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The Journal of Immunology, 2003, 171: 5148-5156.
Copyright © 2003 by The American Association of Immunologists

Cyclopentenone Prostaglandins Induce Lymphocyte Apoptosis by Activating the Mitochondrial Apoptosis Pathway Independent of External Death Receptor Signaling 1

Alessio Nencioni2,*,{ddagger}, Kirsten Lauber2,{dagger}, Frank Grünebach*, Luk Van Parijs{ddagger}, Claudio Denzlinger*, Sebastian Wesselborg3,{dagger} and Peter Brossart3,4,*

Departments of * Hematology, Oncology and Immunology, and {dagger} Internal Medicine I, University of Tübingen, Tübingen, Germany; and {ddagger} Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
15-Deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) is a naturally occurring cyclopentenone metabolite of PGD2 that possesses both peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma})-dependent and PPAR-{gamma}-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ2 as well as synthetic PPAR-{gamma} agonists inhibit lymphocyte proliferation. However, only 15d-PGJ2, but not the specific PPAR-{gamma} activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain-/- or caspase-8-/- Jurkat T cells has no effect on apoptosis induction by 15d-PGJ2. Conversely, overexpression of Bcl-2 or Bcl-xL completely inhibits the initiation of apoptosis, indicating that 15d-PGJ2-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ2 induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential ({Delta}{psi}m) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ2. We also demonstrate that 15d-PGJ2 potently stimulates reactive oxygen species production in Jurkat T cells, and {Delta}{psi}m loss induced by 15d-PGJ2 is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ2 may modulate immune responses even independent of PPAR-{gamma} by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclopentenone PGs of the J series are naturally occurring eicosanoids formed via dehydration within the cyclopentane ring of PGD2 (1). These compounds are produced during an inflammatory event and possess strong anti-inflammatory properties (2, 3, 4, 5, 6, 7). Moreover, they may also play a role in the regulation of lymphocyte function and apoptosis (8, 9, 10, 11, 12, 13, 14). The molecular mechanism(s) underlying the immunomodulatory effects of cyclopentenone PGs are still poorly defined. The discovery that cyclopentenones such as 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2) 5 are activating ligands of the nuclear transcription factor peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) allowed a better understanding of the function of these compounds (15). PPAR-{gamma} is present in adipose tissue, where it plays a key role in the regulation of differentiation and metabolism (16, 17, 18). Moreover, its expression can be detected in several cell types including hemopoietic cells (19), monocytes/macrophages (20), lymphocytes (9, 10), and dendritic cells (21, 22, 23), suggesting a broad spectrum of functions for this transcription factor. PPAR-{gamma} possesses anti-inflammatory properties (5, 6, 7, 24, 25), and modulates lymphocyte proliferative responses and dendritic cell immunogenicity (8, 9, 10, 21). Moreover, it has been reported that this receptor is involved in apoptosis regulation of synoviocytes (26), endothelial cells (27), macrophages (28), and normal as well as malignant lymphocytes (10, 11, 14, 29, 30).

Current knowledge indicates that cyclopentenone PGs may also exert some of their effects via mechanisms unrelated to PPAR-{gamma}. In this context, it was demonstrated that these compounds can directly interfere with NF-{kappa}B signaling at different levels (31, 32, 33, 34). Because NF-{kappa}B controls the expression of inflammation-related genes as well as that of several antiapoptotic proteins such as cIAP1/2 (35), it is conceivable that inhibition of NF-{kappa}B signaling via cyclopentenone PGs accounts, at least in part, for the anti-inflammatory and proapoptotic effects of these compounds. Cyclopentenone derivatives of the J series are also direct inhibitors of the ubiquitin isopeptidase in the proteasome pathway, which was shown to result in the initiation of cell death (36). Finally, cyclopentenone PGs can induce apoptosis in different cell types by a mechanism unrelated to PPAR-{gamma} and involving oxidative stress (37, 38, 39). However, the precise pathway of apoptosis induction by these metabolites is still unclear.

The endogenous suicide program can be initiated via two major signaling pathways: the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Ligation of death receptors (such as CD95, TNFR1, TNFR-related apoptosis-mediating protein, or TNF-related apoptosis-inducing ligand receptors 1 and 2) with their respective ligand or agonistic Abs recruits the adaptor protein Fas-associated death domain (FADD) to the active trimeric death receptor. FADD in turn recruits and activates the death proteases caspase-8 and -10 (40, 41, 42). Caspase-8 and -10, like all other caspases, are cysteine proteases synthesized as catalytically inactive proenzymes that are activated upon proteolytic cleavage. The second death pathway is triggered by the release of proapoptotic factors such as cytochrome c from the mitochondrion into the cytosol (43). The release of cytochrome c can be initiated either through death receptor-mediated activation of the Bcl-2-related protein Bid (44, 45), or independently of this pathway by other proapoptotic Bcl-2 members such as Bcl-xS, Bim, Bad, Bak, Bax, PUMA, and murine Noxa, which may be triggered by apoptotic stimuli such as anticancer drugs and irradiation (45). In the cytosol, cytochrome c together with dATP binds to the adaptor protein Apaf-1, which enables the subsequent binding and activation of caspase-9. Activation of the mitochondrial apoptosis pathway can be inhibited by antiapoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-xL, which block cytochrome c release (46, 47, 48). Activation of one or both of the two pathways via initiator caspase-8, -10, or -9 triggers an amplifying cascade of executioner caspases (such as caspase-3, -6, and -7) that, after cleavage of vital death substrates, leads to the final demise of the cell.

