CD40 Engagement Prevents Peroxisome Proliferator-Activated Receptor Agonist-Induced Apoptosis of B Lymphocytes and B Lymphoma Cells by an N
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免疫学杂志 2005年第7期
Abstract
Peroxisome proliferator-activated receptor (PPAR) is a transcription factor important in fat metabolism and is emerging as an important regulator of immunity and inflammation. We previously demonstrated that normal and malignant B lineage cells express PPAR and die by apoptosis after PPAR agonist exposure. In this study, we used the WEHI-231 mouse B lymphoma and normal mouse spleen B lymphocytes to elucidate the mechanism of PPAR agonist-induced apoptosis, and to determine whether an apoptosis rescue mechanism exists. In WEHI-231 cells, the natural PPAR agonist 15-deoxy-12,14-PGJ2 and the synthetic PPAR agonist ciglitazone induced activation of caspase 3 and caspase 9, a decrease in mitochondrial membrane potential, and caused cleavage of the caspase substrate poly(ADP-ribose) polymerase. We next tested whether CD40, whose engagement delivers a potent prosurvival signal for B cells, could protect B cells from PPAR agonist-induced apoptosis. CD40 engagement with CD40L significantly blunted the ability of PPAR agonists to induce apoptosis of B lymphocytes and prevented the inhibition of NF-B mobilization by 15-deoxy-12,14-PGJ2 and ciglitazone. Interestingly, PPAR agonists induced an increase in IB and IB protein levels, which was prevented with CD40 engagement. The rescue mechanism induced by CD40 engagement was dependent on NF-B, as an NF-B inhibitor prevented rescue. Apoptosis induction by PPAR ligands may be important for immune regulation by killing B lymphocytes as a rapid means to dampen inflammation. Moreover, the ability of PPAR agonists to kill malignant B lineage cells has implications for their use as anti-B lymphoma agents.
Introduction
There are three forms of peroxisome proliferator-activated receptor (PPAR)3 including PPAR, PPAR (also called PPAR), and PPAR, each encoded by separate genes and having distinct tissue distribution (1). PPAR has been of particular interest as it is a ligand-activated receptor important for regulating the storage and metabolism of dietary fats. PPAR agonists include the thiazolidinedione class of synthetic anti-type 2 diabetes drugs, and the naturally occurring endogenous ligands 15-deoxy-12,14-PGJ2 (15d-PGJ2) and lysophosphatidic acid (2, 3, 4, 5). The 15d-PGJ2 is a product of the cyclooxygenase enzymes and is formed as a consequence of the spontaneous dehydration of PGD2 (6). Several different types of cells are known to produce PGD2, including mast cells, APCs, and certain activated T cells (7, 8, 9). The synthesis of 15d-PGJ2 in vivo can occur in macrophages, as well as during certain chronic inflammatory states (10, 11). 15d-PGJ2 in humans has not been well studied except that it can be found in human atherosclerotic lesions (10) and in human urine (12).
An emerging feature of PPAR agonists is their anti-inflammatory properties. For example, 15d-PGJ2 inhibits the production of proinflammatory mediators TNF- and IL-1 in monocytes, and inducible NO synthase and matrix metalloproteinase-9 in macrophages (13, 14). PPAR agonists can also inhibit cyclooxygenase-2 induction (15). Several in vivo studies have confirmed the anti-inflammatory effects of PPAR agonists. In animal models, PPAR agonists attenuate inflammation in experimental autoimmune encephalomyelitis, inflammatory bowel disease, and adjuvant induced arthritis (16, 17, 18). Our laboratory has demonstrated the expression of PPAR and induction of apoptosis with PPAR agonists in both human and mouse B and T lymphocytes (19, 20, 21). PPAR agonists may therefore play an important role in immune regulation.
Apoptosis is a mechanism of cell death characterized by shrinkage of the cell, chromatin condensation, DNA fragmentation, caspase activation, and packaging of cellular components into membrane-bound apoptotic bodies (22). Caspases are cysteine proteases that cleave substrates after specific aspartic acid residues. These enzymes exist as inactive proenzymes and are activated when a cell receives an apoptotic stimulus. By breaking down cellular components, caspases induce the morphologic features of apoptosis (22). Understanding the mechanism of PPAR agonist-induced apoptosis will provide insight into agonist anti-inflammatory function and potential use as anti-inflammatory and immune regulatory agents. The NF-B pathway is important for B cell survival, as constitutive NF-B is responsible for maintaining normal cellular functions such as Ig L chain expression (23). Extensive BCR cross-linking by anti-IgM Abs in WEHI-231 immature B lymphoma cells results in apoptosis caused by an inhibition of NF-B and activation of caspases (24, 25, 26). Costimulation through CD40 prevented BCR-induced apoptosis by maintaining or inducing NF-B through the degradation of IB molecules (27, 28).
Our laboratory was the first to show that PPAR agonists influence normal and malignant B cells by inducing apoptosis (19, 20). This effect raised the possibility that natural PPAR agonists may play a role in turning off the cellular and humoral immune response. It also suggested that synthetic PPAR agonists might be used to inhibit abnormal immune responses such as inflammation, autoimmune disease, and malignancies such as B cell lymphoma. In this study, we show that B cells undergo apoptosis through a caspase-mediated process with effects on mitochondrial depolarization. If natural PPAR agonists have a physiologic role in the immune response, we hypothesize that there is likely a rescue pathway to protect B cells from PPAR agonist-induced apoptosis to prevent unchecked B cell depletion during the response. We report that PPAR agonists induce apoptosis by inhibiting NF-B activation through the up-regulation of IB and IB and that CD40 activation blocks this process. As such, T cell-B cell interaction with its resultant CD40L-CD40 interaction may inhibit PPAR agonist-induced B cell apoptosis suggesting a possible immunoregulatory pathway.
Materials and Methods
Reagents
Ciglitazone, 15d-PGJ2, WY-14643, and SN50 NF-B inhibitor peptide and SN50 inactive control peptide were purchased from BIOMOL; PGF2, MTT, and DMSO were purchased from Sigma-Aldrich; caspase inhibitor I benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk) was purchased from Calbiochem; caspase substrate for caspases 1, 2, 3, 8, 9, and 10 and for caspase assay buffer were purchased from BioVision; rCD40 ligand membrane was prepared as described (29, 30).
Cells and culture conditions
The WEHI-231 B cell lymphoma is a model for immature B lymphocytes based on surface IgM expression and susceptibility to anti-IgM-induced apoptosis that has been described in detail previously (24, 31). WEHI-231 B lymphoma cells were grown in RPMI 1640 tissue culture media (Invitrogen Life Technologies) supplemented with 5% FBS, 5 x 10–5 M 2-ME (Eastman Kodak), 10 mM HEPES (U.S. Biochemical), 2 mM L-glutamine (Invitrogen Life Technologies), and 50 μg/ml gentamicin (Invitrogen Life Technologies). For apoptosis studies, WEHI-231 cells or mouse spleen B cells were incubated with PPAR agonists or DMSO. For CD40L (CD154) studies, B cells were preincubated with or without recombinant CD40L for 3 h, washed and treated with PPAR agonists. Studies were performed to determine the optimal conditions for the cell rescue assays (data not shown).
B cell purification
Small dense resting B cells were isolated by negative selection from the spleens of 6- to 22-wk-old male BALB/c mice (The Jackson Laboratory). A single cell suspension was prepared by mechanical disruption, and RBC were lysed by incubation in buffered ammonium chloride. Adherent cells were depleted by incubation at 37°C for 2 h. Nonadherent cells were gently washed from the culture, pelleted, and resuspended in a mixture consisting of the following hybridoma supernatants: 30H12 (anti-Thy1.2), GK1.5 (anti-CD4), and 3.155 (anti-CD8). This suspension was incubated on ice for 45 min. T lymphocytes were depleted by the addition of low toxicity baby rabbit complement (Cedarlane) and incubated for 45 min at 37°C. B cells were separated using a discontinuous Percoll gradient (Amersham Pharmacia Biotech). Small dense resting B cells were isolated from the lowest interface. These cells were >98% B220-positive, with <1% of detectable CD3-positive cells as measured by flow cytometry. The B cells were cultured as described for WEHI-231.
Viability assays
Cells were incubated with PPAR agonists or DMSO as a control for 48 h at a density of 6 x 104 cells per well of a 96-well flat-bottom microtiter plate. A solution of 5 mg/ml MTT in PBS was added for the last 4 h of incubation. After 4 h, the plate was centrifuged, the media removed and DMSO was added to each well to dissolve the precipitate. The plate was read at 510 nm on a Benchmark microplate reader (Bio-Rad). The results are presented as the percentage of the DMSO-treated control. Cells treated with CD40L were normalized to account for any increase in MTT metabolism due to CD40 activation. For NF-B inhibition studies, cells were pretreated for 1 h with an NF-B inhibitor peptide, SN50 (32), or an inactive control peptide followed by a 3-h incubation with CD40L. An MTT assay was set up as previously described.
Western blot for poly(ADP-ribose) polymerase (PARP)
Samples were prepared as previously described (33). Briefly, 1 x 107 cells were treated with 15d-PGJ2, ciglitazone, or DMSO for 12 h, washed in PBS, and resuspended in sample buffer (62.5 mM Tris-HCl, (pH 6.8), 4 M urea, 10% glycerol, 2% SDS, 5% 2-ME, and 0.003% bromphenol blue). The cells were lysed by sonication using a Vibra Cell low volume high intensity Ultrasonic Processor (Sonics and Materials) for 30 s on ice. Cell lysate (60 μl) was electrophoresed on a 10% reducing polyacrylamide-stacking gel and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked for 2 h at room temperature in 10% Blotto (PBS/0.1% Tween 20, and 10% milk). A monoclonal mouse anti-PARP Ab, which recognizes the full-length PARP (116 kDa) and the 85 kDa PARP cleavage product (BD Pharmingen), was added at a 1/4000 dilution in 2.5% Blotto for 1 h at room temperature, washed in PBS/0.1% Tween 20, and incubated for 1 h with a 1/2000 dilution of a goat anti-mouse IgG-HRP secondary Ab (Santa Cruz
Biotechnology) for 1 h in 2.5% Blotto. The membrane was washed in PBS/0.1% Tween 20 and developed using a Western Lightning chemiluminescence kit (PerkinElmer Life Sciences).
