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The Journal of Immunology, 2000, 165: 4281-4289.
Copyright © 2000 by The American Association of Immunologists

Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes1

Takuya Nagata*, Hiroyuki Kishi*, Qing Li Liu*, Tomoyasu Yoshino*, Tadashi Matsuda*, Zhe Xiong Jin*, Kimie Murayama{ddagger}, Kazuhiro Tsukada{dagger} and Atsushi Muraguchi2,*

* Department of Immunology and {dagger} Second Department of Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan; and {ddagger} Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Jyuntendo University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR engagement of immature CD4+CD8+ thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the thymus, CD4+CD8+ thymocytes expressing low levels of {alpha}ß-TCR are subjected to both positive and negative selection events (1). Positive selection ensures the survival and differentiation of cells capable of recognizing foreign Ag in the context of self-MHC, whereas negative selection eliminates immature thymocytes expressing self-reactive TCRs by the induction of apoptosis. It is generally believed that the avidity of the interaction between their TCR and the MHC/peptide complex determines the fates of thymocytes for positive or negative selection (2, 3). Concerning molecules or signal transduction pathways leading to positive or negative selection of thymocytes, it was reported that the ZAP-70 and Vav are essential for both positive and negative selection (4, 5). Furthermore, it was reported that the Ras/Raf/mitogen-activated kinase kinase 1/extracellular regulated kinase pathway and the calcineurin pathway are necessary for positive selection (6, 7), whereas the mitogen-activated kinase kinase 6/p38 pathway and c-Jun N-terminal kinase may be involved in the negative selection of thymocytes (8, 9). How these pathways lead to the distinct fates of thymocytes is still unclear.

In apoptotic cells, multiple structural changes, such as plasma and nuclear membrane blebbing, chromatin condensation, and DNA fragmentation, occur (10). Caspases play an inevitable role in an initiation phase as well as an effector phase of apoptosis. Initiator caspases (caspases 8, 9, and 10) cleave and activate effector caspases (caspases 3, 6, and 7). Mitochondria play an important role in the activation of caspases. Some types of apoptotic stimuli induce dysregulation of the mitochondrial transmembrane potential ({Delta}{Psi}m) and the release of cytochrome c from the intermembrane space (11). Free cytochrome c, making complexes with caspase 9 and Apaf-1, activates caspase 3 (12). The resulted activated caspases, in turn, cleave multiple cytoplasmic and nuclear substrates (13). DNA fragmentation factor 40 (DFF40)3/caspase-activated DNase (CAD) exists as a complex with DFF45/inhibitor of CAD (ICAD) in the normal cell, and when activated caspase 3 cleaves DFF45/ICAD, DFF40/CAD is released as an active form and induces nuclear condensation and DNA fragmentation (14, 15). However, mice deficient in the genes encoding the above-mentioned apoptosis-inducing molecules showed no defect of negative selection of thymocytes (16), suggesting that an alternative signaling pathway(s) for negative selection of self-reactive-thymocytes may exist.

It has been reported that stimulation of the CD3/TCR complex of immature thymocytes with anti-CD3 mAb induces DNA degradation and cell death through the endogenous pathway of apoptosis (17). In this study we show that in vivo stimulation of thymocytes with anti-CD3 mAb or a natural ligand such as OVA in DO11.10 TCR-transgenic mice generates activities that cause apoptotic changes and chromosomal DNA fragmentation of naked nuclei as well as the activity to degrade plasmid DNA. We purified the molecule that is responsible for plasmid DNA degradation from the cytosol of in vivo anti-CD3-stimulated thymocytes. Determination of the N-terminal amino acid sequence revealed that its sequence is identical with that of cyclophilin B (Cyp B), a member of cyclophilins that normally localizes in microsome fraction. Our data indicate that stimulation of thymocytes with anti-CD3 mAb induces activation of CAD, which is responsible for generation of internucleosomal DNA fragmentation, as well as the release of Cyp B from the microsome to the cytosolic/nuclear fraction, which directly or indirectly causes chromosomal DNA degradation. Thus, our results pose the possibility that active CAD and Cyp B function in harmony on the cell death of TCR-stimulated thymocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and Ab

ICR mice (4 wk old), C57BL/6 mice (8 wk old), and DO11.10 mice (4 wk old; a gift from Dr. D. Y. Loh, Nippon Roche, Kamakura, Japan) were bred in our animal facility. Anti-mouse CD3{epsilon} Ab (clone 145-2C11) was purified from the culture supernatant of hybridoma cells by protein A-Sepharose column. Anti-mouse Cyp B Ab was a gift from Dr. J. G. Sutcliffe (Research Institute of Scripps Clinic, La Jolla, CA), and anti-mouse CD28 Ab (clone PV-1) was provided by Dr. R. Abe (Science University of Tokyo, Noda, Japan).

Analysis of apoptosis by flow cytometry

Anti-CD3-stimulated or nonstimulated thymocytes (4 x 105/ml) were incubated with FITC-conjugated annexin V (1 µg/ml; Sigma, St. Louis, MO) for 15 min at room temperature or with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3); 40 nM; Aldrich, Milwaukee, WI) for 15 min at 37°C. Stained cells were analyzed by FACSCalibur (Becton Dickinson, San Jose, CA). Acquisition of data was performed without gating on forward light scatter. A minimum of 104 events were acquired for each sample. The staining pattern of the majority of nonstimulated thymocytes was regarded as annexin V negative and DiOC6(3)high (live cell), and the percentage of the annexin V-positive, DiOC6(3)low cell population (apoptotic cells) was calculated.

