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Department of Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to the original model of Bretscher and Cohn 18 , B lymphocytes stimulated through their Ag receptor (signal 1) in the absence of T cell help (signal 2) would be tolerized. The relative lack of T cells in the bone marrow that could provide a "second signal" to immature B cells exposed to self Ag suggested that the negative selection of immature B cells occurred because of a lack of T cell help. However, lack of T cell help does not totally account for the relative inability of immature lymphocytes to proliferate in response to Ag; highly purified populations of mature B cells proliferate in response to anti-Ig stimulation in vitro in the absence of T cell help, while immature B cells readily undergo apoptosis under the same stimulatory conditions 19, 20, 21 . However, addition of cytokines, such as IL-4, or ligation of CD40 makes it possible to rescue immature lymphocytes from anti-Ig-induced death, indicating that the availability of T cell help can certainly influence the outcome of negative selection 19, 22, 23 . It has also been suggested that the dichotomy in responses elicited by BCR engagement in immature (IgMhighIgDdull) vs mature (IgMlowIgDhigh) B cells could be due to differential signaling through sIgM and sIgD 24 . However, we have recently reported that ligation of IgD on immature, transitional B cells readily induces apoptosis and fails to protect from anti-IgM-induced apoptosis 20 , indicating that the differential responses of immature and mature B cells cannot be attributed to a "protective" signaling mechanism transduced through sIgD. These results are consistent with studies done in transgenic mice that express either anti-hen egg lysozyme IgM or IgD 25 . In these studies, exposure to Ag led to efficient tolerization in both IgM-only and IgD-only expressing mice, conclusively demonstrating that signaling through IgD does not protect immature B cells from negative selection. Taken together, these observations suggest that the differential sensitivity to negative selection of the immature and mature B lymphocyte must be due to intrinsic, developmentally regulated differences in the Ag-receptor-mediated signal transduction pathways.
Ag-receptor ligation of mature B cells leads to the activation of
multiple signaling pathways, including those involving tyrosine kinases
and phosphatases, Ras and mitogen-activated protein kinases
(MAPKs), and phosphoinositide-3 kinase 26 . Activation of the src
family kinases following BCR engagement leads to the eventual tyrosine
phosphorylation and subsequent activation of phospholipase C 
27, 28, 29 , resulting in the breakdown of polyphosphoinositide bis
phosphate (PIP2) into the second messengers inositol
triphosphate (IP3) and diacylglycerol (DAG) 30, 31 .
IP3 is believed to be responsible, at least in part, for
the release of calcium from intracellular stores, while DAG can
activate a subset of PKC isoenzymes, including the conventional
isoenzymes, PKC-
,- ß, and - and the "novel" isoenzymes,
PKC-
and PKC-
32 . While it is not known what roles individual
PKC isoenzymes may play in B cell activation, it is clear that they are
not completely interchangeable. PKC-ß-deficient mice exhibit a
phenotype that is similar to the xid immunodeficient mouse
strain in that they display a reduced primary response to T
cell-dependent Ags, as well as a decreased response to T
cell-independent type II Ags 33 . Although these mice presumably
express other PKC isoenzymes, they appear to be unable to replace
PKC-ß in signaling through the BCR, thus demonstrating a unique
requirement for PKC-ß in B cell activation.
Previous studies from our laboratory have demonstrated that although mature B cells respond to BCR cross-linking by increasing PIP2 hydrolysis and elevating intracellular-free calcium levels, immature B cells increase intracellular calcium levels in the relative absence of PIP2 hydrolysis 34, 35 . Such findings suggest that while DAG-responsive PKC isoenzymes may be activated in mature B cells following sIg cross-linking, they are unlikely to be activated in response to BCR-induced signal transduction in immature B cells. These findings led us to propose a hypothesis in which an imbalance in BCR-induced signal transduction due to the relative inability of immature B cells to activate DAG-responsive PKC isoenzymes may be responsible for the BCR-induced apoptosis of immature B cells. In this report, we present evidence that suggests that activation of PKC can rescue immature B cells from BCR-induced apoptosis. Furthermore, mature B cells that are either depleted of PKC or are stimulated in the presence of PKC inhibitors are rendered susceptible to BCR-induced apoptosis, suggesting that a relative inability to activate PKC following BCR engagement is responsible for the apoptotic response. These results suggest that an uncoupling of PKC from BCR-induced signal transduction may, therefore, play an integral role in the negative selection of developing B lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice used in experiments were BALB/c, obtained from The
Jackson Laboratory (Bar Harbor, ME), and maintained in our colony. For
autoreconstitution studies, adult mice 8–10 wk of age were subjected
to 500 rad of whole-body irradiation, then the peripheral lymphoid
compartments were allowed to reconstitute over a period of 13–15 days,
as described 7 . F(ab')2 fragments of polyclonal rabbit
anti-mouse IgM were created in our laboratory and are described
previously 36 . PMA, phorbol dibutyrate (PDBu), 4- and 4ß-
phorbol didecanoate (PDD), and thapsigargin were purchased from Sigma
(St. Louis, MO), and ionomycin was purchased from Calbiochem (SanDiego,
CA). Polyclonal anti-PKC, -PKCß, -PKC, -PKC, and -PKC
were purchased from Life Technologies (Gaithersburg, MD).
