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J. Biol. Chem., Vol. 277, Issue 9, 6838-6845, March 1, 2002

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Nuclear Receptor Peroxisome Proliferator-activated Receptor  (PPAR) Is Expressed in Resting Murine Lymphocytes

THE PPAR IN T AND B LYMPHOCYTES IS BOTH TRANSACTIVATION AND TRANSREPRESSION COMPETENT^*

Dallas C. Jones^§, Xiaohong Ding, and Raymond A. Daynes^¶

From the  Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132 and the ^¶ Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, Salt Lake City, Utah 84112

Received for publication, July 20, 2001, and in revised form, October 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the nuclear hormone receptor superfamily. PPAR and PPAR ligands have been demonstrated to exert anti-inflammatory activities in macrophages by repressing the activities of several transcription factors. PPAR is expressed in T lymphocytes and may play a role in cytokine production, cellular proliferation, and susceptibility to apoptosis. Herein, we demonstrate that T and B lymphocytes constitutively express PPAR. PPAR represents the predominant isoform expressed in lymphocytes, whereas PPAR dominates in all cell types of the myeloid lineage. PPAR expression was down-regulated following T-cell activation while PPAR expression increased under the same activating conditions. PPAR expression in T cells may be regulated by microenvironmental factors, because Peyer's patch T cells expressed far greater levels of PPAR than T cells isolated from peripheral lymphoid organs. Exposure to specific ligand determined that PPAR in lymphocytes can effectively transactivate a peroxisome proliferator response element reporter construct. PPAR's ability to regulate endogenous genes, however, required treatment with histone deacetylase inhibitors. Finally, ligand activation of lymphocyte PPAR antagonized NF-B. Our observation that a functional PPAR exists within T cells and B lymphocytes suggests an expanding role for this nuclear receptor in cells of the immune system.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PPARs¹ are ligand-inducible transcription factors that belong to the nuclear hormone receptor superfamily. To date, three PPAR subtypes have been identified: PPAR, PPAR (also known as PPAR or NUC-1), and PPAR (1-3). The PPAR isoforms exhibit a high level of sequence and structural homology, but each displays a divergent pattern of tissue-specific expression and ligand-binding specificity (4, 5). PPAR is expressed at relatively high levels in tissues that utilize fatty acids as the primary energy source, including hepatocytes, cardiac myocytes, and proximal tubular epithelial cells of the kidney (5, 6). PPAR expression is highest in adipose tissues and is moderately expressed in colonic mucosal epithelium (7, 8). PPAR is ubiquitously expressed in both embryonic and adult tissues with a higher level of expression seen in the placenta and large intestine (5, 9, 10).

In contrast to other nuclear hormone receptors, the ligand-binding domain of the PPARs can accommodate a variety of natural and synthetic ligands. Several ligands, including certain polyunsaturated fatty acids and eicosanoids, have been reported to be pan-agonists that can activate all three PPAR isoforms (11-14). Recent studies have indicated that a number of ligands exist with specificity for distinct PPAR subtypes. The lipoxygenase metabolite 8(S)-hydroxyeicosatetraenoic acid, as well as the natural steroid hormone dehydroepiandrosterone sulfate, have been demonstrated to be specific PPAR activators (15, 16). Furthermore, numerous synthetic compounds exist that are capable of activating PPAR. These include the hypolipidemic agents WY-14,643 and clofibrate, phthalate ester plasticizers, herbicides, and a recently described, highly specific murine PPAR agonist, GW9578 (1, 6, 17, 18).

The PPARs are able to positively regulate gene expression by binding to specific DNA sequences known as a PPRE as a heterodimer with the 9-cis-retinoic acid receptor. In the unliganded state, evidence indicates that the PPARs are associated with a nuclear receptor co-repressor (19). Upon activation, the PPARs undergo a conformational change that results in the dissociation from the co-repressor, enabling the PPAR to bind nuclear receptor co-activators. These co-activators then act to reorganize the chromatin templates allowing the basal transcription machinery to gain access to the promoter regions driving transcription of target genes (20-23). In addition to positively regulating gene expression, activated PPARs have recently been demonstrated to exert anti-inflammatory activities through their ability to antagonize an array of important signaling pathways, including those associated with STATs, AP-1, and NF-B (11, 24-28).

We have recently demonstrated that NF-B is present in an active state in both macrophages and lymphocytes, which reside in the spleen and other secondary lymphoid organs of aged mice (29). This active NF-B was further demonstrated to correlate with the normal constitutive expression of a number of NF-B-regulated genes (29). We subsequently reported that the administration of specific PPAR activators to aged rodents effectively reduced the elevated levels of active NF-B in the spleens of these animals and re-established control over a number of NF-B-regulated genes through a PPAR-dependent process (30). These findings suggest that the cell types residing within the spleen may be direct cellular targets for these PPAR activators. Of the major cell populations that reside within the spleen, only macrophages have been reported to express PPAR (31-33). We therefore questioned if other cell types residing within the spleen, including T cells and B lymphocytes, normally express this nuclear hormone receptor.