Preparation of thymocyte cell extracts

Fifty micrograms of anti-CD3 mAb or control IgG was i.p. injected into ICR mice, or 2.3 µg of OVA or 3.4 µg of BSA was injected into DO11.10 mice. After the indicated periods, the cell extracts of thymocytes were prepared according to the method described by Enari et al. (18) with some modifications. In brief, thymocytes were washed with PBS, pH 7.4, followed by a single wash with 5 ml of cell extract buffer (CEB; 50 mM PIPES (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and 10 mM cytochalasin B) containing a mixture of protease inhibitors (1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 5 µg/ml antipain, and 1 µg/ml chymopain). Cells were spun down and transferred to a Dounce homogenizer (Wheaton, Millville, NJ), allowed to swell by the addition of an adequate volume of CEB, and disrupted by freezing and thawing once. After grinding with the pestle, cell lysis was monitored by staining an aliquot of the cell suspension with methyl green and observation under a microscope. The cell lysate was then transferred to a 1.5-ml microcentrifuge tube and centrifuged at 4°C for 15 min at 700 x g. The supernatant was carefully collected without disturbing the nuclear pellets and was used as cell extracts. The protein concentration of the cell extracts or the purified preparation was measured by protein assay (Bio-Rad, Hercules, CA).

Subcellular fractionation of cell extracts

Cell extract was centrifuged at 7,000 x g at 4°C for 30 min and separated into the pellet of the mitochondria-rich fraction (P7) and the supernatant. Then, the supernatant was ultracentrifuged at 100,000 x g at 4°C for 90 min and separated to the pellet containing the microsomal fraction (P100) and the supernatant. Finally, the supernatant was further ultracentrifuged at 100,000 x g at 4°C for 13 h. After centrifugation, the supernatant was separated into two phases: the lower fraction was orange (LS100), and the upper fraction was colorless (US100). Each fraction was suspended to the same volume with CEB.

Preparation of liver nuclei

All procedures were performed on ice. Livers removed from C57BL/6 mice were minced, put in a 30 ml of homogenization buffer (10 mM HEPES (pH 7.6), 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, and 10% (v/v) glycerol), and homogenized using a motor-driven 30 ml Teflon-glass homogenizer until >90% of the cells were enucleated. The homogenate was diluted to 85 ml with homogenization buffer, layered in three 27-ml aliquots over three 10-ml cushions of the same buffer, and centrifuged at 24,000 rpm for 30 min at 4°C in an SW28 rotor (Beckman Instruments, Palo Alto, CA). The combined nuclear pellets were resuspended in 50 ml of a mixture of homogenization buffer and glycerol (9/1, v/v), using a Teflon-glass homogenizer. This homogenate was layered over two 10-ml cushions as described above and centrifuged under the same conditions. Pelleted nuclei were resuspended in 0.5 ml of nuclei storage buffer (10 mM PIPES (pH 7.40, 80 mM KCl, 20 mM NaCl, 250 mM sucrose, 5 mM EGTA, 1 mM DTT, 0.5 mM spermidine, 0.2 mM spermine, and 50% (v/v) glycerol) at a concentration of 1 x 106 nuclei/µl and stored at -80°C.

Assay of apoptosis-inducing activity

To investigate the nuclear DNA fragmentation-inducing activity, reaction buffer (1 mM HEPES (pH 7.0), 4 mM ß-glycerophosphate, 5 mM NaCl, 2 mM ATP, 1 mM creatine phosphate, and 5 µg/ml creatine kinase), various amounts of the cell fractions, CEB, and 2 x 106 nuclei in a final volume of 200 µl were incubated at 37°C for various time periods. After incubation, nuclei were collected by centrifugation for 10 min at 10,000 x g, then resuspended in 20 µl of resuspension buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% (w/v) sodium lauroyl sarkosinate, and 0.5 mg/ml proteinase K), and incubated at 50°C for 2 h. Ten microliters (0.5 mg/ml) of RNase A was added to each sample and incubated at 50°C for an additional 2 h. Samples were heated to 70°C, and 10 µl of 1% (w/v) low gelling temperature agarose containing 10 mM EDTA (pH 8.0), 0.25% (w/v) bromophenol blue, and 40% (w/v) sucrose was mixed with each sample before loading into the dry wells of a 2% (w/v) agarose gel containing 0.1 µg/ml ethidium bromide. To investigate plasmid DNA degradation activity, various amounts of the cell fractions and 1 µg plasmid DNA in a final volume of 20 µl were incubated at 37°C for 30 min and assayed with 1.5% (w/v) agarose gel electrophoresis. DNA fragmentation- and DNA degradation-inducing activities were assessed by the analysis of densitograph (Atto, Tokyo, Japan). For examining the morphology of apoptotic nuclei, 2 x 106 nuclei were incubated with the cell lysates, and an aliquot (6 µl) of the nuclei was stained with 10 µg/ml 4,6-diamino-2-phenylindole (Sigma) in 200 mM sucrose, 5 mM MgCl2, 15 mM PIPES (pH 7.4), 80 mM KCl, 15 mM NaCl, 5 mM EDTA, and 3.7% (v/v) formaldehyde. The nuclei were observed under a fluorescence microscope (Olympus, Tokyo, Japan).

Construction of expression vectors

The open reading frame of mouse Cyp B cDNA with the coding region of Flag tag was amplified by PCR from cDNA synthesized from total RNA of mouse thymocytes and cloned into the EcoRI and XhoI sites of pME18S (gift from Dr. Maruyama, University of Tokyo, Tokyo, Japan) to produce Flag-tagged Cyp B expression vector (Cyp B-Flag). N-terminal signal sequence-deleted Cyp B was also amplified and cloned into the EcoRI site of the pGEX-4T-1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) to produce the GST fusion protein of Cyp B (GST-Cyp B). Mouse ICAD cDNA was provided by Dr. S. Nagata (Osaka University, Osaka, Japan). ICAD cDNA was inserted into the EcoRI site of pGEX-4T-1 vector to produce the GST-ICAD protein. We also obtained pcDNA3-HA-CAD and pcDNA-3-Flag-DFF45 vectors from Dr. Núñez (University of Michigan, Ann Arbor, MI).