Caspase activity assay
Cells (107) were treated with PPAR agonists, DMSO control, with PPAR agonist and the general caspase inhibitor Z-VAD-fmk, or with a PPAR agonist and CD40L for 6 h. The cells were washed in PBS and lysed in 50 μl of cell lysis buffer (BioVision). The protein concentration in the cell lysate was quantified using the bicinchoninic acid protein assay (BCA Assay kit; Pierce). Caspase activity was assayed by a colorimetric method using the amino acid substrate for a specific caspase linked to the chromophore p-nitroanilide (pNA). Activity was assayed for caspase 3 using DEVD-pNA as substrate and caspase 9 with LEHD-pNA used as substrate (BioVision). Briefly, 100 μg of total protein was incubated with 2x reaction buffer (BioVision), 10 mM DTT, and 200 μM caspase substrate for 1 h at 37°C. The appearance of cleaved pNA was monitored at 405 nm on a Benchmark microtiter plate reader (Bio-Rad). The results are presented as fold increase in activity over DMSO-treated control cell lysates.
Propidium iodide analysis of DNA content
WEHI-231 cells (106) were exposed to 15d-PGJ2 or ciglitazone for up to 12 h. The cells were washed in 1x PBS, fixed in 70% EtOH for at least 2 h and stored at –20°C until the time of analysis. After fixation, the cells were washed in 1x PBS and resuspended in a solution containing 0.1% Triton X-100, 0.2 mg/ml RNase A (Sigma-Aldrich), and 20 μg/ml propidium iodide (Sigma-Aldrich) in PBS. The cells were incubated for 30 min at room temperature and immediately analyzed on a BD Biosciences FACSCalibur flow cytometer. The percentage of cells with sub-G1 DNA content was determined using CellQuest software (BD Biosciences).
Mitochondrial membrane potential
Cells (106) were treated with 15d-PGJ2 or ciglitazone for up to 12 h. For CD40L rescue assays the cells were first treated with CD40L for 3 h and then exposed to PPAR agonists. The cells were then incubated with 40 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes) for the last 15 min of culture. The cells were harvested, washed in PBS and immediately analyzed on a Becton Dickinson FACSCalibur flow cytometer. Cells with intact mitochondrial membrane potential incorporate DiOC6 into the mitochondria.
EMSA for NK-B
Extracts of nuclear protein were prepared as previously described (34). Cells (5 x 106) were treated with or without CD40L for 3 h, followed by PPAR agonist treatment for 4 h and washed in cold PBS. The cells were incubated on ice in hypotonic buffer A (10 mM HEPES-KOH, (pH 7.9), 1.5 mM KCL, 0.5 mM DTT, 0.5% Nonidet P-40, and 0.2 mM PMSF) for 10 min. The lysates were vortexed, and centrifuged for 15 s. The pellet was resuspended in 80 μl of hypertonic buffer C (20 mM HEPES-KOH, (pH 7.9), 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF) and incubated on ice for 20 min. The lysates were centrifuged for 20 s and supernatant containing the nuclear protein was removed and quantified using a Bio-Rad protein assay kit. The consensus sequence for the NF-B binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega) was labeled with [-32P]ATP using T4 polynucleotide kinase (Invitrogen Life Technologies) and the labeled product was purified on Micro Bio-Spin P-30 Tris Chromatography Columns to remove the unbound nucleotides (Bio-Rad). One microgram of nuclear protein extract was incubated at room temperature with 50,000 counts of labeled oligonucleotide, and binding buffer (10 mM Tris-HCl, (pH 7.5), 50 mM NaCl, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 0.05 mg/ml poly(dI:dC)) for 20 min. The samples were run on a 4% nondenaturing polyacrylamide gel at 100 V, the gel was dried for 1 h on a Savant Slab Gel Dryer SGD 2000 (Savant), and exposed to film overnight.
Western blots for IB, IB, and actin
For IB and IB Western blots, 5 x 106 cells were pretreated with CD40L and then exposed to 15d-PGJ2, ciglitazone, or DMSO control for 1 h. The cells were washed in PBS, lysed in Nonidet P-40 lysis buffer containing a protease inhibitor mix (4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, transepoxysuccinyl-L-leucylamido (4-guanidino) butane, bestatin, leupeptin, and aprotinin) (Sigma-Aldrich), and total protein quantified with a BCA Assay kit. Five micrograms of cell lysate was electrophoresed on 10% denaturing polyacrylamide stacking gel and transferred to nitrocellulose membrane. The membranes were blocked for 2 h at room temperature in 10% Blotto. Anti-IB or anti-IB primary Ab (rabbit polyclonal anti-IB or anti-IB; Santa Cruz Biotechnology) was added at a 1/1000 dilution in 2.5% Blotto for 1 h at room temperature, washed with PBS/Tween 20, and the secondary Ab goat anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories) was added at a 1/2000 dilution for 1 h in 2.5% Blotto. Membranes were washed in PBS/Tween 20 and developed by chemiluminescence using a Western Lightning kit. For loading control, membranes were stripped with 0.2 N NaOH and reprobed with an Ab against actin (monoclonal mouse anti-actin; Oncogene Research Products) and a goat anti-mouse IgM-HRP secondary Ab (Oncogene Research Products). Densitometry was performed using Kodak 1D Image Analysis software (Eastman Kodak). The band intensities were normalized to the actin control and plotted as relative to the untreated sample.
Statistical analysis
Statistical analysis was performed using a two-tailed paired Student’s t test. A value of p < 0.05 was considered statistically significant. Error bars represent the SD from the mean. All data are representative of at least three separate experiments.
Results
spases are activated by PPAR agonists in WEHI-231 cells
It was unknown whether caspase activation occurred as part of the mechanism whereby PPAR agonists induce apoptosis of WEHI-231 cells. We found activation of caspase 3 and caspase 9 after B cell exposure to 15d-PGJ2 or ciglitazone (Fig. 1A). After 6 h of treatment with 15d-PGJ2, caspase 3 increased 11-fold and caspase 9 increased 9-fold over the DMSO control. Ciglitazone induced a similar caspase activity pattern (Fig. 1A). In all cases, coincubation with the caspase inhibitor Z-VAD-fmk prevented caspase activity (Fig. 1A). To test that the caspases were active in the cells, the cleavage of a caspase substrate PARP was determined by Western blot analysis. PARP normally functions as a DNA repair enzyme, but during apoptosis is cleaved and inactivated by caspase 3 (35). PARP cleavage is determined by the appearance of an 85-kDa fragment by Western blot analysis. In the PARP Western blot shown in Fig. 1B, untreated cells contained only the full length (116 kDa) PARP, whereas the 85 kDa cleaved PARP appeared in cells exposed to 15d-PGJ2 or ciglitazone. Apoptosis induction by 15d-PGJ2 and ciglitazone was confirmed by propidium iodide staining of the WEHI-231 cells for cellular DNA content. The appearance of a sub-G1 peak was analyzed by flow cytometry and the results were graphed in Fig. 1C as the percentage of sub-G1 cells. A statistically significant increase in the sub-G1 or apoptotic cells was detected at 4 h and increased over time to 80% at 12 h. Both PPAR agonists induced a similar increase in sub-G1 cells over time. The caspase inhibitor Z-VAD-fmk did not prevent cell death induced by PPAR agonists, suggesting the activation of multiple apoptotic pathways in B lymphocytes (data not shown). From this, we concluded that caspases were activated by PPAR agonists and were functional in WEHI-231 cells.
FIGURE 1. Caspase 3 and caspase 9 are activated by PPAR agonists in WEHI-231 cells. A, WEHI-231 cells were treated for 6 h with 1 μM 15d-PGJ2 or 5 μM ciglitazone. As a control, cells were treated with 15d-PGJ2 and the caspase inhibitor Z-VAD-fmk (25 μM). Caspase activity was measured by cleavage of colorimetric caspase substrates. The results are plotted as fold increase over the DMSO control. The OD 405 nm values for the DMSO controls were 0.032 for caspase 3 and 0.026 for caspase 9. Both ciglitazone and 15d-PGJ2 activated caspase 3 and caspase 9. *, p < 0.05 for 15d-PGJ2 and ciglitazone treatments compared with the DMSO control. B, The caspase substrate PARP is cleaved in cells treated with 15d-PGJ2 and ciglitazone. WEHI-231 cells were treated for 12 h with 1 μM 15d-PGJ2, or 5 μM ciglitazone, the cells were lysed, sonicated, and a Western blot for PARP was performed. The PPAR agonists induced PARP cleavage as visualized by the appearance of an 85 kDa PARP cleavage product. C, A time course analysis of sub-G1 apoptotic WEHI-231 cells was determined by propidium iodide staining after exposure to 1 μM 15d-PGJ2 and 5 μM ciglitazone. *, p < 0.05 for 15d-PGJ2 and ciglitazone treatment as compared with the untreated control.
PPAR agonists induce a loss of mitochondrial membrane potential
In both stress induced apoptosis and death receptor signaling, caspase activation can be associated with mitochondrial damage or caspases can be activated independent of mitochondrial damage (36). To determine whether PPAR agonist-induced caspase activation is associated with mitochondrial damage, we evaluated whether PPAR agonist treatment of WEHI-231 cells resulted in a loss of mitochondrial membrane potential as monitored by the incorporation of the cationic dye DiOC6. Cells with intact mitochondria incorporate DiOC6, whereas cells with damaged mitochondria incorporate less DiOC6. WEHI-231 cells were treated for 12 h with the PPAR agonists 15d-PGJ2 and ciglitazone or with PGF2 or WY-14643 (a PPAR agonist) as negative controls (Fig. 2). When treated with 15d-PGJ2, 18% of the cells at 1 μM, 72% at 2.5 μM, and 96% at 5 μM had a decrease in DiOC6 incorporation, as compared with the DMSO control. The same was true for cells incubated with ciglitazone with 31% at 5 μM, 49% at 10 μM, and 99% of cells at 20 μM with a decrease in DiOC6 incorporation. A time course analysis measuring mitochondrial membrane potential was performed showing that a significant decrease occurs as early as 4 h after PPAR agonist exposure with maximal reduction seen after 12 h of exposure (Fig. 2B). The caspase inhibitor Z-VAD-fmk did not prevent loss of mitochondrial membrane potential suggesting that caspases did not cause the mitochondrial changes (data not shown). These results demonstrate that mitochondrial damage occurs during PPAR agonist-induced apoptosis. Treatment with 10 μM PGF2 or WY-14643 did not decrease DiOC6 incorporation as compared with the DMSO control.
