www.elsevier.com/locate/cellsig

Cellular Signalling 16 (2004) 881–889

Antigen receptor-mediated signaling pathways in

transitional immature B cells

Dorottya Kovesdia, Katalin Pasztyb, Agnes Enyedic, Endre Kissa, Janos Matkoa,Katalin Ludanyid, Eva Rajnavolgyid, Gabriella Sarmaya,*

aDepartment of Immunology, Eotvos Lorand University, Pazmany Peter setany 1/c, H-1117, Budapest, HungarybNational Medical Center, Dioszegi u. 64. H-1113, Budapest, Hungary

cMembrane Research Group of the Hungarian Academy of Sciences, Nador u. 7. H-1051, Budapest, Hungaryd Institute of Immunology, Faculty of Medicine, University of Debrecen, Nagyerdei Korut 98, H-4012, Debrecen, Hungary

Received 4 December 2003; received in revised form 8 January 2004; accepted 8 January 2004

Available online 16 March 2004

Abstract

Engagement of antigen receptors on immature B cells induces apoptosis, while at the mature stage, it stimulates cell activation and

proliferation. The difference in B cell receptor (BCR)-mediated signaling pathways regulating death or survival of B cells is not fully

understood. We aimed to characterize the pathway leading to BCR-driven apoptosis. Transitional immature B cells were obtained from the

spleen of sublethally irradiated and auto-reconstituted mice. We have detected a short-lived BCR-driven activation of mitogen-activated

protein kinases (ERK1/2 and p38 MAPK) and Akt/PKB in transitional immature B cells that correlated with the lack of c-Fos expression,

reduced phosphorylation of Akt substrates and a susceptibility for apoptosis. Simultaneous signaling through BCR and CD40 protected

immature B cells from apoptosis, however, without inducing Bcl-2 expression. The BCR-induced apoptosis of immature B cells is a result of

the collapse of mitochondrial membrane potential and the subsequent activation of caspase-3.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Apoptosis; B lymphocytes; Development; Signal transduction; Transitional immature cells

1. Introduction

B cell maturation proceeds through developmentally

determined checkpoints where the surface immunoglobulin

controls the ability of the cells to discriminate between self

and non-self molecules [1–3]. B cell receptor (BCR)

consisting of immunoglobulin heavy and light chains and

the heterodimer of signal transducing chains, Iga and Igh,is first expressed on immature B cells in the bone marrow.

Engagement of BCR in this environment results in either

the rearrangement of the light chain genes to avoid self-

reactivity or the elimination of the autoreactive B cells by

apoptosis [4–6]. Consequently, the mature B cell popula-

tion becomes tolerant for self-structures, while it reacts to

foreign molecules with cell activation, proliferation and

0898-6568/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cellsig.2004.01.005

* Corresponding author. Tel.: +36-1-209-0555/8662; fax: +36-1-381-

2176.

E-mail address: sarmayg@cerberus.elte.hu (G. Sarmay).

antibody production. Therefore, the immature stage, when

the BCR governs the antigen-dependent negative selection

of B cells, is crucial in avoiding self-reactivity. After

leaving the bone marrow, the B cells are at the transitional

immature stage. These cells colonize the peripheral lym-

phoid organs, where they first encounter foreign antigens.

Transitional B cells can be distinguished from the mature

population by a series of surface markers, such as heat

stabile antigen (HSA/CD24) and high surface IgM and low/

medium CD21 expression [7,8]. Only about 10–30% of

these cells enter the mature B cell pool and the rest go

through negative selection [9,10]. High-affinity interaction

of the antigen with the BCR on mature B cells leads to cell

activation, and eventually, in response to additional signals

such as those mediated by CD40 ligand on T cells and

cytokines, to the differentiation into antibody producing

plasma cells. Recognition of antigen or self molecules with

low affinity and the lack of survival signals drives transi-

tional B cells to apoptosis or, less frequently, may induce

D. Kovesdi et al. / Cellular Signalling 16 (2004) 881–889882

further Ig gene rearrangements (receptor revision) [11]. The

BCR-driven negative selection of transitional immature B

cells helps to maintain peripheral tolerance and to set up the

high-affinity mature B cell repertoire.

B cells are instructed continuously by BCR signals to

make crucial cell-fate decisions at several checkpoints

during their development, the high-affinity interaction of

BCR may rescue the cells from apoptosis [12]. Previous

results have shown that BCR-driven apoptosis is indepen-

dent of the death receptors and Fas–FasL interaction [13],

but the mechanism that directs the transitional immature B

cells toward apoptosis is still poorly understood.