Preparation of recombinant active CAD

Two hundred micrograms of pcDNA3-HA-CAD vector and 50 µg of pcDNA-3-Flag-DFF45 vector were cotransfected into 2 x 107 293T cells by a calcium phosphate method. Twenty-four hours after transfection, 293T cells were harvested and lysed in 1 ml of TBS (25 mM Tris-HCl (pH 7.5) and 150 mM NaCl) containing 1% Nonidet P-40. The cell lysate was incubated with 1 µg of anti-Flag Ab (Upstate Biotechnology, Lake Placid, NY) bound to protein A-Sepharose FF beads (Amersham Pharmacia Biotech). After washing the beads with CEB, 10 µl of them was incubated with appropriate amount of recombinant human caspase 3 (Chemicon, Temecula, CA), and the supernatant was used as an active CAD preparation.

Purification of the LS100 fraction

All purification steps were conducted at 4°C, using a Vision automatic fast protein liquid chromatography station (PE Biosystems Japan, Chiba, Japan). Four hundred fifty milligrams of LS100 from thymocytes stimulated with anti-CD3 mAb for 20 h was applied to 40 ml of Q-Sepharose beads (Amersham Pharmacia Biotech) that was equilibrated with CEB containing 1% (w/v) 3-(1-pyridinio)-1-propanesulfonate (Fluka Chemika, Buchs, Switzerland). After 15-min rotation, the tube was centrifuged at 3000 rpm for 1 min, and the supernatant was applied to 40 ml of hydroxyapatite beads (Bio-Rad). After 15-min rotation, the tube was centrifuged at 3000 rpm for 1 min, and the supernatants were removed. CEB containing 200 mM KCl was added to the beads and rotated for 15 min. Then the tube was centrifuged at 3000 rpm for 1 min, and the supernatants were dialyzed against CEB at 4°C for 3 h. After dialysis, the sample was applied to a Mono S column (Amersham Pharmacia Biotech) equilibrated with CEB and eluted with a 0–1 M linear KCl gradient. Active fraction (eluted at 0.5 M KCl) was loaded onto a Superdex 200 gel filtration column equilibrated and eluted with CEB. After the Superdex 200 column fractionation, each fraction was assayed for DNA degradation activity.

Peptide sequence analysis

The sample was electrophoresed on a 12% SDS-polyacrylamide gel and transferred onto an Immobilon-PSQ membrane (Millipore, Bedford, MA). After staining with Coomassie Brilliant Blue (PhastGel Blue R, Amersham Pharmacia Biotech), a band of 20 kDa was cut out from the membrane and sequenced using a protein sequencer (G1005A, Hewlett Packard, Palo Alto, CA). The amino acid sequence was searched against the databases Swiss Prot and TrEMBL.

Purification of GST-ICAD and GST-Cyp B protein

Escherichia coli, strain DH5, containing GST-ICAD or GST-Cyp B expression plasmid, was grown (37°C) to an OD at 600 nm of 0.5 in 800 ml of Luria-Bertoni medium containing 100 µg/ml of ampicillin. Isopropyl-ß-D-thiogalactopyranoside (1 mM) was added to the culture medium, and the cells were grown for an additional 3 h. Cells were harvested, resuspended in 15 ml of TBS containing 1% Triton X-100 and 10% glycerol, and rotated for 1 h at 4°C. After centrifugation at 14,000 x g for 30 min, the supernatant was mixed with 1 ml of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) and rotated at 4°C overnight. The beads were washed with TBS. Bound GST-ICAD or GST-Cyp B protein was eluted with elution buffer (50 mM Tris-HCl (pH 8.0) and 10 mM glutathione) and dialyzed against CEB.

Transfection of Cyp B into EL4 cells and their TCR stimulation

Cyp B-Flag expression vector (5 µg) was transfected into 5 x 106 of the T lymphoma cell line EL4 by the DEAE-dextran method. Cells were incubated for 12 h in the RPMI medium containing 10% FCS. Transfected cells were stimulated by immobilized anti-CD3 and anti-CD28 mAbs (50 µg/ml) and incubated for an additional 12 h. After incubation, cells were harvested, lysed, and fractionated into nuclear, microsomal (P100), and cytosolic (LS100 + US100) fractions as described.

Western blot analysis

Each subcellular fraction was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was blocked with 5% nonfat milk in 0.1% Tween-20, 25 mM Tris-Cl (pH 7.5), and 150 mM NaCl for 4 h followed by incubation with anti-Flag Ab, M2 (Upstate Biotechnology), for another 2 h at room temperature. The membrane was washed three times with 0.1% Tween-20, 25 mM Tris-Cl (pH 7.5), and 150 mM NaCl and incubated with goat anti-mouse IgG conjugated with HRP (EY Laboratories, San Mateo, CA), developed using chemiluminescence (ECL Plus Western blotting detection reagents, Amersham Pharmacia Biotech), and then exposed on RX-U film (Fuji Film, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell death of thymocytes induced by in vivo TCR stimulation

To elucidate the mechanism of cell death of in vivo TCR-stimulated thymocytes, we i.p. injected anti-CD3 mAb into ICR mice and examined changes in cell surface membrane property, mitochondrial membrane potential, and chromosomal DNA of thymocytes at various times after injection. As shown in Fig. 1GoA, the percentages of annexin V-positive cells and DiOC6(3)low cells gradually increased after anti-CD3 injection. As previously reported (19), internucleosomal DNA fragmentation of thymocytes was detected at 12 h after anti-CD3 stimulation (Fig. 1GoB). It was noteworthy that chromosomal DNA degradation of thymocytes, shown as the smear of DNA electrophoresis pattern, started 24 h after anti-CD3 injection. Thymocytes of control Ab-injected mice did not cause either DNA fragmentation or DNA degradation (Fig. 1GoB). These data show that in vivo TCR stimulation of thymocytes induces apoptotic changes in thymocytes, such as a change in cell surface membrane property, a decrease in membrane potential of mitochondria, and chromosomal DNA fragmentation followed by chromosomal DNA degradation.



