contributes to T lymphocyte apoptosis during sepsis
* Department of Biochemistry I, University Hospital, Johann Wolfgang Goethe-University Frankfurt, Germany;
Department of Anesthesiology, Molecular Cardioprotection and Inflammation Group, Faculty of Medicine, University of Düsseldorf, Germany;
Department of Medicine IV-Experimental Division, Faculty of Medicine, University of Erlangen-Nürnberg, Erlangen, Germany; and
Westpfalz-Klinikum Kaiserslautern, Department of Internal Medicine I, Germany
1 Correspondence: Department of Biochemistry I, University Hospital, Johann Wolfgang Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail: v_knethen{at}zbc.kgu.de
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(PPAR) in provoking apoptosis in activated T lymphocytes. Therefore, we studied whether expression/activation of PPAR might contribute to T cell death during sepsis. We observed PPAR up-regulation in T cells of septic patients. In contrast to controls, PPAR expressing cells from septic patients responded with apoptosis when exposed to PPAR agonists. Cell demise was attenuated by SR-202, a synthetic PPAR antagonist, and specificity was further verified by excluding a proapoptotic response to a PPAR
agonist. We propose that up-regulation of PPAR sensitizes T cells of septic patients to undergo apoptosis. PPAR activation in T cells requires an exogenous PPAR agonist, which we identified in sera of septic patients. Septic sera were used to study reporter gene expression containing a PPAR-responsive element. We conclude that PPAR plays a significant role in T cell apoptosis, contributing to lymphocyte loss in sepsis. Thus, inhibition of PPAR may turn out to be beneficial for patients suffering from lymphopenia during sepsis.
Key Words: cell death • bacterial • inflammation • leukopenia
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T cells are important players of the acquired immune system. Cross-talk with compounds of neighboring cells such as monocytes, macrophages, or dendritic cells may cause activation of T cells. This provokes intracellular activation of transcription factors, e.g., nuclear factor (NF)-
B, activator protein 1, NF of activated T cells (NFAT), as well as expression of proinflammatory cytokines, such as interleukin (IL)-2 and interferon- [2
]. In addition, various factors can be induced to lower T cell responses, inhibiting their proinflammatory phenotype or triggering controlled cell death followed by T cell depletion [3
]. Suppression of immune and inflammatory responses accompanied by leukopenia as a result of the excessive loss of T lymphocytes has been described in sepsis initiation [4
]. Several studies in animal models of sepsis as well as in septic patients identified apoptosis as the underlying reason for lymphocyte cell death during sepsis [5
6
7
8
]. At present, it is unclear which molecular mechanisms are involved in T cell viability during sepsis. In a murine model of sepsis, overexpression of the antiapoptotic protein Bcl-2 and inhibition of caspases have resulted in reduced lymphocyte apoptosis and improved survival of animals [9
, 10
]. Besides established triggers of apoptosis, peroxisome proliferator-activated receptor (PPAR) has been demonstrated to initiate apoptosis in lymphocytes when appropriately activated [11
, 12
]. PPAR is a transcription factor, which upon ligand binding, heterodimerizes with the retinoid X receptor protein and binds to PPAR-response elements (PPREs) found in promoters of target genes [13
]. Known ligands are polyunsaturated fatty acids, 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2), and synthetic antidiabetic thiazolidinediones, such as ciglitazone [14
]. PPAR inhibits T cell activation by scavenging NFAT from binding to the IL-2 promoter [15
] and/or by modulating survival of activated T cells during inflammation via an unknown pathway [11
, 16
].
We demonstrate that leukocytes from peripheral blood of septic donors have reduced counts of CD4+ T cells. Recently, we show that PPAR expression is induced in primary human T cells in response to their activation, thereby sensitizing cells to apoptosis [16
]. Here, we demonstrated that PPAR is up-regulated in septic T cells following microbial infection. In support of our hypothesis, we found that sera of septic patients contain a PPAR-specific agonist using reporter analyses. Activation of PPAR with exogenous agonists provokes apoptosis in septic T cells in vitro, suggesting a possible mechanism for T cell lymphopenia in sepsis. The discovery of the profound molecular mechanisms involved PPAR-mediated T cell death may lead to novel therapeutic, antiapoptotic approaches to improve the immune system during sepsis.
