From the Department of Immunology and Signal
Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo
Metropolitan Organization for Medical Research, Fuchu, Tokyo 183-8526, Japan, the ¶ Cancer Research Institute, Kanazawa University,
Kanazawa 920-0934, Japan, and the
Friedrich Miecher Institute,
CH-4002 Basel, Switzerland
Received for publication, October 10, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Stimulation of B cell antigen receptor (BCR) may
induce proliferation, differentiation, or apoptosis, depending upon the
maturational stage of the cell and the presence or absence of signals
transmitted via coreceptors. One such signal is delivered via CD40; for
instance, ligation of CD40 rescues B cells from BCR-induced apoptosis.
Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not
reverse BCR-induced growth inhibition in the BAL-17 mature B cell line
and CD40 ligation itself inhibited proliferation. This inhibitory
signaling was not observed in CD45-deficient cells. Further analyses
demonstrate that transfection of dominant-negative form of SEK1 or
treatment with SB203580 strongly reduced CD40-induced inhibition of
BAL-17 proliferation, suggesting a requirement for c-Jun
NH2-terminal kinase and p38 in CD40-induced
inhibition of proliferation. Interestingly, CD40-initiated activation
of c-Jun NH2-terminal kinase and p38 was enhanced and
sustained in CD45-deficient cells, and these phenotypes were reversed
by transfecting CD45 gene. However, CD40-mediated induction of cell
surface molecules was not affected in CD45-deficient cells. Taken
collectively, these results suggest that CD45 exerts a decisive effect
on selective sets of CD40-mediated signaling pathways, dictating
B cell fate.
The fate of B cells is determined by the signals from antigen
receptor (BCR).1 These
signals may be influenced by a variety of factors including antigen
binding strength and the maturational stage of the cell. Accumulating
evidence indicates that signals transmitted via coreceptors are also
critical to the final outcome of B cells; one such coreceptor that
appears to be functionally important is CD40 (1-3). CD40, a member of
tumor necrosis factor receptor/nerve growth factor receptor family, has
a cysteine-rich extracellular domain and a short cytoplasmic tail
without enzymatic activity and is expressed on a variety of cells
including B cells, dendritic cells, and epithelial cells. In B cells,
CD40 is known to mediate proliferation, maturation, memory cell
induction, germinal center formation, and class switching of
immunoglobulin (Ig) gene (1-5).
Signaling via CD40 not only protects germinal center B cells and
several B lymphoma cells from spontaneous and BCR-induced apoptosis,
respectively (6-9), but also suppresses the growth of B lymphoma cells
(10-12) as well as mesenchymal-epithelial cells (13-15). Thus, CD40
may transmit both positive and negative signals depending upon the cell
type, implying a complex regulation of CD40 signal transduction.
Numerous studies have been performed in determining specific pathways
of CD40-initiated positive signaling. CD40 ligation has been
demonstrated to activate several different signaling pathways, for
example, activation of tyrosine kinases (Lyn, Fyn, and Syk),
phosphatidylinositol 3-kinase, phospholipase C- In the present study, we show that, in contrast to WEHI-231 cells,
BCR-induced growth inhibition was not rescued by CD40 ligation in
BAL-17 cells; indeed, CD40 ligation itself inhibited proliferation of
BAL-17 cells in a dose-dependent manner. Significantly, in CD45-deficient cells, the CD40-induced inhibition of proliferation was
not observed and activation of JNK and p38 was enhanced and sustained.
The phenotype of CD45-deficient cells was reversed by transfecting CD45
cDNA, suggesting a decisive role for CD45 in these processes.
Further analyses revealed that transfection of dominant-negative form
of SEK1 into BAL-17 or treatment with a p38-specific inhibitor reduced
CD40-induced inhibition of proliferation, suggesting that activation of
JNK or p38 is crucial to CD40-initiated proliferative regulation.