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  		FIGURE 1. Cell death execution of thymocytes after in vivo anti-CD3 stimulation. A, Apoptotic changes in cell membrane and mitochondrial membrane properties in anti-CD3-stimulated thymocytes. Mice were i.p. injected with anti-CD3 mAb or control Ab (50 µg/mouse), and thymocytes were prepared at various times after injection and stained with annexin V or DiOC6(3). Stained cells were analyzed by flow cytometry. The percentages of annexin V positively stained cells and DiOC6(3)low cells of anti-CD3-stimulated thymocytes compared with those of control Ab-stimulated thymocytes is shown. B, Degradation of chromosomal DNA in thymocytes after anti-CD3 stimulation. Mice were i.p. injected with anti-CD3 mAb or control Ab (50 µg/mouse). Chromosomal DNA was extracted from 2 x 106 thymocytes at various times after injection and analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities assessed by densitograph are shown at the bottom.

 
Cell death-inducing molecules in cell lysate from TCR-stimulated thymocytes

To analyze the cell death-inducing molecules in TCR-stimulated thymocytes, we injected anti-CD3 mAb into ICR mice and investigated the abilities of cell extracts of thymocytes to induce changes in morphology and chromosomal DNA in the isolated liver nuclei using a cell-free system. We also investigated the activity of cell extracts to induce the degradation of naked DNA. As shown in Fig. 2GoA, the cell extract of anti-CD3-stimulated thymocytes induced shrinking of nuclei as well as its chromatin condensation. The cell extract of thymocytes from control Ab-injected mice did not induce these morphological changes. The cell extract from anti-CD3-stimulated thymocytes also caused chromosomal DNA fragmentation of the liver nuclei and plasmid DNA degradation (Fig. 2GoB). These activities were gradually increased at 12–24 h after anti-CD3 injection. Cell extracts prepared from the thymocytes of control Ab-injected mice did not induce either nuclear chromosomal DNA fragmentation or plasmid DNA degradation. To test whether the stimulation of thymocytes with more physiological ligand for TCR rather than anti-CD3 mAb generates similar activities, we used DO11.10 TCR-transgenic mice, in which transgene encodes TCR recognizing an OVA peptide in the context of I-Ad (20). We i.p. injected OVA or BSA into DO11.10 mice, prepared the cell extract from the thymocytes at various periods after injection, and examined their abilities to induce changes in nuclear chromosomal DNA or plasmid DNA. As shown in Fig. 2GoC, the cell extract of OVA-stimulated thymocytes at 24 or 48 h after injection induced nuclear DNA fragmentation and plasmid DNA degradation, but the cell extract of thymocytes of BSA-injected mice did not induce these activities. These results indicate that anti-CD3-stimulated cell extracts may reconstitute the death-signaling pathway of the negative selection of immature thymocytes in vitro.



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  		FIGURE 2. Nuclear morphological changes, nuclear DNA fragmentation, and DNA degradation induced by cell extracts of in vivo anti-TCR-stimulated thymocytes. A, Nuclear morphological changes induced by cell extract of anti-CD3-stimulated thymocytes. Mice were injected i.p. with anti-CD3 or control Ab (50 µg/mouse), and cell extracts of thymocytes were prepared at 20 h after injection. Cell extracts (5 mg/ml) were then incubated with liver nuclei for 60 min and stained with 4,6-diamino-2-phenylindole, and the morphology of nuclei was observed under a fluorescence microscope. B, Chromosomal DNA fragmentation of nuclei and plasmid DNA degradation induced by cell extracts of anti-CD3-stimulated thymocytes. Cell extracts (5 mg/ml) were prepared from thymocytes at various times after anti-CD3 or control Ab injection into ICR mice and incubated with liver nuclei for 60 min or with plasmid DNA for 30 min. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. C, Chromosomal DNA fragmentation of nuclei and plasmid DNA degradation induced by cell extracts of OVA-stimulated DO.11.10 thymocytes. Cell extracts (5 mg/ml) were prepared from thymocytes at various times after OVA or BSA injection into DO.11.10 transgenic mice and were incubated with liver nuclei or plasmid DNA as described above. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom.

 
Subcellular localization of cell death-inducing activities

To investigate which subcellular organelles contain these activities, we separated cell extracts, using differential centrifugation methods, into four fractions: mitochondria (P7), microsome (P100), and two cytosolic fractions, LS100 and US100, as described in Materials and Methods (Fig. 3GoA). Electron microscopy revealed that P7 dominantly contained mitochondria, and P100 mainly contained membrane vesicles, endoplasmic reticulum lamellae, and ribosomes (data not shown). Each fraction was incubated with normal liver nuclei or plasmid DNA, and activities that induce nuclear DNA fragmentation or plasmid DNA degradation were investigated. As shown in Fig. 3GoB, both nuclear DNA fragmentation-inducing activity and DNA degradation activity were detected in P100 as well as LS100 of anti-CD3-stimulated thymocytes. Neither nuclear DNA fragmentation-inducing activity nor plasmid DNA degradation activity was found in any fraction of the cell extract of thymocytes from control Ab-injected mice. The corresponding fractions (P100 and LS100) of the cell extracts of thymocytes from OVA-stimulated DO11.10 mice also showed nuclear DNA fragmentation-inducing activity and plasmid DNA degradation activity (Fig. 3GoC), as found in the anti-CD3-stimulated cell extract. The fractions of the cell extracts from thymocytes of BSA-injected DO11.10 mice did not show these activities.



