The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jung, K. C.
Right arrow Articles by Bae, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, K. C.
Right arrow Articles by Bae, Y.
The Journal of Immunology, 2004, 172: 795-802.
Copyright © 2004 by The American Association of Immunologists

TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-Linking1

Kyeong Cheon Jung*, Weon Seo Park{dagger}, Hae Jung Kim{dagger}, Eun Young Choi{ddagger}, Myeong-Cherl Kook{ddagger}, Han-Woong Lee§ and Youngmee Bae2,{dagger}

* Department of Pathology, Hallym University College of Medicine, Chuncheon, Korea; {dagger} Department of Pathology, Kangwon National University College of Medicine, and Clinical Research Institute, Kangwon National University Hospital, Chuncheon, Korea; {ddagger} Department of Pathology, College of Medicine, and Research Division for Human Life Science, Seoul National University, Seoul, Korea; and § Department of Molecular Cell Biology, Samsung Biomedical Research Center, Sungkyunkwan University School of Medicine, Suwon, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During the thymic education, most of the thymocytes (95% or more of the total cells) are eliminated by apoptosis (1). Although the TCR/CD3 complex plays a pivotal role in the induction of apoptosis, other membrane proteins including CD28, CD45, CD90 (Thy-1), CD95 (Fas), and TNFR are also thought to be involved (2, 3, 4, 5).

CD24, also referred to as the heat-stable Ag, is a GPI-linked glycoprotein (6) and is expressed in both lymphoid and neuronal cell lineages (7, 8). During the T cell differentiation in the thymus, CD24 is expressed in the CD4-CD8- double-negative (DN)3 thymocytes, remains in the CD4+CD8+ double-positive (DP) cells, and begins to disappear when thymocytes are further differentiated into single-positive (SP) thymocytes (9). Such a differentiation-dependent expression pattern of CD24 has implied its role in thymocyte development. For example, Hough et al. (10) reported that the numbers of DP and SP thymocytes of transgenic mice, in which CD24 was highly expressed on all subsets of thymocytes, were significantly reduced.

The function of CD24 has been implicated in cell adhesion (11) and lymphocyte activation (12, 13, 14). On APCs, CD24 appears to act synergistically with B7 as a costimulatory molecule for T cell activation (12, 13). CD24 expressed on the activated T cells is also known as a signaling molecule, thus the possibility of homophilic interaction between CD24 molecules on T lymphocytes and APCs was suggested (14). In addition, CD24 appears to be involved in the apoptosis of B cells and thymocytes (3, 15, 16, 17). In fact, in human B cell lines, CD24 engagement by specific Abs induces apoptosis, which does not require B cell receptor signaling (16, 17). In contrast, CD24 ligation in mouse thymocytes has been reported to enhance CD3-mediated apoptosis, but CD24 engagement alone did not cause thymocyte apoptosis (15).

In this study, we developed a new mAb against murine CD24, designated as mT-20, and investigated whether cross-linking of CD24 mediated by mT-20 mAb induces any physiological responses in murine thymocytes. In contrast with the previous report, CD24 ligation by this mAb induced the apoptosis of DN and DP thymocytes as well as the Scid.adh thymic lymphoma cell line in the absence of TCR/CD3 engagement. The CD24-mediated apoptosis proceeded in a caspase-independent manner and was overcome by Bcl-2 overexpression. Thus, our results suggest that, in the absence of TCR signaling, CD24 ligation plays a role in the induction of apoptosis through a caspase-independent pathway.


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

C57BL/6 and BALB/c mice were purchased from Daehan Biolink (Chungbuk, Korea). Fas-defective lpr mice and TNFR p75 knockout mice in C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Wild-type FVB mice and p53 knockout mice in FVB background were also used. All mice were housed in a specific pathogen-free facility at Hallym University (Chuncheon, Korea). All experimental animals were cared for, maintained, and terminated by CO2 inhalation in accordance with the Hallym University Guideline.

Abs and reagents

A hybridoma cell line was generated by fusion of splenocytes from Sprague-Dawley rats immunized with mouse thymocytes and SP2/0-Ag14 myeloma cells and was designated mT-20. The mT-20 mAb was used as either an unconjugated or an FITC-conjugated protein. Anti-CD24 mAb (M1/69) was purchased from BD PharMingen (San Diego, CA). Rabbit anti-apoptosis-inducing factor (anti-AIF) Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the rabbit anti-caspase-3 Ab was from Cell Signaling Technology (Beverly, MA). All other Abs and FITC-labeled annexin V were purchased from DiNonA (Seoul, Korea) or BD PharMingen. 7-Aminoactinomycin D (7-AAD) and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) were obtained from Sigma-Aldrich (St. Louis, MO). N-acetyl-L-cysteine (NAC), z-IETD-fmk, and z-VAD-fmk were obtained from Calbiochem (San Diego, CA), and the mouse rIL-7 was purchased from PeproTech (Princeton, NJ).

Cell preparation and culture

Cell suspensions of thymocytes from 4- to 10-wk-old mice were prepared by mincing thymus tissues through a fine meshed screen, after which dead cells were removed by centrifugation through a Ficoll-Paque gradient (Amersham Bioscience, Buckinghamshire, U.K.). Each thymocyte subset was prepared by magnetic cell sorting using MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as recommended by the manufacturer. Briefly, the CD4+CD8+ DP subset was prepared by positive selection using anti-CD8 microbeads, and anti-CD4 microbeads were used to collect CD4+ SP thymocytes. The CD25+CD4-CD8- DN thymocytes were isolated by elimination of CD4+ or CD8+ thymocytes by MACS and were followed by positive selection of the CD25+ populations. The purity of collected CD4+ T cells, determined by flow cytometry using anti-CD4, anti-CD8, and anti-CD25 mAbs, ranged from 92 to 97%. The purified thymocytes were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10% FBS and 0.05 mM 2-ME. EL-4 and R1.1 cell lines were purchased from American Type Culture Collection (Manassas, VA), and the Scid.adh cell line was obtained from Dr. David L. Wiest (Fox Chase Cancer, Philadelphia, PA). The EL-4 and R1.1 cell lines were cultured in DMEM supplemented with 10% FBS, whereas the Scid.adh cell line was maintained in IMDM (Life Technologies) containing 10% FBS, as previously described (18).