We have reported previously that the kinetics of Erk

phosphorylation is different in transitional immature versus

mature murine B cells, probably as a result of impaired

phosphorylation of Grb2 associated binder protein (Gab1) in

immature cells. The transient Erk signal correlated with the

same kinetics of Elk-1 and CREB phosphorylation and with

the apoptosis of immature B cells [14,15]. However, others

suggested that activation of Erk2 correlates with apoptosis

of WEHI-231 cells, while full activation of all MAP kinases

promotes with cell survival [16]. Our aim here was to

identify the pathway leading to BCR-induced apoptosis of

immature B cells. The results have shown that the duration

of signals mediated by ERK and p38 MAPK as well as by

Akt/PKB has an important impact on life or death decision

of B cells. Transient activation of these kinases cannot

prevent apoptosis triggered by BCR, resulting in a caspase

3-dependent death of transitional immature B cells.

2. Materials and methods

2.1. Reagents and antibodies

Phospho-Akt-specific affinity-purified rabbit polyclonal

antibody against pSer472/473/474 sequences of Akt was

purchased from Pharmingen (Pharmingen, CA). Phos-

pho(ser/thr) Akt kinase substrate antibodies, developed in

rabbit were obtained from Cell Signaling Technology (MA,

USA). Phospho-ERK1/2 rabbit polyclonal antibody specific

for pThr202/pTyr204 and pThr185/pTyr187 residues of

ERK1 and ERK2, respectively, and proteinA–peroxidase

conjugate were purchased from Sigma-Aldrich (USA). The

anti-pan-Erk mouse IgG2a monoclonal antibody, mouse

anti-Akt monoclonal antibody and mouse Bcl-2-specific

antibody from Transduction Laboratories (UK), affinity-

purified anti-SHP-2 polyclonal antibody from Santa Cruz

Biotechnology (CA). Streptavidin–RPE for FACS analysis

was purchased from Southern Biotechnology Associates

(UK), anti-p38 (pT180pY182) antibody was developed in

rabbit and was purchased from BioSource International

(USA). FITC-labeled anti-HSA and anti-CD40 (rat IgG2a)

antibodies were produced in our laboratory by Dr. G.

Laszlo. Goat anti-mouse IgM (FabV)2 fragment was kindly

provided by Dr. J. Haimovich (Tel Aviv University, Tel

Aviv, Israel). Single-chain 7G6scFv antibody specific for

mouse CR1/2 was developed by J. Prechl [17]. Anti-myc

tag antibody (9E10) was purified and biotinylated in our

laboratory. Polyclonal antibody specific both for full length

and cleaved form of poly-(ADP-ribose) polymerase (PARP)

was developed by Biomol Research Laboratories.

2.2. Animals and cell preparation

Adult (6–8 weeks old) BALB/c mice were used in all

experiments. To obtain transitional immature B cells, mice

were irradiated with 500 rad and the autoreconstituted

spleen cells were harvested 14 days after irradiation. B

cells were purified as previously described in Ref [18].

Briefly, spleens were removed from mice killed by cervical

dislocation and the cells were collected and washed in

RPMI 1640 culture medium containing 5% fetal calf

serum, 1 mM Na-pyruvate, 2 mM L-glutamine, 100 U/ml

penicillin, 0.1 mg/ml streptomycin and 5� 10� 5 M h-mercaptoethanol. Red blood cells were lysed in 5 ml Gey’s

solution for 1 min. The remaining cells were washed twice

and resuspended in RPMI 1640 culture medium containing

anti-Thy-1.2 antibodies for 20 min at room temperature

and washed. The cells were further incubated with HBSS

solution containing rabbit complement for 45 min at 37 jCand after washing in HBSS, the cells were centrifuged over

a 50/75% Percoll gradient (Pharmacia, Uppsala, Sweden).

Cells separated at the 50/75% interface were collected and

washed twice in RPMI 1640 culture medium containing

5% fetal calf serum.

2.3. Immunofluorescence

Lymphocytes (106) were washed and incubated for 30 min

at 4 jC with different antibodies (7G6scFv or HSA-FITC)

then washed twice, and 7G6scFv-treated samples were incu-

bated for another 30 min at 4 jC with avidin–phycoerythrin

followed by the addition of biotinylated 9E10 antibody. The

results were quantitated using a Becton–Dickinson flow

cytometer and evaluated by the Cellquest software.

