(Received for publication, June 16, 1995; and in revised form, October 19, 1995)
From the
The B cell surface antigen receptor, surface IgM (sIgM), is
involved in B cell activation and proliferation. CD40 is involved in
regulating IgE production and B cell survival. Cross-linking of B cell
sIgM activates the Ras/Raf/p42pathway. In
contrast, ligation of CD40 by antibody or soluble gp39 (CD40 ligand)
leads to activation of the c-Jun kinase (JNK)/stress-activated protein
kinase pathway. JNK/stress-activated protein kinase activation
correlated with the stimulation of MEK kinase activity. CD40 does not
activate the p42
pathway, and sIgM fails to
regulate the JNK/stress-activated protein kinase pathway in B cells.
Thus, two important cell surface receptors involved in controlling
specific B cell response differentially regulate sequential protein
kinase pathways involving different members of the mitogen-activated
protein kinase family. Anti-CD40 also rescued B cell apoptosis induced
by anti-IgM. CD40 ligation did not affect the sIgM stimulation of
p42
activity. Conversely, sIgM ligation did not
influence CD40 stimulation of JNK/stress-activated protein kinase.
These results suggest that independent, parallel protein kinase
response pathways are involved in the integration of sIgM and CD40
control of B cell phenotype and function.
The B lymphocyte surface antigen receptor, membrane
immunoglobulin, has important functions in the binding and
internalization of antigen as well as in transducing signals through
the plasma membrane that lead to cell activation, differentiation, and
apoptosis(1, 2) . Cross-linking of the receptor
stimulates the Ras/Raf-1/MEK ()cascade with activation of
p42
MAP kinase and
p90
(3) . A second important B cell
surface antigen receptor is CD40. CD40 is a 45-50-kDa
transmembrane glycoprotein expressed on all mature B cells (4) . CD40 is a member of the TNF receptor family and has
homology to the receptors for nerve growth factor(5) ,
TNF-
(6, 7, 8) , Fas(9) , and
CD30(10) . The ligand for CD40 (CD40L, gp39) is expressed on
activated T cells(11) , and activation through CD40 plays an
important role in T cell-dependent immunoglobulin isotype
switching(12, 13) . In contrast to cross-linking of
sIgM, which can cause apoptosis, CD40 can rescue cells from
apoptosis(13, 14, 15, 16, 17) .
The signal transduction pathways through CD40 are not well delineated,
but may induce protein tyrosine phosphorylation of a number of
substrates(18, 19, 20) .
In this paper, we
demonstrate that c-Jun amino-terminal kinases (JNKs)/stress-activated
protein kinases are activated following CD40 ligation of human B cells.
JNKs are members of the MAP kinase family (21) and are
activated by stresses such as UV
irradiation(22, 23, 24) , osmotic
change(25, 26) , and heat shock(21) . In
contrast to CD40, signaling through the antigen receptor activated
p42, but failed to activate JNK.
Figure 1:
Activation of ERK following treatment
with anti-IgM, but not with anti-CD40. A, Ramos cells were
treated with 10 µg/ml anti-IgM antibody or 5 µg/ml anti-CD40
antibody (G28-5) for the indicated times (in minutes), and immunoblot
analysis was performed using monoclonal anti-ERK2 antibody. The
untreated samples contained a single band (42 kDa) reactive with
anti-ERK2 antibody (lane 0`). In samples treated with PMA (100
ng/ml) or anti-IgM for 20 min, a second band with immunoreactivity to
anti-ERK2 antibody appeared (lane PMA and anti-IgM lanes
1`-60`). This lower mobility form represents the activated
form due to phosphorylation(30) . The samples treated with
anti-CD40 demonstrated only a single band throughout the time course. B, ERK activity was evaluated using
EGFR-(662-681)-peptide as a substrate as described under
``Materials and Methods.'' Data are shown as incorporation of P (±S.D.) from separately prepared duplicate
samples from two independent experiments. Statistically significant
differences from control (C; untreated) samples are
represented by an asterisk (p < 0.05). The kinase
activity of samples treated with 100 ng/ml PMA for 20 min and with 10
µg/ml anti-IgM for 5 min was significantly higher than that of
samples from unstimulated (control) cells or cells treated with 1, 5,
or 10 µg/ml anti-CD40 for 20 min. C, immunoblot analysis
of lysates from freshly isolated tonsillar B cells following treatment
with 5 µg/ml anti-CD40 for the indicated times also showed no shift
in ERK2 mobility. Results are representative of at least two
independent experiments.