Genes, transfection, and retroviral transduction

Mouse cDNAs encoding the CD24 Ag and Bcl-2 were amplified from a mouse spleen cDNA library (Clontech Laboratories, Palo Alto, CA) by PCR and were subcloned into the pcDNA3.1 and pRetro plasmids. The pcDNA3.1-CD24 construct and parental plasmid were transfected into 293T cells by using the calcium phosphate precipitation method. The 293GPG packaging cells, obtained as a gift from Dr. R. C. Mulligan (Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA), were used for the production of retrovirus expressing mouse Bcl-2. Transfection of 293GPG cells was performed by calcium phosphate precipitation method. After incubation of 293GPG cells for 16–20 h with coprecipitates of calcium phosphate and plasmid encoding mouse Bcl-2 (pRetro-bcl2), medium was replaced with 5 ml of fresh solution and the cells were incubated for another 24 h. Scid.adh cells were plated onto six-well plates the day before transduction. The viral supernatants harvested from the transfected 293GPG cells and filtered through a 0.45-µm filter to remove cell debris were added to Scid.adh cells.

Flow cytometry

Fresh cell suspensions were prepared in PBS from mouse thymus and cultured cell lines, incubated with appropriate Abs for 30 min at 4°C, and washed with PBS. The flow cytometric analysis was performed using a FACSCalibur (BD Biosciences, Mountain View, CA). For the detection of early and late apoptosis by flow cytometry, Annexin VFITC and the vital dye 7-AAD were used (19). The cells were washed three times with annexin V-binding buffer (0.1 M HEPES/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) and were incubated for 30 min at room temperature in the dark in annexin V-binding buffer containing FITC-conjugated annexin V and 20 µg/ml 7-AAD. Specific cell death (percent cell death) was calculated as a normalized value as follows: [percentage of live cells (unstimulated) - percentage of live cells (stimulated)]/percentage of live cells (unstimulated). To compare thymocyte apoptosis in response to mT-20 signals among experimental groups with different internal controls, individual responses were normalized and expressed as a killing index. The killing index was calculated as follows: (percentage of cell death induced by mT-20 under experimental conditions)/(percentage of cell death induced by mT-20 under control conditions). Killing index = 1.0 means that the indicated condition did not affect mT-20-induced apoptosis.

To evaluate the mitochondrial membrane potential ({Delta}{psi}m) by flow cytometry, the cationic lipophilic fluorochrome DiOC6 was used. A total of 1 x 105 thymocytes or 1 x 104 lymphoma cells was incubated with 20 nM DiOC6 for 15 min at 37°C. Cells were then diluted with PBS to a final volume of 1 ml and were analyzed using a FACSCalibur.

Western blotting

Cell lysates were prepared by dissolving cells in lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM PMSF, and 1% Nonidet P-40. A 50-µg quantity of each lysate was loaded onto SDS-PAGE gels, separated by electrophoresis, and transferred onto polyvinylidene difluoride Immobilon membranes (Millipore, Bedford, MA). The blotted membranes were incubated with primary Ab, followed by peroxidase-conjugated secondary Ab. The specific bands were visualized using an ECL kit from Amersham Pharmacia Biotech (Buckinghamshire, U.K.).

Electron microscopy

Cultured cells were washed with PBS and fixed for 4 h at 4°C in 0.1 M sodium cacodylate buffer (pH 7.3) containing 2.5% glutaraldehyde, 2% sucrose, and 1 mM calcium chloride. This was followed by postfixation with 1% OsO4 in 0.1 M cacodylate-HCl at pH 7.4 for 1 h. The samples were dehydrated in gradient series of ethanol, embedded in Epon812, and examined using an electron microscope.

Fluorometric assay for caspase activity

Caspase-3 activity was calculated by using the Caspase-3 Activity Assay kit (Roche, Mannheim, Germany). Briefly, cytosolic extracts from 2 x 106 cells were incubated in anticaspase-3 Ab-coated plates, followed by washing and adding the fluorescent tetrapeptide substrate (Ac-DEVD-aminomethylcoumarin (AMC)). Free AMC accumulation, which resulted from cleavage of the aspartate-AMC bond, was monitored in each sample in 96-well microtiter plates using a fluorometer at 400-nm excitation and 505-nm emission wavelengths.

Confocal microscopy

Translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy, as indicated previously (20). Briefly, control cells or cells treated with mAb were collected, washed with PBS, and fixed in a solution of 4% paraformaldehyde in PBS. Cell suspensions were then placed onto glass slides previously treated with poly-L-lysine by cytospin. Cells on the glass slides were then washed once with PBS, a drop was added of a 0.1% solution of saponin in PBS containing 5% goat serum and a 1/500 dilution of the anti-AIF antiserum, and they were incubated for 30 min at room temperature in a humidified chamber. Glass slides were then washed once with a 0.1% solution of saponin in PBS and were incubated in the same way with a 1/200 dilution of an anti-rabbit IgG Ab labeled with Cy-3 (Molecular Probes, Eugene, OR). Finally, glass slides were washed sequentially with 0.1% saponin, PBS, and distilled water and were mounted. Preparations were observed with a confocal microscope (Radiance 2000; Bio-Rad, Hercules, CA).

Statistical analysis

Where applicable, values were compared by the Mann-Whitney’s rank sum test using GraphPad Prism version 3.0 software; p values < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mT-20 mAb recognizes murine CD24 protein.