2.4. Stimulation of B cells and preparation of cell lysates

A total of 108 cells were treated with 10 Ag F(abV)2fragment of A-chain-specific goat antibodies for 2, 15, 30,

60 and 120 min, respectively, at 37 jC. Cells were pelletedfor 20 s and immediately frozen in liquid nitrogen. The cells

were solubilized in 0.5 ml of lyses buffer containing 1%

Triton X-100, 50 mM HEPES (pH 7.4), 100 mM NaF, 250

mM NaCl, 10 mM EDTA, 2 mM sodium-o-vanadate, 10

mM sodium pyrophosphate, 10% glycerol, 10 Ag/ml apro-

tinin, 10 Ag/ml pepstatin, 5 Ag/ml leupeptin and 0.2 mM

phenylmethylsulfonyl fluoride. After 30 min of incubation

on ice, cell lysates were centrifuged at 15,000� g for 15

min, at 4 jC, and the supernatants were used in subsequent

experiments.

r Signalling 16 (2004) 881–889 883

2.5. Immunoblotting

Post-nuclear supernatants (250 Al) of detergent extract

obtained from 5� 107 control and anti-IgM-treated B cells

were incubated with 250 Al of reducing SDS–PAGE sample

buffer for 5 min at 95 jC. The samples were subjected to

electrophoresis through 10% SDS–PAGE gel, the proteins

were blotted onto nitro-cellulose membranes (BioRad),

probed with different antibodies and developed by HRP-

conjugated anti-mouse IgG or HRP-conjugated SpA, fol-

lowed by enhanced chemiluminescence detection (ECL

system, Amersham International, Amersham, UK).

2.6. Measurement of caspase-8 activity

Caspase-8 activity was determined using the Ac–IETD–

AFC fluorogenic substrate assay system (Pharmingen Inter-

national). Isolated cells (2� 106/ml) were cultured in me-

dium alone or with 10 Ag antibody specific for A-chain for

different periods of time (0, 3, 6, 20 h). Cells were collected

and washed with ice-cold PBS and lysed in 1 ml cell lysis

buffer (10 mM Tris (pH 7.2), 10 mM NaH2PO4, 130 mM

NaCl, 10 mM Na-pyrophosphate, 1% Triton X-100) for 20

min at 4 jC, then lysates were centrifuged (9000� g, 10

min, 4 jC). For each reaction, 0.5 ml supernatants or cell

lysis buffer only, 0.5 ml protease assay buffer (20 mM

HEPES (pH 7.2), 100 mM NaCl, 1 mM EDTA, 10%

sucrose, 10 mM DTT) and 10 Al Ac–IETD–AFC substrate

(N-acetyl-Ile-Glu-Thr-Asp-Amino-Trifluoromethyl-Couma-

rin) were mixed. Samples were incubated for 60 min at 37

jC. AFC released from the Ac–IETD–AFC molecule was

monitored in a Jobin Yvon (FRA) FluoroMax spectrofluo-

rometer using an excitation wavelength of 400 nm and an

emission wavelength range of 430–550 nm.

2.7. Detection of caspase-3 activity

Isolated cells (106) were incubated in 24-well plates in 1

ml culture medium alone or supplemented with 10 Ag A-chain-specific antibody for different periods of time (0, 3, 6,

20 h). Cells were washed and the proteins were precipitated

by 6% ice-cold trichloracetic acid, resuspended in SDS–

PAGE sample buffer and subjected to electrophoresis on

7.5% polyacrylamide gel by the Laemmli’s procedure [19].

The samples were subsequently electro-blotted, and the

blots were immunostained by polyclonal anti-PARP anti-

body developed by Biomol Research Laboratories.

2.8. Analysis of mitochondrial membrane potential changes

(DWm)

Isolated cells (106) were incubated in culture medium

alone or supplemented with 10 Ag A-chain specific antibody

for different periods of time (0, 3, 6, 20 h). Then the cells

were collected and washed with ice-cold PBS. DWm was

evaluated by staining with the 3,3V dihexyloxacarbocyanine

D. Kovesdi et al. / Cellula

iodide (DiOC6) fluorochrome (Molecular Probes, Eugene,

OR) at a final concentration of 10 nM for 45 min, at 37 jCin the dark. The fluorescence emitted by cells was analyzed

with Becton-Dickinson FACSCalibur flow cytometer using

fluorescence channel 1 (488 nm/520 nm) and the apoptotic

cells were identified by their decreased Wm (DiOC6low).

2.9. Analysis of Bcl-2 gene expression

RNA from immature B cells was isolated using Tri-

Reagent (Sigma, St. Louis, MO, USA) following the firm’s

recommendations. Total RNA (5 Ag/ml) was reverse tran-

scribed with the Superscript II Preamplification Kit (Gibco,

Grand Island, NY, USA). Amplification of the genes was

performed in a total volume of 20 Al with 1 Al of the first-

strand cDNAs as template with the following oligonucleo-

tides: 5V-GCGCTCAGGAGGAGCAATG-3V and 5V-GGCTACAGCTTCACCACCAC-3V (sense and antisense

for h-actin), 5V-TTCGGTGTAACTAAAGACAC-3V and

5V-CTCAAAGAAGGCCACAATCC-3V (sense and anti-

sense for Bcl-2), PCR conditions were 20 cycles of dena-

turation at 95 jC for 45 s, annealing at 53 jC for 30 s and

extension at 72 jC for 30 s (h-actin), 35 cycles of denatur-

ation at 95 jC for 45 s, annealing at 55 jC for 30 s and

extension at 72 jC for 30 s (Bcl-2). PCR products were

separated in 1.5% agarose gels.