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  		FIGURE 3. Subcellular localization of cell death-inducing activity. A, Subcellular fractionation procedure. Cell extracts of thymocytes were fractionated into the mitochondria-rich fraction (P7), the microsome fraction (P100), and two different cytosolic fractions (LS100 and US100) as described in Materials and Methods. B, Nuclear DNA fragmentation or plasmid DNA degradation induced by subcellular fractions from thymocytes of anti-CD3 or control Ab-injected ICR mice (left), or OVA- or BSA-injected DO11.10 mice (right). Cell lysates from thymocytes of ICR mice injected with anti-CD3 or control Ab (left) or those from thymocytes of DO11.10 mice injected with OVA or BSA (right) were prepared after 20 h of injection, and their subcellular fractions were prepared as described above. Two hundred microliters of cell lysate or each subcellular fraction (P7, P100, LS100, and US100) was incubated with liver nuclei for 60 min or plasmid DNA for 30 min. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom.

 
Biochemical characterization of LS100 of cell extract from anti-CD3-stimulated thymocytes

Further biological and biochemical characterization of the molecule(s) that are responsible for nuclear DNA fragmentation and/or plasmid DNA degradation were performed using LS100 from anti-CD3-stimulated thymocytes (LS100-s). As shown in Fig. 4GoA, LS100-s induced nuclear DNA fragmentation and plasmid DNA degradation in a dose-dependent fashion. Kinetics studies showed that nuclear DNA fragmentation by LS100-s was observed at 15 min and became clearer at 30 or 60 min after incubation with the nuclei. DNA degradation by LS100-s was detected at 5 min and became clearer at 15–30 min (Fig. 4GoB). When LS100-s was pretreated with proteinase K or preincubated at 68°C for 10 min, both DNA fragmentation-inducing activity and DNA degradation activity were completely abrogated (Fig. 4GoC), indicating that the molecule(s) responsible for DNA fragmentation and DNA degradation in LS100-s is a heat-labile protein(s).



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  		FIGURE 4. Biochemical characterization of apoptosis-inducing activity in LS100 from anti-CD3-stimulated thymocytes. A, Dose response of DNA fragmentation- or DNA degradation-inducing activity. Various concentrations of LS100 were incubated with liver nuclei or plasmid DNA, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. B, Kinetics of DNA fragmentation or DNA degradation activity. LS100 (50 µg/ml) was incubated with liver nuclei or plasmid DNA for various times at 37°C, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. C, Sensitivity of DNA fragmentation- or DNA degradation-inducing activity to proteinase K or heat treatment. LS100 (50 µg/ml) was pretreated with proteinase K or RNase A beads for 5 min at 4°C (left) or preincubated at 37°C (control) or 68°C for 10 min (right) and then incubated with liver nuclei or plasmid DNA. Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. D, Cation requirements for LS100-induced DNA fragmentation or DNA degradation. LS100 (50 µg/ml) was incubated with liver nuclei or plasmid DNA in the presence of 5 mM EGTA and 5 mM EDTA (shown as -/-), 2 mM Mg2+ and 5 mM EGTA (shown as Mg++), 2 mM Ca2+ and 5 mM EDTA (shown as Ca++), or 2 mM Mg2+ and 2 mM Ca2+ (shown as Mg++/Ca++). Nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom.

 
The cation requirements for a molecule(s) in LS100-s to induce nuclear DNA fragmentation and plasmid-DNA degradation were also investigated. As shown in Fig. 4GoD, both DNA fragmentation and DNA degradation occurred in the presence of Mg2+ with or without Ca2+. They were not induced in the presence of Ca2+ alone. These data show that Mg2+ ions, but not Ca2+ ions, are required for the protein(s) to induce DNA fragmentation and DNA degradation.

Enari et al. recently identified CAD that induces nuclear DNA fragmentation using the cell-free system (15). They also identified ICAD. To examine whether CAD plays a role in the nuclear DNA fragmentation activity of LS100-s, the recombinant GST fusion protein of ICAD (GST-ICAD) was prepared and added to LS100-s, and nuclear DNA fragmentation-inducing activity was analyzed. As shown in Fig. 5GoA, nuclear DNA fragmentation-inducing activity in LS100-s was inhibited in a dose-dependent manner by GST-ICAD, but not by GST. Western blot analysis of LS100-s with anti-CAD Ab revealed a band of CAD protein in LS100-s (data not shown). These results indicate that the molecule responsible for DNA fragmentation of nuclei in LS100-s is CAD. It was worthwhile to note that the plasmid DNA degradation activity in LS100-s was not completely inhibited by GST-ICAD (Fig. 5GoA). Enari et al. previously demonstrated that the DNA degradation activity of CAD was completely inhibited by ICAD (15). By using our cell-free system, CAD also digested plasmid DNA, and its DNA degradation activity was completely inhibited by ICAD at the dose that completely inhibited DNA fragmentation activity of CAD (Fig. 5GoB). These results suggest that a molecule other than CAD is also involved in DNA degradation activity in LS100-s. Thus, we tried to purify the protein that is responsible for DNA degradation in LS100-s.