To characterize the Ag recognized by mAb mT-20, we first analyzed the distribution pattern of the Ag on the four major thymocyte subpopulations distinguished by CD4 and CD8 Ags. The expression of mT-20 was found to be highest on the most immature DN thymocytes, intermediate on the more mature DP cells, and the lowest on the mature CD4 or CD8 SP cells (Fig. 1A). The Ag reactive to mT-20 mAb was rarely expressed in peripheral T cells, whereas most mature B lymphocytes, macrophages, and granulocytes were immunoreactive against mT-20 mAb (data not shown). This expression pattern of mT-20 was nearly identical with that of CD24 (21, 22). Consequently, we compared the molecular mass of Ag recognized by mT-20 mAb with that by known CD24 mAb (M1/69). Both mAbs recognized similarly sized Ags of murine thymocytes (Fig. 1B), suggesting that mT-20 mAb might recognize murine CD24.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 1. The mT-20 Ab recognizes murine CD24 Ag. A, Expression of mT-20 Ag on murine thymocytes. Thymocytes were stained with FITC-conjugated mT-20, Cychrome-conjugated anti-CD4, and PE-conjugated anti-CD8 Abs, followed by flow cytometric analysis. According to the expression pattern of CD4 and CD8, DN, DP, CD4+ SP, and CD8+ SP subsets were defined, and then the expressions of mT-20 Ag in each subset were examined (solid line). B, The mT-20 mAb recognizes the Ag of molecular mass similar to that of murine CD24. Thymocyte lysates from B6 or BALB/c mice were electrophoretically separated on SDS-PAGE gels, and immunoblotting with mT-20 mAb or a known anti-CD24 mAb was performed. C, The mT-20 mAb recognizes GPI-linked protein on thymocytes. Thymocytes were incubated at 37°C in the presence (thick solid line, not filled) or absence (thin solid line, not filled) of PI-PLC (0.5 U/ml). Cells were labeled with the indicated mAbs, followed by flow cytometry. The negative staining profile obtained with an isotype-matched control mAb is shown (filled histogram). D, The mT-20 mAb recognizes murine CD24 Ag. 293T cells were transfected with a plasmid encoding the CD24 molecule (pcDNA-CD24) and were stained with indicated Abs (solid line). Mock-transfected 293T cells (pcDNA) were not reactive to mT-20 or anti-CD24 mAb.

 
CD24 Ag is one of the GPI-anchored proteins and therefore is cleaved by phosphatidylinositol-specific phospholipase (PI-PLC). To determine whether mT-20 mAb recognized the GPI-anchored protein, mouse thymocytes were treated with PI-PLC and were subjected to staining with FITC-conjugated mT-20 mAb. The flow cytometric analysis revealed a decrease in the mean cell surface expression of mT-20 as compared with the control (Fig. 1C), indicating that the mT-20 could also detect GPI-anchored molecules. In parallel samples, the level of CD24 as well as that of another GPI-anchored molecule, Thy-1, was also diminished, whereas the level of a non-GPI-anchored molecule, CD4, was unaffected (Fig. 1C). This result convinced us that the mT-20 mAb could recognize the CD24 molecules, and therefore we tested whether mT-20 mAb was reactive to the 293T cells expressing murine CD24 molecules. As shown in Fig. 1D, successful expression of murine CD24 in 293T cells was confirmed by flow cytometry using a known anti-CD24 mAb. When the same cells were incubated with mT-20 mAb, the mT-20 Ab was reactive to the CD24-transfected 293T cells, whereas neither mT-20 nor M1/69 was reactive to the mock-transfected 293T cells, confirming that the mT-20 mAb recognized murine CD24 Ag.

Cross-linking of CD24 by mT-20 Ab induces apoptosis in thymocyte and Scid.adh cell line

Next, we investigated whether cross-linking of CD24 mediated by mT-20 mAb induced any physiological responses in murine thymocytes. When mouse thymocytes were treated with mT-20 mAb and cross-linked with a secondary Ab for 24 h, more than one-half of the thymocytes underwent apoptosis (Fig. 2A). The apoptosis of thymocytes induced by ligation of CD24 with mT-20 mAb appeared to occur in the absence of CD3 stimulation, whereas CD28 needed CD3 stimulation to cause thymocyte apoptosis (Fig. 2B). In addition, the degree of mT-20-induced apoptosis was not affected by CD3 ligation (Fig. 2B). Apoptosis of thymocytes by the treatment of mT-20 mAb did not occur in the absence of the cross-linking Ab (data not shown), suggesting that cross-linking of CD24 is required to induce apoptosis in mouse thymocytes.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 2. Cross-linking of CD24 by mT-20 mAb induces apoptosis in murine thymocytes. A and B, Total thymocytes were cultured on the PBS- or anti-CD3 (2C11, 10 µg/ml)-coated plates in the presence of the indicated Abs (10 µg/ml) and secondary Abs (20 µg/ml) for 24 h and then were stained with annexin V and 7-AAD, and the apoptotic cells were evaluated by flow cytometry (A). Percentages of dead cells from each sample were quantitated and normalized (B) as described in Materials and Methods. C and D, DN, DP, and CD4+ SP thymocytes were isolated by magnetic sorting and cultured with or without mT-20 mAb and a cross-linking secondary Ab, and the apoptosis was estimated in each population (C). In addition, each thymocyte population was stimulated with mT-20 mAb and cross-linking Ab in the absence or presence of IL-7 (50 ng/ml) to determine whether the addition of exogenous IL-7 is able to protect the thymocytes from apoptosis (D). Percentage of cell death was measured and normalized. All experiments were performed in triplicate, and mean ± SD was presented. Results are representative of at least three separate experiments. *, p < 0.05.

 
It is well understood that the expression level of CD24 Ag varies according to the developmental stage of thymocytes (22). To test which population was susceptible to mT-20-mediated apoptosis, the thymocytes sorted into DN, DP, and SP fractions were incubated with mT-20 and cross-linking Ab. As presented in Fig. 2C, both DN and DP thymocytes, most of which has CD24 expression on their surfaces, underwent apoptosis. In contrast, CD24 engagement by mT-20 mAb did not significantly induce cell death of SP thymocytes, in which CD24 expression is markedly down-regulated (Figs. 1A and 2C).

It is known that the development and survival of DN thymocytes is dependent on the presence of IL-7, and that IL-7 is able to protect the DN thymocytes from spontaneous death (23). Therefore, IL-7 was added in the culture medium to examine whether the CD24-mediated apoptosis of DN thymocytes is inhibited by the addition of IL-7. As shown in Fig. 2D, however, the presence of IL-7 did not block mT-20-induced apoptosis of either DN or DP thymocytes (Fig. 2D).

