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The Journal of Immunology, 2001, 167: 2487-2495.
Copyright © 2001 by The American Association of Immunologists

IFN-{gamma} Induces the Apoptosis of WEHI 279 and Normal Pre-B Cell Lines by Expressing Direct Inhibitor of Apoptosis Protein Binding Protein with Low pI

Hideshi Yoshikawa, Yasuo Nakajima and Kachio Tasaka1

Department of Parasitology and Immunology, Yamanashi Medical University, Yamanashi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} plays a crucial role in induction of Th1 response but is predominantly a negative regulator of B cell differentiation and Th2 response, so it is a key molecule in determining cellular or humoral immunity. In this study, we demonstrate that IFN-{gamma} induces apoptosis in WEHI 279 mouse B cells and IL-7-dependent mouse pre-B cells by disrupting mitochondrial membrane potential and cytochrome c release via down-regulation of Bcl-2 and Bcl-xL. Furthermore, this apoptotic signal is promoted by the de novo synthesis of endogenous direct inhibitor of apoptosis protein binding protein with low pI (DIABLO) by IFN-{gamma} and its release from mitochondria into the cytosol. Inhibition of DIABLO expression by antisense oligonucleotide is sufficient to decrease caspase activities and DNA fragmentation, but not cytochrome c release from mitochondria, suggesting that DIABLO plays a critical role in promoting apoptotic signals downstream of mitochondrial events. Thus, these findings demonstrate a signaling pathway during B cell apoptosis induced by IFN-{gamma} and possible mechanisms by which B cell differentiation is negatively regulated by Th1-type cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is a crucial mediator in the induction of the cell-mediated Th1 response but is predominantly a negative regulator of B cell differentiation and proliferation. Therefore, IFN-{gamma} is a key molecule in determining humoral or cellular immunity. The negative regulation of B cells can be mediated by apoptosis. It has been observed that apoptosis is induced by IFN-{gamma} in IL-7-dependent immature pre-B cells, although it is not known exactly how (1, 2). Recent studies have also shown that cell surface Fas Ag mediates B cell apoptosis, and the susceptibility of B cells to Fas-mediated apoptosis is inhibited by anti-IgM, or IL-4, which is one of the Th2 cytokines (3, 4, 5). Thus, the B cell differentiation and functions are differently regulated by Th1 and Th2 cytokines.

The mechanisms of apoptosis are highly conserved across species. The key enzymes are caspases that are activated sequentially, and the process involves a cascade of initiator and effector caspases (6). One major caspase activation cascade is triggered by the release of cytochrome c from mitochondrial intermembrane spaces (7). Once released to the cytosol, cytochrome c binds to an adaptor molecule, Apaf-1, and forms a complex termed apoptosome, then it recruits the initiator caspase, procaspase-9, and induces its activation (8, 9). This initiator caspase in turn activates downstream effector caspases such as caspase-3, -6, and -7 (8, 10). The adaptor molecules such as Apaf-1 and Fas-associated death domain protein (FADD),2 which mediate apoptotic stimuli by activating cell surface death receptors, are regulated by several families of regulatory molecules. Apoptosis signaled via Apaf-1 can be inhibited by proteins of the Bcl-2 family, and apoptosis signaled by FADD can be inhibited by FADD-like IL-1-converting enzyme inhibitory protein (11, 12).

Members of the Bcl-2 family are major regulators of the caspase-induced activation of the mitochondrial apoptotic pathway (13). The antiapoptotic members of this family, such as Bcl-2, Bcl-xL, and Mcl-1, preserve mitochondrial integrity and prevent the release of cytochrome c (14, 15). In contrast, the proapoptotic members of this family, including Bax, Bad, and Bid, translocate to mitochondria in response to apoptotic stimuli and promote the release of cytochrome c (16). Inhibitors of apoptosis proteins (IAPs) are another family of molecules that directly regulate caspase-activating pathways (17). The mammalian IAPs, cytosolic IAP (cIAP)-1, cIAP-2, and X-linked IAP (XIAP), inhibit apoptosis and are able to directly bind to procaspase-9, caspase-3, and caspase-7 (18). Recently, proapoptotic signaling molecules that bind to IAPs and promote caspase activity have been isolated and named second mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis protein binding protein with low pI (DIABLO) (19, 20, 21). Full-length mouse DIABLO is a 27-kDa protein and undergoes N-terminal processing to yield a 23-kDa active form that interacts with IAPs (20). DIABLO, which localizes to mitochondria in healthy cells, is released from mitochondria into the cytosol and interferes with the activities of IAP family proteins (20). DIABLO is highly expressed in tissues such as heart, liver, testis, and ovary, and weakly expressed in kidney. However, it is not expressed in the immune system, including spleen, thymus, and leukocytes (20).