3. Results

3.1. Characterization of immature and mature B cells

Splenic B cells were isolated from untreated and suble-

thally irradiated mice 14 days after the treatment. Cells were

stained with antibodies specific for sIgM, sIgD, HSA and

type two complement receptor CD21/35. B cells isolated

from the spleen of irradiated, autoreconstituted animals

were highly positive for sIgM and showed a weak IgD

staining, while B cells obtained from untreated mice showed

lower expression of surface IgM and the majority of the

cells were highly positive for IgD [14]. Fig. 1 illustrates that

the B cells isolated from untreated mice can be divided into

mature (HSAint and CD21/35high) and transitional immature

(HSAhigh and CD21/35low/int) populations. In contrast, the

majority of B cells obtained from irradiated and auto-

reconstituted mice corresponds to transitional immature B

cells, the HSAint and CD21/35high mature B cell population

is absent, while an additional HSAhigh CD21low/negative

population appears. The phenotypic characteristics of the

later B cell population resemble transititonal 1 B cells [20].

3.2. BCR-induced phosphorylation of MAP kinases in

mature and immature B cells

The MAPK/Erk signaling cascade is activated by a

wide variety of receptors, including BCR, resulting in

Fig. 1. CR1/2 and HSA expression on B cells obtained from untreated (A) or sublethally irradiated and autoreconstituted mice (B). Cells (106) were incubated

with antibodies, specific for CD21 and HSA and their binding was detected by fluorescein isothiocyanate- or phycoerythrin-labeled secondary antibodies.

Representative of three different experiments is shown.

D. Kovesdi et al. / Cellular Signalling 16 (2004) 881–889884

the transcription of immediate early genes, such as c-Fos

[16,22]. Activation of MAPKs depends on their phosphor-

ylation on tyr/thr/ser residues by dual specific kinases. We

compared the kinetics of Erk1/2 and p38 MAPKs phos-

phorylation in mature and immature B cells using anti-

bodies specific for their phosphorylated motifs. BCR cross-

linking induced phosphorylation of Erk1/2 as well as p38

MAPK both in mature and in immature B cells. However,

we have found a significant difference in the kinetics of

MAPK phosphorylation. A sustained phosphorylation of

both MAPKs was observed in mature B cell extracts,

while phosphorylation of p38 MAPK, similarly to that of

Erk1/2, was only transient in immature B cells, ceasing

after 30–60 min (Fig. 2).

Erk signals induce the expression of the immediate early

gene product c-Fos. Sustained activity of Erk 1 stimulates

Fig. 2. Kinetics of ERK1/2 and p38 MAPKs activation. Cells (108/

samples) were incubated at 37 jC for different time periods (0, 2, 15, 30,

60, 120 min) with 10 Ag/ml A-chain-specific antibody, then lysed in the

presence of 1% Triton X-100. The cell extracts were separated by 10%

SDS–PAGE, transferred onto nitrocellulose membranes and probed with

phospho-ERK1/2, phospho-p38 MAPK, panERK and c-Fos-specific

antibodies, respectively. Representative examples of three independent

experiments are shown.

hyperphosphorylation of c-Fos, which stabilizes the protein,

while transient phosphorylation results in the degradation of

c-Fos [21]. We have compared BCR-induced c-Fos expres-

sion in mature and transitional immature B cells. c-Fos

protein appeared in mature B cell samples at 60 min after

stimulation, and remained stable for 120 min, while in

immature cells, induction of c-Fos expression could not be

observed (Fig. 2).

3.3. Kinetics of Akt/PKB activation upon BCR cross-linking

BCR ligation induces the phosphorylation of Atk/PKB

on serine and threonine in a PI3-K-dependent manner,

which is required for its activation [22]. Since Akt and

its substrates mediate anti-apoptotic signals, we further

examined the kinetics of Akt phosphorylation as well as

the phosphorylation its substrates using phospho-Akt-

specific and phosphorylation site-specific antibodies.