Additionally, induction of cell surface molecules upon CD40 ligation
was not affected in CD45-deficient cells. Thus, these results suggest
that CD45 critically regulates selective sets of signaling events
induced by CD40 ligation, determining the fate of BAL-17 cells.
Cells--
The WEHI-231 and BAL-17 murine B cell lines and the
BAL-17-derived, CD45-deficient clone 44 were all described previously (35, 36). All cells were cultured in RPMI 1640 medium containing 10%
fetal bovine serum, 50 µM 2-mercaptoethanol, 100 µg/ml
streptomycin, and 100 units/ml penicillin (complete medium).
Antibodies and Reagents--
Rat anti-mouse CD40 mAb (FGK46.5)
was a gift from Dr. A. Rolink (Basel Institute for Immunology, Basel,
Switzerland). F(ab')2 fragments of goat anti-mouse IgM
antibody (Ab) and fluorescein isothiocyanate-conjugated (FL) goat
anti-IgM Ab were purchased from ICN Pharmaceuticals, Inc. (Aurora, OH).
Rabbit polyclonal Abs against c-Src, ERK, JNK, and p38 were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-JNK
Ab and anti-phospho-p38 Ab were purchased from Promega (Madison, WI)
and New England Biolab (Beverly, MA), respectively. FL-anti-mouse CD54
mAb, biotin-conjugated anti-mouse CD80 mAb, FL-anti-mouse CD86 mAb, and
biotin-conjugated anti-mouse CD95 mAb were purchased from PharMingen
(San Diego, CA). Anti-I-Ad mAb (MK-D6) was obtained from
American Type Culture Collection (Rockville, MD), and anti-mouse CD45
mAb (104) has been described elsewhere (37). SB203580 was obtained from
Calbiochem-Novabiochem Corp. (La Jolla, CA).
Proliferation Assay--
Proliferation assay was performed as
previously described (35, 36). Briefly, 5-10 × 103
cells were cultured in triplicates in 0.2 ml of complete medium with or
without anti-CD40 mAb or anti-IgM Ab for 24-48 h. To assess DNA
synthesis, 0.5 µCi (18.5 kBq) of [3H]thymidine were
added to each well for the last 4 h. The cells were harvested on
glassfiber filters with a semiautomatic Skatron harvester, and
thymidine incorporation was measured using a Beckman liquid
scintillation counter.
Flow Cytometric Analysis--
To examine surface phenotypes,
cells were incubated with specific Abs for 15 min on ice, washed, and
incubated with FL-secondary Abs for 15 min on ice. FL-protein A
(Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), FL-mouse anti-rat
Ig Electroporation--
To introduce DNA into cells, 2 × 107 cells were washed twice with phosphate-buffered saline
and suspended with 0.4 ml of cytomix buffer (120 mM KCl,
0.15 mM CaCl2, 10 mM
K2HPO4/KH2PO4, pH 7.6, 25 mM HEPES, pH 7.6, 2 mM EGTA, pH 7.6, 5 mM MgCl2, 2 mM ATP, and 5 mM glutathione) containing 40 µg of DNA, and
electroporated at 264 V, 950 microfarads. DNAs introduced were: the
pcDLSR Immunoblot Analysis--
After incubation in complete medium for
1 h at 37 °C, 5-10 × 106 cells were
stimulated with anti-CD40 mAb or anti-IgM Ab. The reactions were
terminated with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and 2 mM
EDTA. The cells were centrifuged and solubilized in lysis buffer (1%
Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 1 mM Na3VO4, and 2 mM EDTA) supplemented with protease inhibitor mixture
(Roche Molecular Biochemicals). The supernatants were separated on
7.5-10% SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane. Blots were incubated with specific Abs for overnight at
4 °C in 0.5% gelatin-Tris buffered saline (20 mM
Tris-HCl, pH 7.5, 0.5 M NaCl) and then incubated with the
corresponding secondary Abs for 1 h; secondary Abs included
alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad), alkaline
phosphatase-conjugated mouse anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA), horseradish peroxidase
(HRP)-conjugated anti-mouse IgG (Santa Cruz Biotechnology), HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated swine anti-goat
IgG (BIOSOURCE International, Camarillo, CA). The
membranes were visualized using alkaline phosphatase-conjugate
substrate kit (Bio-Rad) or ECL detection reagents (Amersham Pharmacia Biotech).