FIGURE 2. PPAR agonists induce a loss of mitochondrial membrane potential. A, WEHI-231 cells were incubated for 12 h with PPAR agonists and 40 nM fluorescent mitochondrial dye DiOC6 was added for the last 15 min of culture. The cells were analyzed for DiOC6 incorporation by flow cytometry. Cells were treated with three different negative controls (upper row): DMSO, 10 μM PGF2, and 10 μM WY-14643. The 15d-PGJ2-treated cells (1, 2.5, and 5 μM) are shown (middle row), and ciglitazone-treated (5, 10, and 20 μM) cells (lower row). A loss of mitochondrial membrane potential, an indicator of apoptosis, was determined by a decrease in DiOC6 fluorescence. The percentage of cells with a decrease in DiOC6 fluorescence is shown on each histogram. The drug treatments (shaded histogram) and the DMSO control (open histogram) are represented. B, A time course analysis of mitochondrial membrane potential for WEHI-231 cells exposed to PPAR agonists. The results are plotted as the percentage of cells with decreased mitochondrial membrane potential. *, p < 0.01 as compared with DMSO control cells.
CD40 engagement partially prevents PPAR agonist-induced cell death of B lymphocytes
CD40 on B cells delivers prosurvival signals and BCR-induced apoptosis can be prevented by engagement of CD40 with CD40L (27). Whether a rescue mechanism exists for PPAR agonist-induced apoptosis of B lymphocytes is unknown. It is also important to determine whether CD40 engagement can blunt the death signal induced by PPAR ligands. Therefore, to determine whether PPAR agonist-induced apoptosis is attenuated by CD40 engagement, WEHI-231 cells and B lymphocytes isolated from mouse spleen were preincubated with CD40L for 3 h and subsequently incubated with 15d-PGJ2, ciglitazone, or DMSO. Cell viability was then determined by MTT assay after 48 h and is presented in Fig. 3 as a percentage of the DMSO control. The protection of WEHI-231 (Fig. 3A) and spleen B lymphocytes (Fig. 3B) by CD40L from cell death elicited by PPAR agonist exposure was dependent on the dose of PPAR agonist. In WEHI-231 cells, CD40L rescued them from 15d-PGJ2-induced cell death at doses <2.5 μM 15d-PGJ2. In contrast CD40L did not rescue cells from death induced by 15d-PGJ2 at doses greater than 2.5 μM (Fig. 3A). The best rescue was observed between 1 and 2 μM 15d-PGJ2 in which incubation with 15d-PGJ2 and CD40L resulted in 95% cell survival in contrast to the 20% cell survival seen for 15d-PGJ2 alone. Cell death with ciglitazone was also prevented dependent on the concentration of ciglitazone (Fig. 3A, right panel). Prevention of cell death was seen at 6 μM ciglitazone, which alone resulted in 35% cell survival, whereas CD40 ligation with ciglitazone increased cell survival up to 80% of the control. In addition, CD40 engagement prevented cell death in freshly purified mouse spleen B lymphocytes, even more significantly than in WEHI-231 cells. With 15d-PGJ2, CD40L cotreatment rescued cell death over a broader range of 15d-PGJ2 concentrations with significant cell death inhibition occurring at doses <5 μM (Fig. 3B). A similar result was obtained with ciglitazone, with the most significant rescue seen at 10 μM at which survival was increased to 90% of the control (Fig. 3B, right panel). As shown in Fig. 3C, CD40 engagement in WEHI-231 cells prevented activation of caspase 3 and caspase 9 suggesting that CD40 signaling prevents apoptosis. DiOC6 staining of the mitochondria was next performed to determine whether CD40 engagement prevented the loss of mitochondrial membrane potential in WEHI-231 cells and spleen B cells. The cells were treated with or without CD40L and PPAR agonist for 12 h at which time incorporation of DiOC6 was measured by flow cytometry. As shown in Table I, CD40 engagement was able to prevent the PPAR agonist-induced loss of mitochondrial membrane potential in both WEHI-231 and normal mouse spleen B lymphocytes. From these results we concluded that CD40 engagement was able to partially rescue PPAR agonist-induced apoptosis. It is evident that at the higher doses of agonist, CD40 costimulation is unable to overcome the apoptotic signals induced by the PPAR agonists.
FIGURE 3. CD40 engagement partially prevents PPAR agonist-induced cell death of B lymphocytes. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h and then exposed to increasing concentrations of 15d-PGJ2 or ciglitazone for 48 h and an MTT assay was performed. The results are graphed as a percentage of the untreated (DMSO) control. *, p < 0.05 for PPAR agonist + CD40L compared with PPAR agonist alone. C, CD40 engagement prevents activation of caspase 3 and caspase 9 by PPAR agonists (1 μM 15d-PGJ2 and 6 μM ciglitazone) in WEHI-231 cells. The results are plotted as fold increase over the DMSO control. DMSO control cells had an OD 405 nm value of 0.035 for caspase 3 and 0.041 for caspase 9. *, p < 0.01 as compared with cells exposed to PPAR agonist alone.
Table I. CD40L prevents PPAR agonist-induced loss of mitochondrial membrane potential in B lymphocytes
PPAR agonist inhibition of NF-B is prevented by CD40 engagement
Inhibition of NF-B, for example by BCR cross-linking, is associated with B cell apoptosis (27, 28). To determine whether NF-B was involved in PPAR agonist-induced apoptosis, gel shift assays were performed for NF-B in WEHI-231 cells and mouse spleen B cells. Cells were incubated with PPAR agonist for 4 h, and 1 μg of nuclear protein was incubated with a radiolabeled probe containing the consensus DNA binding sequence for NF-B. The results of a representative EMSA are shown in Fig. 4. WEHI-231 (Fig. 4A) and normal mouse spleen B cells (Fig. 4B) have a constitutive level of NF-B translocation into the nucleus, which was inhibited dose-dependently by the PPAR agonists 15d-PGJ2 and ciglitazone. NF-B was almost completely inhibited at the highest concentrations of both PPAR agonists. As CD40 activation of B cells induces the degradation of IB and subsequent activation of NF-B (27, 28), we next determined whether CD40 engagement similarly prevented the NF-B inhibition by PPAR agonists. Incubation with CD40L alone increased NF-B translocation and this increase was maintained in the presence of low concentrations of 15d-PGJ2 or ciglitazone, but at higher concentrations of PPAR agonist there was decreased NF-B translocation (Fig. 4). These results indicate that CD40 engagement prevents NF-B inhibition and apoptosis at lower concentrations of PPAR agonists, but is not able to overcome the inhibitory signals at higher PPAR agonist concentrations.
FIGURE 4. PPAR agonist inhibition of NF-B is prevented by CD40 engagement. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h and exposed to 15d-PGJ2 (left panels) and ciglitazone (right panels) for 4 h at the indicated concentrations. An EMSA was performed on 1 μg of nuclear extract using a radiolabeled DNA binding sequence for NF-B. Lane 1 contains only the radiolabeled probe (free probe-FP). The last lane is extract from CD40L-treated cells incubated with an unlabeled probe as a cold competitor (CC) to control for binding specificity.
PPAR agonists induce an increase in IB levels, which is prevented by CD40 engagement
As increased IB expression is one mechanism whereby NF-B activity is inhibited (37), we next determined the effect of PPAR agonists on IB levels. Cells were pretreated with CD40L and then exposed to 15d-PGJ2 or ciglitazone, with or without CD40L for 1 h. A Western blot analysis was performed on whole cell lysates for IB and IB, with actin blots shown as additional loading controls (Fig. 5). WEHI-231 (Fig. 5A) and spleen B cells (Fig. 5B) have a constitutive level of both IB and IB proteins. Upon exposure to 15d-PGJ2 and ciglitazone, the total cellular levels of IB were increased 4- to 5-fold in WEHI-231 cells and 2- to 3-fold in spleen B cells. This increase in IB correlated with PPAR agonist inhibition of NF-B translocation (see Fig. 4). Exposure to CD40L alone significantly reduced IB levels, which remained reduced with the addition of low concentrations of PPAR agonists. However, coincubation with high doses of PPAR agonists resulted in an overall increase in IB levels. These data support the concept that CD40 engagement enhances the degradation of IB in the presence of low concentrations of PPAR agonists thereby attenuating the NF-B inhibition elicited by such low doses of PPAR agonists. In contrast, CD40 engagement in the presence of higher doses of PPAR agonist was unable to overcome the PPAR signal leading to an increase in IB. This may account for our finding that CD40L does not block NF-B inhibition elicited by higher doses of PPAR agonists.
FIGURE 5. PPAR agonists induce an increase in IB levels, which is prevented by CD40 engagement. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h before culture with PPAR agonists for 1 h. Five micrograms of whole cell lysate was loaded into each lane for Western blot. The membrane was first probed for IB, stripped and reprobed for IB and for actin. Densitometry was performed and each band was normalized to actin. The untreated sample was designated an intensity of 1 and the remaining band intensities were plotted as relative to the untreated sample.