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  		FIGURE 5. Effect of ICAD on nuclear DNA fragmentation or DNA degradation induced by LS100 or CAD. A, Effect of ICAD on LS100-induced nuclear DNA fragmentation or plasmid DNA degradation. Various concentrations of LS100 (lane 1, 0 µg/ml; lane 2, 10 µg/ml; lane 3, 25 µg/ml; lane 4, 50 µg/ml) or 50 µg/ml of LS100 together with various concentrations of GST-ICAD or GST (lanes 5 and 8, 1 µg/ml; lanes 6 and 9, 3 µg/ml; lanes 7 and 10, 6 µg/ml) were incubated with liver nuclei or plasmid DNA. Chromosomal DNA fragmentation (upper panel) and plasmid DNA degradation (lower panel) were assessed by agarose gel electrophoresis. B, Effect of ICAD on nuclear DNA fragmentation and plasmid DNA degradation induced by active CAD. Ten microliters of recombinant CAD/ICAD complex was treated with various concentrations of active caspase 3 (lane 1, 0 U/ml; lane 2, 1 U/ml; lane 3, 2 U/ml; lanes 4–6, 3 U/ml), and incubated with liver nuclei (upper panel) or plasmid DNA (lower panel) in the absence (lanes 1–4) or the presence of GST-ICAD (lane 5, 6 µg/ml; lane 6, 12 µg/ml). Chromosomal DNA fragmentation and plasmid DNA degradation were analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom.

 
Purification and characterization of DNA degradation- inducing protein

To purify DNA degradation-inducing protein in LS100-s, LS-100-s (total of 450 mg protein collected from 2000 anti-CD3-injected mice) was fractionated by Q-Sepharose, hydroxyapatite, Mono S, and Superdex 200 columns. The DNA degradation-inducing protein did not bind to the Mono Q column, but bound to hydroxyapatite and Mono S columns. The activity was eluted from hydroxyapatite column with 0.2 M KPO4 and from Mono S column with 0.5–0.6 M KCl. Overall purification of this protein was 631.5-fold, with a yield of 0.1% (Table IGo). After further fractionation by Superdex 200 column chromatography, each fraction was examined for the ability to induce DNA degradation. As shown in Fig. 6Go, DNA degradation activity was detected only in the fractions 39 and 40. When each fraction was electrophoresed on a SDS-polyacrylamide gel, a band around 20 kDa was detected only in the fractions 39 and 40 (Fig. 6Go). However, this band was not detected in the corresponding fractions of LS100 from PBS-injected mouse thymocytes (data not shown).


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  		Table I. Purification of DNA degradation-inducing molecule from LS100

 


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  		FIGURE 6. Purification of DNA degradation-inducing protein in LS100 by Superdex 200 gel filtration chromatography. A, DNA degradation activity in fractions separated by Superdex 200. An aliquot (20 µl) of each fraction was incubated with plasmid DNA, and DNA degradation was analyzed by agarose gel electrophoresis. Nuclease activity was defined as in Table IGo. B, SDS-PAGE of fractions separated by Superdex 200. An aliquot (20 µl) of fractions was electrophoresed on an SDS-polyacrylamide gel, and proteins were visualized by silver staining. Each fraction corresponds to A.

 
The protein of this band was extracted, and the amino acid sequence was determined (NDKKKGPKVT). By using Swiss Prot and TrEMBL databases, it was revealed that this sequence is identical with the sequence of N-terminal of Cyp B.

Cyp B together with CAD induces degradation of chromosomal DNA in apoptotic nuclei

To examine whether Cyp B is involved in the DNA degradation-inducing activity in LS100-s, LS100-s was incubated with anti-Cyp B polyclonal Ab or control Ab and then incubated with plasmid DNA. As shown in Fig. 7GoA, DNA degradation-inducing activity of LS100 was inhibited by anti-Cyp B Ab in a dose-dependent manner but not by control Ab, indicating that Cyp B participates in DNA degradation activity in LS100-s. To delineate the activity of Cyp B in more detail, the GST fusion protein of Cyp B (GST-Cyp B) was prepared, and its activity to induce DNA degradation was investigated. GST-Cyp B induced degradation of chromosomal DNA in nuclei as well as plasmid DNA in a dose-dependent manner, but did not induce internucleosomal DNA fragmentation in nuclei (Fig. 7GoB).



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  		FIGURE 7. Involvement of Cyp B in DNA degradation activity in LS100. A, Inhibition of plasmid DNA degradation activity in LS100 by anti-Cyp B Ab. Various amounts of LS100 (lane 1, 0 µg/ml; lane 2, 10 µg/ml; lane 3, 25 µg/ml; lanes 4–9, 50 µg/ml) were incubated with anti-Cyp B Ab or rabbit IgG (lanes 5 and 8, 1 µg/ml; lanes 6 and 9, 3 µg/ml; lanes 7 and 10, 9 µg/ml) for 30 min at 4°C. After incubation, plasmid DNA (1 µg) was incubated with each mixture for 60 min at 37°C and analyzed by agarose gel electrophoresis. B, Induction of chromosomal and plasmid DNA degradation by recombinant Cyp B. Various concentrations of GST-Cyp B or GST (lanes 1 and 6, 0 µg/ml; lanes 2 and 7, 5 µg/ml; lanes 3 and 8, 10 µg/ml; lanes 4 and 9, 15 µg/ml; lanes 5 and 10, 30 µg/ml) were incubated with liver nuclei or plasmid DNA, and nuclear DNA (upper panel) and plasmid DNA (lower panel) were analyzed by agarose gel electrophoresis. C and D, Degradation of CAD-generated internucleosomal DNA fragments by recombinant Cyp B. Liver nuclei (upper panel) or plasmid DNA (lower panel) was incubated with buffer alone (control), LS100 (50 µg/ml), active CAD (pretreated with 1 U of caspase 3), GST-Cyp B (10 µg/ml), or active CAD and GST-Cyp B (C) or with various concentrations of GST-Cyp B or GST (lanes 1 and 6, 0 µg/ml; lanes 2 and 7, 5 µg/ml; lanes 3 and 8, 10 µg/ml; lanes 4 and 9, 15 µg/ml; lanes 5 and 10, 30 µg/ml) with active CAD (pretreated with 0.5 U of caspase 3; D) for 60 min at 37°C and analyzed by agarose gel electrophoresis. DNA fragmentation- and degradation-inducing activities are shown at the bottom.