In the present study, we have investigated the mechanisms underlying the down-regulatory effects of cyclopentenone PGs on the function of human T lymphocytes. We show that the cyclopentenone PG 15d-PGJ2 reduces the proliferative capacity of lymphocytes by apoptosis induction. In contrast to the results in murine studies, this effect is independent of the cognate receptor PPAR-{gamma}, because synthetic high-affinity PPAR-{gamma} activators fail to induce apoptosis in lymphocytes, although they reduce the proliferative capacity of lymphocytes. Furthermore, we demonstrate that 15d-PGJ2 initiates apoptosis independent of external death receptor signaling via cytochrome c release and activation of the intrinsic mitochondrial pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

The medium used for cell cultures was RPMI 1640 supplemented with 10% inactivated FCS, 50 nM 2-ME, and antibiotics, all purchased from Life Technologies (Grand Island, NY). Caspase-8-deficient and FADD-deficient Jurkat cells and the parental Jurkat cell line A3 were kindly provided by J. Blenis (Harvard Medical School, Boston, MA). Stable transfectants of Jurkat cells overexpressing Bcl-2 and Bcl-xL were a gift of C. Belka (University of Tübingen). Cells were grown at 37°C in a 5% CO2 atmosphere. 15d-PGJ2, {Delta}12-PGJ2, PGA2, PGD2, and PGE2 were from Biomol (Plymouth Meeting, PA). Benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was purchased from Bachem Biochemica (Heidelberg, Germany). BRL49653 was kindly donated by GlaxoSmithKline (Uxbridge, Middlesex, U.K.). Troglitazone and pioglitazone were purchased from Sankyo (Tokyo, Japan) and Takeda (Osaka, Japan), respectively. PMA, ionomycin, PHA-P, N-acetyl-L-cysteine (NAC), 2',7'-dichlorofluorescein diacetate (DCFH-DA), MTT, and 3,3-dihexyloxacarboncyanine iodide (DiOC6) were all obtained from Sigma-Aldrich (St. Louis, MO).

Proliferation assay

PBMC were obtained by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation from buffy coat preparations of healthy donors and seeded at 2 x 105 per well in 96-well microtiter plates. Lymphocyte proliferation was induced by adding PMA (25 ng/ml) and ionomycin (1 µg/ml) or, alternatively, PHA-P (10 µg/ml). Proliferation was measured 48 h later by a 16-h pulse with [3H]thymidine (0.5 µCi/well; Amersham Life Science; Buckingham, U.K.).

Detection of apoptosis

For detection of apoptosis in human T lymphocytes, CD3+ cells were isolated from PBMC using CD3 MicroBeads and MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purity of the isolated CD3+ cells was evaluated by flow cytometry and always exceeded 99%. Cells were seeded in 24-well plates and treated with the respective stimuli. After the indicated time periods, cells were washed and phosphatidylserine externalization of apoptotic cells was visualized by staining with Annexin VFITC and propidium iodide (Annexin V FLUOS staining kit; Roche Diagnostics, Mannheim, Germany) in subsequent flow cytometry. Leakage of fragmented DNA from apoptotic nuclei was measured by the method of Nicoletti et al. (49, 50, 51, 52). In brief, 5 x 104 cells were seeded in 96-well microtiter plates and cultured for the indicated times under different conditions. Cell nuclei were prepared by lysing cells in hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100, and 50 mg/ml propidium iodide) and immediately analyzed by flow cytometry. Nuclei containing hypodiploid DNA were considered to be apoptotic. All flow cytometry analysis was performed on a FACSCalibur (BD Biosciences, Heidelberg, Germany). For the MTT colorimetric viability assay, 105 cells/well were incubated in microtiter plates in medium containing different stimuli for the indicated amount of time. At the end of incubation, 0.1 mg of MTT solution was added to each well and incubated at 37°C for 4 h. Then, the microtiter plates were centrifuged at 1500 rpm for 5 min, the supernatant was aspirated, and the formazan crystals were solubilized by adding 200 µl of DMSO to each well. The optical adsorbance was read immediately at 540 nm on a multiwell scanning spectrophotometer. Results are expressed as surviving fraction of treated cells compared with control cells calculated by the following formula: percent surviving fraction = (mean experimental sample/mean control sample) x 100.

Fluorometric assay of caspase activity

Cytosolic extracts of 5 x 104 cells were prepared in a lysis buffer containing 0.5% Nonidet P-40, 20 mM HEPES (pH 7.4), 84 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM PMSF. Caspase activities were determined by incubation of cell lysates with 50 µM fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin (Ac-DEVD-AMC) (Biomol) in 200 µl of buffer containing 50 mM HEPES (pH 7.3), 100 mM NaCl, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 10 mM DTT. The release of aminomethyl-coumarin was measured in a kinetic by spectrofluorometry using an excitation wavelength of 360 nm and an emission wavelength of 475 nm. Caspase activity was determined as the slope of the resulting linear regressions and expressed in arbitrary fluorescence units per minute.