Rescue of apoptosis by CD40 engagement is prevented by NF-B inhibition
To further support the concept that CD40 activation of NF-B was responsible for apoptosis rescue after exposure of B lymphocytes to low doses of PPAR agonists, the B cells were preincubated with the NF-B inhibitor SN50. SN50 is a cell permeable peptide inhibitor that prevents NF-B translocation into the nucleus (32). After SN50 pretreatment, WEHI-231 and spleen B cells were exposed to CD40L for 3 h and then exposed to PPAR agonists for 48 h in the presence of the NF-B inhibitor, at which time an MTT assay was performed. As shown in Fig. 6, CD40 rescue of PPAR agonist-induced apoptosis was completely inhibited by the addition of SN50 in both the WEHI-231 (Fig. 6A) and spleen B cells (Fig. 6B). An inactive control peptide did not inhibit CD40 rescue of the B cells (data not shown). As shown in Fig. 6C, SN50 reversed the protective effects that CD40L had on mitochondrial membrane potential. The 15d-PGJ2 alone induced a decrease in 77% of WEHI-231 cells and pretreatment with CD40L prevented this decrease in mitochondrial membrane potential (Table I and Fig. 6C). When pretreated with SN50 and CD40L, 15d-PGJ2 still reduced mitochondrial membrane potential in 75% of cells. Therefore, in B lymphocytes NF-B is a key mediator for eliciting apoptosis and for protecting B lymphocytes from apoptosis induced by PPAR agonists.
FIGURE 6. Rescue of PPAR agonist-induced apoptosis by CD40 engagement is prevented by NF-B inhibition. WEHI-231 cells (A) and spleen B lymphocytes (B) were pretreated with the NF-B inhibitor peptide SN50 for 1 h and then CD40L was added for an additional 3 h. An MTT assay was set up and the cells were exposed to 15d-PGJ2 or ciglitazone in the presence of CD40L and SN50 for 48 h. The results are presented as the percentage of the control. *, p < 0.05 for CD40L + PPAR agonist vs PPAR agonist alone; **, p < 0.05 for CD40L + PPAR agonist vs CD40L + SN50 + PPAR agonist. There were no significant differences between PPAR agonist alone and PPAR agonist with CD40L and SN50. C, SN50 prevents CD40L protection from loss of mitochondrial membrane potential. Following pretreatment with CD40L and SN50, WEHI-231 cells were exposed to 1 μM 15d-PGJ2 for 12 h. SN50 alone did not cause a decrease in DiOC6 incorporation (see also Table I for additional controls and data).
Discussion
In the present study, we show that both natural and synthetic PPAR agonists elicit B cell apoptosis as shown by caspase activation, PARP cleavage, and by the loss of mitochondrial membrane potential. In addition, PPAR agonists inhibited NF-B and induced an increase in cellular IB levels not only in WEHI-231 B lymphoma cells but also in freshly isolated normal mouse spleen B lymphocytes. Finally, we demonstrate that CD40 engagement prevents apoptosis and mitochondrial membrane potential loss induced by PPAR agonists in B lineage cells. CD40 signaling prevents the PPAR agonist-induced inhibition of the survival factor NF-B and this rescue event is dependent on NF-B as an NF-B inhibitor reverses the apoptosis rescue.
The mechanism of PPAR agonist-induced apoptosis of B lymphocytes has not been explored and understanding the apoptotic mechanism may have implications for the therapeutic use of PPAR agonists. In this study, we found that caspase 3 and caspase 9 were activated following exposure of B cells to PPAR agonists. Caspase 3 and caspase 9 are of particular importance because their activation supports involvement of mitochondria in the apoptosis pathway. Indeed, we found a reduction in mitochondrial membrane potential as early as 4 h, but caspase activation was not detected until 6 h after PPAR agonist exposure. Because caspase inhibition did not prevent the loss in mitochondrial membrane potential, these data suggest that the reduction in mitochondrial membrane potential did not occur as a result of caspase activation and supports a role for the mitochondria in caspase activation. Activation of caspase 9 only occurs if mitochondria are damaged and cytochrome c is released (36). When released into the cytoplasm, cytochrome c forms a complex with Apaf-1, and this complex associates with pro-caspase 9 and causes caspase 9 activation, which then activates other caspases, such as caspase 3 (36). Caspase 3 is an effector caspase responsible for cleaving cellular substrates during apoptosis, including PARP (36). Caspase 3 has also been shown to cleave and inactivate the p65 subunit of NF-B (38), which may be an additional mechanism by which PPAR agonists caused a significant reduction in NF-B translocation observed in WEHI-231 cells and normal B lymphocytes.
NF-B is an important transcription factor for B cell development and maintenance. In addition to B cell-specific functions, NF-B can also induce expression of antiapoptotic proteins, such as the inhibition of apoptosis proteins that function as caspase inhibitors (39). Therefore, the inhibition of NF-B results in the loss of the cells ability to prevent apoptosis and is one mechanism contributing to PPAR agonist-induced apoptosis of B lymphocytes. The SN50 NF-B inhibitor alone did not kill the B lineage cells suggesting that NF-B inhibition alone is not sufficient to induce apoptosis and that multiple mechanisms contribute to the apoptosis induced by PPAR agonists. It is most likely a combination of caspase activation, mitochondrial damage, and NF-B inhibition that steers the cells down the apoptotic pathway. The existence of multiple pathways may explain why CD40 engagement could not sustain increased NF-B activation at higher concentrations of PPAR agonist and only partially rescued B lymphocytes from PPAR agonist-induced apoptosis. It is probable that activation of NF-B and the subsequent initiation of survival signals by CD40 ligation are not able to fully overcome the potent apoptotic stimuli induced by PPAR agonists in B lymphocytes.
The regulation of NF-B by PPAR agonists is an emerging concept that may have both PPAR-dependent and -independent mechanisms. For example, in mouse macrophage cells, Castrillo et al. (40) reported 15d-PGJ2 inhibited the IB kinase that is responsible for phosphorylating IB. In HeLa cells, this inhibition of IB kinase was independent of PPAR because 15d-PGJ2 was able to inhibit IB kinase in HeLa cells that lack PPAR (41). The PPAR agonist-induced increase in IB levels seen in B lymphocytes could be a result of IB kinase inhibition, which would prevent the degradation of IB. In contrast to reports of PPAR-independent inhibition of NF-B by PPAR agonists, a PPAR-dependent mechanism also exists for NF-B inhibition. In LPS-activated macrophages, PPAR formed a complex with both the p50 and p65 subunits of NF-B, resulting in transrepression of NF-B (42). The interaction of PPAR with NF-B may also be important for regulating shuttling of NF-B to and from the nucleus thereby controlling NF-B activation (43). There is also evidence that PPAR is important in the regulation of NF-B in B lineage cells. In contrast to our findings, Schlezinger et al. (44) reported an increase in NF-B nuclear translocation in mouse pre-B cells after treatment with PPAR agonists. This interesting difference is most likely due to the developmental stage of the B cell and suggests that the effects of PPAR agonists may differ during B cell maturation. In support of our findings of PPAR agonist inhibition of NF-B, B cells from PPAR heterozygous mice (PPAR+/–) exhibited a dysregulation of the NF-B pathway that resulted in increased spontaneous NF-B activation and increased proliferation when compared with wild-type mice (45). These PPAR+/– B cells required higher doses of PPAR agonists to suppress both the LPS-induced proliferation and the increased serum Ab production observed during an Ag-specific response (45). Further studies are needed to better define the PPAR dependency of the effects of PPAR agonists on B lymphocytes. Taken together with the results reported in this study, PPAR agonists appear to play a fundamental role in preventing a pathologic uncontrolled B cell response to Ag in vivo and may do so in part through an NF-B dependent pathway. Finally, such a B cell dampening effect of endogenous PPAR agonists may itself be controlled by CD40 ligation of B cells to prevent B cell depletion.
The findings we report for normal spleen B lymphocytes suggest a pivotal role for PPAR agonists in controlling B cell responses. 15d-PGJ2 and synthetic PPAR agonists may serve to counterbalance the effects of other PGs, such as PGE2, which induces Ig class switching in B cells (46). Although the physiologic levels and importance of 15d-PGJ2 in vivo are under intense investigation, there are several pieces of evidence supporting the in vivo significance of this PG and other newly discovered natural PPAR agonists, such as lysophosphatidic acid (5). For example, 15d-PGJ2 was detected during the resolution phase of rat carrageenin induced pleurisy (11) and also in macrophages from human atherosclerotic plaques (10). The production of 15d-PGJ2 by macrophages, and PGD2 by mast cells, APCs, and activated T cells suggests the possibility that 15d-PGJ2 is produced in lymphoid organs in which B lymphocytes reside and expand during an immune response (7, 8, 9, 10).
We speculate that 15d-PGJ2 or other PPAR agonists contribute to the dampening of B cells especially during the resolution phase of an immune response by inducing their apoptosis as demonstrated for both malignant and normal B lymphocytes. The ability of CD40 engagement to prevent PPAR agonist-induced apoptosis of B lymphocytes suggests the existence of an important antiapoptotic mechanism to allow B cell expansion and survival. Future studies of the in vivo effects of PPAR agonists will be critical in defining the role of 15d-PGJ2 and synthetic PPAR agonists in immune regulation. Finally, our studies clearly point to the potential of natural and synthetic PPAR agonists as therapy for B cell malignancies.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Christine Martey-Ochola for providing technical assistance and Stephen Pollock for preparing reagents.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by United States Public Health Service Grants DE11390, ES01247, a James P. Wilmot Cancer Center Discovery Award, the Environmental Protection Agency Particulate Matter Center, and a Leukemia and Lymphoma Society Translational Research Award. D.M.R. was supported by the Rochester Training Program in Oral Infectious Diseases T32-DE07165. F.A. was supported by the International Union Against Cancer Fellowship Program and the Scientific and Technical Research Council of Turkey (TUBITAK)/North Atlantic Treaty Organization-A2.
2 Address correspondence and reprint requests to Dr. Richard P. Phipps, Box 850, Department of Environmental Medicine, University of Rochester Medical Center, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: richard_phipps@urmc.rochester.edu
3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor ; 15d-PGJ2, 15-deoxy-12,14-PGJ2; PARP, poly(ADP-ribose) polymerase; BCR, B cell receptor.
Received for publication May 21, 2004. Accepted for publication January 19, 2005.