Stimulation of mature B cells via BCR-induced Akt

phosphorylation after 2 min, which remained stable for

2 h. However, phosphorylation of Akt in immature B

cells was transient (Fig. 3a). In line with this result, we

observed sustained phosphorylation of several substrate

proteins of Akt at 116, 85, 58 and 30 kDa apparent

molecular weight, while the phosphorylation of these

proteins was only transient in immature B cells (Fig.

3b,c,d). Moreover, we observed a protein at about 45

kDa, the phosphorylation of which was typical for mature

B cells, while it was undetectable in any samples of

immature B cells.

3.4. The lack of caspase-8 activation upon BCR

cross-linking

Soluble A-chain-specific antibodies usually activate

mitochondrial membrane potential changes and trigger

cytochrome c-dependent activation of caspase-9 and cas-

pase-3, while caspase-8 activation has not been observed

Fig. 3. Kinetics of the activation of Akt/PKB. Cells (108/samples) were

incubated at 37 jC for different time (0, 2, 15, 30, 60, 120 min) with 10

Ag/ml A-chain-specific antibody and lysed, and the cell extracts were

separated by 10% SDS–PAGE, transferred onto nitrocellulose membranes

and probed with antibodies specific either for the phosphorylated form of

Akt/PKB (phosphoSer472/473/474) (A), or for the phospho-(Ser/Thr)

motifs of the substrates phosphorylated by Akt kinase (B, C, D). ECL

detection after different exposure times: 10 s (B), 5 min (C) and 30 s, (D),

respectively, are shown. (E) Demonstrates the equal loading by detecting

Akt.

Fig. 4. BCR cross-linking did not induce Caspase-8 activation in mature

and immature B cells. Caspase-8 activity was monitored by a substrate

peptide becoming fluorescent upon cleavage. The baseline/control cells

(white bar), and the fluorescence levels measured after 1 h (grey bar), 3

h (dark grey bar), 6 h (diagonally hatched bar) and 20 h (black bar) anti-

A-chain antibody treatments are shown for mature (MB) and immature

(IMB) B cells, after background subtraction. As a positive control, 100

AM C2-ceramide (N-acetyl-D-sphingosine) induced caspase-8 activation in

a T helper hybridoma (IP12-7) is also shown (TH). The insert shows the

fluorescent emission spectra for the fluorogenic substrate in buffer

(spontaneous hydrolysis/background; continuous line), for activated (20 h)

immature (dotted line) and mature (dashed line) B cells, as well as for

ceramide treated T cells (11 h; dash–dot–dot line).

D. Kovesdi et al. / Cellular Sign

[23,24]. Others have shown that in the presence of cross-

linked anti-A antibody BCR-mediated apoptosis is asso-

ciated with and depends on caspase-8 activation in a

Burkitt’s lymphoma cell line BL41 [25]. To better un-

derstand the molecular background of BCR-mediated

apoptosis, we investigated the involvement of caspase-

8 activation - an upstream apoptotic signal event - in

untreated and BCR cross-linked mature and transitional

immature B cells. Isolated mature and immature B cells

were cultured in medium alone or in the presence of A-chain specific antibody for the indicated time intervals (0,

3, 6, 20 h). The cells were collected, lysed and following

the addition of synthetic fluorogenic substrates (Ac–

IETD–AFC) the emission of the released AFC was

monitored. We could not detect any caspase-8 activity

after BCR cross-linking either in mature or in immature

B cells indicating that caspase-8 is not involved in BCR

mediated apoptosis (Fig. 4).

3.5. Mitochondrial membrane potential changes upon BCR

cross-linking in mature and immature B cells

Mitochondrial function plays a pivotal role in determin-

ing cellular commitment to survival or apoptosis [26].

Mature and transitionally immature B cells were incubated

in culture media alone or in media supplemented with A-chain-specific antibody for different time periods and the

cells were stained with DiOC6(3) fluorochrome. Incorpora-

tion of this cationic lipophylic dye into the mitochondria is

proportional to the mitochondrial membrane potential. At

the indicated time points, the percentage of cells with

collapsed Wm were estimated by flow cytometry (Fig. 5a).

In immature B cells, BCR cross-linking induced mitochon-

drial membrane depolarization at 6 h after stimulation and

significantly increased the percentage of apoptotic cells after

20 h. In contrast, 6 h stimulation of mature B cells did not

induce programmed cell death; moreover, it protected cells

from spontaneous apoptosis. After 20 h of incubation in the

presence of anti-IgM, the number of dead cells in mature B

cell samples was about half of that detected in immature

cells. Anti-IgM induced apoptosis in a dose-dependent

manner as shown in Fig. 5b.

3.6. Comparison of BCR-induced activation of the effector

caspase-3 in mature and immature B cells

The activation of Caspase-3 was monitored by PARP

cleavage in the cell extracts of mature and immature B cells.