In this study, we report that IFN-{gamma} induces apoptosis in WEHI 279 and normal pre-B cell lines, and the mechanism of this induction involves the expression of DIABLO and its release from mitochondria into the cytosol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

WEHI 279 is an immature B cell line derived from NZC mice and was kindly donated by H. Fujiwara (Osaka University, Osaka, Japan). WEHI 279 cells were cultured in RPMI 1640 medium with 10% FCS and 1 x 10-5 M 2-ME at 37°C in a humidified 5% CO2 atmosphere. IL-7-dependent mouse normal pre-B cells (5-7 cells) derived from BDF1 mice were kindly donated by A. Rolink (Basel Institute, Basel, Switzerland) (1). The 5-7 cells were cultured in RPMI 1640 medium supplemented with IL-7, which was also donated by A. Rolink, under the same conditions as WEHI 279 cells. In the experiments, cells were cultured with 10 U/ml mouse rIFN-{gamma}, which was donated by H. Fujiwara for a given period.

Antibodies

A rat anti-Fas mAb (RMF6) was purchased from MBL (Nagoya, Japan). Rabbit anti-Bcl-2, anti-Bax, and anti-Bad polyclonal Abs were purchased from StressGen (Victoria, Canada). A rabbit anti-Bcl-xL polyclonal Ab was purchased from Transduction Laboratories (Lexington, KY). Rabbit anti-Bid, anti-cIAP1, anti-cIAP2, and anti-caspase-8 polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse anti-Fas ligand (FasL) mAb (K-10), a rabbit anti-caspase-3, and a mouse anti-cytochrome c Ab were purchased from BD PharMingen (San Diego, CA). A rabbit anti-XIAP Ab was purchased from R&D Systems (Minneapolis, MN). A rabbit anti-caspase-9 Ab was purchased from Cell Signaling Technology (Beverly, MA). A rabbit anti-Smac/DIABLO Ab was kindly donated by X. Wang (Howard Hughes Medical Institute, Dallas, TX).

Measurement of apoptosis

We measured apoptosis by flow cytometry. Briefly, cells were incubated with IFN-{gamma} for a specific period. Cells (5 x 105) were washed with PBS and suspended in 500 µl FITC-conjugated annexin V (Caltag Laboratories, Burlingame, CA) and PI (5 µg/ml) in a calcium-containing buffer. After incubation for 10 min at room temperature, the samples were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using CellQuest analysis software. We confirmed DNA fragmentation using flow cytometry after staining with a cell-permeable PI solution. Briefly, cells were dissolved in a hypotonic fluorochrome solution (PI, 50 µg/ml, in 0.1% sodium citrate containing 0.1% Triton X-100). These samples were placed in the dark overnight, and the PI fluorescence of individual nuclei was measured using a flow cytometer. We also confirmed DNA fragmentation by DNA gel electrophoresis.

Measurement of caspase activities

The quantitative measurement of caspase activity was performed with a caspase colorimetric protease assay kit containing caspase-3/7, -8, and -9 using specific substrates, DEVD-pNA, IETD-pNA, and LEHD-pNA, respectively. Briefly, cells were cultured with IFN-{gamma} for a given period. Then, cytosolic protein was extracted and diluted to 200 µg in a volume of 50 µl and incubated with corresponding substrate at 37°C for 2 h. The OD at 405 nm was measured using a Titertek Multiskan Plus microplate reader (Flow Laboratories, McLean, VA).

Measurement of mitochondrial transmembrane potential ({Delta}{Psi}m)

Perturbation in {Delta}{Psi}m was monitored by flow cytometry using JC-1 (Molecular Probes, Engene, OR). Briefly, cells were cultured with IFN-{gamma} for a specific period. After the staining of cells with 5 µg/ml JC-1 for 20 min at 37°C in the dark, fluorescence intensity was estimated by exciting the probes with a laser at 488 nm, and emission was measured through 575/26-nm (red fluorescence) bandpass filters. Logarithmic amplification was used to detect fluorescence intensity.