Next, using two independent CD24 positive tumor cell lines (Scid.adh and R1.1), we examined whether the induction of cell apoptosis by CD24 engagement with mT-20 mAb was reproducible in tumor cell line. In the case of Scid.adh cells, we observed apoptosis, mostly identical results with thymocytes (Fig. 3A). As expected, when the CD24 negative EL-4 cells were similarly examined, treatment with a combination of mT-20 mAb and a cross-linking Ab failed to induce apoptosis (data not shown). However, CD24+ R1.1 cell line was found to be resistant to the mT-20-induced apoptosis (Fig. 3A), although the expression level of CD24 was comparable with that of Scid.adh cells (data not shown). These results suggest that the level of CD24 expression could not be the only factor that determines susceptibility to apoptosis. For example, the expression pattern of apoptosis-related factors such as death receptors (Fas or TNFR), Bcl-2 family proteins, p53, or caspases could be involved in CD24-mediated apoptosis. In fact, we previously reported that the expression level of the mouse Bcl-2 family was varied in thymic lymphoma cell lines (24), and this was further investigated (see CD24 cross-linking leads to the reduction of mitrochondrial membrane potential, {Delta}{psi}m, in a Bcl-2-dependent manner).



View larger version (86K):
[in this window]
[in a new window]
  		FIGURE 3. Induction of apoptosis in Scid.adh thymic lymphoma cells. A, Two CD24+ thymic lymphoma cell lines, Scid.adh and R1.1, were treated with mT-20 or isotype-matched control mAb and cross-linking Ab for 24 h. A representative from three separate experiments is presented. B, Scid.adh cells were stimulated in the same manner as in A, and morphological appearance was examined by electron microscopy. Cells treated with mT-20 Ab showed several features of apoptosis, including cell shrinkage and chromatin condensation, whereas characteristic apoptotic bodies were not detected (original magnification, x3000). C, DNA ladder formation after cross-linking of CD24 was examined. Scid.adh cells were treated with mT-20 Ab or etoposide, and the extracted DNA from each sample was separated by 2% agarose gel electrophoresis. Treatment of etoposide induced the typical DNA ladder formation, whereas it was not detected in mT-20-treated Scid.adh cells. DNA size marker of 100-bp ladders was also presented.

 
Chromatin condensation and the formation of apoptotic bodies, which are associated with the internucleosomal degradation of DNA, are known to be the typical morphologic changes of classical apoptosis (25). Therefore, electron microscopic analysis was performed to investigate whether CD24 engagement induced such morphologic changes in nuclei. As depicted in Fig. 3B, mT-20-treated Scid.adh cells exhibited clumps of partially condensed chromatin, but they did not possess the typical apoptotic bodies (Fig. 3B). In addition, unlike etoposide-treated cells, mT-20-treated Scid.adh cells displayed no internucleosomal DNA fragmentation, which is a hallmark of caspase-3 activity (Fig. 3C), implying that the apoptosis induced by CD24 ligation with mT-20 mAb might not involve caspase-3 activation and the following events.

Caspase activation is not required in CD24-mediated apoptosis of thymocytes

Fas and TNFR p75 are well-known apoptosis-inducing membrane proteins (26). Especially in thymocytes, TCR-independent apoptosis can result from TNFR p75 and Fas activation (3). Thus, to assess whether these TNFR superfamily members and the related signaling pathways are involved in mT-20 mAb-mediated apoptosis, we compared the impact of CD24 ligation on thymocytes from wild-type mice with that of thymocytes from Fas-deficient lpr mice or TNFR p75 knockout mice. When the thymocytes from Fas- or TNFR p75-deficient mice were treated with mT-20 mAb and cross-linking secondary Ab, they were still susceptible to apoptosis (Fig. 4A). Furthermore, p53 did not seem to be involved in the mT-20-induced apoptosis of thymocytes, in that the degree of thymocyte apoptosis in p53 knockout mice was comparable with that of the wild type (Fig. 4A). These data suggest that the CD24-mediated signaling pathway is distinctly different from Fas, TNFR p75, and p53.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4. Caspase activation is not required in CD24-mediated apoptosis of thymocytes and Scid.adh cells. A, CD24-mediated apoptosis of thymocytes is not mediated by Fas, TNFR p75, or p53. Total thymocytes were isolated from the mice indicated (wild-type B6, Fas-deficient lpr, and TNFR p75- and p53-deficient strains) and cultured in the presence of mT-20 and cross-linking Ab for 24 h, and the incidence of apoptosis was examined. To compare thymocyte apoptosis from different mouse strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as a killing index (see Materials and Methods). B, Caspase inhibitors z-IETD-fmk and z-VAD-fmk do not prevent CD24-mediated cell death. Thymocytes were stimulated with the mT-20 and cross-linking Ab for 8 h in the presence of 50 µM caspase inhibitors, z-IETD-fmk or z-VAD-fmk, and cell death was examined on flow cytometry. The individual responses were normalized to the control (DMSO), and the killing index was depicted. Values are the mean ± SD from experiments performed in triplicate. C, Activation of caspase-3 was measured upon the treatment of mT20 mAb or etoposide. Thymocytes were incubated with mT20 and cross-linking Ab or etoposide, and the cellular extracts were collected after 8 or 18 h. Caspase-3 activity was assayed by fluorometric method as described in Materials and Methods. Caspase-3 activity indicates fluorescence (excitation 400 nm/emission 505 nm). D, Protein lysates were prepared from thymocytes exposed to mT-20 mAb and cross-linking Ab or etoposide for indicated periods. Each lysate was electrophoretically separated on SDS-PAGE gels, and both proform (Pro) and the active form (p17) of caspase-3 were visualized by immunoblotting with anti-caspase-3 Ab. Results are representative of three separate experiments. *, p < 0.05.

 
DNA fragmentation and ordered disintegration of the cell organelles were established hallmarks of the cells undergoing apoptosis, especially the apoptosis associated with recruitment of caspase-8 to the receptors’ death domain, such as cell death after ligation of Fas and TNFR (25, 27). However, recent studies have suggested that apoptosis may also proceed independently of caspase activation (28, 29, 30, 31, 32) and that apoptosis without DNA fragmentation is one of many characteristic features of caspase-independent apoptosis (28, 33). Therefore, we next questioned whether caspase activation is required for CD24-mediated apoptotic pathways in thymocytes and thymic lymphoma cell lines. To determine the role of caspases in the mT20 mAb-mediated thymocyte death signaling, we first incubated the thymocytes or Scid.adh cells with 50 µM caspase-8 inhibitor z-IETD-fmk for 30 min before the addition of mT-20 mAb and cross-linking Ab, and then they were further cultured for another 8 h. Flow cytometric analyses using annexin V and 7-AAD staining revealed that z-IETD-fmk did not interfere with the apoptosis responses of thymocytes (Fig. 4B) and Scid.adh cells (data not shown) to the CD24 ligation with mT-20 mAb, supporting the notion that CD24-induced apoptosis of thymocytes is independent of Fas and TNFR signaling (Fig. 4A). In addition, we found no interference of a pan-caspase inhibitor, z-VAD-fmk, with the ability of mT-20 to induce death of thymocytes (Fig. 4B) and Scid.adh cells (data not shown).