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For analysis of leukocyte subsets, we purified freshly drawn blood from healthy and septic donors by incubating samples in 5 vol erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA), followed by centrifugation (300 g, 10 min, 24°C) and two washing steps by adding lysis buffer.
For apoptosis measurements on lymphocytes, we isolated cells from donors using Vacutainer CPTTM cell preparation tubes (BD Biosciences, Heidelberg, Germany) containing the anticoagulant sodium heparin. After gel and gradient centrifugation, cells and serum were transferred to a 15-ml Falcon tube (BD Biosciences). Cells were pelleted, and serum was shock-frozen in liquid nitrogen and stored at –70°C until needed for transactivation reporter assays. Cells were washed twice in phosphate-buffered saline (PBS), and CD3+ T cells were further isolated using the magnetic cell sorting (MACS) technology (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometry confirmed 95–98% purity of isolated cells. We used cells for the experiments after 2 h of recovery. The experimental setup of our study is shown in Figure 1 .
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Figure 1. Experimental setup. Schematic representation of the experimental setup of the study. Blood was drawn from healthy donors and septic patients. Cells and sera were processed as described. PCR, Polymerase chain reaction;  , mitochondrial membrane potential; DiOC6(3), dihexaoxacarbocyanine iodide dye; PI, phosphatidylinositol; wt, wild-type; d/n, dominent negative.
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FAS-ab; Santa Cruz Biotechnology, Heidelberg, Germany), and FAS (Beckman Coulter, Krefeld, Germany) in water and phytohemagglutinin (PHA; Sigma Chemical Co., Deisenhofen, Germany) in PBS.
Analysis of leukocyte subsets
Analysis of white blood cells was performed with a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) using antibodies for cell type-specific antigens: granulocytes CD66b+, monocytes CD14+CD66b–, T cells CD3+, and B cells CD19+, respectively. To differentiate CD4+ and CD8+ T cells, staining using CD3/CD4, CD3/CD8, or CD4/CD8 antibody cocktails was performed. Stained cells were analyzed by two-color immunofluoresence analyses on a FACSCalibur flow cytometer. Quantification was performed with the CellQuestPro software. The fluorescein isothiocyanate (FITC)-labeled CD3, CD4, CD14, and CD19 antibodies as well as the phycoerythrin (PE)-labeled CD4 and CD8 antibodies were obtained from Immunotools (Frisoythe, Germany). The CD66b-FITC antibody was from BD Biosciences.
RNA extraction and real-time quantitative PCR (QPCR)
We extracted RNA from purified cells using peqGOLD RNAPure (Peqlab, Erlangen, Germany) according to the distributor’s manual. For the reverse transcriptase (RT) reactions of human PPAR and ß2-microglobulin transcripts, we used the Advantage RT-for-PCR kit (Clontech, BD Biosciences). The real-time QPCR was performed using a MyiQ real-time PCR system (Bio-Rad, München, Germany) and the Absolute QPCR SYBR Green mix (Abgene, Hamburg, Germany) according to the manufacturer’s instructions. Moreover, we used the following primer sequences (Metabion GmbH, Planegg, Germany): PPAR (213–495), TA = 52°C: 5'-ACT TTG GGA TCA GCT CCG-3', 5'-GCC ATG AGG GAG TTG GAA-3'; ß2-microglobulin (69–368), TA = 52°C: 5'-CTC CGT GGC CTT AGC TGT-3', 5'-TTC ACA CGG CAG GCA TAC-3'. We calculated annealing temperatures using the primer design program Oligo (Molecular Biology Insights, Cascade, CO). Controls of isolated RNA omitting RT during PCR were used to guarantee genomic DNA-free RNA preparations (data not shown). Quantification of real-time PCR results was performed using the Gene Expression Macro (V1.1) from Bio-Rad, taking ß2-microglobulin expression as the internal control.
