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The Journal of Immunology, 2003, 170: 252-260.
Copyright © 2003 by The American Association of Immunologists

Pre-B Cell Antigen Receptor-Mediated Signal Inhibits CD24-Induced Apoptosis in Human Pre-B Cells1

Tomoko Taguchi*,{dagger}, Nobutaka Kiyokawa2,*, Kenichi Mimori*, Toyo Suzuki*, Takaomi Sekino*, Hideki Nakajima*, Masahiro Saito*, Yohko U. Katagiri*, Nobutake Matsuo{ddagger}, Yoshinobu Matsuo§, Hajime Karasuyama and Junichiro Fujimoto*

* Department of Developmental Biology, National Research Institute for Child Health and Development, Tokyo, Japan; {dagger} Department of Pediatrics, School of Medicine, Keio University, Tokyo, Japan; {ddagger} National Center for Child Health and Development, Tokyo, Japan; § Fujisaki Cell Center, Hayashibara Biochemical Labs Inc., Okayama, Japan; and Department of Immune Regulation, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that the cross-linking of cluster of differentiation (CD)24 induces apoptosis in Burkitt’s lymphoma cells and that this phenomenon can be enhanced by a B cell Ag receptor (BCR)-mediated signal. In this study, we extend our previous observation and report that CD24 also mediated apoptosis in human precursor-B acute lymphoblastic leukemia cell lines in the pro-B and pre-B stages accompanying activation of multiple caspases. Interestingly, simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. We also observed that mitogen-activated protein kinases (MAPKs) were involved in the regulation of this apoptotic process. Pre-BCR cross-linking induced prompt and strong activation of extracellular signal-regulated kinase 1, whereas CD24 cross-linking induced the sustained activation of p38 MAPK, following weak extracellular signal-regulated kinase 1 activation. SC68376, a specific inhibitor of p38 MAPK, inhibited apoptosis induction by CD24 cross-linking, whereas anisomycin, an activator of p38 MAPK, enhanced the apoptosis. In addition, PD98059, a specific inhibitor of MEK-1, enhanced apoptosis induction by CD24 cross-linking and reduced the antiapoptotic effects of pre-BCR cross-linking. Collectively, whether pre-B cells survive or die may be determined by the magnitude of MAPK activation, which is regulated by cell surface molecules. Our findings should be important to understanding the role of CD24-mediated cell signaling in early B cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bcell development is a dramatic process that generates clones that produce Ig having greater affinity for exogenous Ags. In the process, B cells proceed through multiple developmental stages that determine whether they will survive or die, changing their phenotype in a developmental-stage-specific manner (1, 2). In mature B cells, signals transduced through the B cell Ag receptor (BCR),3 which consists of the µ H chain, conventional {kappa} or {lambda} L chain, Ig{alpha}, and Ig{beta}, play an essential role in B cell activation and terminal differentiation. In contrast to mature B cells, B cell precursors do not possess the complete form of BCR, but already have alternate Ag receptor complexes. For example, pre-B cells that have successfully accomplished rearrangement of H chain genes start to express a premature form of the Ag receptor, namely, pre-BCR, consisting of the µ H chain, surrogate L chain (VpreB and {lambda}5), and the Ig{alpha}/Ig{beta} heterodimer (3, 4). A number of works have shown the vital importance of pre-BCR as a mediator of pre-B cell differentiation signals (5, 6).

In addition to the Ag receptors, a number of B cell differentiation Ags have been found to mediate signal transduction that leads to proliferation and differentiation upon binding with their specific ligands. Investigation of the stimuli mediated by these surface molecules should therefore provide an approach to understanding the molecular basis of B cell development. Cluster of differentiation (CD)24, also referred to as heat-stable Ag (HSA) in mice, is a B cell differentiation Ag (7). During B cell ontogeny, CD24 is already expressed in very early stage B cell precursors (8), remains expressed on mature resting B cells, and begins to disappear when B cells are activated and induced to further maturation (7, 9, 10, 11, 12). This differentiation-dependent expression pattern has implied a role for CD24 in B cell development, and indeed, several lines of evidence suggest functional involvement of CD24 in B cell development. First, CD24 is known to mediate signal transduction, including phosphorylation of intracellular proteins and intracellular calcium mobilization (13, 14, 15). In addition, cross-linking of CD24 induces apoptosis in mouse B cell precursors (16, 17). These findings suggest that CD24 is a signal-transducing molecule that acts as a potent negative regulator of B cell development.