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Peroxisome proliferator-activated receptor (PPAR) is a transcription factor important in fat metabolism and is emerging as an important regulator of immunity and inflammation. We previously demonstrated that normal and malignant B lineage cells express PPAR and die by apoptosis after PPAR agonist exposure. In this study, we used the WEHI-231 mouse B lymphoma and normal mouse spleen B lymphocytes to elucidate the mechanism of PPAR agonist-induced apoptosis, and to determine whether an apoptosis rescue mechanism exists. In WEHI-231 cells, the natural PPAR agonist 15-deoxy-12,14-PGJ2 and the synthetic PPAR agonist ciglitazone induced activation of caspase 3 and caspase 9, a decrease in mitochondrial membrane potential, and caused cleavage of the caspase substrate poly(ADP-ribose) polymerase. We next tested whether CD40, whose engagement delivers a potent prosurvival signal for B cells, could protect B cells from PPAR agonist-induced apoptosis. CD40 engagement with CD40L significantly blunted the ability of PPAR agonists to induce apoptosis of B lymphocytes and prevented the inhibition of NF-B mobilization by 15-deoxy-12,14-PGJ2 and ciglitazone. Interestingly, PPAR agonists induced an increase in IB and IB protein levels, which was prevented with CD40 engagement. The rescue mechanism induced by CD40 engagement was dependent on NF-B, as an NF-B inhibitor prevented rescue. Apoptosis induction by PPAR ligands may be important for immune regulation by killing B lymphocytes as a rapid means to dampen inflammation. Moreover, the ability of PPAR agonists to kill malignant B lineage cells has implications for their use as anti-B lymphoma agents.
Introduction
There are three forms of peroxisome proliferator-activated receptor (PPAR)3 including PPAR, PPAR (also called PPAR), and PPAR, each encoded by separate genes and having distinct tissue distribution (1). PPAR has been of particular interest as it is a ligand-activated receptor important for regulating the storage and metabolism of dietary fats. PPAR agonists include the thiazolidinedione class of synthetic anti-type 2 diabetes drugs, and the naturally occurring endogenous ligands 15-deoxy-12,14-PGJ2 (15d-PGJ2) and lysophosphatidic acid (2, 3, 4, 5). The 15d-PGJ2 is a product of the cyclooxygenase enzymes and is formed as a consequence of the spontaneous dehydration of PGD2 (6). Several different types of cells are known to produce PGD2, including mast cells, APCs, and certain activated T cells (7, 8, 9). The synthesis of 15d-PGJ2 in vivo can occur in macrophages, as well as during certain chronic inflammatory states (10, 11). 15d-PGJ2 in humans has not been well studied except that it can be found in human atherosclerotic lesions (10) and in human urine (12).
An emerging feature of PPAR agonists is their anti-inflammatory properties. For example, 15d-PGJ2 inhibits the production of proinflammatory mediators TNF- and IL-1 in monocytes, and inducible NO synthase and matrix metalloproteinase-9 in macrophages (13, 14). PPAR agonists can also inhibit cyclooxygenase-2 induction (15). Several in vivo studies have confirmed the anti-inflammatory effects of PPAR agonists. In animal models, PPAR agonists attenuate inflammation in experimental autoimmune encephalomyelitis, inflammatory bowel disease, and adjuvant induced arthritis (16, 17, 18). Our laboratory has demonstrated the expression of PPAR and induction of apoptosis with PPAR agonists in both human and mouse B and T lymphocytes (19, 20, 21). PPAR agonists may therefore play an important role in immune regulation.
Apoptosis is a mechanism of cell death characterized by shrinkage of the cell, chromatin condensation, DNA fragmentation, caspase activation, and packaging of cellular components into membrane-bound apoptotic bodies (22). Caspases are cysteine proteases that cleave substrates after specific aspartic acid residues. These enzymes exist as inactive proenzymes and are activated when a cell receives an apoptotic stimulus. By breaking down cellular components, caspases induce the morphologic features of apoptosis (22). Understanding the mechanism of PPAR agonist-induced apoptosis will provide insight into agonist anti-inflammatory function and potential use as anti-inflammatory and immune regulatory agents. The NF-B pathway is important for B cell survival, as constitutive NF-B is responsible for maintaining normal cellular functions such as Ig L chain expression (23). Extensive BCR cross-linking by anti-IgM Abs in WEHI-231 immature B lymphoma cells results in apoptosis caused by an inhibition of NF-B and activation of caspases (24, 25, 26). Costimulation through CD40 prevented BCR-induced apoptosis by maintaining or inducing NF-B through the degradation of IB molecules (27, 28).
Our laboratory was the first to show that PPAR agonists influence normal and malignant B cells by inducing apoptosis (19, 20). This effect raised the possibility that natural PPAR agonists may play a role in turning off the cellular and humoral immune response. It also suggested that synthetic PPAR agonists might be used to inhibit abnormal immune responses such as inflammation, autoimmune disease, and malignancies such as B cell lymphoma. In this study, we show that B cells undergo apoptosis through a caspase-mediated process with effects on mitochondrial depolarization. If natural PPAR agonists have a physiologic role in the immune response, we hypothesize that there is likely a rescue pathway to protect B cells from PPAR agonist-induced apoptosis to prevent unchecked B cell depletion during the response. We report that PPAR agonists induce apoptosis by inhibiting NF-B activation through the up-regulation of IB and IB and that CD40 activation blocks this process. As such, T cell-B cell interaction with its resultant CD40L-CD40 interaction may inhibit PPAR agonist-induced B cell apoptosis suggesting a possible immunoregulatory pathway.
Materials and Methods
Reagents
Ciglitazone, 15d-PGJ2, WY-14643, and SN50 NF-B inhibitor peptide and SN50 inactive control peptide were purchased from BIOMOL; PGF2, MTT, and DMSO were purchased from Sigma-Aldrich; caspase inhibitor I benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk) was purchased from Calbiochem; caspase substrate for caspases 1, 2, 3, 8, 9, and 10 and for caspase assay buffer were purchased from BioVision; rCD40 ligand membrane was prepared as described (29, 30).
Cells and culture conditions
The WEHI-231 B cell lymphoma is a model for immature B lymphocytes based on surface IgM expression and susceptibility to anti-IgM-induced apoptosis that has been described in detail previously (24, 31). WEHI-231 B lymphoma cells were grown in RPMI 1640 tissue culture media (Invitrogen Life Technologies) supplemented with 5% FBS, 5 x 10–5 M 2-ME (Eastman Kodak), 10 mM HEPES (U.S. Biochemical), 2 mM L-glutamine (Invitrogen Life Technologies), and 50 μg/ml gentamicin (Invitrogen Life Technologies). For apoptosis studies, WEHI-231 cells or mouse spleen B cells were incubated with PPAR agonists or DMSO. For CD40L (CD154) studies, B cells were preincubated with or without recombinant CD40L for 3 h, washed and treated with PPAR agonists. Studies were performed to determine the optimal conditions for the cell rescue assays (data not shown).
B cell purification
Small dense resting B cells were isolated by negative selection from the spleens of 6- to 22-wk-old male BALB/c mice (The Jackson Laboratory). A single cell suspension was prepared by mechanical disruption, and RBC were lysed by incubation in buffered ammonium chloride. Adherent cells were depleted by incubation at 37°C for 2 h. Nonadherent cells were gently washed from the culture, pelleted, and resuspended in a mixture consisting of the following hybridoma supernatants: 30H12 (anti-Thy1.2), GK1.5 (anti-CD4), and 3.155 (anti-CD8). This suspension was incubated on ice for 45 min. T lymphocytes were depleted by the addition of low toxicity baby rabbit complement (Cedarlane) and incubated for 45 min at 37°C. B cells were separated using a discontinuous Percoll gradient (Amersham Pharmacia Biotech). Small dense resting B cells were isolated from the lowest interface. These cells were >98% B220-positive, with <1% of detectable CD3-positive cells as measured by flow cytometry. The B cells were cultured as described for WEHI-231.
Viability assays
Cells were incubated with PPAR agonists or DMSO as a control for 48 h at a density of 6 x 104 cells per well of a 96-well flat-bottom microtiter plate. A solution of 5 mg/ml MTT in PBS was added for the last 4 h of incubation. After 4 h, the plate was centrifuged, the media removed and DMSO was added to each well to dissolve the precipitate. The plate was read at 510 nm on a Benchmark microplate reader (Bio-Rad). The results are presented as the percentage of the DMSO-treated control. Cells treated with CD40L were normalized to account for any increase in MTT metabolism due to CD40 activation. For NF-B inhibition studies, cells were pretreated for 1 h with an NF-B inhibitor peptide, SN50 (32), or an inactive control peptide followed by a 3-h incubation with CD40L. An MTT assay was set up as previously described.
Western blot for poly(ADP-ribose) polymerase (PARP)
Samples were prepared as previously described (33). Briefly, 1 x 107 cells were treated with 15d-PGJ2, ciglitazone, or DMSO for 12 h, washed in PBS, and resuspended in sample buffer (62.5 mM Tris-HCl, (pH 6.8), 4 M urea, 10% glycerol, 2% SDS, 5% 2-ME, and 0.003% bromphenol blue). The cells were lysed by sonication using a Vibra Cell low volume high intensity Ultrasonic Processor (Sonics and Materials) for 30 s on ice. Cell lysate (60 μl) was electrophoresed on a 10% reducing polyacrylamide-stacking gel and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked for 2 h at room temperature in 10% Blotto (PBS/0.1% Tween 20, and 10% milk). A monoclonal mouse anti-PARP Ab, which recognizes the full-length PARP (116 kDa) and the 85 kDa PARP cleavage product (BD Pharmingen), was added at a 1/4000 dilution in 2.5% Blotto for 1 h at room temperature, washed in PBS/0.1% Tween 20, and incubated for 1 h with a 1/2000 dilution of a goat anti-mouse IgG-HRP secondary Ab (Santa Cruz
Biotechnology) for 1 h in 2.5% Blotto. The membrane was washed in PBS/0.1% Tween 20 and developed using a Western Lightning chemiluminescence kit (PerkinElmer Life Sciences).