Next, to confirm that CD24-mediated apoptosis is independent of caspase activity, we measured the caspase-3 activity by using fluorometric method (Fig. 4C) and the Western blot analysis (Fig. 4D). As depicted in Fig. 4, C and D, CD24 ligation through the mT-20 epitope failed to activate caspase-3, whereas etoposide-treated thymocytes showed caspase-3 activity. Taken together, CD24-mediated cell death apparently does not require caspase activation and also proceeds independently of Fas and TNFR, but by a distinct pathway.

CD24 cross-linking leads to the reduction of mitochondrial membrane potential, {Delta}{psi}m, in a Bcl-2-dependent manner

It is almost generally accepted today that the mitochondria constitute the center of death control during both caspase-dependent and caspase-independent apoptosis (4, 28). Before classical signs of cell death become manifest, a reduction in the mitochondrial membrane potential, {Delta}{psi}m, is known to occur as an early event that marks irreversible commitment to apoptosis (34, 35). In this study, we found that treatment of thymocytes or Scid.adh cells with mT-20 mAb also induced a significant reduction in {Delta}{psi}m, as determined by DiOC6 staining (Fig. 5A), suggesting that CD24-induced apoptosis is associated with the collapse of mitochondrial membrane potential.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 5. Reduction in {Delta}{psi}m in CD24-mediated apoptosis of thymocytes and Scid.adh cells. Thymocytes or Scid.adh cells were incubated with mT-20 and cross-linking Ab and were stained with DiOC6, which reveals {Delta}{psi}m. A, Bcl-2 overexpressing cell lines as well as wild-type Scid.adh cell line were stimulated in the same manner as in A, and the incidence of cell death was examined on flow cytometry after DiOC6 (B) or annexin V and 7-AAD (C) staining. Overepxression of Bcl-2 protects the Scid.adh from CD24-mediated cell death. Values are the mean ± SD from experiments performed in triplicate. Results are representative of three separate experiments. *, p < 0.05.

 
The mitochondrial membrane potential is regulated by members of the Bcl-2/Bax family (36). Bcl-2-like anti-apoptotic proteins are constitutively present in mitochondria and would favor the maintenance of normal mitochondrial function, whereas Bax-like pro-apoptotic proteins, which translocate to mitochondria only in conditions of apoptosis induction, would favor irreversible loss of mitochondrial function. In addition, it has been reported that transcripts of bcl-2 were abundant in the R1.1 cell line, which is resistant to mT-20-induced apoptosis, but scanty in the apoptosis-susceptible Scid.adh cell line (24). Therefore, we questioned whether Bcl-2 is involved in the CD24-mediated death of thymic lymphoma cell lines. To assess this possibility, a mouse Bcl-2 gene was introduced into the Scid.adh cell line using a retroviral vector, and two cell lines (Scid.bcl-2 #4 and #7) that stably expressed Bcl-2 were selected (data not shown). Overexpression of Bcl-2 suppressed the reduction of {Delta}{psi}m by spontaneous death (Fig. 5B). In addition, when they were treated with mT-20 mAb, the Scid.adh cells overexpressing Bcl-2 reproducibly inhibited the {Delta}{psi}m decline, and this effect contributed to the decreased apoptosis of cells overexpressing Bcl-2, compared with that of wild-type Scid.adh cells (Fig. 5, B and C). Altogether, these data confirm that CD24 ligation induces apoptosis of thymocytes and thymic lymphoma cell line Scid.adh by modulating {Delta}{psi}m in a Bcl-2-dependent manner.

CD24 cross-linking leads to the production of reactive oxygen species (ROS) and the release of AIF

The data shown above indicate that the mitochondria might participate in the process of CD24-mediated apoptosis of thymocytes and the Scid.adh cell line, and a possible cause of mT-20 mAb-mediated cell death might be ROS production by the mitochondria, which could be involved in cell death by various apoptotic signals (34, 37, 38). To determine the implication of ROS in the CD24-mediated death of thymocyte, we have cross-linked CD24 on thymocytes with mT-20 mAb in the presence of NAC, a well-characterized ROS scavenger (39). The addition of NAC could diminish CD24-mediated cell death in a dose-dependent manner (Fig. 6), indicating a role for ROS in the CD24-mediated apoptotic pathway.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 6. ROS scavenger NAC protects thymocytes from CD24-mediated apoptosis. Thymocytes were stimulated with mT-20 mAb and cross-linking Ab in the absence or presence (2 or 10 µg/ml) of NAC, and cell death was assessed by flow cytometry. A, The figure illustrates the typical dot blots obtained from each experiment. B, Values are the mean ± SD from experiments performed in triplicate. Results are representative of three separate experiments. *, p < 0.05.

 
To further elucidate the role of mitochondria in the CD24-induced apoptosis of thymocytes and Scid.adh cells, we tested the possible involvement of AIF, a protein released from mitochondria during apoptosis that especially exerts its proapoptotic activity in a caspase-independent manner (20, 40). During apoptosis, AIF released from mitochondria rapidly translocates to the nucleus, where it induces peripheral chromatin condensation (40). This translocation could be tracked by confocal microscopy using a specific antiserum (Fig. 7). Given the small size of the cytoplasm in these cells, it was difficult to distinguish the punctuated staining corresponding to the typical mitochondrial localization. However, when cells were treated with mT-20 mAb, the significant translocation of AIF to the nucleus was evident. In contrast, AIF was still retained in the cytoplasm in the Bcl-2-overexpressing cells after CD24 stimulation. These data clearly demonstrate a role for AIF in CD24-induced apoptosis.