In correlation with these findings, we recently reported that CD24 also mediates apoptosis in human Burkitt’s lymphoma (BL) cells, which are thought to be related to germinal-center B cells (18). In the findings, we observed a synergism between the cross-linking of CD24 and that of BCR in the effect on apoptosis induction in BL cells, suggesting an interaction between CD24-mediated signaling and that of BCR (18). Because pre-BCR and BCR are structurally related, it is reasonable to expect that CD24 is also closely correlated with the pre-BCR-mediated signaling system in pre-B cells, although the details are unknown.

To clarify the function of CD24 in early B cell development, we analyzed the effect of cross-linking the molecule in human precursor-B acute lymphoblastic leukemia (ALL) cell lines derived from B cell precursors in bone marrow (BM). Our findings show that cross-linking of CD24 induces apoptosis in two distinct classes of precursor-B ALL cells, namely, pre-B and pro-B ALL cells. They also indicate that the cell signaling that affects mitogen-activated protein kinases (MAPKs) is involved in this apoptotic process. Interestingly, unlike the BCR expressed in BL cells, we further observed that a pre-BCR-mediated signal can inhibit CD24-induced apoptosis in pre-B ALL cells.


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

Pre-B ALL-derived cell lines, including HPB-NULL, NALM-6, NALM-17 (19), and P30/OHK (20); Pro-B ALL-derived cell lines, including NALM-16, NALM-20, NALM-27 (19), LC4-1 (21), and KM-3 (22); and CD24+ BL-derived cell line BALM-24 were used. Cells were cultured in RPMI 1640 supplemented with 10% FCS at 37°C in a humidified 5% CO2 atmosphere.

The mouse mAbs used were: anti-CD24 (L30) (9); anti-µ (DA4.4) from American Type Culture Collection (Manassas, VA); antiextracellular signal-regulated kinase 1 (ERK1) and anti-caspase-2, -3, and -7 from BD Transduction Laboratories (Lexington, KY); anti-caspase-8 and anti-inhibitor of caspase-activated DNase (ICAD) from Medical & Biological Laboratories (Nagoya, Japan); and anti-poly(ADP-ribose) polymerase (PARP) from Biomol (Plymouth Meeting, PA). The polyclonal Abs used were: anti-p38 MAPK and anti-stress-activated protein kinase (SAPK) from New England Biolabs (Beverly, MA); anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA); anti-GST from Boehringer Mannheim (Indianapolis, IN). All anti-phospho-specific Abs and all anticleaved caspase Abs were purchased from New England Biolabs. Secondary Abs, including fluorescein-conjugated and enzyme-conjugated Abs, were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Biotinylation and FITC-conjugation of mAb was performed as described previously (10). To cross-link CD24, the combinations of either purified L30 (5 µg/ml) and secondary anti-mouse Ig Ab (10 µg/ml) or biotinylated L30 (10 µg/ml) and avidin (20 µg/ml) (Sigma-Aldrich, St. Louis, MO) were used. To cross-link pre-BCR, purified DA4.4 (5 µg/ml) was used. In these cases, Abs were dialyzed in medium before being introduced to culture to remove additives. A peptide inhibitor for a broad spectrum of caspases, z-Val-Ala-Asp-fmk (z-VAD-fmk), was obtained from Bachem (Torrence, CA). The MEK-1 inhibitors PD98059 and U0126 were purchased from Calbiochem (La Jolla, CA) and New England Biolabs, respectively. The p38 MAPK inhibitor SC68376 and p38 MAPK activator anisomycin were purchased from Calbiochem. The GST-ATF-2 and GST-ELK-1 fusion proteins were purchased from New England Biolabs. All chemical reagents were obtained from Wako Pure Chemical (Osaka, Japan), unless otherwise indicated.

Immunofluorescence study and detection of apoptosis

Cells were stained with FITC-labeled mAbs and analyzed by flow cytometry (EPICS-XL; Beckman Coulter, Fullerton, CA) as described previously (10). To quantify the incidence of apoptotic cells, cells were stained with FITC-labeled annexin V using a MEBCYTO-Apoptosis kit (Medical & Biological Laboratories) and then analyzed by flow cytometry according to the manufacturer’s protocol. Experiments were performed in triplicate, and the means ± SDs of the cells that bound annexin V are shown. Caspase-3 activity was assessed with a PhiPhiLux G1D2 kit (Medical & Biological Laboratories) and analyzed by flow cytometry according to the manufacturer’s protocol.