RT-PCR analysis

Cells were cultured with IFN-{gamma}, and total RNA was isolated using the guanidium isothiocyanate method. Then, 5 µg total RNA was reverse transcribed with murine leukemia virus reverse transcriptase. The products obtained by reverse transcription were amplified by PCR on a Zymoreactor thermal cycler (Atto, Tokyo, Japan). Amplification was done at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min. Each cDNA was amplified for 30 cycles. The following sense and antisense primer sets were used: bcl-2, 5'-TGCACCTGACGCCCTTCAC-3' and 5'-TAGCTGATTCGACCATTTGCCTGA-3'; bcl-xL, 5'-TGGTCGACTTTCTCTCCTAC-3' and 5'-GAGATCCACAAAAGTGTCCC-3'; mcl-1, 5'-TGCTCCGGAAACTGGACATT-3' and 5'-AATCCTGGGCAGCTTCAAGT-3'; bax, 5'-ACAGATCATGAAGACAGGGG-3' and 5'-CAAAGTAGAAGAGGGCAACC-3'; bad, 5'-CAGAGTATGTTCCAGATCCC-3' and 5'-AGGACTGGATAATGCGCGTC-3'; bid, 5'-TCTGGCTGTACTCGCCAAGA-3' and 5'-CCAAGTTCCTCACATAGGAG-3'; cIAP-1, 5'-ACCGTCAGTGACCTCGTTAT-3' and 5'-TACAGATGGGACACTTCCTC-3'; cIAP-2, 5'-AGAGAGCTTATTGACACCGT-3' and 5'-AGATGGGGCACTTCCTTAGA-3'; XIAP, 5'-CCAGAATCCTATGGTGCAAG-3' and 5'-CGTAATGACGGTGTAGCACA-3'; DIABLO, 5'-ACGCGCTGATTGAAGCAATC-3' and 5'-ACTGTTAGCTCTGCAGAGCT-3'; and {beta}-actin, 5'-CATCACTATTGGCAACGAGC-3' and 5'-ACGCAGCTCAGTAACAGTCC-3'.

Subcellular fractionation

Cells were washed in PBS, resuspended in isotonic HIM buffer (200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES, pH 7.5), supplemented with a protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO), and homogenized using a Polytron homogenizer (KINEMATICA, Luzern, Switzerland). Nuclei and unbroken cells were separated by centrifugation at 120 x g for 5 min. The supernatant was centrifuged at 10,000 x g for 10 min to collect the heavy membrane pellet that is enriched with intact mitochondria. This supernatant was centrifuged at 100,000 x g for 30 min to yield the light membrane pellet that contains the endoplasmic reticulum and plasma membrane, and a final soluble fraction that represents the cytosol (22).

Western blot analysis

Concentrations of proteins obtained by SDS lysis or subcellular fractionation were determined by the Bradford method (Bio-Rad, Richmond, CA). Proteins (10 µg) were separated on 15% polyacrylamide gel. After transfer to a polyvinylidene difluoride membrane and incubation overnight at 4°C with 5% BSA in PBS to block nonspecific Igs, the membrane was incubated for 1 h at room temperature with each Ab. After a wash, the membrane was incubated with HRP-conjugated secondary Ab for 1 h at room temperature, and specific bands were detected using ECL, according to the manufacturer’s protocol (Amersham, Arlington Heights, IL).

Antisense oligonucleotide treatment

Cells were treated with morpholino-oligonucleotide (5'-TCACCCAACTTCTCAGAGCCGCCAT-3') complementary to the 25-base mouse DIABLO mRNA sequence (antisense oligonucleotide), or an invert control (5'-TACCGCCGAGACTCTTCAACCCACT-3') of the antisense oligonucleotide for DIABLO, according to the manufacturers’ protocol for 3 h, and incubated with IFN-{gamma} for 18 h; then each experiment was performed.

Statistics

The statistical significance was analyzed using Student’s t test. Data are presented as means ± SD. Differences were considered to be significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma} induces apoptosis in WEHI 279 cells without using the Fas/FasL system

We evaluated the apoptosis induced by IFN-{gamma} in WEHI 279 cells using three different methods. After incubation with IFN-{gamma}, the percentage of apoptotic cells was determined by flow cytometry using double staining with FITC-conjugated annexin V and PI. Early apoptotic events (annexin V+PI-) are shown in the upper left quadrants of each panel in Fig. 1GoA. At 10 U/ml, IFN-{gamma} effectively induced apoptosis after 18-h culture. In addition, we evaluated apoptosis based on the DNA fragmentation of IFN-{gamma}-treated cells. As shown in Fig. 1GoB, the percentage of apoptotic cells containing fragmented DNA was measured by flow cytometry after cell-permeable PI staining. It was markedly increased 18 h after addition of IFN-{gamma}. This apoptosis was confirmed by gel electrophoresis of DNA extract, as shown in Fig. 1GoC. It was significant after 18-h culture with IFN-{gamma}, confirming the results obtained by flow cytometric analysis. A previous report showed that IFN-{gamma}-induced apoptosis was mediated by enhanced expressions of Fas and FasL in other systems (23, 24, 25, 26), so we evaluated the change of cell surface expression of Fas and FasL on WEHI 279 cells. As shown in Fig. 2Go, Fas expression was not changed and FasL was not detected on either unstimulated or IFN-{gamma}-stimulated WEHI 279 cells, suggesting that IFN-{gamma} induces apoptosis in WEHI 279 cells via mechanisms other than Fas/FasL interaction.