Measurement of
We incubated samples with 40 nM DiOC6(3) for 30 min (Molecular Probes, Leiden, The Netherlands) and measured cell death in response to particular cell treatments by flow cytometry [16
]. At least 10,000 cells were accumulated for analysis. Results are expressed as percentage of total cells with breakdown (dead cells).
Annexin V-FITC/PI staining
Following incubations, 2 x 105 cells were labeled with 5 µl annexin V-FITC and 2.5 µl PI in 100 µl binding buffer for 15 min on ice in the dark to differentiate between apoptotic and necrotic cell death using an annexin V-FITC/PI-staining kit (Immunotech, Krefeld, Germany). Afterwards, 150 µl binding buffer was added, and cell samples were analyzed immediately using a FACSCalibur flow cytometer and CellQuestPro software. Apoptosis was assessed when cells were annexin V-FITC-positive (early apoptosis) or annexin V-FITC/PI-positive (late apoptosis). Accordingly, only PI-positive cells were considered as necrotic. A minimum of 10,000 cells was analyzed.
PPRE reporter gene assay
The p(A-Ox3)-TKL plasmid, containing three copies of the PPRE site derived from the human acyl coenzyme A oxidase gene promoter cloned upstream of the thymidine kinase minimal promoter and the luciferase gene, was transfected into the Jurkat T cells using jetPEI (Biomol), according to the manufacturer’s instructions. Simultaneously, a PPAR wt vector (pcDNA3-PPAR wt) or a PPAR d/n-encoding plasmid (pcDNA3-PPAR AF2), containing two amino acid exchanges (Leu468Ala/Glu471Ala) and preventing ligand binding and concomitant activation of PPAR, were transfected as well. Cells were treated as indicated. After harvesting and lysing cells, extracts were assayed for luciferase activity.
Statistical analysis
We used two-tailed statistical analysis to evaluate the data. Results are expressed as the mean ± SD. As a result of the relative small sample size, the Mann-Whitney U-test was used for statistical analysis of intergroup comparisons of primary cells. Jurkat cell experiments were evaluated using the Student’s t-test. We considered P values 
0.05 as significant.
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2:1 (Fig. 2A
, right panel, and Table 1
). Staining the cells for CD4 and CD8 demonstrated a reduction of CD4+ T cells in septic patients (Fig. 2B
, right panel) compared with healthy controls (Fig. 2B
, left panel). Therefore, the decrease of CD4+ T cells was shown in whole blood after lysing erythrocytes and also after gel and density gradient centrifugation. This reduction corroborates a massive deprivation of lymphocytes in septic patients. This clearly indicates that depletion of CD4+ cells contributes to lymphopenia in sepsis. |
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Table 1. Changes in the Number and the Phenotype of Peripheral Blood Cellsa
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Figure 2. Leukocyte populations in peripheral blood. (A) Ratio of lymphocytes (R2) and monocytes (R1) following gel and gradient depletion of granulocytes (left panel, healthy donor; right panel, septic patient). (B) Proportion of CD4+ and CD8+ T cells (left panel, healthy donor; right panel, septic patient) using the lymphocyte gate (R2).
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in septic T cells is a potential key regulator in apoptosis of activated T cells [16
, 18
]. Therefore, we focused our work on this transcription factor. To test the hypothesis that PPAR increased T cell susceptibility to activation-induced cell death in sepsis, we performed real-time QPCR to examine PPAR mRNA expression in CD3+ T lymphocytes. Real-time PCR revealed an 12-fold higher PPAR expression in T lymphocytes of patients with sepsis than in healthy donors (Fig. 3
). The finding of increased PPAR mRNA in septic CD3+ T cells supported the hypothesis of PPAR-dependent T cell apoptosis accounting for lymphocyte loss in sepsis.