Antibodies specific for both the intact and the cleaved form

alling 16 (2004) 881–889 885

Fig. 5. Mitochondrial membrane potential changes (DWm) after BCR cross-

linking in mature and immature B cells. (A) Isolated mature and immature

B cells (106) were incubated in culture medium alone (5) or supplemented

with 10 Ag/ml anti-A-chain-specific antibody (n) for different times (0, 3, 6,

20 h). Representative example of three independent experiments is shown.

(B) Dependence of Wm in immature B cells on the dose of anti-A antibodies

(0, 0.03, 0.3, 3 Ag/ml) at the indicated time points of treatment. DWm was

analysed by staining cells with DiOC6 at a final concentration of 10 nM.

The subpopulations with low DiOC6 staining (left) represent the cells with

collapsed Wm. (C) BCR cross-linking induced PARP cleavage in immature

B cells. Isolated cells (106) were incubated in culture medium alone or

supplemented with 10 Ag A-chain-specific antibody for different intervals

(0, 3, 6, 20 h). The proteins were precipitated by 6% ice-cold TCA and

electrophoresed on 7.5% polyacrylamide gel. The samples were subse-

quently blotted, and the blots were probed with polyclonal PARP specific

antibodies, either for full length PARP (116 kDa) or its cleaved fragment

(85 kDa).

Fig. 6. Signaling through CD40 may rescue B cells from apoptosis. (A) B

cells (108/samples) were incubated at 37 jC in the presence of with 10 Ag/ml A-chain-specific, 10 Ag/ml anti-CD40 antibody, or the combination of

the two for the indicated time. Percentages of apoptotic cells were estimated

as described at Fig. 5. (B) Samples of cells treated with the combination of

anti-IgM and anti-CD40 for the indicated time intervals were analyzed for

the activation of Akt/PKB and Erk1/2 by Western blot experiments using

antibodies specific for the phosphorylated form of Akt and Erk1/2,

respectively. Representative example of three independent experiments is

shown.

D. Kovesdi et al. / Cellular Signalling 16 (2004) 881–889886

of PARP detected the degree of cleavage. Inducible caspase-

3 activation was detected in the anti-IgM-treated samples of

immature B cells at 3 h, increased after 6 h, and intensive

cleavage was found at 20 h, while the cleaved fragment of

PARP was undetectable in mature B cells even at 6 h. PARP

cleavage, albeit at a lower degree, was also observed in

mature B cells at 20 h after stimulation (Fig. 5c). These data

indicate that the BCR-induced apoptosis of transitional

immature B cells is caspase-3 dependent.

3.7. Rescue of immature B cells from apoptosis by

anti-CD40

CD40 signaling was shown to mediate rescue from anti-

IgM-induced cell death [17,27]. In WEHI-231 cell line, this

was reported to correlate with the expression of the anti-

apoptotic protein Bcl-xL [28]. To see whether triggering via

CD40 can protect transitional immature B cells from apo-

ptosis here, we stimulated mature and immature B cells with

anti-IgM, anti-CD40 and with both reagents, and we mon-

itored cell death and/or survival by detecting mitochondrial

membrane depolarization. Signaling via CD40 rescued both

mature and immature B cells from apoptosis (Fig. 6a). We

also monitored the kinetics of Erk1/2 and Akt activation in

the same samples. Both Akt and Erk1/2 exhibited transient

phosphorylation kinetics in samples of anti-IgM- and anti-

CD40-treated immature B cells, while showed sustained

activation in mature B cells. Phosphorylation of GSK-3, an

important substrate of Akt was observed only in mature but

not in immature B cells (Fig. 6b).

The expression of anti-apoptotic molecule, Bcl-2 was

tested in parallel samples both at protein and at mRNA

levels. Western blot experiments have shown that Bcl-2

protein expression in mature B cells increased above the

control level 30 min after simultaneous stimulation through

BCR and CD40, while the same treatment did not induce

Fig. 7. Cross-linking of BCR and CD40 induces Bcl-2 expression on

mature but not on immature B cells. (A) Cells (108/samples) were

incubated at 37 jC for different times (0, 2, 15, 30, 60, 120 min) in

combination with 10 Ag/ml A-chain-specific and 10 Ag/ml anti-CD40

antibody, and lysed, and the cell extracts were separated by 10% SDS–

PAGE, transferred onto nitrocellulose membranes and probed with

antibodies specific for Bcl-2. (B) Bcl-2 RNA expression in immature B

lymphocytes. Cells (107) were incubated in culture medium alone, in the

presence of with 10 Ag/ml A-chain-specific antibody or in combination of

anti-A and 10 Ag/ml anti-CD40 antibody for different intervals (0, 1, 3, h).