Cell extracts and immunoblotting

Cleavage of caspases and the caspase substrate poly(ADP-ribose) polymerase (PARP) was detected by immunoblotting as described previously (49, 50, 51, 53). Cells (2 x 106) were seeded in 24-well plates and treated with the respective apoptotic stimuli. After the indicated time periods, cells were washed and lysed in 1% Triton X-100, 50 mM Tris (pH 7.6), and 150 mM NaCl containing 3 µg/ml aprotinin, 3 µg/ml leupeptin, 3 µg/ml pepstatin A, and 2 mM PMSF. Subsequently, proteins were separated under reducing conditions on a SDS-polyacrylamide gel and electroblotted to a polyvinylidene difluoride membrane (Amersham, Braunschweig, Germany). Membranes were blocked for 1 h with 5% nonfat dry milk powder in TBS and then immunoblotted for 1 h with rabbit polyclonal Abs against PARP (Roche Molecular Biochemicals, Mannheim, Germany) or mouse mAbs directed against caspase-8 (BioCheck, Münster, Germany) or caspase-3 (Transduction Laboratory, Heidelberg, Germany). Membranes were washed with TBS/0.02% Triton X-100 and incubated with the respective peroxidase-conjugated affinity-purified secondary Ab for 1 h. Following extensive washing, the reaction was developed by ECL staining using ECL reagents (Amersham).

Determination of reactive oxygen species (ROS) generation

For determination of intracellular H2O2 production, 2 x 106 Jurkat cells were seeded in 2 ml of medium in the presence of different stimuli. After 15 min, cells were harvested, washed, and incubated for 15 min in PBS containing 5 µM DCFH-DA. Thereafter, cells were washed and analyzed by flow cytometry (55).

Flow-cytometric assay of mitochondrial transmembrane potential ({Delta}{psi}m)

For quantitation of cells with reduced {Delta}{psi}m, 2 x 106 cells/well were incubated in 0.5 ml of culture medium in 24-well plates in the presence of different stimuli. At different time points, cells were harvested, washed, and incubated for 15 min in culture medium containing 20 nM DiOC6. {Delta}{psi}mlow cells were enumerated by flow cytometry (54).

Measurement of cytochrome c release

Relocalization of cytosolic cytochrome c was determined as previously described (49). In brief, 107 cells were collected by centrifugation, washed with PBS, and resuspended in 5 vol of buffer A containing 250 mM sucrose, 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were homogenized with 12 strokes in a douncer, and the homogenates were centrifuged at 1,000 x g for 10 min at 4°C to remove cell nuclei. The supernatants were transferred to a fresh tube and centrifuged at 10,000 x g for 10 min at 4°C to deplete mitochondria. The resulting supernatants were boiled for 5 min in SDS-loading buffer (62.5 mM Tris (pH 6.8), 6.5% glycerol, 2% SDS, and 100 µg/ml bromophenol blue) and subsequently loaded on a 15% SDS-polyacrylamide gel. Cytochrome c release was analyzed by immunoblotting with the mouse mAb 7H8.2C12 (BD PharMingen, Hamburg, Germany)

Statistical analysis

The significance of the results was evaluated by unpaired t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR-{gamma} agonists inhibit lymphocyte proliferative responses

Recent reports indicate that PPAR-{gamma} regulates lymphocyte activation by modulating autologous IL-2 production and subsequent induction of proliferation (8, 9). In the first series of experiments, we exposed human purified CD3+ cells to three synthetic PPAR-{gamma} ligands, the thiazolidinediones troglitazone, BRL49653, and pioglitazone, as well as to the naturally occurring ligand 15d-PGJ2. Subsequently, primary T cells were stimulated with PMA/ionomycin or, alternatively, with PHA. As shown in Fig. 1, all of the PPAR-{gamma} agonists inhibited lymphocyte proliferation in a concentration-dependent fashion. A similar inhibition of lymphocyte proliferation by PPAR-{gamma} agonists and 15d-PGJ2 was also observed in mixed leukocyte reaction in which allogeneic PBMC or monocyte-derived dendritic cells were used as stimulators (data not shown).



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  		FIGURE 1. PPAR-{gamma} activators inhibit lymphocyte proliferation. PBMC were seeded at 2 x 105 per well in microtiter plates and preincubated for 6 h with PPAR-{gamma} agonists at the indicated concentrations. Thereafter, proliferation was stimulated either by 25 ng/ml PMA and 1 µg/ml ionomycin (A) or, alternatively, 10 µg/ml PHA-P (B). Thymidine incorporation was measured after 48 h by a 16-h pulse with 0.5 µCi/well [3H]thymidine. The mean value with SD of triplicate cultures is shown. Significance of the results was determined by unpaired t test compared with the control (none). *, p < 0.05.