Assays for MAPKs--
Activation of JNK and p38 was examined by
immunoblot analysis with rabbit anti-phospho-JNK and anti-phospho-p38
Abs. The protein amount of JNK and p38 was assessed by immunoblot with
anti-JNK and anti-p38 Abs. To assess JNK activity, lysates were
incubated for 2 h at 4 °C with glutathione
S-transferase-c-Jun-Sepharose. The resultant precipitates
were washed three times with a washing buffer (20 mM HEPES,
pH 8.0, 2.5 mM MgCl2, 0.1 mM EDTA,
50 mM NaCl, and 0.05% Triton X-100) and then rinsed once
with kinase reaction buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM
CD40 Ligation Does Not Rescue BCR-mediated Inhibition of
Proliferation but Suppresses Proliferation in BAL-17 B Cells--
To
elucidate the function of CD40 in B cell signaling, we examined how
CD40 ligation affects B cell behaviors using mature B cell line BAL-17,
as compared with immature B cell line WEHI-231. WEHI-231 and BAL-17
cells were cultured with 1-20 µg/ml anti-CD40 mAb in the presence or
absence of F(ab')2 fragments of anti-IgM Ab for 48 h,
and DNA synthesis was assayed. As shown in Fig.
1, stimulation with 1-20 µg/ml
anti-CD40 mAb did not significantly affect the proliferative capacity
of WEHI-231 cells and completely reversed anti-IgM-induced growth
arrest, as reported previously (9). In BAL-17 cells, by contrast,
anti-CD40 mAb stimulation did not rescue cells in anti-IgM-induced
growth inhibition, but actually enhanced it. Moreover, CD40 ligation
itself significantly and dose-dependently inhibited DNA
synthesis of BAL-17 cells (Fig. 1).
CD40-induced Inhibition of BAL-17 Proliferation Is Strictly
Dependent on CD45--
CD40 ligation is known to inhibit proliferation
not only in B lineage cells (10-12) but also in nonlymphoid cells
(13-15). However, mechanisms underlying these phenomena are still not
completely understood. We attempted to examine the role of CD45 in
CD40-mediated signaling in BAL-17 cells. For this purpose, we utilized
a CD45-deficient clone previously generated from BAL-17 cells (36).
Flow cytometric analysis showed that, in clone 44 cells, expression of
CD45 was completely absent, but that surface IgM levels and CD40
expression were comparable to those of the parent cells (Fig.
2). Expression of other molecules
including sIgD, MHC class I and class II, Src family protein-tyrosine
kinases (Lyn, Fyn, Blk, Lck), and Syk was also comparable in clone 44 to that in parental cells (36). No significant differences in protein
tyrosine phosphorylation induced by CD40 ligation were observed between
BAL-17 and clone 44 cells (data not shown).
We first asked whether CD40-evoked inhibition of DNA synthesis was
observed in CD45-deficient, clone 44 cells, and as shown in Fig. 1,
anti-CD40 mAb had no effect on their proliferation. In WEHI-231 cells,
however, CD40-mediated effects on proliferation were not altered in
CD45-deficient cells (data not shown). To confirm that the effect of
anti-CD40 mAb in BAL-17 cells was mediated by CD45, we transiently
transfected Src-CD45 cDNA or an empty vector back into clone 44 cells. We used Src-CD45 cDNA, because it has been demonstrated that
Src-CD45 is sufficient for reconstituting the function of
CD45-deficient T cell clones (38) and is more efficiently expressed
than the full length. The transfectants efficiently expressed the CD45,
as assessed by immunoblotting with anti-c-Src Ab (Fig.