Figure 2:
Activation of JNK following treatment with
anti-IgM or anti-CD40. JNK activity was measured by solid-phase kinase
assay using GST-c-Jun-(1-79) as a substrate following treatment
with anti-IgM antibody or anti-CD40 antibody (G28-5) in Ramos cells
(three independent experiments) and in tonsillar B cells (two
independent experiments). The level of P incorporation
(±S.D.) into the substrate from at least two experiments was
also evaluated by the PhosphorImager and then illustrated as the ratio
of JNK activity to that of untreated samples. Statistically significant
differences from control (untreated) samples are represented by an asterisk (p < 0.05). A, shown is the time
course of JNK activation after treatment of Ramos cell with 1 µg/ml
anti-CD40. B, shown is the dose response of
anti-CD40-activated JNK in Ramos cells treated with various
concentrations of anti-CD40 for 15 min. C, shown is the dose
response of anti-CD40 in tonsillar B cells treated for 15 min. D, soluble gp39 activates JNK. Dilutions of culture
supernatants containing soluble gp39 were added to Ramos cells for 15
min. E, anti-gp39 antibody prevents activation of JNK by
soluble gp39. Dilutions of culture supernatants containing soluble gp39
were preincubated with anti-gp39 antibody (2 µg/ml) for 5 min prior
to addition to Ramos cells. JNK activation by UV irradiation was
unaffected by the presence of the antibody. F and G,
anti-IgM fails to activate JNK. Anti-IgM antibody was added to Ramos
cells (F) or tonsillar B cells (G) for different time
periods. mAb, monoclonal antibody.
JNK activity was not increased following surface IgM cross-linking even in the presence of 10 µg/ml anti-IgM antibody in Ramos cells (Fig. 2F) and tonsillar B cells (Fig. 2G). These anti-IgM antibody concentrations were effective in ERK activation (Fig. 1A). The results demonstrate that JNK is activated by anti-CD40, but not anti-IgM, indicating that anti-CD40 activates JNK through a different signaling pathway than that which mediates ERK activation by anti-IgM. We therefore investigated the signaling pathways that lead to ERK and JNK activation following treatment with anti-IgM or anti-CD40 antibody.
Figure 3:
Activation of Ras following treatment
with anti-IgM, but not with anti-CD40. Metabolically P-labeled Ramos cells were untreated (control) or treated
with 10 µg/ml anti-IgM or 5 µg/ml anti-CD40 for 1, 5, and 10
min. Ras was immunoprecipitated using the Y13-259 anti-Ras
antibody, and radioactive GTP and GDP bound to Ras were measured as
described under ``Materials and Methods.'' The data were
quantitated by a PhosphorImager, and shown are the GTP/GDP + (1.5)
GDP ratios (in percent) for each condition. Results are representative
of three separate experiments. The data indicate means ± S.D.,
and statistically significant differences from control (untreated)
samples are represented by an asterisk (p <
0.05).
Figure 4:
Activation of Raf-1 following treatment
with anti-IgM, but not with anti-CD40. Ramos cells were untreated
(control (C)) or treated with 100 ng/ml PMA, 10 µg/ml
anti-IgM, or 5 µg/ml anti-CD40 for the indicated times (in
minutes). Raf-1 was immunoprecipitated, and a kinase assay was
performed as described under ``Materials and Methods.'' After
SDS-PAGE, the proteins were transferred onto nitrocellulose membranes. P incorporation into catalytically inactive MEK (KMMEK)
was measured by autoradiography (upper panel). The membrane
was also probed with the same anti-Raf-1 antibody used for
immunoprecipitation, and immunoreactivity was visualized by the
alkaline phosphatase system to verify similar loading of
immunoprecipitated Raf-1 (lower panel). Results are
representative of two separate experiments.
Figure 5:
Activation of MEKK following treatment
with anti-CD40 antibody. Ramos cells were treated with 2 µg/ml
anti-CD40 antibody for the indicated times (in minutes). MEKK was
immunoprecipitated, and a kinase assay was performed as described under
``Materials and Methods.'' After SDS-PAGE, the proteins were
transferred onto nitrocellulose membranes. P incorporation
into JNKK was measured by autoradiography (upper panel). COS
cells transfected with full-length MEKK were used to localize JNKK. The
level of
P incorporation (±S.D.) into the substrate
from three independent experiments was evaluated by the PhosphorImager
and then illustrated as the ratio of MEKK activity to that of untreated
samples (lower panel). Statistically significant differences
from untreated (lane 0`) samples are represented by an asterisk (p < 0.05).
Figure 6:
Rescue from anti-IgM-induced apoptosis by
anti-CD40 antibody. Ramos cells were untreated (control) or treated
with 10 µg/ml anti-IgM antibody or 2 µg/ml anti-CD40 antibody;
costimulation of cells consisted of a 30-min preincubation with
anti-CD40 antibody followed by anti-IgM antibody. A, after an
18-h culture, DNA breaks derived from anti-IgM-induced apoptosis were
evaluated using an in situ terminal
deoxynucleotidyltransferase assay as described under ``Materials
and Methods.'' B, p42 activation
(5 min following treatment) and JNK activation (15 min following
treatment) were evaluated under identical conditions as described under
``Materials and Methods.'' The level of
P
incorporation (±S.D.) from two independent experiments was
evaluated by the PhosphorImager and then illustrated as the ratio of
JNK activation to that of untreated samples. Statistically significant
differences from untreated samples are represented by an asterisk (p < 0.05).