 
PGD2-derived cyclopentenone PGs, but not thiazolidinediones, induce apoptosis in human T lymphocytes and Jurkat T cells

Using the MTT viability/growth assay, we observed that 15d-PGJ2 inhibits the growth of PMA/ionomycin-stimulated primary CD3+ T cells to a higher extent than the PPAR-{gamma} agonist BRL49653. Moreover, a decrease in viable cells was also noted in resting T cells exposed to 15d-PGJ2 but not to BRL49653 (Fig. 2A). Hence, we evaluated whether 15d-PGJ2 and the PPAR-{gamma}-specific ligand BRL49653 would induce apoptosis in human T cells. To this purpose, CD3+ T lymphocytes were incubated with 15d-PGJ2 or BRL49653, and apoptosis was evaluated at different time points. As shown in Fig. 2B, 15d-PGJ2 was found to strongly increase the number of annexin V-positive cells already after a 24-h exposure, whereas this effect was not reproduced by BRL49653. Activating stimuli such as the combination PMA/ionomycin are known to induce activation-induced cell death in T cells. In our experiments, a slight increase in the number of apoptotic cells was detected after exposure to these mitogens, and the rate of apoptotic cells was strongly increased by 15d-PGJ2 but not by BRL49653 (Fig. 2B). Phosphatidylserine exposure, an early event in the apoptotic process detectable by staining with Annexin-VFITC, was demonstrated on both resting and activated T cells after a 24-h exposure to 15d-PGJ2. Cell membrane disruption, as detected by positivity of propidium iodide cell staining, usually appeared within the following 24 h (Fig. 2C). Resting and activated primary T cells showed similar susceptibility to 15d-PGJ2-induced apoptosis (Fig. 2D). Because 15d-PGJ2 precursors were also demonstrated to possess proapoptotic effects (34), we evaluated apoptosis induction in primary human T cells in response to different stimuli including the 15d-PGJ2 precursors {Delta}12-PGJ2 and PGD2. As a positive control for apoptosis induction, CD3+ lymphocytes were exposed to the potent kinase inhibitor staurosporine (49, 50, 51, 52). As shown in Fig. 2E, lymphocyte apoptosis was induced by 15d-PGJ2 and, to some extent, by {Delta}12-PGJ2 and PGD2. In contrast, PGE2, PGA2, as well as the PPAR-{gamma}-specific agonists failed to mediate an increase of apoptotic cells.



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  		FIGURE 2. Cyclopentenone derivatives of PGD2, but not thiazolidinediones, induce lymphocyte apoptotic cell death. A, T lymphocytes were enriched from PBMC by immunomagnetic selection of CD3+ cells (purity, >99%). Subsequently, the cells were seeded at 105 per well in microtiter plates and cultured in the presence or in the absence of the PPAR-{gamma} agonists BRL49653 or 15d-PGJ2 at the indicated concentrations. Cell viability was determined after 24- and 48-h incubation by MTT colorimetric assay. The mean values of triplicates are shown. *, p < 0.05. B, A total of 106 purified CD3+ cells per well were seeded in 24-well plates and treated with or without (none) BRL49653 (40 µM) or 15d-PGJ2 (40 µM). Cells were either left unstimulated or activated by PMA (25 ng/ml)/ionomycin (500 ng/ml). At the indicated time points, cells were harvested, washed, and stained with FITC-conjugated annexin V. Annexin-V+ cells were enumerated by flow cytometry. C, Resting or PMA/ionomycin-activated CD3+ cells were cultured in the presence or absence of 15d-PGJ2 (40 µM). Cells were harvested after 24 and 48 h, washed, stained with annexin V/propidium iodide, and analyzed by flow cytometry. D, Isolated CD3+ cells were cultured in the presence or absence (none) of the mitogens PMA/ionomycin. Concomitantly, these cells were exposed to different concentrations of 15d-PGJ2. Twenty-four hours later, cells were harvested, washed, and analyzed by flow cytometry after staining with annexin V. E, Purified CD3+ lymphocytes were treated for 20 h with medium, 2.5 µM staurosporine, or 40 µM respective PGs or thiazolidinediones troglitazone, BRL49653, or pioglitazone for 20 h. Thereafter, cells were washed, stained with Annexin VFITC, and analyzed by flow cytometry.

 
In the subsequent experiments, apoptosis induction was evaluated in the T lymphocytic cell line Jurkat, which lacks PPAR-{gamma} expression (8, 13, 19). Again, apoptosis was determined by 15d-PGJ2 and by high concentrations of PGD2 (data not shown) but not by synthetic PPAR-{gamma} agonists (Fig. 3A). Apoptosis induction in Jurkat cells by 15d-PGJ2 was blocked by the broad-spectrum caspase inhibitor zVAD-fmk.



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  		FIGURE 3. 15d-PGJ2 induces apoptosis in Jurkat T cells. A, Jurkat cells were seeded at 5 x 104 per well and pretreated with medium ({blacksquare}) or 100 µM zVAD-fmk ({square}) for 1 h. Subsequently, the cells were stimulated with medium or one of the indicated PPAR-{gamma} agonists (40 µM) for 20 h. Percentage of apoptotic nuclei was determined by flow cytometry of propidium iodide-stained hypodiploid nuclei. B, Jurkat cells were stimulated for 10 h with 15d-PGJ2 at the indicated concentrations. After 10 h of treatment, cell lysates were prepared, incubated with the fluorogenic caspase substrate DEVD-AMC, and measured in a spectrofluorometer. Caspase activity is given in arbitrary units. C, Jurkat cells (2 x 106) were incubated for 1 h with medium or zVAD-fmk (100 µM) and subsequently exposed to 15d-PGJ2 at the indicated concentrations for 10 h. Thereafter, caspase activity was detected by cleavage of caspase-3, caspase-8, and the caspase-specific substrate PARP. Filled arrowheads ({blacktriangleleft}) indicate the uncleaved form, and open arrowheads ({triangleleft}) indicate the cleaved form of the respective protein.