12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), a
PKC-ß1-specific activator was purchased from Biomol Research Labs
(Plymouth Meeting, PA), while the PKC inhibitors bisindolylmaleimide I
and Ro-32-0432 were purchased from Calbiochem.
B lymphocyte purification
Splenic B cells were prepared as previously described 19 . Briefly, adult mice were killed by cervical dislocation at 8–10 wk of age. Spleens were removed aseptically and placed in HBSS + 2% FCS. Single cell suspensions were prepared by grinding spleens between the frosted ends of glass slides. To remove T cells, the cells were treated with anti-Thy1.2 (HO-13-4) 37 , rabbit complement, and DNase I. RBC were lysed by treatment with Gey’s solution, and the cells were size fractionated by passage over a 50/75% Percoll gradient (Pharmacia Biotech, Piscataway, NJ). B cells from autoreconstituting mice were isolated in an analogous manner. In autoreconstituting mice, B cell populations are regenerated in a relatively synchronous wave 7, 8 . Late-stage immature or transitional-stage B cells (IgMhigh, IgDlow, B220int, heat-stable Ag (HSA)high) can be found in the spleens of autoreconstituting mice as early as day 13 and persist as long as day 18. After this time, the cells begin to acquire the phenotypic characteristics of mature B cells (IgMlow, IgDhigh, B220high, HSAlow) and after day 21, respond functionally as do mature cells (L.B.K., unpublished observation).
Apoptosis assay
Purified B cells were cultured under various conditions overnight in either sterile 2-ml click-cap tubes or 96-well plates. When utilized, PKC inhibitors were added 15 min before the addition of the stimulant. After 14–18 h in the case of immature B cells or 24 h in the case of mature B cells, the cells were harvested and washed in PBS containing 2% FCS. Cells were fixed and permeabilized by resuspending in 70% ethanol made in 1x PBS and incubating at -20°C for at least 2 h. After incubation, the cells were washed twice with PBS + 2% FCS and finally resuspended in a staining solution made in 1x PBS containing 50 µg/ml of RNase A, 0.01% sodium azide, and 10 µg/ml of propidium iodide (PI). The cells were stained at 4°C overnight and analyzed by flow cytometry on a Becton Dickinson (Mountain View, CA) FACScan using LYSYS II software. The mean percentages of apoptotic cells and the SEs for each culture condition were determined and significance assessed by the Student’s two-tailed t test.
Measurement of intracellular calcium
Purified B cells were washed in HBSS without phenol red and supplemented with 2% FCS (HBSS + 2% FCS). For loading, cells were resuspended in HBSS without phenol red and supplemented with 10% FCS (HBSS + 10% FCS). Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) was added to a final concentration of 1 µM from a 1 mM stock made in DMSO. Cells were incubated in light tight tubes for 30 min at 30°C with occasional inversion. Cells were then washed twice with HBSS + 2% FCS and resuspended at a concentration of 3.3 x 107/ml in HBSS + 10% FCS. Fluorometric analysis of dyed cells was performed using a luminescence spectrometer (model LS50B; Perkin-Elmer, Norwalk, CT). Excitation was at 340 nM and 380 nM with emission monitored at 510 nM. B cells were diluted to 2 x 106/ml in HBSS and equilibrated for 2 min, at which time a baseline spectrum was read. The cells were stimulated with rabbit anti-mouse IgM F(ab')2 fragments (50 µg/ml) and the response measured. Cells were then lysed with Triton X-100 to determine Fmax, and Fmin was measured by the chelation of extracellular calcium with the addition of EGTA. Calcium traces were calculated by the Intracellular Biochemistry Application (Perkin-Elmer, Norwalk, CT) using these values.