Many growth factors and cytokines activate MAP kinase family members, including ERKs and JNKs. Many growth factors have been shown to activate ERKs, and the signaling cascade has been well characterized. Similar to other members of the MAP kinase family, JNKs are activated through phosphorylation at conserved Thr and Tyr residues (41) . The pathways leading to JNK activation are less well understood and may function as a protective response against environmental stresses and may influence the apoptotic response.
We
previously showed (3) and confirmed here that following
triggering of B cells through the surface antigen receptor, Ras, Raf-1,
and MEK are all activated and participate in p42 activation. Following stimulation with anti-IgM, Ras activation
was observed, and the ability of Raf-1 to phosphorylate recombinant and
kinase-inactive MEK was increased. In parallel, MEK activity toward
kinase-active or -inactive recombinant MAP kinase also increased. Under
conditions where anti-IgM increased phosphorylation of
p42
, we were unable to detect any activation of JNK in
either a B lymphoblastoid cell line or freshly isolated tonsillar B
cells.
In contrast to anti-IgM, the addition of anti-CD40 antibody
or recombinant soluble gp39 activated JNK in a dose- and time-dependent
fashion. Qualitatively, the results were similar when studied in Ramos
cells or tonsillar B cells. The higher level of JNK activation in Ramos
cells may reflect higher levels of CD40 expression on the B cell line
compared with freshly isolated tonsillar B cells(42) . As
reported previously (43) for activation of NF-B or B cell
proliferation, soluble gp39 resulted in greater increases in JNK
activation than did the addition of anti-CD40 antibody. This may be due
to the fact that soluble gp39 is highly aggregated, resulting in
oligomerization of CD40 and in a stronger signal. Activation of B
lymphocytes through CD40 did not result in any detectable increase in
phosphorylation or activation of p42
or its downstream
substrate, p90
. Furthermore, the addition of anti-CD40
failed to affect Ras or Raf-1 activation. These data indicate that
signaling of B cells through CD40 leads to JNK activation by a
Ras-independent pathway.
These results are similar to what has been
described for TNF- stimulation of PC12 cells(40) . In
these cells, activation of JNK by epidermal growth factor or nerve
growth factor was dependent on Ha-Ras activation, while activation by
TNF-
was Ras-independent. It appears that Ras activates two
kinases, Raf-1 and MEK kinase (MEKK1)(44) . Although Raf-1
contributes to ERK but not JNK activation(40) , MEKK is
involved in JNK activation(40, 45) . In the absence of
measurable Ras activation, as observed with TNF-
in PC12 cells and
signaling through CD40 in B cells, the pathway leading to MEKK
activation is presently unclear(34, 38) .
The B
cell antigen receptor (surface immunoglobulin) is important for binding
and internalization of antigen as well as transducing signals through
the plasma membrane, resulting in cell activation and
differentiation(1, 2) . Anti-Ig antibodies trigger the
rapid activation of phospholipase C and several tyrosine kinases,
increases in cytosolic Ca concentrations, and
increased transcription of a number of early genes including Egr, c-fos, and
c-myc(46, 47, 48, 49, 50, 51, 52, 53, 54) .
Ligation of CD40 appears to initiate a distinct series of events with
no increase in cytosolic Ca
concentrations and
inconsistent results from different groups on Src kinase
activation(18, 19, 20) . Together with the
absence of Ras activation, it appears that CD40 uses signal
transduction pathways resulting in MEKK and JNK activation distinct
from responses controlled by sIgM. Whether these pathways may involve
other GTP-binding proteins such as Rac, which has been shown to
regulate the JNK pathway(55, 56) , is not known.
The role of JNK activation in CD40-mediated B cell survival is
unclear. CD40 is also involved in B cell activation,
differentiation(57, 58) , and Ig class
switching(12, 13) . It is likely that CD40 signaling
involves more than the activation of JNK sequential protein kinase
pathways. Nonetheless, the identification of JNK as a CD40-regulated
kinase allows, for the first time, for the characterization of
cytoplasmic signal transduction pathways that can influence sIgM
control of the B cell phenotype. This activity of CD40 was independent
of the sIgM activation of p42. In neurons, dominant
negative c-Jun has been shown to block serum deprivation-induced
apoptosis(39) . This finding infers that stimulation of JNK
would contribute to the apoptotic response. This is not the case in B
lymphocytes, where JNK activation is not observed in sIgM-stimulated
apoptosis. Rather, JNK is activated in association with the protective
response mediated by CD40 and even possibly augmented to some degree in
the presence of anti-IgM antibody. The apparent independence of CD40
and sIgM signaling involving MAP kinase pathways suggests that the
integration of the JNK pathway with sIgM responses dramatically alters
the functional response of the B cell. Genetic manipulation of JNK
activation in B cells, including altering the apoptotic response to
sIgM ligation, will define whether this is a dominant pathway in CD40
function.