View larger version (121K):
[in this window]
[in a new window]
  		FIGURE 7. AIF translocation during CD24-mediaed apoptosis. Wild-type or Bcl-2-overexpressing (bcl-2 #4) Scid.adh cells were treated with mT-20 mAb or isotype-matched control mAb for 24 h. Then the cells were sequentially incubated with a specific anti-AIF Ab and a Cy3-labeled anti-rabbit IgG, and fluorescence was analyzed by confocal microscopy. Stimulation with mT-20 induced translocation of AIF into the nucleus (upper left and right), and this translocation was inhibited by bcl-2 gene overexpression (lower left and right). Original magnification, x1000. The pictures shown are representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD24 is one of the costimulatory molecules that amplifies signals derived through the TCR in the thymocytes (9, 12). In this study, we demonstrate that cross-linking of CD24 with mT20 mAb induced the apoptosis of thymocytes even in the absence of TCR signals. Under these conditions, thymocytes and a thymic lymphoma cell line, Scid.adh, showed the several characteristic features of apoptosis, namely 1) exposure of phosphatidylserine in the cell membrane (annexinv V+) with or without incorporation of vital dye (7-AAD), 2) reduction in {Delta}{psi}m (DiOC6low), 3) production of ROS, and 4) release of AIF from mitochondria. In addition, Bcl-2 overexpression appeared to protect from the apoptosis, possibly by inhibiting the release of AIF from the mitochondria. In contrast with classical caspase-dependent apoptosis, however, the CD24-mediated apoptosis of thymocytes and Scid.adh cells did not lead to internucleosomal fragmentation of DNA and was not prevented by caspase inhibitors. It is now well demonstrated that caspases are not the only molecules that are able to induce apoptosis (28, 33). For instance, CD99-mediated (31) or superantigen-induced T cell death (28) appeared to be caspase independent. Likewise, the CD24-induced apoptosis seemed to be caspase-independent in thymocytes and the thymic cell line.

In addition to the TCR-CD3 complex, several surface molecules have been found to affect apoptosis during thymocyte development. In the thymus, a number of cell surface molecules including CD5, CD28, and CD43 cooperate with TCR to kill thymocytes, whereas a few molecules on the surface of thymocyte are able to induce the apoptosis of thymocytes in the absence of TCR ligation (3). Two members of the TNFR family, Fas and TNFR, are well-known receptors that induce the apoptosis of thymocytes in a TCR-independent manner (3). In addition, it has been known that thymocytes undergo cell death after CD45 cross-linking (29). In this work, we show that CD24 is another candidate molecule that is able to induce the apoptosis of thymocytes in a TCR-independent manner (Fig. 2). Our present findings differ from those of Hitsumoto et al. (15) and Punt et al. (3), who found that CD24 ligation only enhanced the CD3-mediated death of thymocytes. We assume that this could come from different approaches applied in our system for the detection of apoptotic cells. For example, in the previous studies, staining of vital dyes such as 4',6'-diamidino-2-phenylindole and ethidium bromide was used to detect apoptotic thymocytes. Therefore, they might fail to detect early apoptotic cells that are annexin V+/7-AAD-. In our system, upon CD24 ligation, a number of annexin V+/7-AAD- early apoptotic cells were observed, and in fact they were the major targets of CD24-induced apoptosis in murine thymocytes (Figs. 2A and 6A). To investigate whether other CD24 mAbs are also able to induce apoptosis, we treated murine thymocytes or Scid.adh cells with a known anti-CD24 Ab (M1/69) and a cross-linking Ab and found that this treatment induced the exposure of annexin V on the surface in both murine thymocytes and Scid.adh cells (data not shown). This opens the possibility that CD24 Ag could act as a death receptor in the murine system.

Apoptosis of thymocytes occurs through specific death receptors, including TNFR and Fas, or DNA damages (19, 27). However, the classical apoptotic signaling pathways were not involved in the CD24-mediated apoptosis. The thymocytes from the mice deficient in Fas, TNFRp75, or p53 still revealed the same characteristic death response to the mT-20 mAb treatment. In addition, the CD24-mediated apoptosis of thymocytes did not reveal any evident induction of classical DNA fragmentation (Fig. 3C) and was not inhibited by the addition of caspase inhibitors z-VAD-fmk or z-IETD-fmk (Fig. 4B). In fact, the proapoptotic effect of CD24 itself was also documented in human B cell lines (16, 17). However, in the case of human B cell apoptosis via CD24 stimulation, multiple caspases were activated and the apoptotic process was inhibited by the addition of caspase inhibitors (17). Thus, it appeared that different death mechanisms might be involved in mouse thymocytes and human B cells during the course of CD24-mediated apoptosis. We do not yet have a clue whether the discrepancy is due to the differences of species or the cell types studied.

Although receptor-mediated death signaling plays a major role in classical apoptosis, an increasing number of Ags are implicated in the nonclassical programmed cell death characterized by the pathways proceeding independently of caspase activation (31). Among these, ligation of CD45 molecules has been shown to induce apoptosis of murine thymocytes by a pathway with a close resemblance to CD24-mediated death signaling (29). For instance, the CD45-mediated death of thymocytes is characterized by several events, including the exposure of phosphatidylserine residues, incorporation of vital dyes, a reduction in {Delta}{psi}m, production of ROS, and a lack of DNA degradation (29). However, the CD24-mediated apoptosis is different from that by CD45 in two ways. First, susceptibility to the CD45-mediated death is acquired during the transition of early CD4-CD8-TCR- DN T cell precursors into CD4+CD8+TCR- thymocytes and is increased with further acquisition of surface TCR on these cells, whereas both DN and DP thymocytes are similarly susceptible to the CD24-mediated apoptosis (29). Second, the CD45-mediated cell death is not blocked by Bcl-2 overexpression (29). In contrast, the Bcl-2 overexpression in Scid.adh cells inhibited both loss of {Delta}{psi}m and exposure of phosphatidylserine by CD24 cross-linking (Fig. 5). Thus, the CD24-induced death of thymocytes seems to rely on a different apoptotic pathway from that activated by CD45.

