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Published online before print December 4, 2003
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agonists on T lymphocytes derived from MS patients
* Department of Neurology, University of Bonn, Germany; and
Department of Anesthesiology, University of Illinois, Chicago
1 Correspondence: Neurologische Universitätsklinik Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. E-mail: m.heneka{at}uni-bonn.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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isoform is expressed in human T lymphocytes, and oral administration of PPAR- agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, suggesting a potential role for PPAR- agonists in the treatment of autoimmune diseases. To assess a potential therapeutic role of PPAR- agonists in multiple sclerosis, we compared the immunomodulatory effects of the thiazolidinedione (TZD) drugs pioglitazone (PIO) and ciglitazone and the non-TZD PPAR- agonist GW347845 on human T leukemia cells (Jurkat cells) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) derived from 21 multiple sclerosis patients and 12 healthy donors. PIO, ciglitazone, and GW347845 suppressed PHA-induced T cell proliferation by 40–50% and secretion of interferon- and tumor necrosis factor 
, by 30–50%. Inhibition of proliferation was increased to 
80% and that of proinflammatory cytokine secretion, to 80–90% when PBMCs were first preincubated with PPAR- agonists and re-exposed at the time of PHA stimulation, indicating a sensitizing effect of PPAR- agonists. Inhibition of proliferation was also observed in the tetanus toxoid-specific T cell line KHS.TT2, albeit to a lesser extent. The antiproliferative effects of PIO and GW347845 were accompanied by a decrease of cell viability. Electron microscopy and Western blot analysis revealed DNA condensation and down-regulation of bcl-2, suggesting the induction of apoptosis in activated T lymphocytes. In summary, the data support the potential use of PPAR- agonists as immunomodulatory, therapeutic agents for autoimmune diseases.
Key Words: EAE • IFN- • TNF-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have examined the possibility that the recently discovered anti-inflammatory and antiproliferative properties of insulin-sensitizing, oral antidiabetic agents might provide benefit in animal models of MS [4
5
6
]. The first showed that the thiazolidinedione (TZD) derivative troglitazone provided some improvement in the clinical symtpoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein (MOG) peptide [4
]. In our studies [5
], we observed significant improvement in the clinical course of MOG-induced EAE with oral administration of the TZD pioglitazone (PIO) as well as the investigational drug GW347845 (GW). These drugs, which are structurally distinct agonists of the peroxisome proliferator-activated receptor- (PPAR-), reduced the incidence and severity of clinical symptoms when given before EAE induction and attenuated clinical signs when provided after disease onset [5
]. The beneficial effects of PIO and GW were paralleled by a decrease of perivascular infiltrates and demyelination, as well as by reduced chemokine mRNA levels in brain. The third study [6
] reported that the PPAR- TZD agonist ciglitazone was protective in active and adoptive forms of EAE in SJL mice. In our studies and the ciglitazone study, effects on T cell function were observed. For example, we demonstrated that PIO reduced the capacity of T cells to synthesize and secrete IFN- [5
].
Ligand-dependent activation of PPAR-, a member of the nuclear hormone receptor superfamily of transcription factors [7
], reduces inflammatory gene expression and protects neurons from immunostimulated cell death [8
9
10
11
]. As murine and human T cells express PPAR- [12
13
14
15
16
17
], and TZDs can inhibit interleukin (IL)-2 production [17
] and mitogen-induced proliferation of human T and Jurkat cells, it seems likely that PPAR- is involved in the immunoregulation of T lymphocytes [17
]. In the present study, we investigated the immunomodulatory effects of PIO, ciglitazone, and GW on human T cells derived from MS patients and healthy donors (HDs) as well as Jurkat cells and determined T cell proliferative responses in one MS patient treated with oral PIO.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and culture of peripheral blood mononuclear cells (PBMCs) and Jurkat cells
PBMCs were isolated by Ficoll standard density gradient centrifugation (Nycomed, Oslo, Norway) and were cultured at 1 x 105 cells per well in RPMI-1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin (all from Gibco, Berlin, Germany) and 5% autologous serum in the presence of phytohemaglutinin (PHA; 5 µg/ml). Jurkat cells (DSMZ, Braunschweig, Germany) were grown in supplemented RPMI 1640 with 10% fetal calf serum at 1 x 105 cells per well.
As previous studies demonstrated antiproliferative effects of the lower-affinity PPAR- agonists ciglitazone and troglitazone on human T cells in vitro [17
], further experiments focused on higher-affinity PPAR- ligands PIO and GW.
To investigate a possible effect of short-term versus long-term exposure of PBMCs to PPAR- agonists, four different types of culture conditions were designed: PHA stimulation and concomitant administration of PIO (30 µM) or GW (1 µM) for 48 h; preincubation of PBMCs with PIO (15 µM) or GW (0.5 µM) for 48 h and additional administration of PIO (15 µM) or GW (0.5 µM) at the time of PHA stimulation for 48 h; PHA stimulation of PBMCs and administration of PIO (30 µM) or GW (1 µM) after 6, 12, 24, and 48 h; and preincubation of PBMCs with PIO (15 µM) or GW (0.5 µM) for 48 h and PHA stimulation and additional administration of PIO (15 µM) or GW (0.5 µM) after 6, 12, 24, and 48 h.
Isolation of antigen-specific T cell lines
A tetanus toxoid (TT)-specific T cell line (KHS.TT2) of a HD was generated using the "split-well" cloning technique as described previously [20
]. TT was purchased from Behringwerke (Marburg, Germany).
To assess a possible sensitizing effect of PPAR- agonists observed after PHA stimulation, antigen-specific restimulation was performed with concomitant administration of PIO (10 µM) and GW (1 µM) as well as after preincubation of TT-specific T cells with PIO (5 µM) and GW (0.5 µM) for 48 h. Another dose of PIO (5 µM) and GW (0.5 µM) was added at the time of the antigen-specific restimulation for 48 h.
Proliferation assays
T cell proliferative responses were determined using a nonradioactive proliferation assay measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) as described [21
, 22
]. BrdU (Boehringer Mannheim, Germany) was added to the cultures for 24 h. Absorbance was measured at 450 nm on an Optimax enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA). All proliferation assays were performed in duplicate.
Viability assays
Cell viability was assessed by tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide bromide (MTT) assay. For MTT assays, PBMCs were seeded at 1 x 105/cells per well and cultured as described above. MTT (10% vol/vol; 5 mg/ml phosphate-buffered saline) was added for 2 h. After washing with dimethyl sulfoxide, the absorbance was measured at 550 nm on an Optimax ELISA reader (Molecular Devices).
ELISA
Cell-culture supernatants were collected 24, 48, and 72 h after PHA stimulation, centrifuged, and stored at –80°C until assayed. Cytokine measurements were performed using commercially available ELISAs (R&D Systems, Minneapolis, MN) following the manufacturer’s instructions. The detection limits were 10 pg/ml for IL-4, 4.4 pg/ml for tumor necrosis factor (TNF-), and 8 pg/ml for IFN-. All measurements were performed in duplicate, and mean values of the two measurements were used for statistical analysis. Supernatants were used undiluted for the determination of IL-4 and were diluted tenfold for the determination of TNF- and IFN-. Absorbances were measured at 450 nm on an Optimax ELISA reader (Molecular Devices).
Western blot analysis
Western blot analysis was performed by using standard procedures. For the detection of PPAR- expression, incubation with primary anti-PPAR- antibody #210-118 (Alexis Biochemicals, Grünberg, Germany) was performed at 1:500 dilution overnight at 4°C. The secondary antibody [horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) from Sigma, Deisenhofen, Germany] was diluted at 1:2500 and incubated at room temperature for 1 h. For the detection of Bcl-2, incubation with primary antibody (monoclonal mouse anti-human Bcl2 antibody, Transduction Laboratories, San Diego, CA) was performed at 1:100 dilution for 2 h at room temperature. The secondary antibody (HRP-conjugated goat anti-mouse IgG from Dianova, Hamburg, Germany) was diluted at 1:30,000 for 1 h at room temperature. Antibody binding was detected by enhanced chemiluminescence (ECL) using Hyperfilm-ECL (Pharmacia Biotech, Freiburg, Germany).
Electron microscopy
Cell pellets were fixed overnight at 4°C in 4% paraformaldehyde and 0.1% glutaraldehyde with 0.1 M hepes buffer at pH 7.5, dehydrated with ethanol, and embedded with Spurr. Ultrathin sections were examined using a Zeiss 900 electron microscope (Zeiss, Jena, Germany).
Immunofluoresence
After PHA stimulation, T cells were transferred to 10 cm dishes (Costar, Cambride, MA) at 1 x 106/ml and were fixed with acetone/methanol (1:1) at 4°C for 5 min. Monoclonal mouse anti-human cytochrome C IgG antibody (Promega, Madison, WI) at a 1:50 dilution was applied for 60 min at 37°C. The secondary rhodamine (Texas Red)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was diluted at 1:200 and added for 30 min at room temperature. Stained cells were covered with Dabco (Aldrich, Taufkirchen, Germany) and Moviol 4-88 (Calbiochem, La Jolla, CA), and immunofluorescence of cytochrome C was visualized using a Nikon microscope (Nikon, Düsseldorf, Germany).
Statistical analysis
Group comparisons were performed using Student’s unpaired t-test (two-sample test) or one-way ANOVA with subsequent post-hoc Tuckey tests (multiple comparison test). Calculations were performed with the GraphPad Prism software package (GraphPad Software, San Diego, CA). Results are given as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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agonists PIO and GW on PHA-stimulated human T cells in vitro was demonstrated by Western blotting in T lymphocytes derived from MS patients and HDs (not shown). PHA-induced proliferation of PBMCs and proliferative responses of Jurkat cells were inhibited by ciglitazone and PIO in a concentration-dependent manner (Fig. 1
). Control experiments with vehicle revealed no significant effect on PHA-induced T cell proliferation (not shown). The antiproliferative effects of PIO and GW on PHA-induced proliferation were significantly increased when PBMCs were preincubated with PIO and GW before PHA stimulation, followed by a second dose at the time of PHA stimulation (Fig. 2
). This sensitizing effect was also observed in a series of preincubation experiments demonstrating that re-exposure to PIO and GW as late as 48 h after PHA stimulation still resulted in a significant inhibition of T cell proliferative responses (Fig. 3
).
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treatment were also confirmed in one MS patient who had been treated with PIO (15 mg tid) over a period of 2 months. T cell proliferation of this patient was only moderately affected by exposure to PPAR- agonists in vitro before PIO treatment. However, after 2 months of oral PIO medication, the inhibitory effect on T cells was significantly increased (Fig. 4
).
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agonist PIO on the antigen-specific human T cell line KHS.TT2
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and TNF- secretion of PHA-stimulated T cells by PPAR- agonists PIO and GW and TNF- secretion, which again, was most pronounced after preincubation of PBMCs with PIO and GW (Fig. 6
). IL-4 levels were below detection level in all samples tested.
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agonists PIO and GW agonists PIO and GW were associated with decreased viability as demonstrated by MTT assays over time (Fig. 7
) and were accompanied by a release of cytochrome C from the mitochondria as shown by immunofluorescence (Fig. 8C
and 8D
). Moreover, condensation of nuclear DNA in PHA-stimulated T cells indicated that induction of apoptosis contributes to the antiproliferative effects of PPAR- agonists on human T cells in vitro (Fig. 8A)
. Apoptotic cell death was further suggested by down-regulation of Bcl-2 (Fig. 8B)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ligands PIO, GW, and ciglitazone inhibit proliferative responses and proinflammatory cytokine secretion of mitogen-stimulated PBMCs derived from MS patients and HDs. Additionally, antiproliferative effects were demonstrated in Jurkat cells and the TT-specific T cell line KHS.TT2. Inhibition of proliferation as well as IFN- and TNF- secretion were most pronounced after preincubation of PBMCs with PIO and GW, suggesting a sensitizing effect of these drugs. In support of this assumption, the antiproliferative effects of PIO in vitro were increased in one MS patient treated with PIO (15 mg tid) for 2 months. The induction of apoptosis seems to contribute to the antiproliferative and anti-inflammatory action of PPAR- agonists in vitro.
PPAR- was primarily characterized in adipocytes as a key regulator of lipid metabolism [7
, 23
] but was also identified in murine and human T cells, suggesting a possible involvement in the regulation of T cell activation [12
13
14
15
16
, 24
]. Recent experimental work has focused on the clinical and immunomodulatory effects of PPAR- agonists on EAE [4
5
6
]. As protection from EAE is paralleled by a significant decrease of perivascular infiltrates within the CNS [4
, 5
], it is likely that PPAR- agonists exert their immunomodulatory effects, at least in part, by interfering with the activation of T cells.
In the present study, constitutive expression of PPAR- was demonstrated in T cells derived from MS patients and HDs in accordance with previous reports [12
, 13
, 15
, 17
]. Here, we demonstrate a profound antiproliferative effect of two structurally distinct and highly selective PPAR- agonists, PIO and GW, on PHA-induced T cell proliferation. As the TZD PIO and the non-TZD GW exert antiproliferative effects of a similar magnitude, it is likely that these effects are PPAR--mediated. Induction of apoptosis seems to contribute significantly to these anti-inflammatory effects [13
14
15
]. Although the most pronounced, antiproliferative effects of the TZD derivatives ciglitazone and PIO peaked at concentrations of 10–100 µM as described previously [12
13
14
15
16
17
], induction of apoptotic cell death by PIO in single T cells was observed at 1 µM PIO, indicating that treatment effects of PPAR- agonists are already detectable at low concentrations.
Inhibition of proliferation was paralleled by a significant decrease of proinflammatory cytokine secretion such as IFN- and TNF-. Previous studies demonstrated that PPAR- agonists inhibit IL1-ß, IL-6, and TNF- expression in human periperal blood monocytes [25
], decrease IL-2 and IFN- production in mitogen-stimulated murine splenocytes [12
, 13
, 17
, 26
], and down-regulate IFN--induced chemokine expression in endothelial cells [27
].
As a principal and new finding, we demonstrate that the extent of the immunomodulatory effects of PPAR- agonists was significantly enhanced when PBMCs were pretreated with PIO or GW, suggesting that prestimulation of PPAR- increases further ligand-induced receptor activation. It is interesting that this sensitizing effect of PPAR- agonists does not seem to be restricted to the in vitro situation, as an increased, antiproliferative effect of PPAR- agonists was observed in one MS patient who received oral PIO medication for 2 months. Although this remains a single observation, translation into a therapeutic scenario for MS patients suggests that long-term treatment with PPAR- agonists might be used to down-regulate peripheral T cell responses, opening the possibility of further medication in the case of acute clinical deterioration. In line with this hypothesis, we demonstrate that pretreatment of PBMCs with PIO and GW not only increased the magnitude of the antiproliferative effects but also extended the maximal time span during which significant antiproliferative effects are achievable when a second dose of PIO and GW is given after PHA stimulation. However, the molecular mechanisms of this sensitizing effect remain to be elucidated. One might speculate that ligand-dependent stimulation of PPAR- may result in auto-induced up-regulation of the receptor, resulting in an enhanced DNA-binding activity. Ongoing experiments using Western blot detection and electrophoretic mobility shift assay to detect changes in PPAR- expression and altered DNA binding of PPAR- derived from nuclear protein extracts of T cells pretreated with PPAR- agonists will help to clarify this issue.
In conclusion, this is the first study demonstrating significant antiproliferative effects of PPAR- agonists on human T cells derived from MS patients. Given the pathogenetic importance of activated T cells in MS and the anti-inflammatory and clinically beneficial effects of PIO and GW in various murine EAE models, PPAR- agonists may provide a novel, therapeutic principle in MS treatment.
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ACKNOWLEDGEMENTS |
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Received August 26, 2003; revised October 9, 2003; accepted October 14, 2003.
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R. Cunard, Y. Eto, J. T. Muljadi, C. K. Glass, C. J. Kelly, and M. Ricote Repression of IFN-{gamma} Expression by Peroxisome Proliferator-Activated Receptor {gamma} J. Immunol., June 15, 2004; 172(12): 7530 - 7536. [Abstract] [Full Text] [PDF]
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