3B). Control transfection with
a vector containing enhanced green fluorescence protein revealed that
transfection efficiency was 25-30%. Once CD45 gene was transfected,
clone 44 cells were as susceptible to CD40-induced inhibition of
proliferation as were BAL-17 cells (Fig. 3A).
Thus, in contrast to WEHI-231 cells, where CD40 transmits positive
signals thereby reversing BCR-induced growth inhibition, in BAL-17
cells, CD40 exerts a negative effect on proliferation and on
BCR-induced growth inhibition. More significantly, the data show that
CD40-induced inhibition of BAL-17 proliferation was mediated through CD45.
CD40-induced Up-regulation of Cell Surface Molecules Is Not
Controlled by CD45--
CD40 ligation by CD40 mAb has been shown to
up-regulate a variety of cell surface molecules. Therefore, to
investigate signaling pathways governed by CD45, we first examined the
capacity of anti-CD40 mAb to induce cell surface molecules on BAL-17
and clone 44 cells. When the cells were cultured with 10 µg/ml
anti-CD40 mAb for 2 days and changes in the expression of CD54, CD80,
CD86, CD95, and I-A molecules were assessed by flow cytometry, no
difference in the degree to which these molecules were up-regulated by
CD40 ligation in CD45-positive and CD45-deficient cells were found (Table I). In addition, reverse
transcriptase-polymerase chain reaction analysis revealed that
induction of the dual-specificity phosphatase, Pac-1, was also not
significantly different in BAL-17 and clone 44 cells (data not
shown).
CD40-induced Activation of JNK and p38 Is under the Control of
CD45--
To examine the extent to which CD45 regulates activation of
MAPK family members, BAL-17 and clone 44 cells were cultured with 10 µg/ml anti-CD40 mAb for 5-60 min, and the lysates were subjected to
immunoblotting with Abs against phosphorylated (activated) forms of
ERK, JNK, and p38. CD40 ligation only marginally elevated ERK
activation in BAL-17 cells (data not shown). However, as shown in Fig.
4A, CD40 ligation induced
activation of JNK with a peak at 10 min in BAL-17. Significantly,
CD40-induced activation of JNK was enhanced and sustained in clone 44 (Fig. 4A). CD40 ligation also induced p38 activation in
BAL-17 with a peak at 5 min. In CD45-deficient cells, however,
activation peak was shifted to 20 min (Fig. 4B). Thus CD45
appears to attenuate CD40-mediated activation of both JNK and p38,
particularly at later phases.
To further confirm that this negative effect is exerted by CD45,
Src-CD45 cDNA was introduced into clone 44 cells (Fig.
5). Twenty-four hours later, the
transfected cells were cultured with 10 µg/ml CD40 mAb, after which
the activation of JNK and p38 was assayed by kinase assays with
glutathione S-transferase-c-Jun as a substrate and
immunoblotting with anti-phospho-p38 Ab, respectively. As shown in Fig.
5A, in CD45 transfectants, enhanced CD40-mediated JNK
activation was reduced to the level seen in BAL-17, whereas cells
transfected with an empty vector were minimally affected. It was also
observed that activation of p38 in CD45-deficient cells was decreased
to the level of parental cells by transfecting CD45 cDNA (Fig.
5B). These results suggest that CD45 indeed exerts negative
effects on CD40-induced activation of JNK and p38.
Activation of JNK and p38 Is Required for CD40-induced Inhibition
of Proliferation--
To evaluate the direct contribution of JNK and
p38 to CD40-induced inhibition of proliferation, BAL-17 cells were
transfected with a plasmid containing a dominant-negative form of SEK1
(DN-SEK1) (39), an upstream activator of JNK, or an empty vector.
Twenty-four hours later, the transfected cells were cultured with
anti-CD40 mAb, and DNA synthesis and activation of JNK were assayed.