 
To examine the possibility that Cyp B and CAD synergistically execute apoptotic nuclear changes, we characterized the nuclear DNA degradation activity and plasmid DNA degradation activity of LS100-s, CAD, Cyp B, or CAD plus Cyp B. As shown in Fig. 7GoC, LS100-s caused internucleosomal DNA fragmentation in nuclei as well as plasmid DNA degradation. A low concentration of active CAD induced internucleosomal DNA fragmentation stronger than LS100, but hardly degraded plasmid DNA. In the presence of both CAD and Cyp B, they caused not only internucleosomal DNA fragmentation but also degradation of chromosomal DNA and plasmid DNA (Fig. 7Go, C and D). These data showed that not only CAD but also Cyp B is required to reconstitute DNA fragmentation and DNA degradation activity in LS100-s.

Cyc B is released from microsomal fraction to cytosolic/nuclear fractions of a T cell line by TCR stimulation

It was reported that premature Cyp B contained a signal sequence targeting the protein to the endoplasmic reticulum and existed in microsome fraction in rat hepatocytes (21). To examine whether Cyp B is released from microsome fraction to cytosol and moves into nuclei by stimulation of TCR-CD3 complexes, we constructed an expression vector for Flag-tagged Cyp B (Cyp B-Flag) and transfected it into a T lymphoma cell line, EL4. EL4 cells were then stimulated with immobilized anti-CD3 and anti-CD28 mAbs, and subcellular distributions of Cyp B were examined by Western blot analysis using anti-Flag Ab (Fig. 8Go). In the fractions of nonstimulated EL4 cells, a high level of Cyp B was detected in the microsome fraction, but lower levels of Cyp B were found in the cytosolic and nuclear fractions. On the contrary, when EL4 cells were stimulated with anti-CD3/CD28 mAb, higher levels of Cyp B were detected in the nuclear and cytosolic fractions compared with those in the microsome fraction. These data show that Cyp B is released from the microsome fraction to the cytosolic/nuclear fraction by the signaling from TCR-CD3 complexes on EL4 cells.



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  		FIGURE 8. Release of Cyp B from cytoplasmic microsome fraction to cytosol/nuclear fractions of EL4 T cells by TCR stimulation. EL4 cells (5 x 106) transfected with Cyp B-Flag expression vector were incubated in the presence or the absence of immobilized-anti-CD3 and anti-CD28 mAbs for 12 h. After incubation, cells were fractionated into nuclear, microsomal, and cytosolic fractions. Total cell lysate and each fraction (50 µg of protein) were separated on SDS-PAGE, and the blot was probed with anti-Flag Ab.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, using a cell-free system, we showed that the cell extracts from anti-CD3 mAb and natural Ag (OVA)-stimulated thymocytes contain activities to induce morphological changes in naked nuclei and two distinct endonuclease activities ( Figs. 2–5GoGoGoGo), one that induced internucleosomal nuclear DNA fragmentation and the other that induced DNA degradation. With regard to internucleosomal DNA fragmentation activity, Enari et al. recently reported that CAD caused internucleosomal DNA fragmentation in nuclei in vitro (15). DNA fragmentation activity in TCR-activated thymocyte extracts was completely inhibited by ICAD (Fig. 5Go), indicating that CAD was activated by TCR stimulation in thymocytes and may be responsible for DNA fragmentation activity in TCR-simulated thymocytes. In agreement with our results, Clayton et al. demonstrated that an inhibitor of caspases inhibited apoptosis of thymocytes using a TUNEL assay (22). As the TUNEL assay should reflect DNA fragmentation in apoptotic cells, their results indicated the involvement of caspases in the activation of nucleases. Concerning the DNA degradation activity, Enari et al. (15) demonstrated that activated CAD showed DNase activity to degrade plasmid DNA. However, Toh et al. showed that a human homologue of CAD, DFF40, alone could not cause degradation of plasmid DNA (23). In the present study we demonstrated that a low dose of CAD induced internucleosomal DNA fragmentation, but hardly degrades the chromosomal DNA into smaller pieces when incubated with naked nuclei (Fig. 7GoC).

Interestingly enough, ICAD did not completely inhibit the plasmid DNA degradation activity in the cell extracts of TCR-stimulated thymocytes, suggesting that a DNase(s) other than CAD is also activated by TCR stimulation in the thymocytes (Fig. 5GoA). We purified this protein and revealed that its N-terminal amino acid sequence is identical with that of Cyp B (Fig. 6Go). Cyclophilin, cyclosporin A-binding protein, has five family members: Cyp A (18 kDa) (24), Cyp B (21 kDa) (21), Cyp C (23 kDa) (25), Cyp D (19 kDa) (26), and Cyp 40 (40 kDa) (27). With respect to the nuclease activity, Montague et al. demonstrated that the nuclease purified from glucocorticoid-treated rat thymocytes was highly homologous to Cyp A (28) and that recombinant Cyp A, Cyp B, and Cyp C degrade plasmid DNA (29) in a Ca2+- or Mg2+-dependent manner. They showed that Cyp C could generate 50-kb DNA fragments when incubated with naked nuclei; however, they did not show whether Cyp A, B, or C is involved in DNA degradation in apoptotic cells and especially they did not demonstrate whether Cyp B could degrade chromosomal DNA in nuclei. Thus, the role of Cyp B in thymocyte apoptosis has been obscure. In this study we showed that Abs to Cyp B dose dependently inhibited DNA degradation activity in cytosolic fraction prepared from anti-CD3-activated thymocytes, and that the recombinant Cyp B degrades chromosomal DNA in nuclei as well as plasmid DNA (Fig. 7Go). Our results demonstrated, for the first time, the possible involvement of Cyp B in the DNA degradation activity in TCR-stimulated thymocytes.