The bcl-2 gene expression was tested by RT-PCR.

D. Kovesdi et al. / Cellular Signalling 16 (2004) 881–889 887

Bcl-2 expression in immature B cells (Fig. 7a). This result

was confirmed by controlling Bcl-2 gene expression in anti-

IgM- plus anti-CD40-treated immature B cells, as compared

to the appropriate control samples (Fig. 7b). These data

suggest that CD40-mediated signals may rescue transitional

immature cells from BCR-induced apoptosis independently

of Bcl-2 upregulation.

4. Discussion

Cross-linking of BCR on the surface of both mature and

immature cells rapidly activates Src family kinases and

initiates complex signaling cascades involving adaptor pro-

teins, kinases and transcriptional factors. The Grb-2 associ-

ated binder (Gab1) adaptor/scaffolding protein, after being

recruited to the cell membrane via its PH domain and

phosphorylated by Lyn and Syk, is involved in PI-3K-

dependent activation of the Akt/GSK3 pathway, as well as

in the activation of SHP-2 and Erk [29,30]. We have shown

previously that in contrast to mature B cells, Gab1 was

constitutively phosphorylated in transitional immature B

cells, which is not modified by BCR ligation. We observed

a transient Erk activation and short-lived phosphorylation of

the transcription factors Elk-1 and CREB in the same

samples [14,15].

In this study, we reveal that the duration of BCR-

triggered Erk-, p38MAPK- and Akt/PKB-mediated signals

are considerably shorter in transitional immature B cells

than in mature B cell population. The transient signals are

not sufficient to induce the expression of immediate early

genes such as c-Fos and cannot protect immature B cells

from programmed cell death. Signaling via CD40, al-

though it does not induce Bcl-2 gene expression, neither

modifies the kinetics of Erk and Akt activation, may

rescue immature B cells from BCR-induced apoptosis.

BCR cross-linking drives transitional immature B cells to

programmed cell death as a result of the disruption of

mitochondrial transmembrane potential and the consecutive

activation of caspase-3.

The Ras/MAPK signaling network regulates diverse

biological responses such as proliferation, differentiation,

migration or survival [31], and the duration of Erk signaling

may determine the cell’s response. It was recently shown

that the sensor of the duration of Erk phosphorylation in

fibroblasts is c-Fos. Docking of activated Erk on c-Fos

results in hyperphosphorylation, which stabilizes and phos-

phorylates the protein and enables c-Fos to induce gene

transcription and eventually, cell proliferation. Under con-

ditions of transient Erk signaling, the newly formed c-Fos

cannot be phosphorylated, thus, it becomes degraded [21].

We observed a significant difference in the duration of Erk

and p38 kinase phosphorylation between immature and

mature B cells. In contrast to the sustained phosphorylation

of both kinases in mature B cells stimulated through the

BCR, the activity of these MAP kinases faded away in

samples of immature B cells after 30 min. This difference

was particularly characteristic for Erk1. Activated Erk

migrates to the nucleus where it phosphorylates transcrip-

tion factors, such as Elk-1 and CREB and induces the

expression of early response genes, such as c-Fos [26,34].

In mature B cells, c-Fos expression was clearly detected at

60 min after stimulation through the BCR, while induction

of c-Fos was not detected in samples of immature B cells.

These findings together indicate that the transient Erk1/2

and p38 MAPK-mediated signals induced by BCR in

transitional immature B cells are not sufficient to induce

the prolonged expression of immediate early genes, which is

a prerequisite of cell proliferation.

Cell survival requires the active inhibition of apoptosis,

which is accomplished either by inhibiting caspases or by

preventing their activation. Inhibition of caspase-9 through

phosphorylation by Erk was observed recently [32]. Thus,

we suppose that short-term Erk activation in transitional

immature B cells is not sufficient for the inhibition of

caspase-9 and the subsequent formation of the apoptosome,

thus, the cells are still driven to apoptosis.