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  		FIGURE 1. Apoptosis induction in WEHI 279 cells by IFN-{gamma}. WEHI 279 cells were cultured with mouse rIFN-{gamma} (10 U/ml) for the indicated periods. A, The early apoptotic events (annexin V+PI-) are shown in the upper left quadrants (FL1-HhighFL2-Hlow) of each panel. B, The percentage of apoptotic cells was determined by flow cytometry after cell-permeable PI staining, as described in Materials and Methods. A representative of three independent experiments is shown in A and B. C, DNA was extracted and separated on 2% agarose gel by electrophoresis and visualized by staining with ethidium bromide. M indicates the DNA size marker.

 


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  		FIGURE 2. Expression of cell surface Fas and FasL on WEHI 279 cells. Cells were cultured with or without IFN-{gamma} (10 U/ml) for 12 h, and cell surface Fas and FasL were stained by single-color indirect immunofluorescence. Cells cultured under each condition were incubated with anti-mouse Fas mAb (bold line) or an isotype control of rat IgG (light line), or with anti-mouse FasL mAb (bold line) or an isotype control of mouse IgG (light line) in the presence of protease inhibitors for 1 h. After washing, FITC-conjugated goat F(ab')2 of anti-rat IgG or FITC-conjugated goat F(ab')2 of anti-mouse IgG was added and incubated another 1 h. Then flow cytometric analyses were performed with a FACSCalibur flow cytometer. A representative of three experiments is shown.

 
IFN-{gamma} induces apoptosis in WEHI 279 cells via decrease of {Delta}{Psi}m and cytochrome c release to the cytosol

To evaluate the mechanisms by which IFN-{gamma} induced apoptosis in WEHI 279 cells, we determined the signaling pathway of the apoptosis. We first evaluated the caspase activation by Western blotting and activities using cleavage of specific substrates. After incubation with IFN-{gamma}, the cleavage of procaspase-3, -7, -8, and -9 and activities of caspase-3/7, -8, and -9 were measured. As shown in Fig. 3GoA, cleavage of procaspase-9 preceded that of procaspase-3 and -7, and activation of procaspase-8 was not observed. The activities of caspase-3/7 and -9, but not caspase-8, were increased 18 h after addition of IFN-{gamma}, as shown in Fig. 3GoB, confirming the results obtained by Western blotting. These findings suggest that IFN-{gamma}-induced apoptosis of WEHI 279 cells is mediated by a mitochondrial pathway. So we next evaluated the change of {Delta}{Psi}m using a specific probe (JC-1) by flow cytometry. As shown in Fig. 4Go, a slight decrease of {Delta}{Psi}m was observed 12 h after addition of IFN-{gamma}, and the decrease became prominent later. At the same time, we detected the release from mitochondria of cytochrome c, which was thought to bind Apaf-1 and activate procaspase-9 in the cytosol. As shown in Fig. 5Go, the cytochrome c in the mitochondrial fraction decreased gradually and translocated to the cytosol 18 h after addition of IFN-{gamma}. These kinetic studies confirmed the mitochondrial pathway in IFN-{gamma}-induced apoptosis of WEHI 279 cells.



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  		FIGURE 3. Activation of caspases during IFN-{gamma}-induced apoptosis in WEHI 279 cells. Cells were cultured with IFN-{gamma} for the period indicated, cell extract was prepared, and activation of caspase-8, -9, -7, and -3 was determined by Western blot analysis using corresponding Abs to detect cleavages of the active form of each caspase (A) or with a caspase colorimetric protease assay kit using specific substrates, IETD-pNA for caspase-8, LEHD-pNA for caspase-9, and DEVD-pNA for caspase-3 and -7, respectively (B). The OD at 405 nm was measured using a microplate reader. Data represent means ± SD of three replicates.