Immunoblotting and in vitro kinase assay

Cell lysates were prepared by dissolving cells in lysis buffer, and the protein concentration of each cell lysate was determined as described previously (23). A 50-µg quantity of each whole cell lysate was electrophoretically separated on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane by a semidry transblot system (Bio-Rad, Hercules, CA). Immunoblotting was performed as described previously (23).

To test p38 MAPK activity, 500 µg of cell lysate was incubated with 2 µg of anti-p38 MAPK Ab in the presence of 30 µl of 50% protein G-agarose (Boehringer Mannheim) for 1 h. After intensive washing, the immunoprecipitates were mixed with 2 µg of GST-ATF-2 fusion protein as exogenous substrate. Transphosphorylation activity of p38 MAPK was determined by two types of assay. First, the mixture was incubated for 20 min at room temperature in 30 µl of kinase assay buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 1 mM DTT; 1 mM EGTA; 100 µM ATP) with 10 µCi of [{gamma}-32P]ATP (specific activity >3000 Ci/mM; NEN, Boston, MA). Reactions were stopped by adding 6 µl of 6x SDS-sample loading buffer (23). After separation on a 10% SDS-PAGE gel, phosphorylated proteins were visualized with autoradiography as described previously (23). Alternatively, the kinase reaction was performed as described above but without the use of [{gamma}-32P]ATP. Subsequent incorporation of nonisotopic phosphates into substrate was determined by immunoblotting using anti-phospho-ATF-2 Ab. The ERK1 kinase activity was examined similarly by using GST-ELK-1 fusion protein as substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cross-linking of CD24 induces apoptosis in pre-B and pro-B ALL cell lines

First, we tested whether cross-linking of CD24 induces apoptosis in precursor-B ALL cells, including pre-B and pro-B ALL cells, expressing CD24 (Fig. 1GoA), as is the case with murine pre-B cells (17). When pre-B HPB-NULL cells were exposed to anti-CD24 mAb in the presence of secondary rabbit polyclonal anti-mouse Ig Ab, a significant number of annexin-V-bound cells appeared in a time-dependent manner (Fig. 1GoB). We also examined combinations of biotinylated anti-CD24 mAb and avidin, and obtained identical results (Fig. 1GoC). These phenomena were specific to CD24 because other mAbs, such as anti-CD22 and anti-CD72, both of which bind to HPB-NULL cells, did not induce apoptosis (data not shown). We similarly examined other pre-B and pro-B ALL cell lines and obtained essentially the same results, but pro-B NALM-27 and NALM-16 cells showed much less sensitivity to induction of CD24-mediated apoptosis than was observed in the other cells (Fig. 1Go, D and E). These findings suggest that induction of apoptosis upon CD24 cross-linking is a feature common to pre-B and pro-B ALL cell lines expressing CD24, but that the magnitude of cell death is variable. By contrast, when KM-3 pro-B cells, which do not express CD24, were similarly examined (Fig. 1GoF), treatment with a combination of anti-CD24 mAb and rabbit anti-mouse Ig Ab failed to induce apoptosis (Fig. 1GoF), indicating that CD24 cross-linking specifically induces apoptosis in CD24-expressing cells, and that this is not due to the nonspecific binding of either mouse Ig or secondary rabbit polyclonal anti-mouse Ig Ab.



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  		FIGURE 1. Induction of apoptosis mediated by cross-linking of CD24 in pre-B and pro-B ALL cells. A, Expression of CD24 on pre-B and pro-B ALL cell lines was examined by flow cytometry. The histograms obtained (dark lines) have been superimposed on those of the negative control (cells stained with isotype-matched control mouse Ig, light lines) and displayed. x-axis, fluorescence intensity; y-axis, relative cell number. B, Pre-B ALL HPB-NULL cells (2.5 x 105 cells in 500 µl of medium) were exposed to either anti-CD24 mAb L30 ({alpha}CD24, upper panels) or isotype-matched control mouse Ig (MsIg, lower panels) in the presence of secondary rabbit anti-mouse Ig Ab (R{alpha}M) for the periods indicated. At the end of the culture period, HPB-NULL cells were stained with FITC-conjugated annexin V and analyzed by flow cytometry. C, HPB-NULL cells were treated with either biotinylated anti-CD24 mAb L30 ({alpha}CD24-B, upper panels) or biotinylated isotype-matched control mouse Ig (MsIg-B, lower panels) in the presence of avidin (Av) and examined as in B. D, The pre-B ALL cell lines indicated were treated as in B for 24 h, and the annexin-V-bound cells were detected in a similar manner. E, The pro-B ALL cell lines indicated were examined as in D. F, CD24- pro-B ALL KM-3 cells were examined as in A and D.