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Figure 3. Expression of PPAR in T cells from healthy donors and septic patients. PPAR expression in T cells (open bar, healthy donors, n=7; solid bar, septic patients, n=7) determined by real-time QPCR as described in Materials and Methods. *, P 0.05, versus control donors. Experiments were performed three times in triplicate. Relative PPAR expression of healthy donors was set as 1.
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activation agonists on their own, exogenously provided PPAR ligands might affect cell viability and act proapoptotic in septic T cells [16
]. To show T cell apoptosis in response to PPAR agonists, we incubated T cells derived from septic blood and healthy donors with the synthetic PPAR-specific agonist ciglitazone [14
]. Taking into consideration that humans express three known isoforms of PPARs, namely PPAR, PPAR
, and PPAR [19
], each recognizing an identical DNA motif, specificity was verified using the PPAR agonist WY14643 [20
], and DMSO was included as a vehicle control. Ciglitazone, WY14643, or DMSO did not affect cell viability of T cells from healthy donors (Fig. 4A
). In contrast, in septic CD3+ T cells, the PPAR agonist ciglitazone significantly increased cell death from 11 ± 2.4% in untreated controls to 26 ± 5.6% in response to PPAR activation (Fig. 4A)
. WY14643 and DMSO controls did not affect septic CD3+ T cell survival (Fig. 4A)
. In these experiments, cell death was determined by DiOC6(3) staining, which is incorporated into the mitochondrial membrane when the is intact. Cells, not showing a green fluorescence, have lost their , thus indicating cell death. To verify results obtained by the DiOC6(3) method, we analyzed annexin V-FITC/PI staining in parallel to assess apoptotic (early apoptosis shows annexin V-FITC-positive, whereas late apoptosis shows annexin V/PI-positive) and necrotic (only PI-positive) cells. Results shown in Figure 4B
4C
4D
4E
, are in line with our DiOC6(3) data. In addition, they suggest apoptosis as the underlying mechanism of cell death. T cells from healthy donors did not show increased apoptosis in response to PPAR activation by ciglitazone (11.1% Fig. 4B
vs. 14% Fig. 4C
). However, in CD3+ T cells derived from septic patients, ciglitazone promoted apoptosis, provoking an increase of apoptotic cells from 12.41% in untreated cells to 28.11% in response to the PPAR agonist (Fig. 4D
vs. Fig. 4E
). We conclude from these data that apoptosis is the underlying principle of cell death in septic T cells in response to PPAR agonists.
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Figure 4. PPAR agonist triggered apoptosis in septic CD3+ T cells. (A) T cells (open bars, control donors, n=10; solid bars, septic patients, n=15) were incubated with 10 µM ciglitazone, 10 µM WY14643, or appropriate amounts of DMSO for 4 h or remained as controls. Percentage of apoptosis was analyzed using DiOC6(3) as described in Materials and Methods. *, P 0.05, versus untreated control. (B–E) Annexin V-FITC/PI staining was performed by flow cytometry as described in Materials and Methods. T cells from healthy donors (B, C) or septic patients (D, E) were incubated for 4 h. Samples were treated with 10 µM ciglitazone (C, E) or remained as controls (B, D). Data are representative of n = 8 healthy donors and n = 8 septic patients.