 


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  		FIGURE 4. The {Delta}{Psi}m during IFN-{gamma}-induced apoptosis in WEHI 279 cells. Cells were cultured with IFN-{gamma} (10 U/ml) for the period indicated. After staining of the cells using 5 µg/ml JC-1, red fluorescence intensity (FL-2H) was measured by flow cytometer. The percentage of decreased {Delta}{Psi}m, which indicates a lower intensity of red fluorescence, is shown. A representative of three independent experiments is shown.

 


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  		FIGURE 5. Subcellular distribution of cytochrome c and DIABLO during IFN-{gamma}-induced apoptosis in WEHI 279 cells. Cells were cultured with IFN-{gamma} (10 U/ml) for the period indicated and were suspended in isotonic buffer, homogenized, and separated into each fraction, as described in Materials and Methods. The fractions were separated on 15% polyacrylamide gel and analyzed by Western blotting with anti-cytochrome c Ab or anti-Smac/DIABLO Ab.

 
IFN-{gamma} induces down-regulation of Bcl-2 and Bcl-xL, but does not change the expression of other Bcl-2 family and IAP family members

To evaluate the mechanisms by which stimulation of IFN-{gamma} decreased {Delta}{Psi}m of WEHI 279 cells, we examined the expression of mitochondria-related Bcl-2 family proteins, such as Bcl-2, Bcl-xL, Mcl-1, Bax, Bad, and Bid, using RT-PCR and Western blotting. As shown in Fig. 6Go, A and B, no significant change in the expression of proapoptotic molecules such as Bax, Bad, Bid, or Bcl-xS was observed on stimulation with IFN-{gamma} at either the mRNA or protein level. However, a decrease in the expression of antiapoptotic molecules such as Bcl-2 and Bcl-xL was observed in WEHI 279 cells 12 h after addition of IFN-{gamma}, and the decrease most likely induced a decline in {Delta}{Psi}m and the release of cytochrome c from mitochondria to the cytosol. To analyze the mechanisms of IFN-{gamma}-induced B cell apoptosis, we next evaluated the expression of IAP family proteins that were thought to directly bind caspases and inhibit their activation. As shown in Fig. 6Go, A and B, RT-PCR analysis and Western blotting indicated no change in the expression of IAP proteins, including cIAP-1, cIAP-2, and XIAP.



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  		FIGURE 6. Expression of apoptosis-related proteins in WEHI 279 cells. A, WEHI 279 cells were cultured with IFN-{gamma} (10 U/ml) for the period indicated. Total RNA (5 µg) was extracted, reverse transcribed, and amplified by PCR, as previously described. Products obtained using specific primers were separated on 2% agarose gel and visualized by staining with ethidium bromide. B, Cells were cultured with IFN-{gamma} (10 U/ml) for the period indicated, and protein extracts (10 µg) were prepared and separated on 15% polyacrylamide gel. Specific bands were detected by Western blotting using ECL.

 
DIABLO is synthesized de novo on stimulation with IFN-{gamma} and contributes to enhanced caspase activities and DNA fragmentation during IFN-{gamma}-induced B cell apoptosis

As mentioned above, the expression of IAP family proteins was not changed by IFN-{gamma}. Recently, a molecule that bound to IAP members and promoted apoptosis was cloned and named DIABLO in mouse (20). So we examined the expression of DIABLO on stimulation with IFN-{gamma} using RT-PCR and Western blotting. As shown in Fig. 5Go, DIABLO was newly produced 6 h after the stimulation. We detected only the 23-kDa active form of DIABLO in the cytosol, but both the full-length and active form in mitochondria. The expression of mRNA of DIABLO was observed 3 h after the stimulation, supporting the results obtained by Western blotting. To investigate the role of DIABLO in the regulation of IFN-{gamma}-induced apoptosis, WEHI 279 cells were treated with antisense or control oligonucleotide for DIABLO and incubated with IFN-{gamma}. To confirm the specificity and efficacy of the antisense oligonucleotide corresponding to mouse DIABLO mRNA, we first analyzed the expression of cytoplasmic DIABLO protein using both antisense and control oligonucleotide after 18 h of stimulation with IFN-{gamma}. Immunoblotting demonstrated a decrease in the protein level in WEHI 279 cells pretreated with antisense oligonucleotide (Fig. 7GoA). In contrast, no significant effect was observed on treatment with control oligonucleotide, suggesting that the antisense oligonucleotide specifically and efficiently inhibits translation of DIABLO protein. We next measured the activation and activity of caspase-3, -7, -8, and -9 treated with IFN-{gamma} in the presence of antisense or control oligonucleotide. As shown in Fig. 7GoB, activation of caspase-3, -7, and -9 was inhibited in the presence of antisense oligonucleotide for DIABLO. The activities of caspase-3/7 and -9 in IFN-{gamma}-treated cells were inhibited by addition of antisense oligonucleotide for DIABLO (Fig. 7GoC). Moreover, as shown in Fig. 7GoD, DNA fragmentation was completely inhibited by treatment with antisense oligonucleotide for DIABLO in cells stimulated by IFN-{gamma}. These results suggest that the expression of DIABLO is necessary and sufficient for the promotion of IFN-{gamma}-induced B cell apoptosis via the functional inhibition of IAP family proteins and promotion of caspase activity.