Though Fas and TNFR have been implicated in death signaling of developing thymocytes (3, 41), the requirement of CD95 and TNFR in T cell selection has also been questioned (42, 43). Notably, nonclassical forms of apoptosis may be important in T cell selection because programmed cell death in developing thymocytes can proceed independently of the caspase enzyme activity (28). On the basis of these results, receptors linked to the caspase-independent signaling pathway in thymocytes, such as CD45 and CD99, were suggested as candidate molecules for the induction of thymocyte apoptosis (29, 31). In the present study, we have found that CD24 signals also induced the apoptosis of thymocytes in a caspase-independent pathway. In addition, CD24 engagement resulted in the apoptosis of both DN and DP thymocytes in a TCR-independent manner. As a consequence, these results raised the possibility that CD24 signals might affect death by neglect during {beta} and/or positive selection(s).

It is still unclear how CD24 cross-linking could be achieved in vivo and whether thymocyte development is affected by CD24 cross-linking. In transgenic mice, the constitutive expression of CD24 on all subsets of thymocytes resulted in a pronounced reduction in the numbers of DP and SP thymocytes, suggesting that CD24 might provide signals for negative selection (10). In contrast, no developmental abnormality was found in the thymus from CD24-deficient mice (44). One possible answer could be that the CD24 engagement signals a redundant pathway that can be provided through other Ag(s) present on mouse thymocytes. Therefore, to understand the role of CD24 in thymic development, more sophisticated mouse models using a single TCR in the background of both recombination-activating gene and CD24 gene-deficient mice would be required for further investigation.


   Acknowledgments
 
We thank Dr. David L. Wiest for the Scid.adh cell line. We thank Jae Nam Seo, Jin Sil Choi, Seung Hee Lee, and Ju Hyun Kim (Department of Pathology, Hallym University College of Medicine, Chuncheon, Korea) for their kind technical assistance.


   Footnotes
 
1 This work was supported by a Korea Research Foundation Grant (KRF-2001-002-F0006) and by Grant R11-2002-098-00000-0 from the Korea Sciences and Engineering Foundation through the Rheumatism Research Center at Catholic University (Seoul, Korea). Back

2 Address correspondence and reprint requests to Dr. Youngmee Bae, Department of Pathology, Kangwon National University College of Medicine, 192-1 Hyoja-dong Chuncheon, Kangwon-Do 200-701, Korea. E-mail address: ymbae{at}kangwon.ac.kr Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; AIF, apoptosis-inducing factor; 7-AAD, 7-amino actinomycin D; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; NAC, N-acetyl-L-cysteine; {Delta}{psi}m, mitochondrial membrane potential; AMC, aminomethylcoumarin; PI-PLC, phosphatidylinositol-specific phospholipase; ROS, reactive oxygen species. Back