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requires an exogenously provided agonist [16
]. Therefore, we were looking for a potential PPAR activator during sepsis. This would provide evidence for the clinical importance of T cell death in septic patients. As a possible source of PPAR ligands, we tested sera of septic patients on their ability to activate PPAR in a Jurkat cell line model. Jurkat cells express only low levels of PPAR [16
]. To enhance the sensitivity of this system, we transiently transfected these cells with the PPRE-containing luciferase reporter plasmid and in addition, with a PPAR wt or PPAR d/n expression plasmid. Transfected Jurkat cells were incubated for 6 h with sera of septic patients or healthy volunteers. Detection of luciferase expression revealed a threefold induction in response to sera of septic patients compared with sera of healthy donors (Fig. 5A
, cross-hatched bars vs. shaded bars) in cells transfected with the PPAR wt plasmid. These data suggest a PPAR-activating compound in the sera of sepsis patients. For further proof of the involvement of PPAR in PPRE-dependent gene induction, Jurkat cells were transfected with the PPAR d/n expression plasmid. This leads to a PPAR mutant protein (AF2), containing two amino acid changes (Leu468Ala/Glu471Ala), which prevent agonist binding and significantly reduced PPAR-transactivation capability [21
]. In these cells, we failed to see a PPRE-dependent gene expression in response to the PPAR agonist ciglitazone (Fig. 5B
, open bars) as well as by adding sera of septic patients (Fig. 5B
, cross-hatched bars) compared with luciferase activity relative to untreated controls (Fig. 5B
, solid bars). These results enforced the presence of a specific PPAR agonist in sera of septic patients. Taking these results into consideration, we exposed PPAR wt- and accordingly, d/n-transfected Jurkat cells with sera of sepsis patients and healthy donors. As shown in Figure 5C
, sera of sepsis patients provoked cell death in PPAR wt-transfected cells (Fig. 5C
, left panel, cross-hatched bar, 39.8±25.3%), whereas sera of healthy donors did not alter cell survival of PPAR wt-transfected cells (Fig. 5C
, left panel, shaded bar, 13.7±0.8%). In accordance with our hypothesis that sera of sepsis patients contain a PPAR agonist provoking PPAR-dependent apoptosis, in Jurkat cells expressing a PPAR d/n mutant (AF2), cell death in response to sera of septic patients was inhibited (Fig. 5C
, right panel, cross-hatched bar, 15.6±8.2%). In line, the specific PPAR agonist ciglitazone induced cell death in PPAR wt-transfected cells, which was significantly decreased in PPAR d/n-transfected Jurkat cells (Fig. 5C
, left panel, open bar, 38.9±6%, vs. right panel, open bar, 16.7±5%).
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Figure 5. Sera of septic patients activate PPRE, and PPRE activity is given as fold induction of luciferase reporter activity (mean±SD) measured in Jurkat cells, which were transfected with a PPRE-reporter construct [p(A-Ox3)-TKL]. (A) A PPAR wt vector was cotransfected. Twenty-four hours after transfection, cells were incubated for 6 h with sera of septic patients versus healthy donors (solid bars, control donors, n=10; cross-hatched bars, septic patients, n=15) or 10 µM ciglitazone as positive control (open bars). Each experiment was performed three times in duplicate. *, P 0.05. Relative luciferase activity of healthy donors was set as 1. (B) A PPAR d/n (AF2) vector was cotransfected with the PPRE reporter plasmid. Twenty-four hours after transfection, cells were treated with 10 µM ciglitazone (open bars, n=3) or septic sera (cross-hatched bars, n=15) or remained as control (solid bars, n=3) for 6 h. Each experiment was performed three times in duplicate. Relative luciferase activity of the untreated control was set to 1. (C) A PPAR wt vector or a PPAR d/n (AF2) vector was transfected. Cells were treated for 4 h with 10 µM ciglitazone (open bars, n=3), septic sera (cross-hatched bars, n=10), or sera from healthy donors (shaded bars, n=5) or remained as controls (solid bars, n=3). Each experiment was performed three times. *, P 0.05.
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-transactivating capabilities and reduced T cell apoptosis agonists might provoke apoptosis by a PPAR-independent mechanism [22
23
24
], we concentrated our experiments on the question of whether T cell apoptosis in sepsis demands PPAR. We set up an experimental system to prove the potency of SR-202, a known PPAR antagonist [25
], to repress the transactivating capability of PPAR in PHA-pretreated Jurkat T cells, which were transfected with a PPRE-containing luciferase reporter plasmid. PHA was used as an established activator of T cells to provoke PPAR expression in our cell culture model [16
]. The addition of ciglitazone to PHA-preincubated cells elicited a significant increase of luciferase activity (Fig. 6A
). This promoter activity was repressed with SR-202 being present. Based on the pharmacological inhibitor approach with SR-202 and postulating that PPAR is involved in provoking cell death, we predicted higher cell viability in septic CD3+ T lymphocytes when applying a combination of PPAR agonist plus SR-202. Indeed, SR-202 significantly blocked PPAR-mediated cell death in response to ciglitazone (14.3±3.7% vs. 27.6±4.9%; Fig. 6B
).