PI-3-kinase activity is essential for the prevention of

apoptosis in a number of cell types [33–36], and this appears

to be mediated by Akt/PKB [37,38]. Akt/PKB mediates

survival signals by the phosphorylation of its apoptosis-

regulating substrates [39–41]. BCR-induced apoptosis in

immature B cells was previously studied in a variety of

models resulting in conflicting data [25,42,43]. The different

D. Kovesdi et al. / Cellular Signalling 16 (2004) 881–889888

outcome of these experiments may be the consequence of

various cell sources and the different conditions of activa-

tion. Deciphering the signaling pathways leading to apopto-

sis of transitional immature and mature splenic B cells of

mice, we first detected a striking difference in the kinetics of

Akt phosphorylation in these two cell types. Similarly to Erk

and p38 MAPK, duration of AKT signal was shorter in

immature than in mature B cells. Akt signals directly

influence mitochondrial functions, and it has been proposed

that Akt prevents apoptosis by phosphorylating Bad, inhib-

iting thereby its interaction with Bcl-xL, which, in turn, can

exert its anti-apoptotic effects [38,39]. Akt phosphorylates

caspase-9 and prevents its proteolytic activation and may

also prevent apoptosis by phosphorylating forkhead family

transcription factors [40]. Phosphorylation of these transcrip-

tion factors promotes their export from the nucleus and

prevents them from inducing expression of pro-apoptotic

genes, such as Fas ligand. Another major downstream target

of Akt is glycogen synthase kinase-3 (GSK-3), a constitu-

tively active serine/threonine kinase, whose activity is

inhibited by Akt. GSK-3 is important in the regulation of a

number of cellular processes [44].

As a consequence of the short-lived activity of Akt in

immature B cells, its substrates have also shown a transient

phosphorylation. Several substrates at 30, 58, 84 and 116

kDa, whose phosphorylation was constant up to 2 h after

stimulation of mature B cells via BCR, showed a decreased

signal in immature cells after 30–60 min. Most importantly,

one protein at around 45 kDa was not phosphorylated at all

upon BCR ligation on immature cells. This difference in the

phosphorylation of Akt substrates indicates impaired anti-

apoptotic signaling in transitional immature B cells.

Activation of CD40 is essential for thymus-dependent

humoral immune responses and for rescuing B cells from

apoptosis [45]. This is a key event of cognate B cell–T cell

interaction in germinal centers. CD40 has been shown to

induce anti-apoptotic signals by inducing Bcl-xL and Bcl-2

expression in murine and human B cells and in WEHI-231

cell lines [46–48]. We have shown here that anti-CD40

treatment partially protected transitional B cells from apo-

ptosis, without affecting the level of Bcl-2 tested both at the

mRNA and protein levels. This CD40-triggered anti-apo-

ptotic signal with most likeliness is not mediated entirely

through Akt, since we could not detect phosphorylation of

one of the major Akt substrate, GSK-3. It has been sug-

gested previously that multiple survival pathways are trig-

gered via CD40 and they are not relying entirely on the

induction of known anti-apoptotic molecules [49]. In agree-

ment with this finding, we suggest that CD40 signaling

protects transitional B cells from apoptosis in the absence of

GSK-3 phosphorylation and upregulation of Bcl-2 expres-

sion, with a yet unclear mechanism.

During apoptotic signaling, mitochondrial changes result

in enhanced production of reactive oxygen species (ROS),

calcium cycling and disruption of the inner mitochondrial

potential [50,51]. Measuring DWm, we have shown that

BCR ligation induced depolarization of mitochondria mem-

brane in transitional immature B cells after 6 h. The

correlation between the BCR-induced collapse of mitochon-

drial membrane potential and the lack of sustained Akt

activity suggests that Akt may be directly involved in the

protection of mitochondria in mature B cells.

Depolarization of the mitochondrial membrane leads to

MOMP (mitochondrial outer membrane permeabilization)

and release of cytochrome C and a consequent formation of

the caspase-3 activating complex. Caspase-3 is one of the

key effectors of apoptosis and activated caspases-3 cleaves

PARP [52]. The kinetics of the cleavage of caspase-3

substrate, PARP in BCR-stimulated transitional immature

B cells correlated with the decrease of mitochondrial mem-

brane potential, indicating that the apoptotic process was

induced in a caspase-3-dependent manner, independently of

the caspase-8 death signal pathway.

This finding obtained with primary murine B cells is

apparently controversial with some previous data claiming

that apoptosis of immature B cells—namely the immature B

lymphoma cell line WEHI-231 [53]—is independent of

caspase-3. The discrepancy might be explained with the

different sources and features (phenotype, signaling machin-

ery) of B cells.

Taken together, we describe here that BCR ligation on

primary immature B cells results in a transient activation of

Erk, p38 and Akt, resulting in the lack of induction of early

response genes and an impaired function of the anti-apo-

ptotic substrates of Akt. Thus, Akt is unable to protect

mitochondria from the loss of membrane potential, leading

to the release of cytochrome c and the subsequent activation

of caspase-3. The pathway described here supposed to be

responsible for the negative selection of immature B cells

and may have an importance in maintaining peripheral

tolerance.

Acknowledgements

This work was supported by the Hungarian National

Science and Research Developmental Foundation (OTKA

T029535, T034493, T034536, TS044711, D38465) and by

the Foundation for the Development of Graduate Education

and Research (FKFP 0155/200).

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