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  		FIGURE 7. Specific inhibition of the expression and function of DIABLO by antisense oligonucleotide in IFN-{gamma}-treated WEHI 279 cells. After pretreatment with antisense or control oligonucleotide for 3 h, cells were cultured with or without IFN-{gamma} (10 U/ml) for 18 h. A, Protein extracts (10 µg) were prepared, and specific bands of cytochrome c and DIABLO were detected by Western blot analysis using specific Abs. B, Cell extract was prepared, and activation of caspase-8, -9, -7, and -3 was determined by Western blot analysis using corresponding Abs to detect cleavages of the active form of each caspase. C, Cell extract was prepared, and caspase activities were measured with a caspase colorimetric protease assay kit using specific substrates, IETD-pNA for caspase-8, LEHD-pNA for caspase-9, and DEVD-pNA for caspase-3 and -7, respectively. The OD at 405 nm was measured using a microplate reader. Data represent means ± SD of three replicates. Statistical significance, *, p < 0.05; #, p < 0.05 as compared with control. D, The percentage of apoptotic cells containing fragmented DNA was determined by flow cytometry after cell-permeable PI staining. Data represent means ± SD of three replicates. Statistical significance, *, p < 0.05 as compared with control.

 
DIABLO contributes to the apoptosis induced by IFN-{gamma} in IL-7-dependent normal mouse pre-B cells

To confirm the induction of apoptosis by IFN-{gamma} in immature B cells, we used IL-7-dependent normal mouse pre-B cells. After incubation with IFN-{gamma}, the percentage of apoptotic cells was determined by flow cytometry. As shown in Fig. 8Go, apoptotic cells increased 48 h after addition of IFN-{gamma}, but not without IFN-{gamma}. To evaluate the mechanisms by which IFN-{gamma} induced apoptosis in IL-7-dependent pre-B cells, we examined the signaling pathway of IFN-{gamma}-induced apoptosis in IL-7-dependent pre-B cells by detecting the expression of mitochondria-related molecules such as Bcl-2, IAP family proteins, cytochrome c, and DIABLO. As shown in Fig. 9GoA, although DIABLO protein was constitutively expressed in low levels in mitochondria as well as mRNA, it increased after IFN-{gamma} stimulation and was translocated into the cytosol (Fig. 9GoB). Western blotting showed a decrease in the expression of Bcl-2, as observed in IFN-{gamma}-treated WEHI 279 cells. After the translocation of cytochrome c and DIABLO from mitochondria into the cytosol, procaspase-3, -7, and -9, but not procaspase-8, were activated, as shown in Fig. 9GoC, suggesting a mitochondrial pathway of IFN-{gamma}-induced apoptosis in IL-7-dependent pre-B cells. Finally, we confirmed the contribution of DIABLO protein to the activation of caspase and fragmentation of DNA during IFN-{gamma}-induced apoptosis using antisense oligonucleotide for DIABLO. We measured the activities of caspase-3/7, -8, and -9 in IL-7-dependent pre-B cells treated with IFN-{gamma} in the presence of antisense or control oligonucleotide. As shown in Fig. 10GoA, activities of caspase-3/7 and -9 in IFN-{gamma}-treated cells were inhibited by addition of antisense oligonucleotide for DIABLO. Moreover, as shown in Fig. 10GoB, DNA fragmentation induced by IFN-{gamma} was also significantly inhibited on treatment with antisense oligonucleotide. These findings confirmed that the enhanced expression and translocation of DIABLO into the cytosol play a critical role in the promotion of IFN-{gamma}-induced apoptosis of IFN-{gamma}-sensitive B cells.



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  		FIGURE 8. Apoptosis induction in IL-7-dependent normal pre-B cells by IFN-{gamma}. IL-7-dependent normal pre-B cells were cultured with or without IFN-{gamma} (10 U/ml) in the presence of IL-7 for the periods indicated. The percentage of apoptotic cells was measured by flow cytometry after cell-permeable PI staining, as described in Materials and Methods. A representative of three experiments is shown.