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Figure 6. SR-202 attenuates transactivating capabilities of PPAR and T cell death. (A) PPRE activity is given as fold induction of luciferase reporter activity (mean±SD) measured in Jurkat cells transfected with a PPRE reporter construct [p(A-Ox3)-TKL]. Cells were stimulated with 10 µg/ml PHA for 15 h, incubated with SR-202 for 1 h, and afterwards, treated for 4 h with ciglitazone as indicated. Relative luciferase activity of the PHA-treated sample was set to 1. *, P 0.05, ciglitazone versus SR-202/ciglitazone. (B) Percentage of cells (mean±SD) with decreased  . T cells of septic patients (n=6) were incubated with 10 µM ciglitazone, with a combination of 10 µM ciglitazone and 200 µM SR-202, or with 200 µM SR-202 alone or remained as controls. Incubations went for 4 h, and 1 h SR-202 was preincubated. Experiments were performed three times in duplicate. *, P 0.05, ciglitazone versus SR-202/ciglitazone.
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-mediated apoptosis-dependent apoptosis in T cells is unclear. With regard to the induction of apoptosis in T cells, Fas (Apo-1/CD95) and tumor necrosis factor receptor (TNFR) gained extensive attention [4
]. In sepsis, the death receptor as well as the mitochondrial pathways have been desrcibed to be involved [26
27
28
]. To elucidate the pathway responsible for PPAR-dependent apoptosis in septic T cells, we aimed at clarifying the role of Fas (Apo-1/CD95) in a final set of experiments. The participation of death domain receptors in apoptosis upon PPAR activation was proposed recently for epithelial cells [29
]. Addition of anti-Fas-ab inhibited Fas-mediated apoptosis from 66.8 ± 6.7% to 17.4 ± 3.8% but left the ciglitazone response in septic T cells unaltered (26.2±3.5% vs. 25.4±4.5%; Fig. 7A
). Representative annexin V-FITC/PI stainings are shown in Figure 7B
7C
7D
7E
. Evidently, apoptosis constitutes the underlying principle in Fas and in line with Figure 4E
, also in PPAR-dependent cell death. As a result of these experiment, we propose a Fas (Apo-1/CD95)-unrelated but PPAR-dependent mechanism leading to T cell apoptosis during sepsis.
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Figure 7. Fas (Apo-1/CD95)-unrelated T cell apoptosis. (A) T cells of septic patients (n=6) were incubated for 4 h with 10 µM ciglitazone, 10 µM ciglitazone and 0.5 µg/ml anti-Fas-ab, 10 ng/ml FAS, 10 ng/ml FAS and 0.5 µg/ml anti-Fas-ab, or 0.5 µg/ml anti-Fas-ab or remained as controls. was analyzed to determine cell death. *, P 0.05, versus control treated with anti-Fas-ab only. Experiments were performed in duplicate. (B–E) Annexin V-FITC/PI staining was performed by flow cytometry as described in Materials and Methods. Incubation was carried out for 4 h: (B) treatment with 10 ng/ml FAS, (C) incubations with 0.5 µg/ml anti-Fas-ab and 10 ng/ml FAS, (D) addition of 10 µM ciglitazone, and (E) coincubation with 0.5 µg/ml anti-Fas-ab and 10 µM ciglitazone. Data are representative of n = 4.
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, as determined by real-time PCR, and die in response to its activation by apoptosis. Second, sera of septic patients contain a PPAR-specific ligand, as shown by reporter analyses and decreased cell viability. Our data strongly suggest the involvement of PPAR in T cell apoptosis during sepsis, thus contributing to lymphopenia. We speculate that antagonizing PPAR might prove beneficial in attenuating T lymphocyte apoptosis in the therapy of sepsis, thereby contributing to improved T cell survival.