 
Activation of caspases in the course of CD24-mediated apoptosis

A number of studies have indicated that caspases are essential as effector molecules in the apoptotic process in most cases (24). Therefore, we investigated whether caspases are activated during the apoptotic process induced by cross-linking CD24 in HPB-NULL cells. As shown in Fig. 2Go, A and B, immunoblot analysis revealed cleavage of caspases, including caspases-8, -3, -2, and -7, in HPB-NULL cells after CD24 cross-linking in parallel with the appearance of annexin-V-bound cells (Fig. 1GoC), indicating activation of the caspases. Cleavage of PARP and ICAD, which are known to be substrates of caspases, was also consistently observed (Fig. 2Go, A and B). We also tested the activity of caspase-3 in individual cells by using PhiPhiLux G1D2. As shown in Fig. 2GoC, a significant increase in fluorescence activity was observed after CD24 cross-linking. These findings indicate that multiple caspases are activated during the course of CD24-mediated apoptosis in pre-B ALL cells, and evidence that this apoptotic process is inhibited by z-VAD-fmk (Fig. 2GoD), a specific peptide inhibitor of caspases, further supports this idea.



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  		FIGURE 2. Activation of caspases in the course of CD24-mediated apoptosis in HPB-NULL cells. A, In parallel with the experiment presented in Fig. 1GoC, cell lysates were prepared. The proforms of each caspase and caspase substrate were detected by immunoblotting with Abs as indicated. Cnt, treated with biotinylated isotype-matched control mouse Ig and avidin; {alpha}CD24-Bio, biotinylated anti-CD24 mAb; Casp-, caspase. B, In parallel with A, the cleaved forms of each caspase and PARP were detected with Abs as indicated. Cl-, cleaved. C, Caspase-3 activity in the same sample preparation as in Fig. 1GoB was examined using PhiPhiLux G1D2. D, After 1-h preincubation with different concentrations of z-VAD-fmk, a peptide inhibitor of caspases, HPB-NULL cells were exposed to a combination of anti-CD24 mAb L30 ({alpha}CD24) and secondary rabbit anti-mouse Ig Ab (R{alpha}M) as in Fig. 1GoD. The subsequent incidence of apoptotic cells was determined.

 
Activation of MAPK mediated by cross-linking of CD24

In other cell types, it has been reported that cross-linking of CD24 results in intracellular signaling events such as the activation of protein kinases (13, 18, 25, 26, 27, 28). It has also been shown that MAPKs are involved in determining whether a cell survives or undergoes apoptosis in several cases (29, 30). Thus, we attempted to examine the changes in MAPK activity after CD24 cross-linking in pre-B cells.

When the total cell lysates prepared from CD24-cross-linked HPB-NULL cells were examined by immunoblotting with Abs that specifically recognize phosphorylated MAPKs, clear increases in the phosphorylations of p38 MAPK and ERK1 were detected (Fig. 3GoA), while the protein amounts of these kinases did not change during the course of stimulation (Fig. 3GoA). In vitro kinase assay revealed that the phosphorylations of these kinases were indeed accompanied by an elevation of the kinase activity (Fig. 3Go, B and C). These findings suggest that CD24 cross-linking activates both kinases. Interestingly, the activation of these kinases occurred in a nonsynchronous manner. As shown in Fig. 3GoA, the phosphorylation of ERK1 peaked at 30 min after CD24 cross-linking and then decreased to the resting level at 120 min. By contrast, the peak activation of p38 MAPK was observed at 60 min after CD24 cross-linking, and it remained activated at 120 min (Fig. 3GoA). Activation of SAPK (Fig. 3GoA) and ERK5 (data not shown) was not detected after CD24 cross-linking.