Densitometric analysis revealed that introduction of DN-SEK1 reduced
activation of JNK1 and JNK2 by 52% and 70%, respectively, as compared
with vector control (Fig. 6A).
Under this condition, inhibition of DNA synthesis was also
significantly attenuated in transfectants of DN-SEK1 but not in cells
transfected with an empty vector (Fig. 6B).
We then examined the role of p38 using a specific inhibitor for p38,
SB203580. BAL-17 cells were cultured with 5-20 µM
SB203580 for 1 h, after which the inhibitor was removed and DNA
synthesis of pretreated cells was assessed at 24 h after CD40
ligation. As shown in Fig. 6C, the treatment with a
p38-specific inhibitor blocked CD40-induced inhibition of proliferation
in a dose-dependent manner. Activation of p38 was inhibited
10%, 35%, and 50% by 5, 10, and 20 µM SB203580,
respectively, as revealed by Western blot analysis with
anti-phospho-p38 Ab (data not shown). Taken together, activation of JNK
and p38 is required for CD40-initiated inhibition of proliferation in
BAL-17 cells.
There have been several reports that negative signaling evoked by
CD40 ligation inhibits proliferation of B lymphoma cells (10-12).
However, compared with the role played by CD40 in B cell survival and
rescue from apoptosis, the molecular mechanisms by which CD40 inhibits
proliferation have not been extensively studied. In the present study,
we demonstrated that, in contrast to WEHI-231 cells, CD40 ligation in
the mature BAL-17 cells does not reverse BCR-initiated inhibition of
proliferation, but instead inhibits proliferation further (Fig. 1).
Interestingly, CD40 ligation itself induced inhibition of BAL-17 cell
proliferation and such inhibition was not observed in CD45-deficient
clone 44 cells (Fig. 1). Transfection of the CD45 gene into the clone
reversed this phenotype (Fig. 3), indicating a decisive role for CD45
in CD40-induced inhibition of BAL-17 cell proliferation.
To elucidate the mechanisms by which CD45 exerts its regulatory effects
on CD40-induced inhibition of proliferation, we examined several
signaling events mediated by CD40 ligation in BAL-17 and its
CD45-deficient clone. As one of the activation events, CD40 ligation by
anti-CD40 mAb was shown to up-regulate a number of cell surface
molecules (24-29), and our findings suggest that CD45 is not involved
in the CD40-induced up-regulation of CD54, CD80, CD86, CD95, and MHC
class II (Table I). On the other hand, CD40-mediated activation of JNK
and p38 was augmented and sustained in the CD45-deficient clone (Fig.
4), and introduction of the CD45 gene resulted in recovery of the
parental phenotype (Fig. 5). Thus, CD45 negatively regulates
CD40-mediated activation of JNK and p38 in BAL-17 cells.
The question now arises as to whether activation of JNK and p38
directly contributes to CD40-induced inhibition of proliferation in
BAL-17 cells. Our results showed that inhibition of JNK and p38,
respectively, by transfecting DN-SEK1 into BAL-17 cells and treatment
with a p38-specific inhibitor, SB203580, blocks proliferation inhibition triggered by anti-CD40 mAb (Fig. 6), suggesting that activation of JNK or p38 is required for CD40-initiated inhibition of
proliferation in BAL-17 cells. Given that CD40 ligation did not induce
inhibition of proliferation in clone 44 cells, where JNK and p38
activities were enhanced and sustained, it is possible that the level
and duration of JNK and p38 activation may need to fall within a narrow
window for optimal CD40 signaling, meaning that CD40-induced inhibition
of proliferation would be blocked when JNK and p38 are activated either
above or below a certain threshold at a certain time. It has been shown
that activation of JNK by CD40 ligation requires TRAF2 (40, 41). One
signaling cascade leading to JNK activation is believed to proceed from membrane proximal molecule, Rac Role of CD45 in CD40 signaling has been investigated previously
using CD45 mAb. One such study demonstrated that cross-linking CD45
inhibits CD40-induced proliferation of human peripheral blood B cells
and small tonsillar B cells, but has no effect on large tonsillar B
cells (46), suggesting that CD45 exerts its inhibitory effects on
CD40-induced growth regulation in a cell type-dependent manner. Another study showed that CD45 mAb blocked tyrosine
phosphorylation evoked by CD40 ligation in human Raji cells (16).
However, these experiments do not enable one to draw any conclusion as
to how CD45 may affect CD40 signaling. It was reported recently that CD40-induced proliferation was partially impaired in splenic B cells
isolated from CD45 knockout mice (47). Given that CD45 is not involved
in the regulation of CD40 signaling in WEHI-231 cells, the effect of
CD45 on CD40 signaling may be dependent on the cell type or the
maturational stage of B cells. One possible factor for differential
effects is NF- In summary, our results demonstrate that, in mature BAL-17 B cells,
CD40-mediated signaling is unable to reverse BCR-induced inhibition of
proliferation, and that CD40 ligation itself inhibits proliferation.
Such inhibition is strictly regulated by CD45. Furthermore,
CD40-initiated growth inhibition is mediated through activation of JNK
and p38 MAPK, and CD40-induced activation of JNK and p38, but not
induction of cell surface molecules, is under strict control of CD45.
Thus, CD45 is involved in the regulation of selective sets of
CD40-induced signaling pathways, determining the final outcome of
BAL-17 B cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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2, Jak3-signal transducer and activators of transcription 3, and nuclear factor
B
(NF-
B) (16-21). CD40 signaling also enhances the expression of
Pac-1, Bcl-xL, Cdk4, and Cdk6 (22, 23), as well as various membrane
molecules including CD23 (Fc
RII) (24), CD54 (ICAM-1) (25), CD80
(B7-1) (26), CD86 (B7-2) (27), CD95 (Fas) (28), and MHC class II
(29). Furthermore, members of mitogen-activated protein kinase (MAPK)
family are differentially activated by CD40 ligation. Three MAPK
members have been identified: extracellular signal-regulated kinase
(ERK), c-Jun NH2-terminal kinase (JNK, or stress-activated
protein kinase), and the p38 MAPK. Although CD40-mediated activation of
ERK is dependent on the cell types, JNK and p38 are activated in B
cells and B cell lines by ligation via CD40 (30-33), and p38 is
reported to be required for CD40-induced B cell proliferation and
NF-
B activation in B cell lines and tonsillar B cells (34). However,
the molecular basis for the negative effects of CD40 remains to be elucidated.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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chain mAb (Zymed Laboratories Inc., San
Francisco, CA) and FL-avidin (ICN Pharmaceuticals) were used as
secondary Abs. The FL-labeled cells were then analyzed with an Epics
ELITE flow cytometer (Coulter, Miami, FL). For induction of membrane
protein expression, 5 × 104 cells were stimulated
with anti-CD40 mAb for 2 days before staining.
expression vector containing Src-CD45 (a chimeric cDNA
encoding the intracellular murine CD45 preceded by a short
amino-terminal sequence from p60c-src) (38) or
the pcDLSR
empty vector (a gift from Dr. Y. Minami, Kobe University,
Kobe, Japan), and pEFII expression vector containing a gene encoding a
hemagglutinin-tagged dominant-negative SEK1 mutant (SEK-AL) (39) (a
gift of Dr. Jim Woodgett, Ontario Cancer Institute, Toronto, Canada),
which had been excised from pcDNA3 Zeo, or the empty vector (a gift
from Dr. G. Koretzky, University of Pennsylvania, Philadelphia, PA).
The cells were then resuspended in complete medium and incubated for
24-36 h. After removing dead cells by using Lympholyte-M (Cedarlane,
Ontario, Canada), the remaining cells were subjected to functional assays.
-glycerophosphate, 0.1 mM
Na3VO4, and 2 mM dithiothreitol).
The kinase reactions were elicited in 40 µl of the reaction buffer
containing 20 µM cold ATP and 5 µCi (185 kBq) of
[32P]ATP. The reactions were stopped by adding Laemmli
sample buffer and boiling for 5 min. The samples were then separated on
10% SDS-polyacrylamide gels and subjected to autoradiography. The signals were quantified as a function of the density of the bands using
a Bio-Rad Imaging Densitometer; measurements were normalized to the
quantities of proteins applied.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Effects of anti-CD40 mAb on proliferation of
WEHI-231, BAL-17, and clone 44 cells. Cells were cultured in
triplicate for 48 h in the presence or absence of indicated
concentrations (in µg/ml) of anti-mouse CD40 mAb and/or goat
anti-mouse IgM Ab. To assess DNA synthesis, 0.5 µCi (18.5 kBq) of
[3H]thymidine was added to each well for the last 4 h of culture. The results are expressed as mean percentage of
control ± standard error (S.E.) of five independent
experiments.
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Fig. 2.
Expression of sIgM and CD40 on BAL-17 and its
CD45-deficient clone 44 cells. The left two
panels show the expression level of CD45 detected by
staining with anti-CD45 mAb and FL-protein A. Background
(Bgd) represents staining with FL-protein A alone. The
right two panels show the expression
of sIgM and CD40 assessed by staining with FL-anti-IgM Ab and with
anti-CD40 mAb and FL-mouse anti-rat Ig mAb. Bgd
represents staining with FL-mouse anti-rat Ig
mAb alone.
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Fig. 3.
Introduction of CD45 gene into clone 44 cells
reverses CD40-induced proliferation pattern. A, a
vector containing Src-CD45 cDNA and an empty vector were
transiently transfected into clone 44. Proliferation inhibition induced
by 10 µg/ml anti-CD40 mAb was then assessed in BAL-17
(BAL), clone 44 (44), CD45-transfected clone 44 (S45), and vector-transfected clone 44 (Vec). The
results are representative of three separate experiments and expressed
as percentage of inhibition of DNA synthesis ± S.E. B,
expression of Src-CD45 was examined in untreated clone 44 ( ),
CD45-transfected clone 44 (S45), and vector-transfected
clone 44 (Vec) by immunoblotting with anti-c-Src Ab.
Exogenous CD45 was detected only in S45 cells, as indicated by an
arrow.
Anti-CD40-mediated induction of cell surface molecules in BAL-17
and its CD45-deficient clone
) of 10 µg/ml anti-CD40 mAb. After
culture, cells were labeled with mAbs against CD54, CD80, CD86, CD95,
and I-Ad, and then subjected to flow cytometric analysis. The
results are expressed as mean fluorescence intensities and are
representative of three separate experiments.
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Fig. 4.
CD40-induced activation of JNK and p38
in BAL-17 and clone 44 cells. Five million cells were stimulated
for indicated times with 10 µg/ml anti-CD40 mAb, and the lysates were
subjected to Western blot analysis with antibodies against JNK and the
phosphorylated form of JNK (pJNK) (A) and p38 and the
phosphorylated form of p38 (pp38) (B). The results are
representative of four separate experiments. Numbers shown
below indicate -fold increases of activated JNK and p38
after CD40 ligation calculated by densitometry, with the intensity of
unstimulated group arbitrarily set to 1.
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Fig. 5.
Enhanced CD40-induced JNK and p38 activation
in clone 44 cells is reversed by transfection of CD45 cDNA.
A, a vector containing Src-CD45 cDNA and an empty vector
were transiently transfected into clone 44, and 24 h later
transfected cells were stimulated with 10 µg/ml anti-CD40 mAb for 20 min. JNK activity was then assessed by in vitro kinase
assays with glutathione S-transferase-c-Jun as a substrate.
Relative JNK activity of CD40-stimulated BAL-17 (BAL), clone
44 (44), Src-CD45-transfected clone 44 (S45), and
vector-transfected clone 44 (Vec) was calculated from the
density of each band, with the value of unstimulated cells being
arbitrarily assigned as 1. B, after transfection performed
as above, cells were stimulated with 10 µg/ml anti-CD40 mAb for 10 min and subjected to immunoblotting with anti-phospho-p38 Ab. Data were
treated as in A. C, expression of Src-CD45 was
assessed in BAL-17, untreated clone 44 ( ), Src-CD45-transfected clone
44 (S45), and vector-transfected clone 44 (Vec)
by immunoblotting with anti-c-Src Ab. The results are representative of
three independent experiments.
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Fig. 6.
Activation of JNK and p38 is required for
CD40-induced inhibition of proliferation. A, BAL-17
cells were transfected with a plasmid containing DN-SEK1 or an empty
vector, and 24 h later the transfected cells were stimulated with
10 µg/ml anti-CD40 mAb for 10 min. Activation of JNK was then
assessed by immunoblotting with anti-phospho-JNK and anti-JNK Abs.
Numbers shown below indicate -fold increases of
phospho-JNK upon CD40 ligation, with the intensity of unstimulated
group set to 1. B, BAL-17 cells, untreated and transfected
with DN-SEK1 or with an empty vector, were stimulated with 10 µg/ml
anti-CD40 mAb for 24 h, and DNA synthesis was measured as
described in Fig. 1. The results are shown as percentage of inhibition
of DNA synthesis ± S.E. and are representative of three
experiments. C, BAL-17 cells were pretreated with 5-20
µM SB203580 for 1 h and then stimulated with 10 µg/ml anti-CD40 mAb for 24 h, after which DNA synthesis was
assessed. The results are shown as mean percentage of inhibition of DNA
synthesis ± S.E. of three experiments.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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MEKK1
SEK1/MKK4
JNK;
where and how the signal from TRAF2 converges into this pathway is
still unknown. The signal from CD40 is also capable of activating
NF-
B through TRAF2 and NF-
B inducing kinase to the I
B
kinase/I
B complex (20, 42-44). In addition, MEKK1 has been reported
to be a common upstream activator of both JNK and NF-
B (45). It is
thus also possible that CD45-generated signals leading to proliferation inhibition may not be mediated solely by JNK or p38 but by a concerted action of multiple signaling molecules including MAPKs and NF-
B, for example.
B. The NF-
B complexes in WEHI-231 and BAL-17 cells
consist predominantly of c-Rel/p50 (48) and
c-Rel/p65,2 respectively, and
the specific combination of NF-
B family members may strongly affect
total transcriptional activity. Indeed, transcriptional activity was
different between WEHI-231 and BAL-17 cells.2 Additionally,
our preliminary studies indicate that CD40 ligation-mediated recruitment of TRAFs differs in WEHI-231 and BAL-17 cells. These differences may contribute to the final outcome of CD40 signaling in
the two cell lines.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Rolink for anti-CD40 mAb, Dr. J. Woodgett for DN-SEK1, and Dr. G. Koretzky and Dr. Y. Minami for vectors.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research and for international scientific research from the Japanese Ministry of Education, Science, Sports and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Microbiology and Immunology, Tokyo Women's Medical University, School of Medicine, Tokyo 162-8666, Japan.
** To whom correspondence should be addressed: Tokyo Metropolitan Inst. for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Tel.: 81-42-325-3881; Fax: 81-42-321-8678; E-mail: yakura@tmin.ac.jp.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M009242200
2 Y. Arimura and H. Yakura, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
BCR, B cell antigen
receptor;
JNK, c-Jun NH2-terminal kinase;
MAPK, mitogen-activated protein kinase;
NF-B, nuclear factor
B;
TRAF, tumor necrosis factor receptor-associated factor;
HRP, horseradish
peroxidase;
MHC, major histocompatibility complex;
Ab, antibody;
mAb, monoclonal antibody;
ERK, extracellular signal-regulated kinase.
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