Caspase activity assay
Cells (107) were treated with PPAR agonists, DMSO control, with PPAR agonist and the general caspase inhibitor Z-VAD-fmk, or with a PPAR agonist and CD40L for 6 h. The cells were washed in PBS and lysed in 50 μl of cell lysis buffer (BioVision). The protein concentration in the cell lysate was quantified using the bicinchoninic acid protein assay (BCA Assay kit; Pierce). Caspase activity was assayed by a colorimetric method using the amino acid substrate for a specific caspase linked to the chromophore p-nitroanilide (pNA). Activity was assayed for caspase 3 using DEVD-pNA as substrate and caspase 9 with LEHD-pNA used as substrate (BioVision). Briefly, 100 μg of total protein was incubated with 2x reaction buffer (BioVision), 10 mM DTT, and 200 μM caspase substrate for 1 h at 37°C. The appearance of cleaved pNA was monitored at 405 nm on a Benchmark microtiter plate reader (Bio-Rad). The results are presented as fold increase in activity over DMSO-treated control cell lysates.
Propidium iodide analysis of DNA content
WEHI-231 cells (106) were exposed to 15d-PGJ2 or ciglitazone for up to 12 h. The cells were washed in 1x PBS, fixed in 70% EtOH for at least 2 h and stored at –20°C until the time of analysis. After fixation, the cells were washed in 1x PBS and resuspended in a solution containing 0.1% Triton X-100, 0.2 mg/ml RNase A (Sigma-Aldrich), and 20 μg/ml propidium iodide (Sigma-Aldrich) in PBS. The cells were incubated for 30 min at room temperature and immediately analyzed on a BD Biosciences FACSCalibur flow cytometer. The percentage of cells with sub-G1 DNA content was determined using CellQuest software (BD Biosciences).
Mitochondrial membrane potential
Cells (106) were treated with 15d-PGJ2 or ciglitazone for up to 12 h. For CD40L rescue assays the cells were first treated with CD40L for 3 h and then exposed to PPAR agonists. The cells were then incubated with 40 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes) for the last 15 min of culture. The cells were harvested, washed in PBS and immediately analyzed on a Becton Dickinson FACSCalibur flow cytometer. Cells with intact mitochondrial membrane potential incorporate DiOC6 into the mitochondria.
EMSA for NK-B
Extracts of nuclear protein were prepared as previously described (34). Cells (5 x 106) were treated with or without CD40L for 3 h, followed by PPAR agonist treatment for 4 h and washed in cold PBS. The cells were incubated on ice in hypotonic buffer A (10 mM HEPES-KOH, (pH 7.9), 1.5 mM KCL, 0.5 mM DTT, 0.5% Nonidet P-40, and 0.2 mM PMSF) for 10 min. The lysates were vortexed, and centrifuged for 15 s. The pellet was resuspended in 80 μl of hypertonic buffer C (20 mM HEPES-KOH, (pH 7.9), 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF) and incubated on ice for 20 min. The lysates were centrifuged for 20 s and supernatant containing the nuclear protein was removed and quantified using a Bio-Rad protein assay kit. The consensus sequence for the NF-B binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega) was labeled with [-32P]ATP using T4 polynucleotide kinase (Invitrogen Life Technologies) and the labeled product was purified on Micro Bio-Spin P-30 Tris Chromatography Columns to remove the unbound nucleotides (Bio-Rad). One microgram of nuclear protein extract was incubated at room temperature with 50,000 counts of labeled oligonucleotide, and binding buffer (10 mM Tris-HCl, (pH 7.5), 50 mM NaCl, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 0.05 mg/ml poly(dI:dC)) for 20 min. The samples were run on a 4% nondenaturing polyacrylamide gel at 100 V, the gel was dried for 1 h on a Savant Slab Gel Dryer SGD 2000 (Savant), and exposed to film overnight.
Western blots for IB, IB, and actin
For IB and IB Western blots, 5 x 106 cells were pretreated with CD40L and then exposed to 15d-PGJ2, ciglitazone, or DMSO control for 1 h. The cells were washed in PBS, lysed in Nonidet P-40 lysis buffer containing a protease inhibitor mix (4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, transepoxysuccinyl-L-leucylamido (4-guanidino) butane, bestatin, leupeptin, and aprotinin) (Sigma-Aldrich), and total protein quantified with a BCA Assay kit. Five micrograms of cell lysate was electrophoresed on 10% denaturing polyacrylamide stacking gel and transferred to nitrocellulose membrane. The membranes were blocked for 2 h at room temperature in 10% Blotto. Anti-IB or anti-IB primary Ab (rabbit polyclonal anti-IB or anti-IB; Santa Cruz Biotechnology) was added at a 1/1000 dilution in 2.5% Blotto for 1 h at room temperature, washed with PBS/Tween 20, and the secondary Ab goat anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories) was added at a 1/2000 dilution for 1 h in 2.5% Blotto. Membranes were washed in PBS/Tween 20 and developed by chemiluminescence using a Western Lightning kit. For loading control, membranes were stripped with 0.2 N NaOH and reprobed with an Ab against actin (monoclonal mouse anti-actin; Oncogene Research Products) and a goat anti-mouse IgM-HRP secondary Ab (Oncogene Research Products). Densitometry was performed using Kodak 1D Image Analysis software (Eastman Kodak). The band intensities were normalized to the actin control and plotted as relative to the untreated sample.
Statistical analysis
Statistical analysis was performed using a two-tailed paired Student’s t test. A value of p < 0.05 was considered statistically significant. Error bars represent the SD from the mean. All data are representative of at least three separate experiments.
Results
spases are activated by PPAR agonists in WEHI-231 cells
It was unknown whether caspase activation occurred as part of the mechanism whereby PPAR agonists induce apoptosis of WEHI-231 cells. We found activation of caspase 3 and caspase 9 after B cell exposure to 15d-PGJ2 or ciglitazone (Fig. 1A). After 6 h of treatment with 15d-PGJ2, caspase 3 increased 11-fold and caspase 9 increased 9-fold over the DMSO control. Ciglitazone induced a similar caspase activity pattern (Fig. 1A). In all cases, coincubation with the caspase inhibitor Z-VAD-fmk prevented caspase activity (Fig. 1A). To test that the caspases were active in the cells, the cleavage of a caspase substrate PARP was determined by Western blot analysis. PARP normally functions as a DNA repair enzyme, but during apoptosis is cleaved and inactivated by caspase 3 (35). PARP cleavage is determined by the appearance of an 85-kDa fragment by Western blot analysis. In the PARP Western blot shown in Fig. 1B, untreated cells contained only the full length (116 kDa) PARP, whereas the 85 kDa cleaved PARP appeared in cells exposed to 15d-PGJ2 or ciglitazone. Apoptosis induction by 15d-PGJ2 and ciglitazone was confirmed by propidium iodide staining of the WEHI-231 cells for cellular DNA content. The appearance of a sub-G1 peak was analyzed by flow cytometry and the results were graphed in Fig. 1C as the percentage of sub-G1 cells. A statistically significant increase in the sub-G1 or apoptotic cells was detected at 4 h and increased over time to 80% at 12 h. Both PPAR agonists induced a similar increase in sub-G1 cells over time. The caspase inhibitor Z-VAD-fmk did not prevent cell death induced by PPAR agonists, suggesting the activation of multiple apoptotic pathways in B lymphocytes (data not shown). From this, we concluded that caspases were activated by PPAR agonists and were functional in WEHI-231 cells.
FIGURE 1. Caspase 3 and caspase 9 are activated by PPAR agonists in WEHI-231 cells. A, WEHI-231 cells were treated for 6 h with 1 μM 15d-PGJ2 or 5 μM ciglitazone. As a control, cells were treated with 15d-PGJ2 and the caspase inhibitor Z-VAD-fmk (25 μM). Caspase activity was measured by cleavage of colorimetric caspase substrates. The results are plotted as fold increase over the DMSO control. The OD 405 nm values for the DMSO controls were 0.032 for caspase 3 and 0.026 for caspase 9. Both ciglitazone and 15d-PGJ2 activated caspase 3 and caspase 9. *, p < 0.05 for 15d-PGJ2 and ciglitazone treatments compared with the DMSO control. B, The caspase substrate PARP is cleaved in cells treated with 15d-PGJ2 and ciglitazone. WEHI-231 cells were treated for 12 h with 1 μM 15d-PGJ2, or 5 μM ciglitazone, the cells were lysed, sonicated, and a Western blot for PARP was performed. The PPAR agonists induced PARP cleavage as visualized by the appearance of an 85 kDa PARP cleavage product. C, A time course analysis of sub-G1 apoptotic WEHI-231 cells was determined by propidium iodide staining after exposure to 1 μM 15d-PGJ2 and 5 μM ciglitazone. *, p < 0.05 for 15d-PGJ2 and ciglitazone treatment as compared with the untreated control.
PPAR agonists induce a loss of mitochondrial membrane potential
In both stress induced apoptosis and death receptor signaling, caspase activation can be associated with mitochondrial damage or caspases can be activated independent of mitochondrial damage (36). To determine whether PPAR agonist-induced caspase activation is associated with mitochondrial damage, we evaluated whether PPAR agonist treatment of WEHI-231 cells resulted in a loss of mitochondrial membrane potential as monitored by the incorporation of the cationic dye DiOC6. Cells with intact mitochondria incorporate DiOC6, whereas cells with damaged mitochondria incorporate less DiOC6. WEHI-231 cells were treated for 12 h with the PPAR agonists 15d-PGJ2 and ciglitazone or with PGF2 or WY-14643 (a PPAR agonist) as negative controls (Fig. 2). When treated with 15d-PGJ2, 18% of the cells at 1 μM, 72% at 2.5 μM, and 96% at 5 μM had a decrease in DiOC6 incorporation, as compared with the DMSO control. The same was true for cells incubated with ciglitazone with 31% at 5 μM, 49% at 10 μM, and 99% of cells at 20 μM with a decrease in DiOC6 incorporation. A time course analysis measuring mitochondrial membrane potential was performed showing that a significant decrease occurs as early as 4 h after PPAR agonist exposure with maximal reduction seen after 12 h of exposure (Fig. 2B). The caspase inhibitor Z-VAD-fmk did not prevent loss of mitochondrial membrane potential suggesting that caspases did not cause the mitochondrial changes (data not shown). These results demonstrate that mitochondrial damage occurs during PPAR agonist-induced apoptosis. Treatment with 10 μM PGF2 or WY-14643 did not decrease DiOC6 incorporation as compared with the DMSO control.