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  		FIGURE 3. Activation of MAPKs in HPB-NULL cells mediated by CD24 cross-linking. A, Protein lysates were prepared from HPB-NULL cells exposed to (lanes 2–6) or not exposed to (lane 1) the combination of biotinylated anti-CD24 mAb and avidin (CD24 cross-link) for the periods indicated. Each cell lysate was electrophoretically separated on 10% SDS-PAGE gels and immunoblotted with specific Abs against the MAPKs indicated. P-p38, phosphorylated p38 MAPK; P-ERK, phosphorylated ERK; P-SAPK, phosphorylated SAPK. B, The immunoprecipitates with anti-p38 MAPK Ab were prepared in triplicate from cell lysates described in A. In vitro kinase assay was performed using GST-ATF-2 as a substrate. In addition to conventional kinase assay using [{gamma}-32P]ATP (32-P/ATF2), transphosphorylation activity of p38 MAPK was also assessed by immunoblotting using a combination of nonisotopic ATP and specific Ab against phosphorylated ATF-2 (P-ATF2). After stripping the Abs, the membrane was reproved with anti-GST Ab to test the protein amounts of the substrate in each reaction ({alpha}GST). The p38 MAPK proteins in each immunoprecipitate were detected by immunoblotting (p38). C, Transphosphorylation activity of ERK1 was also assessed by in vitro kinase assay as in B using GST-ELK-1 as a substrate.

 
Simultaneous cross-linking of pre-BCR inhibits CD24-mediated apoptosis in HPB-NULL cells

As previously reported (19), and shown in Fig. 4GoA, HPB-NULL cells express a considerable amount of pre-BCR on their cell surface. Because pre-BCR is thought to mediate the proliferation and differentiation signals in pre-B cells (31), we tested the effect of simultaneous cross-linking of pre-BCR on the apoptosis induction mediated by CD24 and found that the CD24-mediated apoptosis was significantly inhibited in the presence of anti-µ mAb (Fig. 4GoB). We also tested other pre-B ALL lines (Fig. 4Go, A and B and data not shown) and observed mostly identical results. As shown in Fig. 4GoC, when HPB-NULL cells previously exposed to an excess of anti-µ mAb were stained with FITC-conjugated anti-CD24 mAb, no significant reduction in consequent fluorescein intensity was observed. Therefore, the inhibition of CD24-mediated apoptosis by anti-µ mAb is not merely the result of inhibition of the binding of anti-CD24 mAb to the cells.



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  		FIGURE 4. Effect of simultaneous cross-linking of pre-BCR with CD24 on the induction of apoptosis in pre-B ALL cells. A, Expression of the µ H chain on pre-B ALL cell lines was examined as in Fig. 1GoA. B, Pre-B ALL cell lines HPB-NULL and NALM-17 were exposed to and not exposed to anti-CD24 mAb ({alpha}CD24) and anti-µ H chain mAb ({alpha}µ) in the presence or absence of secondary rabbit anti-mouse Ig Ab (R{alpha}M) as indicated. After 24-h cultivation, cells were examined as in Fig. 1GoD. C, HPB-NULL cells preincubated with an excess amount (40 µg/ml) of either {alpha}µ or isotype-matched control mouse Ig (MsIg) were stained with FITC-conjugated {alpha}CD24 and analyzed by flow cytometry as in Fig. 1GoA.

 
To address the mechanism of pre-BCR-mediated inhibition of CD24-induced apoptosis, we examined the intracellular signaling events following the cross-linking of pre-BCR. As shown in Fig. 5GoA, immunoblotting analysis revealed prompt and strong activation of ERK1 after pre-BCR cross-linking. The phosphorylation of ERK1 increased to its maximal level at 5 min after stimulation and decreased quickly thereafter. By contrast, p38 MAPK and SAPK presented very weak and transient phosphorylation after pre-BCR cross-linking (Fig. 5GoA).



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  		FIGURE 5. Activation of MAPKs mediated by pre-BCR in pre-B cells and by BCR in BL cells. A, Protein lysates were prepared from pre-B HPB-NULL cells exposed (lanes 2–6) or unexposed (lane 1) to anti-µ H chain mAb for the periods indicated. Each cell lysate was analyzed by immunoblotting with the Abs indicated. P-p38, phosphorylated p38 MAPK; P-ERK, phosphorylated ERK; P-SAPK, phosphorylated SAPK. B, Expression of CD24 (a) and the µ H chain (b) on BL BALM-24 cells was examined by flow cytometry as in Fig. 1GoA. BALM-24 cells were exposed to and not exposed to suboptimal doses of anti-CD24 mAb ({alpha} CD24-S, 2.5 µg/ml) and anti-µ H chain mAb ({alpha} µ-S, 0.1 µg/ml) in the presence of 5 µg/ml of secondary rabbit anti-mouse Ig Ab as indicated (c–d). After 24-h cultivation, the subsequent incidence of apoptotic cells was examined as in Fig. 1GoB. C, Activation of MAPKs after BCR cross-linking in BALM-24 cells were examined as in A.

 
Involvement of MAPKs in apoptosis induction induced by CD24 cross-linking and pre-BCR-mediated inhibition of the apoptosis

Because it was recently reported that MAPKs have opposite effects on the induction of apoptosis and that ERK1 induces cell survival signals in a variety of cell types, whereas p38 MAPK and SAPK mediate apoptotic signals (29, 30), we examined whether change in p38 MAPK activity has any effect on CD24-mediated apoptosis. As shown in Fig. 6GoA, pretreatment with SC68376, a selective p38 MAPK inhibitor (32, 33), inhibited CD24-mediated apoptosis in a dose-dependent manner. In parallel, immunoblotting analysis revealed that SC68376 indeed inhibited the activation of p38 MAPK induced by CD24 cross-linking (Fig. 6GoB). By contrast, pretreatment with anisomycin, a strong activator of p38 MAPK (34, 35), activated p38 MAPK (Fig. 6GoD) and enhanced CD24-mediated apoptosis in a dose-dependent manner (Fig. 6GoC).



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  		FIGURE 6. Effect of kinase inhibitors and activator on CD24-mediated apoptosis in pre-B ALL cells. A, HPB-NULL cells were preincubated with p38 MAPK inhibitor SC68376 for 1 h. After being exposed to anti-CD24 mAb in the presence of secondary rabbit anti-mouse Ig Ab (R{alpha}M) for 24 h, annexin-V-bound cells were detected as in Fig. 1GoD. B, HPB-NULL cells were preincubated (lanes 3–5) or not incubated (lanes 1 and 2) with the indicated amount of SC68376 for 1 h. After being exposed or unexposed to the combinations of Abs indicated for 60 min, cell lysates were prepared and immunoblotting analysis was performed as in Fig. 3Go. P-p38, phosphorylated p38 MAPK. C, HPB-NULL cells were preincubated with indicated concentrations of p38 MAPK activator anysomycin for 1 h. After being exposed to the combination of suboptimal doses of anti-CD24 mAb ({alpha}CD24-S, 3.5 µg/ml) and secondary R{alpha}M for 24 h, annexin-V-bound cells were detected as in Fig. 1GoD. D, Protein lysates were prepared from HPB-NULL cells treated with anysomycin (50 ng/ml) for the periods indicated, and immunoblotting analysis was performed as in Fig. 3Go. E, HPB-NULL cells were preincubated with MEK-1 inhibitor PD98059 for 1 h. After being exposed to the combinations of Abs indicated for 24 h, annexin-V-bound cells were detected as in Fig. 1GoD. F, HPB-NULL cells were preincubated (lanes 3–5) or not incubated (lanes 1 and 2) with the indicated amount of PD98059 for 1 h. After being exposed or unexposed to anti-CD24 mAb in the presence of R{alpha}M ({alpha}CD24) for 30 min, cell lysates were prepared and immunoblotting analysis was performed as in Fig. 3Go. P-ERK, phosphorylated ERK. G, HPB-NULL cells were preincubated with PD98059 for 1 h as in D. After being exposed or unexposed to anti-µ H chain mAb for 5 min, cell lysates were prepared and immunoblotting analysis was performed as in Fig. 3Go.

 
We next examined whether inhibition of ERK1 activity has any effect on CD24-mediated apoptosis. As shown in Fig. 6GoE, pretreatment with PD98059, an MEK-1 inhibitor (36, 37), clearly enhanced CD24-mediated apoptosis in a dose-dependent manner. In parallel, immunoblotting analysis revealed that PD98059 indeed inhibited the activation of ERK1 induced by CD24 cross-linking (Fig. 6GoF). It is noteworthy that pretreatment with PD98059 also markedly inhibited the activation of ERK1 induced by pre-BCR cross-linking (Fig. 6GoG), and that simultaneous cross-linking of pre-BCR was no longer sufficient to effectively inhibit CD24-mediated apoptosis (Fig. 6GoE). Essentially identical results were obtained when another inhibitor of MEK-1/2, U0126, was used (data not shown).