 
Consistently, a concentration-dependent activation of caspases was observed by monitoring caspase activity with the fluorogenic substrate Ac-DEVD-AMC and the cleavage of the caspase substrate PARP as well as of procaspase-8 and procaspase-3 (Fig. 3, B and C).

Apoptosis induction by 15d-PGJ2 is mediated by activation of the mitochondrial apoptosis pathway

We further focused on 15d-PGJ2 and examined the mechanism underlying the initiation of apoptosis by this compound. For this purpose, we used a number of different Jurkat cell clones that either were deficient for individual elements involved in apoptosis regulation or overexpressed proteins blocking the apoptosis machinery. To analyze the role of death receptor signaling in apoptosis induction by 15d-PGJ2, we used Jurkat A3 wild type, Jurkat A3 FADD-/-, and Jurkat A3 caspase-8-/- (49). The deficiency of FADD prevents apoptosis induced via death receptors, i.e., CD95 ligation, whereas mitochondrial death stimuli such as anticancer drugs are not affected (49, 51). Similar properties are shared by the caspase-8-/- Jurkat cells, which are protected from CD95 ligand (CD95L)-induced apoptosis but not from anticancer drugs (49, 51). To investigate a potential role of 15d-PGJ2 in activating the mitochondrial signaling pathway, we made use of Jurkat cells stably overexpressing Bcl-2 or Bcl-xL. As previously demonstrated, overexpression of Bcl-2 or Bcl-xL prevents cytochrome c release and apoptosis mediated by stimuli like anticancer drugs, while only partially interfering with the death receptor pathway (49).

In this study, we found that incubation of FADD-/- or caspase-8-/- Jurkat cells with 15d-PGJ2 had no effect on apoptosis induction by this PG (Fig. 4A), suggesting that the apoptotic effects of 15d-PGJ2 are independent of the death receptor pathway. On the contrary, induction of apoptosis was significantly reduced in Jurkat cells overexpressing Bcl-2 or Bcl-xL (Fig. 4, B and C), thus indicating that 15d-PGJ2 induces apoptosis via the mitochondrial death pathway. Consistent with these results, stimulation by 15d-PGJ2 induced the cleavage of the caspase substrate PARP in FADD-/- and caspase-8-/- Jurkat to levels comparable with those of the wild-type control (Fig. 5). Conversely, PARP, caspase-3, and caspase-8 activation was completely prevented in Bcl-xL-overexpressing Jurkat (Fig. 6).



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  		FIGURE 4. Apoptosis via 15d-PGJ2 is inhibited by overexpression of Bcl-2 and Bcl-xL, but not in caspase-8-/- or FADD-/- Jurkat. A total of 5 x 106 caspase-8- or FADD-deficient Jurkat cells or the parental cell line Jurkat A3 (wild type) (A) or Jurkat cells overexpressing Bcl-2 (B) or Bcl-xL (C) with the respective vector control cells were pretreated with medium ({blacksquare}) or 100 µM zVAD-fmk ({square}) for 1 h and subsequently stimulated with medium or 40 µM 15d-PGJ2 for 20 h. Apoptosis was determined by flow-cytometric assessment of apoptotic hypodiploid nuclei. The mean values of triplicate cultures with SD are shown.

 


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  		FIGURE 5. Caspase activation by 15d-PGJ2 is not prevented in caspase-8-/- and FADD-/- Jurkat cells. A total of 2 x 106 caspase-8- or FADD-deficient Jurkat cells or the parental cell line Jurkat A3 (wild type) were treated for the indicated times with 40 µM 15d-PGJ2. For the longest time point, a 100 µM zVAD-fmk control (preincubation for 1 h) was included. PARP and caspase-8 cleavage were detected by immunoblotting. Filled arrowheads ({blacktriangleleft}) indicate the uncleaved form, and open arrowheads ({triangleleft}) indicate the cleaved form of the respective protein. As a control, cells were stimulated with 1 µg/ml anti-CD95 or 25 µg/ml etoposide for the indicated times.

 


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  		FIGURE 6. Overexpression of Bcl-xL results in inhibition of 15d-PGJ2-induced caspase activation. A total of 2 x 106 Jurkat cells stably transfected with the vector alone (Vector) or with Bcl-xL were exposed for the indicated times to 40 µM 15d-PGJ2. A 100 µM zVAD-fmk control was included for the longest time point. PARP, caspase-3, and caspase-8 cleavage were detected by immunoblotting. Filled arrowheads ({blacktriangleleft}) indicate the uncleaved form, and open arrowheads ({triangleleft}) indicate the cleaved form of the respective protein.

 
Interestingly, in FADD-/- cells, caspase-8 was found to be cleaved following exposure to 15d-PGJ2. In this case, caspase-8 activation reasonably happened downstream of cytochrome c release and activation of the mitochondrial apoptosis pathway.

15d-PGJ2 promotes {Delta}{psi}m loss and cytochrome c release in human T cells

Given the inhibition of 15d-PGJ2-induced apoptosis in the Jurkat cells overexpressing Bcl-2 or Bcl-xL, which prevent mitochondria disassemblage, we evaluated whether exposure to 15d-PGJ2 may indeed produce mitochondrial damage. Loss of mitochondrial transmembrane potential was determined by means of the cationic lipophilic fluorochrome DiOC6, which accumulates in the mitochondrial matrix driven by the {Delta}{psi}m (54). We first monitored {Delta}{psi}m in primary T lymphocytes exposed to 15d-PGJ2 or BRL49653. As expected, 15d-PGJ2 rapidly induced mitochondrial depolarization, this effect being complete within 8–12 h (Fig. 7A). Conversely, no significant effect on the {Delta}{psi}m was produced by the PPAR-{gamma} ligand BRL49653, suggesting that 15d-PGJ2 acts independently of the PPAR-{gamma}. Surprisingly, when evaluating the effect of zVAD-fmk on {Delta}{psi}m loss induced via 15d-PGJ2, we found that the broad spectrum caspase inhibitor reduces the effect of this PG (Fig. 7B).



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  		FIGURE 7. {Delta}{psi}m disruption in lymphocytes exposed to 15d-PGJ2. A, Purified human CD3+ cells were stimulated with medium, BRL49653 (40 µM), or 15d-PGJ2 (40 µM). Cells were harvested at the indicated time points, stained with 20 nM DiOC6, and analyzed by flow cytometry. B, Purified CD3+ cells were pretreated with medium ({blacksquare}) or 100 µM zVAD-fmk ({square}) for 1 h and subsequently stimulated with medium, BRL49653, or 15d-PGJ2 at the indicated concentrations for 12 h. Subsequently, cells were stained by DiOC6, and {Delta}{psi}mlow cells were enumerated by flow cytometry.

 
According to a previous report showing cytochrome c freeing induced by 15d-PGJ2 in macrophages (37), we found that stimulation of primary T lymphocytes with 15d-PGJ2 induces release of cytochrome c into the cytosol (Fig. 8A). This effect was detected in both resting and activated T cells and was reduced by zVAD-fmk. Similarly, 15d-PGJ2 determined cytochrome c freeing in Jurkat T cells within a few hours from the beginning of exposure (Fig. 8, B and C). No cytosolic cytochrome c could be detected in the Bcl-xL-overexpressing Jurkat cells upon treatment with 15d-PGJ2 (data not shown).



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  		FIGURE 8. Cytochrome c release induced via 15d-PGJ2. A, A total of 107 human primary CD3+ cells were pretreated for 1 h with medium or 100 µM zVAD-fmk. Subsequently, cells were exposed to 40 µM 15d-PGJ2 or left unstimulated. Ten hours later, cells were harvested and homogenized, and the release of cytochrome c into the cytosol was evaluated by immunoblotting. As a control for equal protein loading, the membranes were reprobed with an anti-{beta}-tubulin Ab. B, A total of 107 Jurkat cells were pretreated with medium or zVAD-fmk (100 µM) for 1 h and then incubated for 10 h in the presence or absence of zVAD-fmk and 15d-PGJ2 at the indicated concentrations. Cells were then harvested, and the release of cytochrome c into the cytosol was determined by immunoblotting. A nonspecific (ns) immune band was used as a loading control. C, A total of 107 Jurkat cells exposed to 15d-PGJ2 (40 µM) were harvested at the indicated time points, and cytosolic cytochrome c was determined by immunoblotting. D, Cell homogenates were obtained from 107 caspase-8-/- Jurkat cells and the respective wild-type control cells, which were exposed for different amounts of time to 40 µM 15d-PGJ2. Cytochrome c released into the cytosol was evaluated by immunoblotting.

 
Subsequently, we tested the hypothesis that zVAD-fmk may reduce the mitochondrial damage produced by 15d-PGJ2 by blocking positive amplification loops involving caspase-8/Bid activation, which have previously been suggested (49). However, this mechanism was ruled out, because no reduction of cytosolic cytochrome c was observed in the caspase-8-/- cells as compared with the wild-type control cells after exposure to 15d-PGJ2 (Fig. 8C).

15d-PGJ2-induced mitochondrial damage is dependent on oxidative stress

It has recently been proposed that the proapoptotic effects of 15d-PGJ2 may be related to ROS production (37, 38, 39). Indeed, ROS such as H2O2 rapidly induce loss of mitochondrial transmembrane potential and cytochrome c release-dependent apoptosis (Ref.55 and data not shown). Hence, we tested the hypothesis that 15d-PGJ2 may produce mitochondrial damage in T cells by ROS. In line with previous reports (37, 38, 39), we found that 15d-PGJ2 acts as a potent stimulator of ROS production, because it raises intracellular H2O2 levels in Jurkat T cells as measured by detecting DCFH-DA oxidation (Fig. 9A). This effect was found to occur very rapidly, H2O2 production being already detectable after a few minutes, and to be independent of caspase activity, because zVAD-fmk does not prevent H2O2 formation (data not shown).



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  		FIGURE 9. Evidence for a role of ROS in 15d-PGJ2-mediated mitochondrial injury. A, A total of 2 x 106 Jurkat T cells were cultured for 15 min in medium with or without H2O2 (100 µM) or 15d-PGJ2 (40 µM). Thereafter, cells were washed and incubated for additional 15 min in normal PBS (none) or PBS containing 5 µM DCFH-DA to measure H2O2 formation. Finally, cells were washed and analyzed by flow cytometry. B, A total of 2 x 106 Jurkat cells were preincubated for 1 h with or without (none) NAC (5 mM) or zVAD-fmk (100 µM). Thereafter, cells were treated with or without (control) 15d-PGJ2 (40 µM). Cells were harvested 20 h later and stained by DiOC6, and {Delta}{psi}mlow cells were enumerated by flow cytometry.

 
The role of oxidative stress in the mitochondrial insult was tested, making use of the ROS scavenger NAC, whereas the role of caspases was evaluated in the presence of zVAD-fmk. Fig. 9B shows the results of a representative experiment in which 15d-PGJ2 induces loss of {Delta}{psi}m in a large majority of treated Jurkat cells. This effect is significantly reduced, even if not completely abolished by NAC, indicating that ROS are likely to play a key role in the mitochondrial alterations determined by 15d-PGJ2. Again, the caspase inhibitor zVAD-fmk reduced 15d-PGJ2-mediated {Delta}{psi}m loss.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Deletion of unwanted cells by means of apoptosis plays a fundamental role in lymphocyte homeostasis. First, apoptosis is involved in the clonal deletion of autoreactive thymocytes. Thereafter, in the periphery, it is required to control the expansion of activated T lymphocytes during an immune response and to eliminate self-reacting T cells. Following activation, T cells acquire enhanced susceptibility to apoptosis, which probably involves signaling through the CD95/TNFR2 pathway, ROS, or perforin (40, 56). In this study, we demonstrate that the endogenous suicide program in T cells can also be activated by cyclopentenone PGs like 15d-PGJ2. Because these metabolites are synthesized in the context of an inflammatory response, it is conceivable that they might also contribute to the resolution of inflammation-induced immune responses (2, 3, 4).

Interestingly, cyclopentenone PGs induced apoptosis of human T lymphocytes by a mechanism independent of the corresponding receptor PPAR-{gamma}. We found that specific high-affinity PPAR-{gamma} agonists, while down-regulating T-lymphocyte proliferative responses, failed to induced apoptosis, even when applied at high concentrations. Conversely, apoptosis was induced by 15d-PGJ2, {Delta}12-PGJ2, and, to some extent, high concentrations of PGD2, indicating that cyclopentenone derivatives of PGD2 possess cytotoxic properties unrelated to PPAR-{gamma}. Our results are in line with other reports indicating PPAR-{gamma}-independent anti-inflammatory and proapoptotic effects of cyclopentenone PGs (22, 31, 32, 33, 34, 36, 38). Wang et al. (12) reported that PPAR-{gamma} might even favor T lymphocyte survival by enhancing mitochondrial transmembrane potential. Several authors (10, 12, 14) report about the induction of apoptosis in primary T cells by 15d-PGJ2. However, in two recent studies, the authors (11, 13) failed to detect any apoptosis upon exposure of primary human T cells to 15d-PGJ2. The data by Cippitelli et al. (13) even suggest that 15d-PGJ2 may protect T lymphocytes from activation-induced cell death by down-regulating CD95L expression. These data are in contrast with our results, because we failed to detect any protective effect of 15d-PGJ2 on PMA/ionomycin-activated T cells. The cyclopentenone PG promoted cytochrome c release and apoptosis in activated as well as in resting T lymphocytes. Conversely, apoptosis induced by 15d-PGJ2 was prevented by Bcl-2 and Bcl-xL. These molecules are up-regulated in T cells in response to costimulation via CD28 or to autocrine/paracrine signals such as IL-2 (14, 57, 58, 59). In the light of these data, we propose that cyclopentenone PGs may play a role in T cell homeostasis, thereby acting as an inhibitory/proapoptotic factor counterbalanced by activatory signals.

With respect to the induction of apoptosis, several mechanisms have been proposed for cyclopentenone PGs, including activation of PPAR-{gamma}, inhibition of NF-{kappa}B signaling, induction of intracellular oxidative stress, and blocking of the ubiquitin isopeptidase activity in the proteasome pathway. In this study, we show for the first time that external death receptor signaling via CD95 or related receptors is not involved in 15d-PGJ2-induced apoptosis, because blocking this pathway in FADD-/- and caspase-8-/- Jurkat T cells did not prevent caspase activation and apoptosis. On the contrary, the inhibition of apoptosis induction by 15d-PGJ2 in cells overexpressing Bcl-2 or Bcl-xL, strongly suggests an involvement of the intrinsic mitochondrial apoptotic pathway in apoptosis initiated by cyclopentenone PGs. However, because Bcl-2 localizes to the endoplasmic reticulum and nucleus in addition to mitochondria (60), we cannot exclude the possibility that other pathways like endoplasmic reticulum stress might also be involved. Indeed, consistent with a primary involvement of the mitochondrial pathway in cyclopentenone PG-mediated apoptosis, we demonstrate that 15d-PGJ2 induces {Delta}{psi}m loss and cytochrome c release into the cytosol. This effect is likely to depend, at least in part, on oxidative stress, because 15d-PGJ2 potently stimulated ROS production in Jurkat T cells, and 15d-PGJ2-mediated mitochondrial injury was significantly down-regulated in the presence of an ROS scavenger.