FIGURE 2. PPAR agonists induce a loss of mitochondrial membrane potential. A, WEHI-231 cells were incubated for 12 h with PPAR agonists and 40 nM fluorescent mitochondrial dye DiOC6 was added for the last 15 min of culture. The cells were analyzed for DiOC6 incorporation by flow cytometry. Cells were treated with three different negative controls (upper row): DMSO, 10 μM PGF2, and 10 μM WY-14643. The 15d-PGJ2-treated cells (1, 2.5, and 5 μM) are shown (middle row), and ciglitazone-treated (5, 10, and 20 μM) cells (lower row). A loss of mitochondrial membrane potential, an indicator of apoptosis, was determined by a decrease in DiOC6 fluorescence. The percentage of cells with a decrease in DiOC6 fluorescence is shown on each histogram. The drug treatments (shaded histogram) and the DMSO control (open histogram) are represented. B, A time course analysis of mitochondrial membrane potential for WEHI-231 cells exposed to PPAR agonists. The results are plotted as the percentage of cells with decreased mitochondrial membrane potential. *, p < 0.01 as compared with DMSO control cells.
CD40 engagement partially prevents PPAR agonist-induced cell death of B lymphocytes
CD40 on B cells delivers prosurvival signals and BCR-induced apoptosis can be prevented by engagement of CD40 with CD40L (27). Whether a rescue mechanism exists for PPAR agonist-induced apoptosis of B lymphocytes is unknown. It is also important to determine whether CD40 engagement can blunt the death signal induced by PPAR ligands. Therefore, to determine whether PPAR agonist-induced apoptosis is attenuated by CD40 engagement, WEHI-231 cells and B lymphocytes isolated from mouse spleen were preincubated with CD40L for 3 h and subsequently incubated with 15d-PGJ2, ciglitazone, or DMSO. Cell viability was then determined by MTT assay after 48 h and is presented in Fig. 3 as a percentage of the DMSO control. The protection of WEHI-231 (Fig. 3A) and spleen B lymphocytes (Fig. 3B) by CD40L from cell death elicited by PPAR agonist exposure was dependent on the dose of PPAR agonist. In WEHI-231 cells, CD40L rescued them from 15d-PGJ2-induced cell death at doses <2.5 μM 15d-PGJ2. In contrast CD40L did not rescue cells from death induced by 15d-PGJ2 at doses greater than 2.5 μM (Fig. 3A). The best rescue was observed between 1 and 2 μM 15d-PGJ2 in which incubation with 15d-PGJ2 and CD40L resulted in 95% cell survival in contrast to the 20% cell survival seen for 15d-PGJ2 alone. Cell death with ciglitazone was also prevented dependent on the concentration of ciglitazone (Fig. 3A, right panel). Prevention of cell death was seen at 6 μM ciglitazone, which alone resulted in 35% cell survival, whereas CD40 ligation with ciglitazone increased cell survival up to 80% of the control. In addition, CD40 engagement prevented cell death in freshly purified mouse spleen B lymphocytes, even more significantly than in WEHI-231 cells. With 15d-PGJ2, CD40L cotreatment rescued cell death over a broader range of 15d-PGJ2 concentrations with significant cell death inhibition occurring at doses <5 μM (Fig. 3B). A similar result was obtained with ciglitazone, with the most significant rescue seen at 10 μM at which survival was increased to 90% of the control (Fig. 3B, right panel). As shown in Fig. 3C, CD40 engagement in WEHI-231 cells prevented activation of caspase 3 and caspase 9 suggesting that CD40 signaling prevents apoptosis. DiOC6 staining of the mitochondria was next performed to determine whether CD40 engagement prevented the loss of mitochondrial membrane potential in WEHI-231 cells and spleen B cells. The cells were treated with or without CD40L and PPAR agonist for 12 h at which time incorporation of DiOC6 was measured by flow cytometry. As shown in Table I, CD40 engagement was able to prevent the PPAR agonist-induced loss of mitochondrial membrane potential in both WEHI-231 and normal mouse spleen B lymphocytes. From these results we concluded that CD40 engagement was able to partially rescue PPAR agonist-induced apoptosis. It is evident that at the higher doses of agonist, CD40 costimulation is unable to overcome the apoptotic signals induced by the PPAR agonists.
FIGURE 3. CD40 engagement partially prevents PPAR agonist-induced cell death of B lymphocytes. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h and then exposed to increasing concentrations of 15d-PGJ2 or ciglitazone for 48 h and an MTT assay was performed. The results are graphed as a percentage of the untreated (DMSO) control. *, p < 0.05 for PPAR agonist + CD40L compared with PPAR agonist alone. C, CD40 engagement prevents activation of caspase 3 and caspase 9 by PPAR agonists (1 μM 15d-PGJ2 and 6 μM ciglitazone) in WEHI-231 cells. The results are plotted as fold increase over the DMSO control. DMSO control cells had an OD 405 nm value of 0.035 for caspase 3 and 0.041 for caspase 9. *, p < 0.01 as compared with cells exposed to PPAR agonist alone.
Table I. CD40L prevents PPAR agonist-induced loss of mitochondrial membrane potential in B lymphocytes
PPAR agonist inhibition of NF-B is prevented by CD40 engagement
Inhibition of NF-B, for example by BCR cross-linking, is associated with B cell apoptosis (27, 28). To determine whether NF-B was involved in PPAR agonist-induced apoptosis, gel shift assays were performed for NF-B in WEHI-231 cells and mouse spleen B cells. Cells were incubated with PPAR agonist for 4 h, and 1 μg of nuclear protein was incubated with a radiolabeled probe containing the consensus DNA binding sequence for NF-B. The results of a representative EMSA are shown in Fig. 4. WEHI-231 (Fig. 4A) and normal mouse spleen B cells (Fig. 4B) have a constitutive level of NF-B translocation into the nucleus, which was inhibited dose-dependently by the PPAR agonists 15d-PGJ2 and ciglitazone. NF-B was almost completely inhibited at the highest concentrations of both PPAR agonists. As CD40 activation of B cells induces the degradation of IB and subsequent activation of NF-B (27, 28), we next determined whether CD40 engagement similarly prevented the NF-B inhibition by PPAR agonists. Incubation with CD40L alone increased NF-B translocation and this increase was maintained in the presence of low concentrations of 15d-PGJ2 or ciglitazone, but at higher concentrations of PPAR agonist there was decreased NF-B translocation (Fig. 4). These results indicate that CD40 engagement prevents NF-B inhibition and apoptosis at lower concentrations of PPAR agonists, but is not able to overcome the inhibitory signals at higher PPAR agonist concentrations.
FIGURE 4. PPAR agonist inhibition of NF-B is prevented by CD40 engagement. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h and exposed to 15d-PGJ2 (left panels) and ciglitazone (right panels) for 4 h at the indicated concentrations. An EMSA was performed on 1 μg of nuclear extract using a radiolabeled DNA binding sequence for NF-B. Lane 1 contains only the radiolabeled probe (free probe-FP). The last lane is extract from CD40L-treated cells incubated with an unlabeled probe as a cold competitor (CC) to control for binding specificity.
PPAR agonists induce an increase in IB levels, which is prevented by CD40 engagement
As increased IB expression is one mechanism whereby NF-B activity is inhibited (37), we next determined the effect of PPAR agonists on IB levels. Cells were pretreated with CD40L and then exposed to 15d-PGJ2 or ciglitazone, with or without CD40L for 1 h. A Western blot analysis was performed on whole cell lysates for IB and IB, with actin blots shown as additional loading controls (Fig. 5). WEHI-231 (Fig. 5A) and spleen B cells (Fig. 5B) have a constitutive level of both IB and IB proteins. Upon exposure to 15d-PGJ2 and ciglitazone, the total cellular levels of IB were increased 4- to 5-fold in WEHI-231 cells and 2- to 3-fold in spleen B cells. This increase in IB correlated with PPAR agonist inhibition of NF-B translocation (see Fig. 4). Exposure to CD40L alone significantly reduced IB levels, which remained reduced with the addition of low concentrations of PPAR agonists. However, coincubation with high doses of PPAR agonists resulted in an overall increase in IB levels. These data support the concept that CD40 engagement enhances the degradation of IB in the presence of low concentrations of PPAR agonists thereby attenuating the NF-B inhibition elicited by such low doses of PPAR agonists. In contrast, CD40 engagement in the presence of higher doses of PPAR agonist was unable to overcome the PPAR signal leading to an increase in IB. This may account for our finding that CD40L does not block NF-B inhibition elicited by higher doses of PPAR agonists.
FIGURE 5. PPAR agonists induce an increase in IB levels, which is prevented by CD40 engagement. WEHI-231 cells (A) and spleen B lymphocytes (B) were preincubated with CD40L for 3 h before culture with PPAR agonists for 1 h. Five micrograms of whole cell lysate was loaded into each lane for Western blot. The membrane was first probed for IB, stripped and reprobed for IB and for actin. Densitometry was performed and each band was normalized to actin. The untreated sample was designated an intensity of 1 and the remaining band intensities were plotted as relative to the untreated sample.