Activation of MAPKs is mediated by the cross-linking of BCR in BL cells

We previously reported that the cross-linking of CD24 induces apoptosis in BL cells (18). In this phenomenon, however, a synergism was observed between the cross-linking of CD24 and that of BCR in their effect on apoptosis induction. As shown in Fig. 5GoB, both anti-CD24 mAb (e) and anti-µ H chain mAb (d) failed to induce significant apoptosis in CD24+ BL cell line BALM-24 at suboptimal doses (1.25 and 0.1 µg/ml, respectively). When suboptimal doses of both anti-CD24 mAb and anti-µ H chain mAb were mixed, however, a significant level of apoptosis was induced in BALM-24 cells (Fig. 5GoBf). To address the difference between the regulatory mechanism of CD24-mediated apoptosis by BCR-mediated signals in BL cells and that by pre-BCR in pre-B ALL cells, we examined the changes in MAPK activity after BCR cross-linking in BL cells. Like the example of pre-BCR cross-linking in pre-B ALL cells (Fig. 5GoA), an immunoblotting analysis revealed the prompt and strong phosphorylation of ERK1 in BALM-24 BL cells following BCR cross-linking (Fig. 5GoB). Simultaneously, the distinct phosphorylation of both p38 MAPK and SAPK was observed in BALM-24 cells after BCR cross-linking (Fig. 5GoB). This phenomenon was quite unlike the case of pre-BCR cross-linking in pre-B ALL cells, in which both kinases were phosphorylated only faintly (Fig. 5GoA).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings clearly indicate that the cross-linking of CD24 induces apoptosis in human precursor-B ALL cells, including pro-B and pre-B ALL cells. Consistent with our observations, Chappel et al. demonstrated that the cross-linking of HSA, a mouse homolog of CD24, induces apoptosis in murine B cell precursors in BM (17) and concluded that HSA acts as a negative regulator of B cell precursors in a physiological context. Ligation of HSA may assist in the elimination of undesirable B cell precursors, such as cells with aberrant or nonproductive H chain and L chain gene rearrangements, or cells that display self-specificity (17). Our research allows their hypothesis to be expanded to include a potential role for CD24 as a negative regulator of B cell precursors in humans.

In addition to B cell precursors, CD24 was found to affect other B-lineage cells as a negative regulator, but its effect depends on the stage of B cell differentiation. For example, Chappel et al. observed an inhibitory, but not apoptotic, effect of HSA cross-linking on proliferation of murine mature resting B cells (17), while we recently reported that CD24 cross-linking induces apoptosis in BL cells, which are thought to be related to germinal center B cells in lymphoid follicles (18). Among B cell precursors in mice, IL-7-responsive clonogenic progenitors, consisting mainly of pre-B cells, have been found to display the greatest sensitivity to HSA-mediated apoptosis despite no significant difference in cell surface HSA expression compared with other B-lineage cell populations in BM (16, 17). Consistent with these observations, we found that all pre-B ALL lines tested exhibited significant induction of apoptosis after CD24 cross-linking, whereas some pro-B ALL lines are less sensitive to CD24-mediated apoptosis. All of these findings suggest differential effects of CD24 cross-linking at different stages of B cell development.

We observed activation of multiple caspases in the process of CD24-mediated apoptosis in precursor-B ALL cells. The caspases are thought to be essential as effector molecules in the many cases of apoptotic process (24). Caspases exist in the cells as inactive proenzymes and become activated upon cleavage and the subsequent heterotetramerization of the cleaved subunits. Caspases themselves have also been shown to form a regulatory cascade that transduces apoptotic signals. Apoptotic stimuli mediated by cell surface molecules induce the activation of upstream caspases, such as caspase-8 and -6, which subsequently cleave downstream caspases, such as caspase-3, -2, and -7 (38). The downstream caspases go on to cleave various cellular substrates, including PARP, fodrin, lamin, and ICAD, all of which are responsible for apoptosis (38). Our findings suggest that CD24 cross-linking initiates just such a caspase cascade. Additional experiments to better define the mechanism by which CD24 initiates this process are now under way.

We noted the activation of MAPKs, including ERK1 and p38 MAPK, but not SAPK, after CD24 cross-linking in pre-B cells. It was recently shown that ERK1 induces cell proliferation and differentiation signals in a variety of cell types, while activation of p38 MAPK and SAPK mediate apoptotic signaling (29, 30). For example, ERK1 signaling leads to the promotion of cell survival in NGF-differentiated PC-12 cells, whereas activation of p38 MAPK and SAPK induces apoptosis (29). Activation of the ERK1 cascade in T cells is sufficient to provide positive selection signals, whereas the p38 MAPK signaling pathway has been found to be critical in inducing negative selection of developing T cells in mouse thymocytes (30). These observations suggest that ERK1 and p38 MAPK may antagonize each other by a direct or indirect mechanism, and that the dynamic balance between these MAPKs may be important in determining whether a cell survives or undergoes apoptosis (29). In view of this evidence, it is reasonable to hypothesize that ERK1 and p38 MAPK are also involved in determining the survival or death of pre-B cells. As shown by this study, CD24 cross-linking induced the activation of ERK1 and p38 MAPK. But the kinetics and magnitude of the activation of these kinases were different, and the activations of p38 MAPK are delayed and/or sustained. Thus, the death signal mediated by p38 MAPK may overcome the survival signal mediated by ERK1 in these cells. The fact that both inhibition of ERK1 activity by MEK-1 inhibitor PD98059 and activation of p38 MAPK activity by anisomycin enhance CD24-mediated apoptosis, while inhibition of p38 MAPK activity by a selective inhibitor such as SC68376 retards apoptosis, appears to support this notion.