Received for publication June 16, 2003. Accepted for publication November 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shortman, K., L. Wu. 1996. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14:29.[Medline]
  2. Fujita, N., N. Kodama, Y. Kato, S. H. Lee, T. Tsuruo. 1997. Aggregation of Thy-1 glycoprotein induces thymocyte apoptosis through activation of CPP32-like proteases. Exp. Cell Res. 232:400.[Medline]
  3. Punt, J. A., W. Havran, R. Abe, A. Sarin, A. Singer. 1997. T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus. J. Exp. Med. 186:1911.[Abstract/Free Full Text]
  4. Loeffler, M., G. Kroemer. 2000. The mitochondrion in cell death control: certainties and incognita. Exp. Cell Res. 256:19.[Medline]
  5. McKean, D. J., C. J. Huntoon, M. P. Bell, X. Tai, S. Sharrow, K. E. Hedin, A. Conley, A. Singer. 2001. Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation. J. Immunol. 166:3468.[Abstract/Free Full Text]
  6. Kay, R., F. Takei, R. K. Humphries. 1990. Expression cloning of a cDNA encoding M1/69-J11d heat-stable antigens. J. Immunol. 145:1952.[Abstract]
  7. Alterman, L. A., I. N. Crispe, C. Kinnon. 1990. Characterization of the murine heat-stable antigen: a hematolymphoid differentiation antigen defined by the J11d, M1/69 and B2A2 antibodies. Eur. J. Immunol. 20:1597.[Medline]
  8. Rougon, G., L. A. Alterman, K. Dennis, X. J. Guo, C. Kinnon. 1991. The murine heat-stable antigen: a differentiation antigen expressed in both the hematolymphoid and neural cell lineages. Eur. J. Immunol. 21:1397.[Medline]
  9. Kishimoto, H., J. Sprent. 1997. Negative selection in the thymus includes semimature T cells. J. Exp. Med. 185:263.[Abstract/Free Full Text]
  10. Hough, M. R., F. Takei, R. K. Humphries, R. Kay. 1994. Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen. J. Exp. Med. 179:177.[Abstract/Free Full Text]
  11. Kadmon, G., M. Eckert, M. Sammar, M. Schachner, P. Altevogt. 1992. Nectadrin, the heat-stable antigen, is a cell adhesion molecule. J. Cell Biol. 118:1245.[Abstract/Free Full Text]
  12. Liu, Y., B. Jones, W. Brady, C. A. Janeway, Jr, P. S. Linsley, P. S. Linley. 1992. Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen. Eur. J. Immunol. 22:2855.[Medline]
  13. Enk, A. H., S. I. Katz. 1994. Heat-stable antigen is an important costimulatory molecule on epidermal Langerhans’ cells. J. Immunol. 152:3264.[Abstract]
  14. Hubbe, M., P. Altevogt. 1994. Heat-stable antigen/CD24 on mouse T lymphocytes: evidence for a costimulatory function. Eur. J. Immunol. 24:731.[Medline]
  15. Hitsumoto, Y., D. S. Song, M. Okada, F. Hamada, S. Saheki, N. Takeuchi. 1996. Enhancement of CD3-mediated thymocyte apoptosis by the cross-linkage of heat-stable antigen. Immunology 89:200.[Medline]
  16. Suzuki, T., N. Kiyokawa, T. Taguchi, T. Sekino, Y. U. Katagiri, J. Fujimoto. 2001. CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system. J. Immunol. 166:5567.[Abstract/Free Full Text]
  17. Taguchi, T., N. Kiyokawa, K. Mimori, T. Suzuki, T. Sekino, H. Nakajima, M. Saito, Y. U. Katagiri, N. Matsuo, Y. Matsuo, H. Karasuyama, J. Fujimoto. 2003. Pre-B cell antigen receptor-mediated signal inhibits CD24-induced apoptosis in human pre-B cells. J. Immunol. 170:252.[Abstract/Free Full Text]
  18. Wiest, D. L., W. H. Burgess, D. McKean, K. P. Kearse, A. Singer. 1995. The molecular chaperone calnexin is expressed on the surface of immature thymocytes in association with clonotype-independent CD3 complexes. EMBO J. 4:3425.
  19. Donner, K. J., K. M. Becker, B. D. Hissong, S. A. Ahmed. 1999. Comparison of multiple assays for kinetic detection of apoptosis in thymocytes exposed to dexamethasone or diethylstilbesterol. Cytometry 35:80.[Medline]
  20. Pardo, J., P. Pérez-Galán, S. Gamen, I. Marzo, I. Monleón, A. A. Kaspar, S. A. Susin, G. Kroemer, A. M. Krensky, J. Naval, A. Anel. 2001. A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis. J. Immunol. 167:1222.[Abstract/Free Full Text]
  21. Bruce, J., F. W. Symington, T. J. McKearn, J. Sprent. 1981. A monoclonal antibody discriminating between subsets of T and B cells. J. Immunol. 127:2496.[Abstract]
  22. Crispe, I. N., M. J. Bevan. 1987. Expression and functional significance of the J11d marker on mouse thymocytes. J. Immunol. 138:2013.[Abstract]
  23. Kim, K., C. K. Lee, T. J. Sayers, K. Muegge, S. K. Durum. 1998. The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J. Immunol. 160:5735.[Abstract/Free Full Text]
  24. Chae, J. S., H. J. Kim, W. S. Park, Y. Bae, K. C. Jung. 2002. The differential staging of murine thymic lymphoma cell lines, Scid.adh, R1.1 and EL-4. Immune Network 2:217.
  25. Rathmell, J. C., C. B. Thompson. 1999. The central effectors of cell death in the immune system. Annu. Rev. Immunol. 17:781.[Medline]
  26. Wallach, D., E. E. Varfolomeev, N. L. Malinin, Y. V. Goltsev, A. V. Kovalenko, M. P. Boldin. 1999. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu. Rev. Immunol. 17:331.[Medline]
  27. Ashkenazi, A., V. M. Dixit. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  28. Bidère, N., A. Senik. 2001. Caspase-independent apoptotic pathways in T lymphocytes: a minireview. Apoptosis 6:371.[Medline]
  29. Lesage, S., A. M. Steff, F. Philippoussis, M. Page, S. Trop, V. Mateo, P. Hugo. 1997. CD4+CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway. J. Immunol. 159:4762.[Abstract]
  30. Pettersen, R. D., K. Hestdal, K. K. Olafsen, S. O. Lie, F. P. Lindberg. 1999. CD47 signals T cell death. J. Immunol. 162:7031.[Abstract/Free Full Text]
  31. Pettersen, R. D., G. Bernard, M. K. Olafsen, M. Pourtein, S. O. Lie. 2001. CD99 signals caspase-independent T cell death. J. Immunol. 166:4931.[Abstract/Free Full Text]
  32. Mateo, V., E. J. Brown, G. Biron, M. Rubio, A. Fischer, F. L. Deist, M. Sarfati. 2002. Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization. Blood 100:2882.[Abstract/Free Full Text]
  33. Jäättelä, M., J. Tschopp. 2003. Caspase-independent cell death in T lymphocytes. Nat. Immunol. 4:416.[Medline]
  34. Zamzami, N., P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, S. A. Susin, P. X. Petit, B. Mignotte, G. Kroemer. 1995. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182:367.[Abstract/Free Full Text]
  35. Zazami, N., P. Marchetti, M. Castedo, C. Zanin, J. L. Vayssière, P. X. Petit, G. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181:1661.[Abstract/Free Full Text]
  36. Gross, A., J. M. McDonnell, S. J. Korsmeyer. 1999. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev. 13:1988.
  37. Hilderman, D. A., T. Mitchell, T. K. Teague, P. Henson, B. J. Day, J. Kappler, P. C. Marrack. 1999. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10:735.[Medline]
  38. Fleury, C., B. Mignotte, J. L. Vayssière. 2002. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84:131.[Medline]
  39. Maianski, N. A., D. Roos, T. W. Kuijpers. 2003. Tumor necrosis factor {alpha} induces a caspase-independent death pathway in human neutrophils. Blood 101:1987.[Abstract/Free Full Text]
  40. Susin, S. A., H. K. Lorenzo, N. Zamzami, I. Marzo, B. E. Snow, G. M. Brothers, J. Mangion, E. Jacotot, P. Costantini, M. Loeffler, et al 1999. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441.[Medline]
  41. Yonehara, S., Y. Nishimura, S. Kishil, M. Yonehara, K. Takazawa, T. Tamatani, A. Ishii. 1994. Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes. Int. Immunol. 6:1849.[Abstract/Free Full Text]
  42. Singer, G. G., A. K. Abbas. 1994. The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1:365.[Medline]
  43. Page, D. M., E. M. Roberts, J. J. Peschon, S. M. Hedrick. 1998. TNF receptor-deficient mice reveal striking differences between several models of thymocyte negative selection. J. Immunol. 160:120.[Abstract/Free Full Text]
  44. Nielsen, P. J., B. Lorenz, A. M. Müller, R. H. Wenger, F. Brombacher, M. Simon, T. von der Weid, J. Langhorne, H. Mossmann, G. Köhler. 1997. Altered erythrocytes and a leaky block in B-cell development in CD24/HSA-deficent mice. Blood 89:1058.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
E. Sagiv, A. Starr, U. Rozovski, R. Khosravi, P. Altevogt, T. Wang, and N. Arber
Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or Small Interfering RNA
Cancer Res., April 15, 2008; 68(8): 2803 - 2812.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. K. Nussbaum and J. L. Whitton
The Contraction Phase of Virus-Specific CD8+ T Cells Is Unaffected by a Pan-Caspase Inhibitor
J. Immunol., December 1, 2004; 173(11): 6611 - 6618.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jung, K. C.
Right arrow Articles by Bae, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jung, K. C.
Right arrow Articles by Bae, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS