data not shown). As measured by [3H]thymidine incorporation, treatment of human ECs with the natural PPARγ ligand reduced T-lymphocyte proliferation (Figure 7), thus illustrating the functional consequence of inhibition of MHC-II antigens by PPARγ ligands.
In summary, we demonstrate in these in vitro experiments that different PPARγ ligands repress MHC-II antigen induction by IFN-γ in atheroma-associated cells and describe the mechanism of this effect through repression of promoter IV of the MHC-II transactivator CIITA. PPARγ activators are commonly used in treatment of type II diabetes. In a preliminary study, treatment with the PPARγ ligand troglitazone was found to reduce carotid intimal-medial thickness,39 a marker of early stages of atherosclerosis. Because there is increasing evidence that immunoinflammatory interactions play important roles during early stages of atherogenesis, our results may provide a scientific rationale for the beneficial effects of antidiabetic glitazone drugs on the development of atherosclerosis. The discovery of immunomodulatory effects of PPARγ ligands may thus lead to new strategies in the clinical management of atherosclerosis. To this end, the development of novel PPARγ ligands processing immunomodulatory and antiatherogenic activities but devoid of antidiabetic activities would be highly desirable. Development of such drugs may also have direct implications for numerous other immunoinflammatory pathologies.
This work was supported by the Swiss National Scientific Research Fund (grant No. 3800-054965.98/1 to F.M and No. 31-53774.98 to E.R.) and by the Fondation Leenaards (to B.K. and F.M.).
Original received August 9, 2001; revision received November 30, 2001; accepted December 21, 2001.
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