We have described how CD24-mediated apoptosis is inhibited by pre-BCR-mediated stimuli in pre-B cells. A series of subsequent analyses of precursor-B cells in normal and mutant mice has revealed the involvement of pre-BCR in several events critical to early B cell development (31, 39, 40, 41), including the differentiation of pre-B cells and the selective amplification of µ H chain-producing pre-B cells by driving the cell cycle. Therefore, the regulation of CD24-mediated apoptosis by pre-BCR-mediated signaling may play a role in early B cell development. Although the precise mechanism by which pre-BCR-mediated signals inhibit CD24-mediated apoptosis remains unclear, our data suggest the involvement of MAPK-mediated signaling in this process. As shown by this study, cross-linking of pre-BCR induces prompt and intensive activation of ERK1, which may inhibit the death signaling by CD24, upon simultaneous stimulation of CD24 and pre-BCR. Indeed, inhibition of ERK1 activity by PD98059 reduces the inhibitory effect of pre-BCR-mediated signaling against the CD24-mediated apoptotic process.

It is noteworthy, however, that CD24-mediated stimuli augmented BCR-mediated apoptosis induction in BL cells, as reported previously (18). The correlation between CD24- and Ag-receptor-mediated stimuli may differ according to the stage of B cell development. Thus, BCR-mediated signals in BL cells and pre-BCR-mediated signals in precursor-B ALL cells have the opposite effect on CD24-mediated apoptosis. Because the expression pattern of signaling molecules in B cells varies according to the developmental stage, even though pre-BCR and BCR are structurally related the signaling molecules located downstream from each Ag receptor should be different. The differences between the regulatory mechanisms of CD24-mediated apoptosis by pre-BCR in pre-B cells and that by BCR in BL cells are presently not known. However, we observed that the cross-linking of BCR activates all three MAPKs, including ERK1, p38 MAPK, and SAPK, in BL cells, whereas pre-BCR activates only ERK1 in pre-B cells. Considering the evidence that ERK1 mediates cell survival signaling whereas p38 MAPK and SAPK mediate apoptotic signaling, our findings concerning the different activation patterns of MAPKs by pre-BCR and BCR may explain the opposite effects of these receptors on CD24-mediated apoptosis, at least in part.

In conclusion, our findings suggest that CD24-mediated apoptosis is a model for the cell death of B cell precursors in BM. Although additional studies are clearly necessary, investigation of the mechanism of CD24-mediated apoptosis and its inhibition by pre-BCR-mediated signaling should provide a new approach to understanding the regulation of early B cell development and lead to the establishment of a new therapeutic strategy for precursor-B ALL.


   Acknowledgments
 
We thank M. Sone and S. Yamauchi for their excellent secretarial work.


   Footnotes
 
1 This work was supported in part by a Grant for Pediatric Research (12C-01) from the Ministry of Health and Welfare of Japan. This work was also supported by a grant from the Japan Health Sciences Foundation for Research on Health Sciences Focusing on Drug Innovation. Additional support was provided by the Program of the Research and Development Promotion Division, Science and Technology Promotion Bureau, Science and Technology Agency for Organized Research Combination System. Back

2 Address correspondence and reprint requests to Dr. Nobutaka Kiyokawa, Department of Developmental Biology, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8567, Japan. E-mail address: nkiyokawa{at}nch.go.jp Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; BL, Burkitt’s lymphoma; CD, cluster of differentiation; HSA, heat-stable Ag; ALL, acute lymphoblastic leukemia; BM, bone marrow; MAPK, mitogen-activated protein kinase; ERK1, extracellular signal-regulated kinase 1; ICAD, an inhibitor of caspase-activated DNase; PARP, poly(ADP-ribose) polymerase; SAPK, stress-activated protein kinase; z-VAD-fmk, z-Val-Ala-Asp-fmk. Back

Received for publication December 6, 2001. Accepted for publication October 29, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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