Our results indicate, for the first time, that lymphocytes express PPAR. We further demonstrate that PPAR is the predominant PPAR isoform present within lymphocytes. This is in contrast to what is observed with macrophages, where PPAR represents the major PPAR subtype. Our findings also suggest that microenvironmental variations within secondary lymphoid organs can influence the cellular level of PPAR expression in T cell but not B cells. Finally, we were able to demonstrate that treatment of T cells with highly specific PPAR activators can up-regulate the expression of endogenous PPAR controlled genes when histone deacetylase inhibitors are utilized.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Experimental Animals-- A colony of DO11.10 TCR transgenic mice was established from breeding pairs originally purchased from Jackson Laboratories (Bar Harbor, ME). The derivation and phenotypic characteristics of these animals have previously been reported (34). C3H/HeN mice were purchased from Charles River Laboratories (Wilmington, MA). Female mice were used for all of the experiments reported herein. All mice were housed in a specific pathogen-free barrier facility at the University of Utah Animal Resource Center, which uses sentinel animals to monitor for the most prevalent murine pathogens and guarantees strict compliance with regulations established by the Animal Welfare Act. Animals used were between 6 and 8 weeks of age, housed in filter-protected cages with a 12-h light-dark controlled cycle, and provided with mouse chow and water ad libitum. Mice were anesthetized with Metofane and sacrificed by cervical dislocation.

Cell Lines and Culture Conditions-- TK.1, Jurkat, EL-4, and RAW 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA). The murine B-cell myeloma cell line, P3X63-Ag8.653, was obtained from Dr. Jerry Spangrude (University of Utah). The T-cell hybridoma, DO11.10, from obtained from Dr. Jerold Woodward (University of Kentucky). Cell lines were maintained in RPMI 1640 (Invitrogen, Gaithersburg, MD) supplemented with 10% FCS (HyClone Laboratories, Logan UT), 200 mM L-glutamine, antibiotics, and 5 × 10⁵ M 2-mercaptoethanol. The murine dendritic cell line, XS-52, was obtained from A. Takashima (University of Texas Southwestern Medical Center) and maintained as described elsewhere (35).

Where indicated, cells were treated with 10 nM trichostatin A (Sigma Chemical Co., St. Louis, MO) alone for 18 h, or with trichostatin A for 18 h followed by a 6-h treatment with the specific murine PPAR ligand, GW9578 (a generous gift from Dr. Peter Brown, Glaxo Wellcome). For T-cell activation, enriched T cells were resuspended at a concentration of 5 × 10⁶ cells/ml and were activated in multiwell plates coated with a solution of 2 µg/ml anti-CD3 or 2 µg/ml anti-CD3 and 2 µg/ml immobilized anti-CD28 (PharMingen, San Diego, CA).

Dynabead Cell Enrichment-- For the preparation of splenic, PLN and PP lymphocytes, freshly isolated lymphoid cells were suspended at a concentration of 2 × 10⁷ cells/ml in RPMI 1640 containing 5% FBS. The erythrocytes present in the cell suspension were lysed by brief treatment with sterile aqueous 0.83% (w/v) ammonium chloride. For T-cell isolation, single cell suspensions were incubated with 2 µg/ml biotinylated anti-CD45 and anti-CD11b antibodies (PharMingen) for 20 min on ice. For B-cell enrichment, the cell suspension was incubated with a mixture consisting of 2 µg/ml biotinylated anti-CD4, biotinylated anti-CD8, and biotinylated CD11b antibodies for 20 min on ice. Following washing with phosphate-buffered saline, cells were resuspended with M-280 magnetic Dynabeads coated with streptavidin (Dynal, New York, NY) incubated at a bead:cell ratio of 1:1 for 20 min with agitation at 4 °C. Cells bound to antibodies were depleted by two rounds of exposure to a magnetic field. The residual cells were collected, washed, and separated for use in culture or for mRNA analysis. The level of purity of the cell preparations was assessed by staining cells with FITC-anti-mouse CD4, FITC-anti-mouse CD8, and FITC-anti-mouse B220. The level of cell purity was routinely >95%.

Quantitative, Real-time PCR-- Reverse transcription was performed as previously described (30). mRNA was isolated by the method of Chomczynski and Sacchi (36), and PCR was performed in a fluorescence temperature cycler (Light Cycler, Idaho Technology) as fully described elsewhere (37). The Light Cycler monitors the cycle-by-cycle accumulation of fluorescently labeled products. The cycle at which the product is first detected is used as an indicator of relative starting copy. Melting curves were acquired to determine specificity of the PCR (38). PCR products for each of the primer sets were confirmed by running samples on an agarose gel. The PCR reaction was carried out in a 10-µl final volume containing 3 mM MgCl₂, 0.2 mM dNTPs, 1:30,000 dilution of SYBR Green I, 5 µM (each) primer, 0.05 unit of Taq polymerase, and 11 ng of TaqStart antibody. Oligonucleotides used for these analyses are as follows: Murine -actin, 5'-GGG TCA GAA GGA CTC CTA TG-3' and 5'-GTA ACA ATG CCA TGT TCA AT-3'; murine PPAR, 5'-GTG GCT GCT ATA ATT TGC TGT G-3' and 5'-GAA GGT GTC ATC TGG ATG GGT-3'; murine PPAR, 5'-CAA GAC TAC CCT TTA AGT GAA-3' and 5'-CTA CTT TGA TCG CAC TTT GGT-3'; murine CPT-1, 5'-ACT TCC ATA TTT CTT CCA AGT TCT C-3' and 5'-TCC AGG AAA TGT GGA GTC AAA TGT G-3'; murine Aco, 5'-CGA CCT TGT TCG GGC AAG TGA GGC GC-3' and 5'-GGA GCT CAG ACG TGT CCC AGG G-3'. -Actin transcript levels were used to normalize the amount of cDNA each sample, and Aco, CPT1, and PPAR transcript levels were reported relative to levels found in the control sample.

Preparation of Nuclear Extracts-- Nuclear extracts were prepared from P3X63-Ag8.653 and EL-4 cell lines following treatment for 24 h with GW9578 or vehicle (0.1% Me₂SO). Briefly, cells were washed twice with ice-cold phosphate buffered saline containing 1 mM PMSF and resuspended in 400 µl of buffer A (10 mM HEPES, pH 7.8, 0.1 mM EDTA, 10 mM KCL, and 1 mM MgCl_2, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM DTT, and 1 mM PMSF and 0.5% Nonidet P-40) and incubated on ice for 15 min. Nuclei were then collected by centrifugation at 20,000 × g for 15 s at 4 °C. 50 µl of buffer C (50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM DTT, and 1 mM PMSF) was added to nuclei and incubated for 20 min on ice. Nuclear debris was removed by centrifugation at 14,000 × g for 30 s. The supernatant was then removed, and protein content was determined by Bradford assay (39).

EMSA-- Equal amounts of nuclear extracts (2 µg of protein as determined by Bradford assay) were incubated with 30,000 cpm of ³²P-labeled NF-B-specific probe (Promega, Madison, WI). Reactions were performed in a 20-µl total volume containing 2 µg of nuclear extract, 4 µl of 5× gel shift binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM MgCl₂, 0.5 mM DTT, 0.5 mM EDTA, and 20% glycerol), 1.5 µg of poly(dI-dC), and 1 µl of probe. For supershift assays, 2 µl of an appropriate anti-NF-B subunit antibody (Santa Cruz Biotechnology, Inc.) was added to each reaction. The reaction was incubated at room temperature for 15 min, loaded on a 4% native polyacrylamide gel, and run in 0.5 × TBE buffer (45 mM Tris, 45 mM Boric Acid 1 mM EDTA). The gel was dried and subjected to autoradiography. NF-B-specific bands were confirmed by competition with a 100-fold excess of an unlabeled NF-B probe, which resulted in no shifted band, or by preparing the reaction with excess labeled nonspecific probe, which did not reduce the intensity of the NF-B band.

Immunofluorescence-- Freshly isolated lymphocytes were plated on 18-mm diameter coverslips that were pretreated with 1 mg/ml polylysine. Cells were then fixed for 30 min at room temperature in 2% paraformaldehyde, washed with TBS, and permeabilized in TBS/0.2% Triton X-100 for 5 min at room temperature. The coverslips were then incubated in 100 µl of rabbit anti-mouse PPAR polyclonal antibody (Affinity Bioreagents, Golden, CO) or a rabbit IgG isotype control antibody (diluted 1:100 in TBS/1% bovine serum albumin) at room temperature for 60 min. After incubation, proteins were visualized using Alexa 594 (Molecular Probes, Eugene, OR) using a Leitz DMR fluorescence microscope.

Transient Transfection and Assay of Luciferase Reporter Constructs-- Transfections were performed as previously described (40). Briefly, 5 × 10⁶ cells were resuspended in 0.65 ml of RPMI 1640 with 10% FCS and. 10 µg of pGL3Basic luciferase reporter construct (Promega) was used in transfecting TK-1 cells and 20 µg of NF-B luciferase reporter construct (Dr. Andrew Thorburn, University of Utah) with 10 µg of murine PPAR expression plasmid (Dr. Ron Evans, Salk Institute) was transfected into Jurkat T cells. One microgram of pRL-TK Renilla luciferase reporter plasmid (Promega) was added to control for transfection efficiency. Cells were incubated with plasmids for 5 min in 0.4-cm electrode gap cuvettes (Invitrogen, Carlsbad, CA) and electroporated at room temperature using the Gene Pulser (Bio-Rad, Hercules, CA) set at 280 V and 960 microfarads (µF) for TK-1 and at 240 V and 960 µF for Jurkat. Cells were incubated for 5 min at room temperature then transferred to 100- × 20-mm tissue culture dishes containing 10 ml of RPMI 1640 with 10% FCS and incubated at 37 °C for 24 h. Luciferase assays were performed using the Dual Luciferase Reporter Assay system (Promega). Briefly, cells were harvested, centrifuged at 200 × g for 5 min, washed twice in phosphate-buffered saline, resuspended in 200 µl of 1× lysis buffer (Promega), and incubated at room temperature for 15 min. Cell debris was pelleted by centrifugation at 13,000 × g for 5 min, and 180 µl of lysate was removed. Cell lysate (10 µl) was loaded into the well of a white opaque microtiter plate, and the dual luciferase assay was performed automatically using the MLX microtiter plate luminometer (Dynex, Chantilly, VA). Serial injection of substrates and monitoring of light emission for 10 s were performed for both firefly and Renilla luciferase. Computer software (Dynex) automatically subtracted background and normalized raw data by calculating the ratio of firefly:Renilla light emission values.

ELISA-- Cytokine concentrations were measured by ELISA, as described previously (41). Monoclonal rat anti-murine IL-2 antibodies and murine recombinant IL-2 cytokine standard were purchased from PharMingen (San Diego, CA).

Statistical Analysis-- Statistical analysis was performed using a Student's t test with p < 0.05 deemed as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPAR Is Expressed in Normal Murine Lymphocytes and Hematopoietic Cell Lines-- To determine whether murine lymphocytes express PPAR, we utilized quantitative, real-time PCR to analyze PPAR mRNA levels in murine T and B lymphocytes. We initially analyzed primary CD4⁺ and CD8⁺ T cells and B cells isolated from the spleen of normal C3H/HeN mice and compared these to splenic macrophages, a cell type already known to express PPAR (31-33). The results of this study (Fig. 1A) demonstrate that splenic T cells (both CD4⁺ and CD8⁺) and splenic B cells express PPAR mRNA at levels that are higher than what is normally observed in splenic macrophages. In the lymphocyte populations, B cells were consistently found to constitutively express higher levels of mRNA for PPAR than T cells. We also evaluated two murine T-cell lines, TK.1 and DO11.10, as well as the B-cell myeloma P3X63-Ag8.653. PCR analysis revealed that these T- and B-cell lines also expressed PPAR (data not shown).

View larger version (77K): [in this window] [in a new window]   Fig. 1.   Lymphocytes express PPAR mRNA and protein. A, freshly isolated splenic CD4+ T cells, CD8+ T cells, and B220+ B cells, and macrophages, were analyzed for PPAR message by quantitative PCR using primers specific for PPAR and GAPDH. The levels of PPAR were determined by normalizing the GAPDH levels in each sample. B, immunofluorescence analysis of PPAR protein in CD4+ T cells reveals that PPAR localization is predominantly cytoplasmic. Nonspecific staining was assessed using a rabbit IgG isotype control. Experiments were repeated at least three times. The results of a representative experiment are shown.

We subsequently utilized immunofluorescence analysis to identify the subcellular localization of PPAR within splenic T and B cells. As shown in Fig. 1B, PPAR protein was excluded from the nucleus in the majority of T cells. A similar localization of PPAR was also observed in B cells (data not shown). The cytoplasmic localization of PPAR in lymphocytes is analogous to what has been reported for unactivated macrophages (31). Similarly, PPAR is also found in the cytoplasm in resting T cells but is shuttled to the nucleus upon T cell activation (42).

It has already been demonstrated that lymphocytes express PPAR and that macrophages can express both PPAR and PPAR (31). We, therefore, compared the relative levels of PPAR and PPAR mRNA within the different lymphoid cell populations. As shown in Fig. 2, the levels of PPAR mRNA were three to five times greater than the level of PPAR mRNA in all lymphocyte populations tested. This is in contrast to what was observed in cells of the myeloid lineage (macrophages, dendritic cells, and mast cells) where PPAR represented the predominant isoform.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Ratio of PPAR and PPAR mRNA in various hematopoietic cells. mRNA was isolated from primary lymphocyte and myeloid cells as well as several cell lines. Real-time quantitative PCR was performed with specific primers for PPAR, PPAR, and glyceraldehyde-3-phosphate dehydrogenase. The relative message levels were obtained by normalizing each sample to glyceraldehyde-3-phosphate dehydrogenase. PCR products were confirmed by running samples on an agarose gel (data not shown). Experiments were repeated three times. The results of a representative experiment are shown.

Cellular Activation Down-regulates PPAR mRNA and Protein Expression in T Lymphocytes-- It has previously been reported that PPAR transcripts in T cells increases following cellular activation (43). To determine if cellular activation alters PPAR in T cells, freshly isolated splenic CD4⁺ T cells were activated with immobilized anti-CD3 or immobilized anti-CD3 plus anti-CD28. PPAR mRNA and PPAR mRNA levels were then analyzed over the subsequent 24-h period. As shown in Fig. 3, PPAR transcript levels were down-regulated in splenic T cells stimulated with immobilized anti-CD3 plus anti-CD28 as early as 3 h post activation and declined further over the next 24-h period. The decline in PPAR expression was contrasted by an observed increase in PPAR message over the same 24-h time period, as has been reported previously (43).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   PPAR mRNA levels decrease after polyclonal T-cell activation. Primary CD4+ T cells were activated with immobilized anti-CD3 plus anti-CD28. mRNA was isolated from cells prior to stimulation and 3, 6, 12, and 24 h following activation. Quantitative PCR was utilized to determine the relative levels of PPAR and PPAR. Experiments were repeated three times. The results of a representative experiment are shown.

T Cells Isolated from Different Secondary Lymphoid Organs Express Varying Levels of PPAR-- To further characterize the presence of PPAR in lymphocytes, we questioned whether the microenvironment of the secondary lymphoid organ in which lymphocytes reside might influence the expression of PPAR mRNA. To address this question, mRNA was isolated from T cells and B lymphocytes purified from the PLN, spleen, and PP of normal C3H/HeN donors. Quantitative, real-time PCR was again used to analyze the relative PPAR transcript levels in the various cell populations. As shown in Fig. 4, PPAR mRNA levels were quite similar from B cells isolated from all the secondary lymphoid organs tested. In contrast, T cells isolated from these same organs expressed varying levels of PPAR mRNA. T cells isolated from the PP were found to express approximately eight times the amount of PPAR mRNA than T cells isolated from the spleen or PLN.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Analysis of PPAR mRNA in lymphocytes isolated from various secondary lymphoid organs. CD3+ T cells and B220+ B cells were isolated from peripheral lymph nodes, spleen, and Peyer's patches using positive selection. Following isolation, mRNA was extracted from the various cell populations and real-time quantitative PCR was used to measure the relative levels of PPAR mRNA in each of the cell populations. Experiments were repeated three times. The results of a representative experiment are shown.

To ensure that the differences in T-cell PPAR mRNA levels observed were not due to differences in the percentages of memory and naive T cells residing within distinct secondary lymphoid organs, an experiment was conducted using CD4⁺ T lymphocytes isolated from the secondary lymphoid organs of DO11.10 TCR transgenic mice. Virtually all the T cells in these mice express a single T-cell receptor with specificity for an ovalbumin peptide and retain a naïve phenotype. T cells isolated from various secondary lymphoid organs of these TCR transgenic mice showed a similar pattern of PPAR mRNA expression to what was observed in wild-type animals, with the CD4⁺ T cells isolated from the PP expressing the greatest levels of PPAR mRNA (data not shown).

Glucocorticoid Treatment Enhances PPAR Transcript Levels in Both B and T Cells-- It has previously been reported that transcription of the PPAR gene is positively regulated by glucocorticoids in vitro and in vivo (44). In addition, we have previously reported that the level of glucocorticoids is higher in the PP than in the other secondary lymphoid organs due to a decreased activity in the PP of 11-hydroxysteroid dehydrogenase, an enzyme that effectively converts glucocorticoids to an inactive 11-keto form (45). We therefore questioned if glucocorticoids might contribute to the differences in PPAR levels between T cells residing within the PP and other secondary lymphoid organs. In an attempt to address this, we treated freshly isolated CD4⁺ T cells and B cells with 10⁷-10⁹ M Dex for 6 and 24 h. Following glucocorticoid treatment, the cells were harvested, and PPAR transcript levels were measured by real-time quantitative PCR. Fig. 5A shows that treatment with 10⁸ M Dex was able to increase the level of PPAR in T cells by 3-fold over vehicle alone. However, a similar enhancement was also obtained with B cells (Fig. 5B) suggesting that an elevation in glucocorticoid influences within the PP might not be responsible for the increased PPAR levels observed only in T cells residing within this secondary lymphoid organ.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Dexamethasone induces PPAR expression in lymphocytes. Freshly isolated CD4+ T cells (A) or B cells (B) were treated for 6 and 24 h with 109, 108 and 107 M dexamethasone or vehicle. Following treatment, PPAR mRNA levels were analyzed by real-time quantitative PCR. Experiments were repeated three times. The results of a representative experiment are shown.

Ligand Activation of PPAR Stimulates Transcription of a PPRE Reporter Construct But Is Unable to Directly Induce the Expression of Endogenous PPAR-regulated Genes in T Cells-- In an attempt to assess the function of PPAR within lymphocytes, we employed a highly specific PPAR ligand, GW9578, to question whether receptor activation would up-regulate the expression of known endogenous PPAR-regulated genes. The CD8⁺ T-cell line, TK.1, was treated for a 24-h period with various levels of GW9578. Following treatment, mRNA was isolated from the treated and control cells, and the mRNA levels of CPT-1, Aco, and PPAR were analyzed by quantitative, real-time PCR. As shown in Fig. 6, exposure of TK-1 T cells to GW9578, was unable to stimulate expression in any of the PPAR-regulated genes above the levels expressed in the vehicle-treated T cells. To ensure that the inability to up-regulate the expression of these genes was not specific to TK.1 cells, this experiment was repeated with the CD4⁺ T-cell lymphoma, DO11.10, as well as with freshly isolated splenic T cells. Similar to what was observed with TK.1, treatment with GW9578 did not increase the levels of these PPAR-driven genes in any of these T-cell populations (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Treatment of the murine T-cell line, TK.1, with GW9578 fails to induce expression of several PPAR-regulated genes. TK-1 T cells were treated with 10 nM, 100 nM, or 1 µM GW9578 or vehicle (0.1% Me2SO) for 24 h. Following treatment, the relative levels of PPAR, CPT-1, and Aco mRNA were determined by quantitative PCR. mRNA was isolated from each of the cell populations, and mRNA levels for the above stated PPAR-regulated genes were analyzed. Experiments were repeated three times. The results of a representative experiment are shown.

To question if the inability of activated PPAR to drive the expression of certain endogenous genes is possibly due to chromatin repression, we utilized a reporter construct that contained the Aco PPRE sequence. This reporter construct was co-transfected into TK.1 along with a pRL-TK Renilla luciferase reporter plasmid to control for transfection efficiency. The transfected cells were then treated with GW9578 or vehicle for 24 h. As shown in Fig. 7, treatment of TK.1 cells with GW9578 was able to induce a dose-dependent increase in the amount of relative luciferase activity. This suggests that the PPAR within lymphocytes is functional and does possess the ability to induce gene transactivation.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Luciferase assays of TK.1 T cells transiently transfected with reporter constructs containing the Aco PPRE sequence. TK.1 T cells were transiently transfected with 10 µg of the PPRE reporter construct as described under "Experimental Procedures." Cells were rested for 24 h following transfection. The cells were then treated for 24 h with 10 nM, 100 nM, or 1 µM GW9578 or vehicle (Me2SO 0.1%). The data are the average from three independent experiments. The asterisks indicate statistically significant differences (p < 0.05) based on Student's t test.

Requirement for Histone Deacetylase Inhibitors to Facilitate the Induction of Genes under PPAR Control in Lymphocytes-- It has previously been reported that the ability of ligand-activated nuclear hormone receptors to drive gene transcription requires the receptor to dissociate from nuclear receptor co-repressors and subsequently complex with nuclear receptor co-activators (20-23). This new complex promotes chromatin acetylation and chromatin remodeling, thereby allowing essential basal transcriptional machinery to gain access to the promoter region of genes under control of the activated nuclear receptor (20-23). We therefore questioned whether the inability of ligand-activated PPAR to up-regulate gene expression within T cells might be due to an inability to properly acetylate histone cores. In an attempt to address this question, TK.1 cells were pretreated with TSA, a known HDAC inhibitor. This was followed by a subsequent exposure to varying doses of GW9578. Treatment of TK.1 cells with TSA alone, although capable of stimulating an increase in the levels of PPAR transcripts, had no effect on CPT-1 or Aco mRNA levels (Fig. 8). The exposure of TSA-treated TK.1 to GW9578 resulted in a dose-dependent increase in mRNA expression of various PPAR-regulated genes (Fig. 8). Similar results were observed when sodium butyrate was employed as the HDAC inhibitor (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   GW9578 up-regulates expression of PPAR regulated genes in T cells pretreated with a HDAC inhibitor. The TK-1 T-cell line was treated with 10 nM TSA for 18 h followed by an additional 6-h treatment with 10 nM, 100 nM, or 1 µM GW9578 or vehicle (0.1% Me2SO). Following treatment, the cells were harvested, mRNA was isolated, and quantitative PCR was performed. Experiments were repeated three times. The results of a representative experiment are shown.

PPAR Activators Decrease the Amount of Nuclear NF-B in Transformed T-cell and B-cell Lines-- To further define possible functions for PPAR within lymphocytes, we questioned whether the activation of PPAR in T and B lymphocytes would lead to the transrepression of NF-B. We previously reported that the supplementation of aged rodents with specific PPAR activators effectively reduced the dysregulated levels of active NF-B in the spleens of these animals (30). Furthermore, treatment of macrophages with PPAR activators in vitro is known to suppress interleukin-6 (IL-6) gene transcription by interfering with NF-B-driven promoter transactivation (32).

To address whether the activation of PPAR in lymphocytes would facilitate the transrepression of NF-B, we evaluated the murine B-cell myeloma, P3X63-Ag8.635, which expresses constitutive nuclear NF-B, and the murine T-cell thymoma, EL-4, which can be induced to express nuclear NF-B following treatment with PMA and ionomycin. As presented in Fig. 9A, the level of nuclear NF-B present in the B-cell myeloma was reduced following a 24-h treatment with GW9578 as determined by EMSA. Similar results were observed in EL-4 cells that had been pretreated with the PPAR agonist GW9578 and activated with PMA and ionomycin for 24 h (Fig. 9B).

View larger version (48K): [in this window] [in a new window]   Fig. 9.   Ligand activation of PPAR decreases NF-B's ability to bind DNA and to transactivate gene expression. A, P3X63-Ag8.653 cells were treated with 1 or 10 µM GW9578 for a 24-h period. B, the murine T-cell line, EL-4, was treated with 1 or 10 µM GW9578 for a 2-h period prior to activation for 24 h with 50 ng/ml PMA and 1 µM ionomycin. In both experiments, cells were harvested, nuclear extracts were prepared, and an NF-B EMSA was performed using 2 µg of nuclear extracts. Extracts were incubated with antibodies recognizing the NF-B subunits p50 and p65 prior to performing the EMSA to confirm NF-B-specific bands. C, EL-4 cells were pretreated with GW9578 or vehicle (0.1% Me2SO) for 2 h followed by a 24-h treatment with 50 ng/ml PMA and 1 µM ionomycin. The levels of IL-2 were then measured in the supernatants by ELISA. D, Jurkat T cells transiently transfected with a plasmid expressing PPAR and a NF-B luciferase-reporter construct were stimulated with 50 ng/ml PMA and 1 µg/ml PHA for 5 h following a 2-h pretreatment with GW9678 or vehicle (0.1% Me2SO). Luciferase expression is shown as the percentage of control. Experiments were repeated three times. The results of a representative experiment are shown. The asterisks indicate statistically significant differences (p < 0.05) based on Student's t test.

To assess if treatment of T cells with a PPAR activator leads to a functional decrease in NF-B, we first analyzed whether GW9578 inhibited production of the NF-B-regulated cytokine IL-2. The level of IL-2 was measured in the supernatant of EL-4 cells that were stimulated with PMA and ionomycin for 24 h, following a 2-h pretreatment with GW9578. As shown in Fig. 9C, treatment of EL-4 T cells with GW9578 led to a significant decrease in IL-2 production compared with control cells. GW9578-treated EL-4 cells were additionally evaluated for cell proliferation. Treatment with the PPAR agonist was found to reduce the proliferation of EL-4 T cells in a dose-dependant manner (data not shown).

To specifically demonstrate that ligand activation of PPAR can inhibit NF-B transactivation, we utilized a NF-B luciferase reporter construct that was transiently transfected into Jurkat T cells along with a murine PPAR expression plasmid. The co-transfected T cells were then treated with various concentration of GW9578 prior to activating the cells with PMA and PHA as described previously (46). As shown in Fig. 9D, pretreatment of Jurkat T cells with GW9578 led to a dose-dependant inhibition of luciferase expression. Together, these experiments suggest that ligand activation of PPAR can suppress NF-B transactivation, possibly by interfering with NF-B's ability to bind to its specific response element.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPAR was first described in the early 1990s as a hormone receptor that could induce peroxisome proliferation in the liver of high dose agonist-treated rodents (6). Over the past decade, the PPAR subfamily of transcription factors has been described to play an active role in many important physiological processes, including adipogenesis, fatty acid metabolism, and inflammation (5-7, 47). The presence and roles of PPARs in hematopoietic cells, however, have only recently been examined. Monocytes and macrophages were the first cells of the immune system in which the physical presence and anti-inflammatory properties of PPARs were first described. Only over the past year has PPAR been reported to exist in other immune cell types of hematopoietic origin, including, dendritic cells, B and T lymphocytes, and mast cells (42, 43, 48-51). In this study, we demonstrate for the first time that murine lymphocytes (both B and T cells) express PPAR transcripts. B and T lymphocytes were also found to contain PPAR protein. The use of real-time quantitative PCR provided a means to quantitatively evaluate the relative levels of PPAR and PPAR mRNA in lymphocytes and in various cell types from the myeloid lineage. We determined that PPAR is the predominant PPAR subtype present within all tested types of murine lymphocytes, whereas PPAR is the major subtype expressed in cells of myeloid origin.

It has previously been reported that, within resting lymphocytes, PPAR is excluded from the nucleus (42). This is in contrast to what has been reported for resting macrophages, were the majority of PPAR is located in the nucleus. We therefore utilized immunofluorescence to determine the subcellular localization of PPAR. Similar to what has been reported for macrophages, the PPAR within lymphocytes is predominantly cytoplasmic. Cellular localization of the PPAR and PPAR isoforms might represent a reflection of the distinct functions of these proteins. It has been reported that lymphocytes undergo apoptosis following their treatment with PPAR agonists but are not affected by treatment with PPAR-specific ligands (42). Likewise, agonist activation of the cytosolic PPAR does not induce apoptosis in macrophages unless the cells are treated in the presence of tumor necrosis factor- and IFN- (31). This suggests that cytoplasmic localization of PPAR within lymphocytes might preclude this protein from functioning in a way that renders the cells susceptible to apoptosis following agonist treatment alone.

In addition to changing the subcellular localization of PPAR, T cell activation has also been reported to effect the levels of PPAR expression (43). Interestingly, we found that PPAR mRNA was markedly decreased with T-cell activation whereas PPAR mRNA was found to increase. The dynamic flux of PPAR expression within T lymphocytes might suggest that this protein is functional whereas T cells are in a resting state. Activation of PPAR within resting lymphocytes could occur from the presence of an endogenous ligand, or possibly through the ability of PPAR to complex with another protein like Ets-1. The Ets-1 protein is highly expressed in resting T cells and has recently been demonstrated to activate PPAR in a ligand-independent manner (52). Expression of Ets-1 is also rapidly down-regulated upon T-cell activation (53) and thus its ability to interact with PPAR following activation would be limited. Likewise, it has been demonstrated that PPAR is inactivated via phosphorylation provided by the mitogen-activated protein kinase, extracellular signal-regulated kinase (54). This kinase is rapidly up-regulated following T-cell activation and might result in PPAR being inactivated.

We have also determined that the levels of PPAR transcripts in T lymphocytes varies significantly between cells isolated from different secondary lymphoid organs, whereas transcript levels of PPAR within B cells remains unchanged. Expression levels within T cells residing in Peyer's patches are four to six times that seen in the PLNs or spleen. The mechanism responsible for this phenomenon has not yet been elucidated. Presently, only four factors are known to positively regulate PPAR gene expression. These include the hormone actions of glucocorticoids, statins, leptin, and ligand-mediated PPAR activation itself (44, 55, 56). Glucocorticoids and PPAR activation do not appear to be responsible for the observed differences in the level of PPAR mRNA observed between the T-cell populations residing in the PP and other secondary lymphoid organs. Although glucocorticoid treatment of T cells in vitro does result in an up-regulation PPAR mRNA expression, a similar up-regulation was observed in glucocorticoid-treated B cells as well. Treatment of T cells with PPAR-specific ligands was unable to induce an up-regulated expression of PPAR itself and was unable to up-regulate the levels of other endogenous PPAR-regulated genes. These findings suggest that gene transactivation by PPAR may not be responsible for the fluctuations in PPAR mRNA levels seen in T cells residing in the different secondary lymphoid organs.

The inability to induce ligand-activated PPAR transactivation in lymphocytes could be overcome by employing two different approaches. The first utilized transient transfection of a murine T-cell line with a reporter construct containing the Aco PPRE sequence. Treatment of these cells with specific PPAR ligands up-regulated expression of the reporter gene. The second set of conditions capable of inducing effective transactivation by PPAR employed an HDAC inhibitor in conjunction with ligand activation. T cells treated with TSA or sodium butyrate, both known HDAC inhibitors, were rendered susceptible to activation by specific PPAR agonists. Both of these situations suggest that ligand activation of PPAR may not be able to effectively induce expression of certain endogenous genes within lymphocytes, due to an inability to initiate proper chromatin remodeling. It has previously been demonstrated, with cells containing the PR, that ligand treatment effectively induced expression of a transiently transfected reporter construct but failed to induce up-regulation of endogenously expressed genes (57, 58). This phenomenon was reportedly due to chromatin packing of the endogenous genes, not allowing the PR to gain access to the promoter region. Transiently transfected reporter constructs are not associated with chromatin and therefore can be effectively transactivated by PR upon ligand activation. At present, we have only studied a narrow set of PPAR-regulated genes involved in fatty acid metabolism. It is highly possible that activation of PPAR can effectively transactivate other genes within T cells that we have not yet examined.

Although direct ligand activation of PPAR in lymphocytes failed to up-regulate the expression of several PPAR-regulated genes, ligand activation of PPAR did result in the effective transrepression of NF-B, in both transformed T cells and B lymphocytes. We have yet to establish whether the ability of PPAR to transrepress NF-B is achieved through the direct interaction of PPAR with NF-B or through the up-regulation of the IB gene. Both processes represent recently described mechanisms through which activated PPAR can effectively control the level of active NF-B in cells (59). It has been previously demonstrated that the ability of activated PPARs to transrepress the activities of NF-B, as well as a number of other transcription factors, has important physiological consequences within these cells, including anti-inflammatory activities, transcription repression, and cell death (24, 31, 60, 61).

Although we have shown that a functional PPAR receptor is present within T and B lymphocytes, we have yet to establish a role for this receptor in lymphocyte biology. However, we have recently found that T cells isolated from mice taking a functional PPAR receptor (PPAR^/) exhibit dysregulation in activation-induced IFN- production, where PPAR^/ T cells produce much higher levels of IFN- than T cells from PPAR^+/+ mice.² These observations were made in the absence of added exogenous ligand, further suggesting that the endogenous PPAR protein in lymphocytes may already be in an active state. Clearly, additional experimentation is needed to elucidate the mechanism(s) through which PPAR might regulate the expression of inducible cytokines in activated lymphocytes and any additional role(s) this nuclear hormone may play in lymphocyte biology.

	ACKNOWLEDGEMENTS

We thank Dr. Peter Brown for providing the GW9578 compound, Dr. Tom McIntyre for providing the PPRE reporter construct, Dr. Andrew Thorburn for providing the NF-B reporter construct, and Dr. Ron Evans for providing the PPAR expression construct.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants CA25917 and DK55491, by a Browning Foundation grant, and by Department of Veteran's Affairs Medical Research Funds.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by DHHS/NIDDK, National Institutes of Health, Hematology Research Training Grant T32 DK07115.

To whom correspondence should be addressed: Dept. of Pathology, University of Utah, 30 North 1900 East, Salt Lake City, UT 84132-2501. Tel.: 801-581-3013; Fax: 801-581-8946; E-mail: daynes.office@path.utah.edu.

Published, JBC Papers in Press, November 28, 2001, DOI 10.1074/jbc.M106908200

² Jones, D. C., Ding, X., Zhang, T., and Daynes, R. A., manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; Aco, acyl-CoA oxidase; CPT-1, carnitine palmitoyl transferase-1; Dex, dexamethasone; EMSA, electrophoretic mobility shift assay; HDAC, histone deacetylase; NF-B, nuclear factor B; PLN, peripheral lymph node; PMSF, phenylmethylsulfonyl fluoride; PP, Peyer's patch; PPRE, peroxisome proliferator response element; PR, progesterone receptor; TSA, trichostatin A; STAT, signal transducers and activators of transcription; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; DTT, dithiothreitol; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; PMA, phorbol 12-myristate 13-acetate; PHA, phytohemagglutinin; IFN-, interferon .

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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