Regarding nucleases observed in apoptotic cells, at least three types of nucleases were reported. The first is a nuclease that degrades chromosomal DNA into an approximately 50-kb fragment. This includes apoptosis-inducing factor (30) and Cyp C (29). The second is a nuclease that induces internucleosomal DNA fragmentation, a hallmark of an apoptotic nuclear change, including DNase II and CAD (15, 31). The last is a nuclease that breaks chromosomal DNA into smaller pieces. This involves DNase I (32) and Cyp B in this study. Recently, Wu et al. demonstrated that NUC-1, a Caenorhabditis elegans DNase II homologue, is involved in DNA degradation in apoptotic cells and that activation of NUC-1 may degrade internucleosomally fragmented DNA in apoptotic cells (33). They observed more TUNEL-reactive nuclei in NUC-1-deficient embryos than in the wild-type embryos. Thus, DNA degradation is one of the major aspects of the apoptotic process. In this respect we observed the chromosomal DNA degradation after internucleosomal DNA fragmentation in apoptotic thymocytes induced by the anti-CD3-stimulation (Fig. 1GoB), indicating that Cyp B is involved in this process.

Cyp B contains N-terminal signal sequence (21), is produced in endoplasmic reticulum, and is secreted into biological fluids such as milk and plasma (34, 35). Spik and his colleagues demonstrated that peripheral blood T lymphocytes possess binding sites of Cyp B that internalize extracellular Cyp B into cells (36). To date, the fate and the function of internalized Cyp B are not clear. In Fig. 8Go it is shown that Cyp B is released from microsome fraction into cytosol as well as the nuclear fraction of a T cell line by signals from TCR. This result coincides with an assumption that Cyp B may be involved in chromosomal DNA degradation in apoptotic thymocytes stimulated by TCR. How is Cyp B in the microsome fraction translocated into cytoplasm and nuclei? Concerning this question, Peitsch et al. demonstrated that DNase I, produced in endoplasmic reticulum, is involved in nuclear DNA degradation during apoptosis and proposed that the mechanism by which DNase I gains access to nuclei is through the breakdown of the endoplasmic reticulum and nuclear membrane during apoptosis (32). In the case of cytochrome c, several models were proposed for its release of cytochrome c from mitochondria during the process of apoptosis (37): 1) cytochrome c is released as a result of the rupture of outer mitochondrial membrane; and 2) cytochrome c is released by the formation of a pore in the outer membrane. With regard to the latter case, it was demonstrated that the Bcl-2 family, including Bax and Bcl-2, possesses a pore-forming ability (38, 39) and that Bax could release cytochrome c from isolated mitochondria (40). Bcl-2 is shown to be localized not only on mitochondrial membrane, but also on endoplasmic reticulum (41). Thus, it may be possible to assume that a proapoptotic Bcl-2 family forms a pore on the membrane of endoplasmic reticulum to release Cyp B during apoptosis, although this is merely a speculation.

In the beginning of this study we unexpectedly observed DNA fragmentation activity in the microsomal fraction of TCR-stimulated thymocytes in addition to the cytosolic fraction. This activity may also be attributed to a CAD-like molecule(s), since this activity was inhibited by ICAD (data not shown). CAD was purified from the cytoplasm of apoptotic cells (18), and at the present time it is not clear whether CAD is localized in the microsomal fraction. In this respect, it has been shown that procaspase 12 is localized in the microsomal fraction and released to the cytoplasm by endoplasmic reticulum stress (42). We are now investigating the relationship between CAD in the cytoplasm and CAD-like molecule in the microsomal fraction as well as its physiological role in apoptotic cells.

Based on these results, we propose a model for chromosomal DNA degradation that was seen in TCR-stimulated thymocytes as follows. TCR engagement of immature thymocytes induces activation of CAD. The engagement also induces the release of Cyp B from microsome fraction to cytosol fraction. Activated CAD and Cyp B are translocated into nuclei and exert their activities in harmony on degrading chromosomal DNA after they are translocated into the nucleus (Fig. 9Go). Further biochemical analysis is necessary to elucidate the signal transduction from TCR causing the release of Cyp B from endoplasmic reticulum as well as the release of cytochrome c from mitochondria.



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  		FIGURE 9. A model for the DNA degradation mechanism in TCR-stimulated thymocytes. Signals from TCR activate caspase 3. Active caspase 3 cleaves the ICAD/CAD complex, and free CAD is localized in nuclei to induce chromosomal DNA fragmentation. Simultaneously, Cyp B localized in endoplasmic reticulum (ER) is released into cytosol by the signals from TCR, translocated into nuclei, and finally degrades CAD-fragmented chromosomal DNA into small pieces.

 


   Acknowledgments
 
We thank H. Hirano for helpful suggestions about purifying Cyp B protein, R. Mineki and N. Shindo for determining the protein sequence, D. Y. Loh for providing the DO11.10 mice, Dr. J. G. Sutcliffe for Ab to Cyp B, Dr. R. Abe for anti-mouse CD28, Dr. S. Nagata for mouse ICAD cDNA, Dr. K. Maruyama for pME18S vector, and Dr. G. Núñez for HA-CAD and Flag-DFF45 expression vectors.


   Footnotes
 
1 This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Atsushi Muraguchi, Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. Back

3 Abbreviations used in this paper: DFF40, DNA fragmentation factor 40; CAD, caspase-activated DNase; ICAD, inhibitor of caspase-activated DNase; Cyp B, cyclophilin B; CEB, cell extract buffer; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; LS100-s, LS100 from anti-CD3-stimulated thymocytes. Back

Received for publication May 1, 2000. Accepted for publication July 24, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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