 


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  		FIGURE 9. Subcellular distribution of mitochondria-related molecules and caspase activation during IFN-{gamma}-induced apoptosis in IL-7-dependent pre-B cells. Cells were cultured with IFN-{gamma} (10 U/ml) for the periods indicated and were fractionated into mitochondria (A) and cytosol (B and C), as described in Materials and Methods. The fractions were separated on 15% polyacrylamide gel and analyzed by Western blotting with specific Abs. A representative of repeated experiments is shown.

 


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  		FIGURE 10. Specific inhibition of DIABLO function by antisense oligonucleotide in IFN-{gamma}-treated IL-7-dependent pre-B cells. After pretreatment with antisense or control oligonucleotide for 3 h, cells were cultured with or without IFN-{gamma} for 36 h (A) or 60 h (B). A, Cell extract was prepared, and caspase activities were measured with a caspase colorimetric protease assay kit using specific substrates. The OD at 405 nm was measured, and data represent means ± SD of three replicates. B, The percentage of apoptotic cells was determined by flow cytometry after cell-permeable PI staining. Data represent means ± SD of three replicates. Statistical significance, *, p < 0.05; #, p < 0.05 as compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we first demonstrated that IFN-{gamma} induced the enhanced expression of endogenous DIABLO in WEHI 279 and normal pre-B cells. DIABLO is highly expressed in tissues such as the heart, liver, testis, and ovary, but not in the immune system, including thymus, spleen, and leukocytes (20). We observed that DIABLO was not expressed in unstimulated WEHI 279 cells and at low levels in normal pre-B cells, but was enhanced on stimulation with IFN-{gamma} and promoted apoptosis. DIABLO and its human homologue Smac are usually located in mitochondria and undergo N-terminal processing to yield smaller active forms (19, 20). We detected both the full-length and active form of DIABLO in mitochondria and only the 23-kDa active form in the cytosol of WEHI 279 and pre-B cells during IFN-{gamma}-induced apoptosis. Expression of DIABLO in WEHI 279 cells treated with IFN-{gamma} was detected as early as 6 h after the stimulation, when the {Delta}{Psi}m was still undisturbed. It has been reported that endogenous DIABLO is only detected as the processed form in healthy cells, indicating that the processing of DIABLO is not dependent on apoptotic signals (20). Our results showed a delay between the up-regulation of DIABLO and the onset of apoptosis, suggesting that the active form of DIABLO was cleaved in mitochondria and both the full-length and active form are present in healthy mitochondria. Taken together, these findings demonstrate that only the active form of DIABLO translocates from mitochondria into the cytosol, the same as cytochrome c upon disruption of {Delta}{Psi}m.

IFN-{gamma} inhibits the growth of many types of cells, and STAT1 that is fully active transcriptionally is required for this effect (27, 28). The inhibition of cell growth correlates with the regulation of several cell cycle regulatory genes by IFN-{gamma}. Expression of the cyclin-dependent kinase inhibitors p21waf1, p27kip1, and c-myc is regulated by IFN-{gamma} (29, 30). The negative regulation of cell growth was mediated by induction of apoptosis. One mechanism of IFN-{gamma}-induced apoptosis is up-regulation of Fas/FasL expression and interaction (23, 24, 25, 26). Besides this receptor-mediated apoptosis, FADD-mediated activation of death receptors, and the expression of caspase-1 also have been reported (31, 32, 33). Caspase-8, which is an initiator of the caspase cascade, is usually activated in those types of apoptosis. However, in B cell apoptosis induced by IFN-{gamma}, neither up-regulation of Fas/FasL nor activation of caspase-8 was observed, suggesting that some other mechanism is involved, such as the usage of a mitochondrial pathway. Indeed, the modulation of expression of Bcl-2 family proteins in IFN-{gamma}-induced apoptosis has been reported in several systems. Down-regulation of both Bcl-2 and Bcl-xL also has been reported in the microglia (34). In melanoma cells, Bcl-xS, but not Bcl-2 or Bcl-xL, is up-regulated, and apoptosis is induced by IFN-{gamma} (35). In contrast, Mcl-1 has been reported to be up-regulated by IFN-{gamma}, and induces resistance to apoptotic stimuli in activated macrophages (36). Thus, IFN-{gamma} induces apoptosis in various types of cells by different mechanisms. In this study, we reported a new mechanism of IFN-{gamma}-induced apoptosis in B cells.