Rescue of apoptosis by CD40 engagement is prevented by NF-B inhibition
To further support the concept that CD40 activation of NF-B was responsible for apoptosis rescue after exposure of B lymphocytes to low doses of PPAR agonists, the B cells were preincubated with the NF-B inhibitor SN50. SN50 is a cell permeable peptide inhibitor that prevents NF-B translocation into the nucleus (32). After SN50 pretreatment, WEHI-231 and spleen B cells were exposed to CD40L for 3 h and then exposed to PPAR agonists for 48 h in the presence of the NF-B inhibitor, at which time an MTT assay was performed. As shown in Fig. 6, CD40 rescue of PPAR agonist-induced apoptosis was completely inhibited by the addition of SN50 in both the WEHI-231 (Fig. 6A) and spleen B cells (Fig. 6B). An inactive control peptide did not inhibit CD40 rescue of the B cells (data not shown). As shown in Fig. 6C, SN50 reversed the protective effects that CD40L had on mitochondrial membrane potential. The 15d-PGJ2 alone induced a decrease in 77% of WEHI-231 cells and pretreatment with CD40L prevented this decrease in mitochondrial membrane potential (Table I and Fig. 6C). When pretreated with SN50 and CD40L, 15d-PGJ2 still reduced mitochondrial membrane potential in 75% of cells. Therefore, in B lymphocytes NF-B is a key mediator for eliciting apoptosis and for protecting B lymphocytes from apoptosis induced by PPAR agonists.
FIGURE 6. Rescue of PPAR agonist-induced apoptosis by CD40 engagement is prevented by NF-B inhibition. WEHI-231 cells (A) and spleen B lymphocytes (B) were pretreated with the NF-B inhibitor peptide SN50 for 1 h and then CD40L was added for an additional 3 h. An MTT assay was set up and the cells were exposed to 15d-PGJ2 or ciglitazone in the presence of CD40L and SN50 for 48 h. The results are presented as the percentage of the control. *, p < 0.05 for CD40L + PPAR agonist vs PPAR agonist alone; **, p < 0.05 for CD40L + PPAR agonist vs CD40L + SN50 + PPAR agonist. There were no significant differences between PPAR agonist alone and PPAR agonist with CD40L and SN50. C, SN50 prevents CD40L protection from loss of mitochondrial membrane potential. Following pretreatment with CD40L and SN50, WEHI-231 cells were exposed to 1 μM 15d-PGJ2 for 12 h. SN50 alone did not cause a decrease in DiOC6 incorporation (see also Table I for additional controls and data).
Discussion
In the present study, we show that both natural and synthetic PPAR agonists elicit B cell apoptosis as shown by caspase activation, PARP cleavage, and by the loss of mitochondrial membrane potential. In addition, PPAR agonists inhibited NF-B and induced an increase in cellular IB levels not only in WEHI-231 B lymphoma cells but also in freshly isolated normal mouse spleen B lymphocytes. Finally, we demonstrate that CD40 engagement prevents apoptosis and mitochondrial membrane potential loss induced by PPAR agonists in B lineage cells. CD40 signaling prevents the PPAR agonist-induced inhibition of the survival factor NF-B and this rescue event is dependent on NF-B as an NF-B inhibitor reverses the apoptosis rescue.
The mechanism of PPAR agonist-induced apoptosis of B lymphocytes has not been explored and understanding the apoptotic mechanism may have implications for the therapeutic use of PPAR agonists. In this study, we found that caspase 3 and caspase 9 were activated following exposure of B cells to PPAR agonists. Caspase 3 and caspase 9 are of particular importance because their activation supports involvement of mitochondria in the apoptosis pathway. Indeed, we found a reduction in mitochondrial membrane potential as early as 4 h, but caspase activation was not detected until 6 h after PPAR agonist exposure. Because caspase inhibition did not prevent the loss in mitochondrial membrane potential, these data suggest that the reduction in mitochondrial membrane potential did not occur as a result of caspase activation and supports a role for the mitochondria in caspase activation. Activation of caspase 9 only occurs if mitochondria are damaged and cytochrome c is released (36). When released into the cytoplasm, cytochrome c forms a complex with Apaf-1, and this complex associates with pro-caspase 9 and causes caspase 9 activation, which then activates other caspases, such as caspase 3 (36). Caspase 3 is an effector caspase responsible for cleaving cellular substrates during apoptosis, including PARP (36). Caspase 3 has also been shown to cleave and inactivate the p65 subunit of NF-B (38), which may be an additional mechanism by which PPAR agonists caused a significant reduction in NF-B translocation observed in WEHI-231 cells and normal B lymphocytes.
NF-B is an important transcription factor for B cell development and maintenance. In addition to B cell-specific functions, NF-B can also induce expression of antiapoptotic proteins, such as the inhibition of apoptosis proteins that function as caspase inhibitors (39). Therefore, the inhibition of NF-B results in the loss of the cells ability to prevent apoptosis and is one mechanism contributing to PPAR agonist-induced apoptosis of B lymphocytes. The SN50 NF-B inhibitor alone did not kill the B lineage cells suggesting that NF-B inhibition alone is not sufficient to induce apoptosis and that multiple mechanisms contribute to the apoptosis induced by PPAR agonists. It is most likely a combination of caspase activation, mitochondrial damage, and NF-B inhibition that steers the cells down the apoptotic pathway. The existence of multiple pathways may explain why CD40 engagement could not sustain increased NF-B activation at higher concentrations of PPAR agonist and only partially rescued B lymphocytes from PPAR agonist-induced apoptosis. It is probable that activation of NF-B and the subsequent initiation of survival signals by CD40 ligation are not able to fully overcome the potent apoptotic stimuli induced by PPAR agonists in B lymphocytes.
The regulation of NF-B by PPAR agonists is an emerging concept that may have both PPAR-dependent and -independent mechanisms. For example, in mouse macrophage cells, Castrillo et al. (40) reported 15d-PGJ2 inhibited the IB kinase that is responsible for phosphorylating IB. In HeLa cells, this inhibition of IB kinase was independent of PPAR because 15d-PGJ2 was able to inhibit IB kinase in HeLa cells that lack PPAR (41). The PPAR agonist-induced increase in IB levels seen in B lymphocytes could be a result of IB kinase inhibition, which would prevent the degradation of IB. In contrast to reports of PPAR-independent inhibition of NF-B by PPAR agonists, a PPAR-dependent mechanism also exists for NF-B inhibition. In LPS-activated macrophages, PPAR formed a complex with both the p50 and p65 subunits of NF-B, resulting in transrepression of NF-B (42). The interaction of PPAR with NF-B may also be important for regulating shuttling of NF-B to and from the nucleus thereby controlling NF-B activation (43). There is also evidence that PPAR is important in the regulation of NF-B in B lineage cells. In contrast to our findings, Schlezinger et al. (44) reported an increase in NF-B nuclear translocation in mouse pre-B cells after treatment with PPAR agonists. This interesting difference is most likely due to the developmental stage of the B cell and suggests that the effects of PPAR agonists may differ during B cell maturation. In support of our findings of PPAR agonist inhibition of NF-B, B cells from PPAR heterozygous mice (PPAR+/–) exhibited a dysregulation of the NF-B pathway that resulted in increased spontaneous NF-B activation and increased proliferation when compared with wild-type mice (45). These PPAR+/– B cells required higher doses of PPAR agonists to suppress both the LPS-induced proliferation and the increased serum Ab production observed during an Ag-specific response (45). Further studies are needed to better define the PPAR dependency of the effects of PPAR agonists on B lymphocytes. Taken together with the results reported in this study, PPAR agonists appear to play a fundamental role in preventing a pathologic uncontrolled B cell response to Ag in vivo and may do so in part through an NF-B dependent pathway. Finally, such a B cell dampening effect of endogenous PPAR agonists may itself be controlled by CD40 ligation of B cells to prevent B cell depletion.
The findings we report for normal spleen B lymphocytes suggest a pivotal role for PPAR agonists in controlling B cell responses. 15d-PGJ2 and synthetic PPAR agonists may serve to counterbalance the effects of other PGs, such as PGE2, which induces Ig class switching in B cells (46). Although the physiologic levels and importance of 15d-PGJ2 in vivo are under intense investigation, there are several pieces of evidence supporting the in vivo significance of this PG and other newly discovered natural PPAR agonists, such as lysophosphatidic acid (5). For example, 15d-PGJ2 was detected during the resolution phase of rat carrageenin induced pleurisy (11) and also in macrophages from human atherosclerotic plaques (10). The production of 15d-PGJ2 by macrophages, and PGD2 by mast cells, APCs, and activated T cells suggests the possibility that 15d-PGJ2 is produced in lymphoid organs in which B lymphocytes reside and expand during an immune response (7, 8, 9, 10).
We speculate that 15d-PGJ2 or other PPAR agonists contribute to the dampening of B cells especially during the resolution phase of an immune response by inducing their apoptosis as demonstrated for both malignant and normal B lymphocytes. The ability of CD40 engagement to prevent PPAR agonist-induced apoptosis of B lymphocytes suggests the existence of an important antiapoptotic mechanism to allow B cell expansion and survival. Future studies of the in vivo effects of PPAR agonists will be critical in defining the role of 15d-PGJ2 and synthetic PPAR agonists in immune regulation. Finally, our studies clearly point to the potential of natural and synthetic PPAR agonists as therapy for B cell malignancies.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Christine Martey-Ochola for providing technical assistance and Stephen Pollock for preparing reagents.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by United States Public Health Service Grants DE11390, ES01247, a James P. Wilmot Cancer Center Discovery Award, the Environmental Protection Agency Particulate Matter Center, and a Leukemia and Lymphoma Society Translational Research Award. D.M.R. was supported by the Rochester Training Program in Oral Infectious Diseases T32-DE07165. F.A. was supported by the International Union Against Cancer Fellowship Program and the Scientific and Technical Research Council of Turkey (TUBITAK)/North Atlantic Treaty Organization-A2.
2 Address correspondence and reprint requests to Dr. Richard P. Phipps, Box 850, Department of Environmental Medicine, University of Rochester Medical Center, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: richard_phipps@urmc.rochester.edu
3 Abbreviations used in this paper: PPAR, peroxisome proliferator-activated receptor ; 15d-PGJ2, 15-deoxy-12,14-PGJ2; PARP, poly(ADP-ribose) polymerase; BCR, B cell receptor.
Received for publication May 21, 2004. Accepted for publication January 19, 2005.
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