Sepsis is often accompanied by lymphopenia [6
, 7
, 30
, 31
]. This was corroborated with material used in our study by showing that lymphocyte loss during sepsis predominantly results from T cell depletion. Particularly, the number of CD4+ T cells is reduced significantly, whereas the CD8+ T lymphocyte count is unaltered. However, it cannot be completely excluded that the CD4+ cell decrease in peripheral blood is not a result of cell death but to cell redistribution, mediated by increased adhesion and/or diapedesis. In accordance with our hypothesis, Roth et al. [7
] has recently described CD4+ T lymphocyte death during sepsis. They provide evidence that T helper cell type 1 (TH1) cells are mainly dying, as the serum level of IL-10, a TH2 cytokine, was enhanced in septic patients, indicating TH2 cell survival. In contrast to their data suggesting activation-induced cell death of T lymphocytes via the CD95 and TNFR1 pathways, we excluded CD95 involvement and hypothesized a role for PPAR in this mechanism of action. Our data strongly supported that PPAR expression was elevated in T cells isolated from septic patients compared with T lymphocytes from healthy donors. Considering that PPAR regulates human T cell function by inhibiting T cell activation [15
] as well as modulating survival of activated T cells during inflammation [11
], we hypothesized that PPAR may be induced in T cells from septic patients in response to microbial infection. In line, Wang et al. [32
] have demonstrated that CD3 and CD3/CD28 activation of primary T cells derived from healthy donors caused an induction of PPAR expression. Therefore, PPAR expression may sensitize septic T cells toward PPAR activation, causing T cell death and hence, lymphopenia. Recently, we reported that activation of PPAR in activated T cells requires the addition of exogenous agonists [16
]. These might be provided by endothelial cells or macrophages under inflammatory or septic conditions [33
, 34
]. It is interesting that enhanced levels of the physiological PPAR agonist 15d-PGJ2 have been described in septic blood [35
]. In our model, PPAR was activated by adding sera of septic patients, as shown by reporter analysis. In line, Jurkat cells transfected with a PPAR wt-encoding vector die in response to sera of septic patients, whereas cells transfected with a PPAR d/n mutant did not. We suggest 15d-PGJ2 as the most likely candidate, being available in septic sera. To finally elucidate the origin of the PPAR activator in sera of septic patients, further experiments are necessary with new methodological approaches presently not available in our lab.
The essential role of PPAR in modulating cell death may be related to the proapoptotic properties of this transcription factor. PPAR is known to regulate inflammation and apoptosis in different cell systems such as macrophages [36
], lung epithelial cells [29
], cancer cells [37
], and lymphocytes [11
, 16
, 18
]. The exact mechanisms underlying the proapoptotic properties are yet unknown. To explore the role of activated PPAR in septic T cell apoptosis, we analyzed cell death using the synthetic PPAR agonist ciglitazone. Our results confirmed that PPAR activation causes T cell death. We found that activation of PPAR in T cells of septic patients with the specific agonist ciglitazone also causes apoptotic cell death, supporting our concept of PPAR-dependent apoptosis.
As lymphocytes express PPAR [38
], it is possible that a mechanism other than PPAR activation is responsible for the cell death observed in activated T lymphocytes. To exclude any unspecific factors, the synthetic PPAR-specific agonist WY14643 [20
] was used, showing no effect on apoptosis in septic T cells, further supporting the concept of a PPAR-dependent mechanism.
In the mouse, it has been shown that the synthetic PPAR antagonist SR-202, belonging to the phosphonophosphate family, decreased PPAR activity in vitro by attenuating adipocyte differentiation as well as in vivo, by protecting animals from high-fat, diet-induced adiposity [25
]. Our transient transfection experiments in activated Jurkat T cells demonstrate that ciglitazone stimulated PPAR-dependent gene transcription twofold, whereas SR-202-inhibited ciglitazone induced transcriptional activity of PPAR almost completely. As further shown by maintaining the , SR-202 blocked in vitro ciglitazone-mediated apoptosis in septic CD3+ T lymphocytes. The role of apoptosis as the underlying mechanism of cell death was confirmed by annexin V/PI staining. Therefore, we propose that PPAR-dependent apoptosis might contribute to lymphopenia during sepsis. To further verify our data, which we obtained with the pharmacological PPAR inhibitor SR-202, RNA interference silencing of PPAR expression in primary T cells will be an aim of future experiments.