Apoptosis induction triggered by Ag receptor ligation in B cells has been well studied in WEHI 231 mouse B cell and human B cell lines (37, 38). In WEHI 231 cells, a correlation between c-Myc as well as the cyclin-dependent kinase inhibitor p27kip1 and apoptosis has been reported (39, 40, 41). In WEHI 231 cells, engagement of the B cell receptor (BCR) leads to an early transient increase in c-myc transcription, and to a drop in c-Myc; this decrease in c-Myc expression results from a drop in NF-{kappa}B/Rel binding, which leads to the immediate induction of apoptosis. At the same time, the cyclin-dependent kinase inhibitor p27kip1 is induced, and this not only leads to growth arrest, but also promotes apoptosis. In contrast, CD40 ligand, by engaging its receptor, controls the activation of c-myc gene transcription and prevents the increase in p27kip1, and WEHI 231 cells are rescued from apoptosis induced by cross-linking of BCR. Recent studies have also shown that caspases play a crucial role in the induction of apoptosis by BCR cross-linking in WEHI 231 cells (42). However, the main caspases involved in this apoptotic signaling differed in each report (43, 44). The effects of BCR cross-linking are completely reversed in other B cells, including WEHI 279 cells (5). In WEHI 279 and other cell lines, ligation of CD40 induces enhanced cell surface expression of and susceptibility to Fas (45, 46). These phenomena are possible regulatory mechanisms of B cell function, which are specific to the stage of differentiation. Moreover, the apoptosis induced by Fas is protected by cross-linking of BCR and Th2 cytokines such as IL-4, suggesting a different regulation by Th1 and Th2 cells during early B cell growth and differentiation (5). According to this hypothesis, our findings that IFN-{gamma}, which represents Th1 cytokines, directly induces apoptosis in WEHI 279 cells and IL-7-dependent normal pre-B cells, suggest one possible mechanism of inhibition of B cell expansion by Th1 cells.

The direct effects of IFN-{gamma} on B cells, especially the induction of apoptosis, are observed in bone marrow-derived immature pre-B cells (1, 2). However, the mechanisms of IFN-{gamma}-induced B cell apoptosis are not well understood. In this study, we proposed one possible mechanism of IFN-{gamma}-induced apoptosis: down-regulation of Bcl-2 and enhanced expression of DIABLO in immature B cells. Previous report suggested that cell cycle arrest preceded the induction of apoptosis by interference with IL-7/IL-7R interaction by IFN-{gamma} in IL-7-responsible pre-B cell in primary bone marrow cultures (2). In our study, cell cycle arrest by IFN-{gamma} was not significant in WEHI 279 and pre-B cell lines (Fig. 8Go). In addition, pre-B cells derived from bcl-2-transgenic mice or bcl-2-transfected pre-B cells were resistant to cell death by IFN-{gamma} or deprivation of growth factor (1, 47), confirming that down-regulation of Bcl-2 is one of the direct targets of IFN-{gamma}-induced B cell apoptosis. However, little is known about the effects of IFN-{gamma} on B cell growth and differentiation in vivo. Analysis of STAT1-deficient mice lacking IFN-{gamma} signals did not reveal any significant abnormalities in B cell differentiation or T cell development (48, 49), suggesting that the phenomena observed in our study are limited to minor populations or restricted to some differential stages of B cell in vivo.


   Acknowledgments
 
We thank Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX) for providing anti-Smac/DIABLO Ab and human recombinant Smac protein, Dr. A. Groenewegen and A. Rolink (Basel Institute for Immunology, Basel, Switzerland) for providing IL-7-dependent normal mouse pre-B cells and an aliquot of IL-7, Dr. H. Fujiwara (Osaka University, Osaka, Japan) for WEHI 279 cells and IFN-{gamma}, and Y. Ohnuma for secretarial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Kachio Tasaka, Department of Parasitology and Immunology, Yamanashi Medical University, 1110 Shimokato, Tamaho-cho, Yamanashi 409-3898, Japan. E-mail address: ktasaka{at}res.yamanashi-med.ac.jp Back

2 Abbreviations used in this paper: FADD, Fas-associated death domain protein; IAP, inhibitor of apoptosis protein; Smac, second mitochondria-derived activator of caspase; DIABLO, direct IAP binding protein with low pI; BCR, B cell receptor; cIAP, cytosolic IAP; XIAP, X-linked IAP; {Delta}{Psi}m, mitochondrial transmembrane potential; FasL, Fas ligand. Back

Received for publication November 27, 2000. Accepted for publication June 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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