{Delta}{psi}m dissipation and cytochrome c release in response to 15d-PGJ2 were also reduced by the broad spectrum caspase inhibitor zVAD-fmk, thus suggesting a role for caspases in mitochondria disassemblage initiated by cyclopentenone PGs. The inhibition of caspase-3 and caspase-8 cleavage and of caspase activity (data not shown) by Bcl-2 and Bcl-xL overexpression places mitochondria upstream of caspases in this apoptotic cascade. However, caspase-2 has been shown to act upstream of mitochondria and to produce mitochondria permeabilization and cytochrome c release (61). Whether the effect of zVAD-fmk on the release of cytochrome c induced by 15d-PGJ2 depends on caspase-2 inhibition has not been addressed in the present study and needs further investigation. Alternatively, zVAD-fmk may reduce the mitochondrial insult by blocking caspases downstream of mitochondria. However, our data indicate that caspase-8, which is cleaved independent of FADD upon exposure to 15d-PGJ2, is unlikely to play a relevant role in cytochrome c relocalization. Finally, a zVAD-fmk-mediated blocking of enzymes other than caspases involved in apoptotic programs such as the cathepsins may also play a role (62). These proteins are released from the lysosomes and participate in apoptotic cell death induced by different stimuli, including death receptor agonists, oxidative stress, and p53 (62). The cathepsins can activate caspases by direct cleavage or by Bid-mediated cytochrome c release. Further investigation is required to explain the mechanism(s) linking ROS production and mitochondria activation in T cells in response to cyclopentenone PGs.

PGD2 levels in the micromolar range have been detected in tissue homogenates of adult rats and the production levels could be strongly increased by addition of exogenous arachidonic acid (64). Hence, it seems possible that concentrations of PGs sufficient to induce lymphocyte apoptosis can be reached in the local microenvironment, i.e., inflamed tissues, due to the rapid conversion of PGD2 into its cyclopentenone derivatives (1). The highest expression levels of PGD2 were detected in bone marrow, spleen, and intestine (63), which constitute the scenario for lymphocyte ontogeny and homeostasis. In this context, it was suggested that the main endogenous producer of PGD2 may be APCs like dendritic cells, Langerhans cells, monocytes/macrophages, and Kupffer cells (64), although this observation still needs further confirmation.

In conclusion, induction of apoptosis in lymphocytes via cyclopentenone PGs may represent an additional mechanism contributing to the down-regulation of an immune response arising in an inflammatory context, whereupon the levels of these compounds are reasonably increased. This effect may act synergistically with the pathways of activation-induced cell death via CD95-CD95L and TNFR2/TNF-{alpha} interactions (65, 66, 67, 68). Lower levels of cyclopentenone PGs produced in secondary lymphoid organs from PGD2 might contribute to lymphocyte homeostasis in the steady state. This goal could eventually be conveyed through more subtle regulatory mechanisms like activation of PPAR-{gamma} and/or direct inhibition of the NF-{kappa}B pathway.


   Acknowledgments
 
We thank Drs. C. Belka, J. Blenis, and K. Schulze-Osthoff for providing many valuable cell lines and reagents. We are thankful to Sylvia Stephan for the excellent technical assistance.


   Footnotes
 
1 This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (SFB510) (to P.B.) and Deutsche Forschungsgemeinschaft (WE-1801/1) (to S.W.), the Federal Ministry of Education, Science, Research and Technology (Fö. 01KS9602) and the Interdisciplinary Center of Clinical Research Center Tübingen (to P.B. and S.W.), and the German Bundesministerium fuer Bildung und Forschung (Hep-Net) and the Landesforschungsschwerpunktprogramm of the Ministry of Science, Research and Arts of the Land Baden-Wuerttemberg (to S.W.). A.N. acknowledges a fellowship from the University of Genova (Genova, Italy) and an Anna Fuller Award (2003) for Research in Molecular Oncology. Back

2 A.N. and K.L. contributed equally to this work. Back

3 P.B. and S.W. share equal senior authorship. Back

4 Address correspondence and reprint requests to Dr. Peter Brossart, Department of Hematology, Oncology and Immunology, University of Tübingen, Otfried-Müller Strasse 10, D-72076 Tübingen, Germany. E-mail address: peter.brossart{at}med.uni-tuebingen.de Back

5 Abbreviations used in this paper: 15d-PGJ2, 15-deoxy-{Delta}12,14-PGJ2; PPAR-{gamma}, peroxisome proliferator-activated receptor {gamma}; FADD, Fas-associated death domain protein; zVAD-fmk, benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone; NAC, N-acetyl-L-cysteine; DCFH-DA, 2',7'-dichlorofluorescein diacetate; DiOC6, 3,3-dihexyloxacarboncyanine iodide; Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; CD95L, CD95 ligand; {Delta}{psi}m, mitochondrial transmembrane potential. Back

Received for publication December 2, 2002. Accepted for publication September 5, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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