Molecular mechanisms initiating PPAR-dependent apoptosis in activated T cells are unclear. Earlier studies pointed to Fas (Apo-1/CD95) in the control of T cell viability. Repeated stimulation of T cells with CD3 antibody resulted in high Fas and Fas ligand expression, and as a consequence, these cells die [39
]. Moreover, Fas (Apo-1/CD95) is associated with PPAR-related apoptosis in A549 lung adenocarcinoma cells [29
]. Our data obtained are in agreement with our previous studies [16
], suggesting that Fas (Apo-1/CD95) does not transmit PPAR-mediated T lymphocyte apoptosis in sepsis, supporting the concept of a receptor-independent initiation of the cell death program.
We conclude that PPAR plays a significant role in T cell apoptosis, contributing to lymphocyte loss during sepsis. Thus, inhibition of PPAR may be beneficial to prevent lymphopenia.
wt and AF2 vectors. We thank Dr. T. Huber and Dr. R. Wendler for providing the septic material and supporting the project. We thank Nadja Wallner for expert technical assistance. Received February 1, 2005; revised July 14, 2005; accepted August 26, 2005.
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expression by peroxisome proliferator-activated receptor J. Immunol. 172,7530-7536 is expressed by mouse T lymphocytes and PPAR agonists induce apoptosis Eur. J. Immunol. 31,1098-1105[CrossRef][Medline] and are killed by PPAR- agonists Clin. Immunol. 103,22-33[CrossRef][Medline] activated by macrophage-derived 12/15-lipoxygenase ligands J. Biol. Chem. 277,3973-3978 expression sensitizes primary human T cells toward apoptosis J. Leukoc. Biol. 73,665-672 agonists induce apoptosis in transformed, but not normal, human T lineage cells Immunology 105,23-34[CrossRef][Medline] (PPAR) mutant is a constitutive repressor and inhibits PPAR-mediated adipogenesis J. Biol. Chem. 275,5754-5759 (PPAR ) ligand, selectively induces the early growth response-1 gene independently of PPAR . A novel mechanism for its anti-tumorigenic activity J. Biol. Chem. 278,5845-5853 12,14-prostaglandin J2 inhibits CD95 ligand gene expression in T lymphocytes: interference with promoter activation via peroxisome proliferator-activated receptor--independent mechanisms J. Immunol. 170,4578-4592 B degradation using a peroxisome proliferator-activated receptor--independent mechanism J. Immunol. 168,6232-6243 antagonist with antiobesity and antidiabetic activity Mol. Endocrinol. 16,2628-2644 transcription factor J. Immunol. 170,887-894 can enhance mitochondrial potential and promote cell survival J. Biol. Chem. 277,31781-31788 12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes J. Biol. Chem. 277,10459-10466 by nitric oxide in monocytes/macrophages down-regulates p47phox and attenuates the respiratory burst J. Immunol. 169,2619-2626 and induces apoptosis of human monocyte-derived macrophages J. Biol. Chem. 273,25573-25580 stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells Oncogene 21,2171-2180[CrossRef][Medline] (PPAR) is expressed in resting murine lymphocytes. The PPAR in T and B lymphocytes is both transactivation and transrepression competent J. Biol. Chem. 277,6838-6845This article has been cited by other articles:
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 S.-H. Jo, C. Yang, Q. Miao, M. Marzec, M. A. Wasik, P. Lu, and Y. L. Wang Peroxisome Proliferator-Activated Receptor {gamma} Promotes Lymphocyte Survival through Its Actions on Cellular Metabolic Activities J. Immunol., September 15, 2006; 177(6): 3737 - 3745. [Abstract] [Full Text] [PDF]
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