Western blot analysis
For analysis of postnuclear extracts, 1 x 107 cells were lysed in 50 µl of Triton-X lysis buffer (0.1% Triton X-100; 10 mM Tris (pH 8.0); 10.8 µg/ml of aprotinin; 1.5 µg/ml each of leupeptin, pepstatin A, chymostatin, and antipain; 2 mM phenylmethylsulfonyl fluoride; and 1 mM N-ethylmaleimide) on ice for 30 min. Lysates were microcentrifuged at 14,000 x g at 4°C for 10 min to remove the Triton X-100 insoluble fraction. Lysates from equivalent cell numbers were fractionated on SDS-PAGE gels and electroblotted onto Immobilon (Millipore, Bedford, MA). Blots were incubated overnight in Tris-buffered saline with Tween (TBST) (10 mM Tris (pH 8), 137 mM NaCl, 0.05% Tween 20) plus 2% BSA to block nonspecific binding. Membranes were washed twice with TBST and probed with the appropriate Ab solutions for 90 min, then washed three times in TBST, followed by secondary incubation with the appropriate horseradish peroxidase-conjugated reagents. Three washes with TBST were conducted before detection with the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Modified TUNEL assay
In some cases, the frequency of apoptotic cells was quantitated using a modified TUNEL assay 38 . Briefly, 1 x 106 cells were fixed in 2.5% formaldehyde in 1x PBS. The fixed cells were permeabilized in 0.2 ml of 0.1% Triton/0.1% citrate for 2 min on ice. After washing, cells were incubated in 50 µl of nick translation reagent, which consists of 5 µl nick translation buffer (500 mM Tris (pH 7.5)/100 mM MgSO4/1 mM DTT), 0.03 µl of fluorescein-dUTP (1 nmol/µl; Boehringer Mannheim, Indianapolis, IN), 1 µl of dTTP (0.7 nmol/µl), 1 µl each of dATP, dCTP, dGTP (1 nmol/µl), and 0.1 µl of DNA polymerase (5 U/µl) from Boehringer Mannheim (Indianapolis, IN), and incubated for at least 1 h at 37°C. Cells were then washed and analyzed by flow cytometry on a Becton Dickinson FACScan.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immature B cells undergo apoptosis in response to BCR cross-linking in vitro
Purified populations (>85% B220+IgM+) of
either immature B cells obtained from the spleens of day 14
autoreconstituting mice or mature B cells isolated from the spleens of
normal adult mice were incubated in the absence or presence of 50
µg/ml of rabbit anti-mouse IgM F(ab')2 fragments
(anti-Ig) for 16–18 h. As we have previously shown 19, 20 ,
addition of anti-Ig to immature B cells results in a substantial
increase in the frequency of apoptotic cells as judged by the increased
number of cells containing subdiploid amounts of DNA (detected by flow
cytometry following propidium iodide staining of ethanol fixed and
permeabilized cells) (Figs. 1
,
A and B). The detection of apoptotic cells using
this assay closely correlates with results obtained using a modified
TUNEL technique that detects nicked DNA 38 (data not shown). In
contrast, overnight culture of mature splenic B cells in the presence
of anti-Ig resulted in only a marginal increase in the frequency of
apoptotic cells over that observed in unstimulated cultures (Figs. 1, C and D). These results reconfirm that, in the
absence of T cell help, immature and mature B cells respond
differentially to BCR signaling and that immature B cells are
susceptible to BCR-induced apoptosis, a mechanism that may account for
their deletion in vivo following exposure to self Ag.
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Mature B cells that proliferate in response to BCR engagement
increase intracellular calcium levels and hydrolyze PIP2 in
response to BCR cross-linking. In contrast, immature B cells are
susceptible to BCR-induced apoptosis and respond to specific
Ag-receptor signaling in the absence of detectable PIP2
hydrolysis 35 . Based on these results, a hypothesis was proposed that
stated that an "imbalance" in BCR-induced signal transduction in
immature B cells in which the activation of DAG-dependent conventional
and novel PKC isoenzymes 39, 40 might be compromised, may result in
their enhanced sensitivity to BCR-induced apoptosis. If the inability
to activate PKC in response to BCR cross-linking was directly linked to
the induction of apoptosis, it would suggest that pharmacologic
activation of PKC in immature B lymphocytes should protect them from
anti-Ig-induced death. To test this hypothesis, isolated immature B
cells were cultured with the phorbol ester, PMA, which bypasses the Ag
receptor and activates DAG-dependent PKC isoenzymes directly 40 .
Late-stage immature B cells were cultured for 18 h in the
presence of medium alone, anti-IgM F(ab')2 fragments
(50 µg/ml), PMA (10 ng/ml) or the combination of anti-Ig and PMA.
After culture, the cells were harvested and assayed for relative DNA
content. As shown in Fig. 2, activation
of PKC in the immature B cell population resulted in significant
protection from anti-IgM-induced apoptosis. The ability of phorbol
esters to activate PKC is critical for the observed response; in
contrast to the biologically active stereoisomer 4ß-PDD, which
efficiently rescued the cells, the inactive stereoisomer 4PDD, did
not (Fig. 2). These observations suggest that while BCR-induced
signaling events can lead to cell death, activation of PKC is
sufficient to rescue the cell from apoptosis.
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B, addition of
DOPPA rescues immature B cells from BCR-induced apoptosis in a
dose-specific manner and at doses that correspond well to the amount
required for PKC-ß activation in vitro 41 . These results suggest
that PKC-ß activation may play an important role in rescuing immature
B cells from BCR-induced apoptosis. However, it must be pointed out
that while some groups have observed that DOPPA maintains its selective
activation of PKC-ß in vivo 42, 43 , other groups have noted the
activation of other PKC isoenzymes, perhaps due to the deacetylation of
DOPPA 44 . Even with this caveat, these data support the hypothesis
that the inability to activate PKC (and perhaps PKC-ß) upon ligation
of the BCR is mechanistically involved in the negative selection of
immature B cells in response to BCR engagement. Depletion of PKC in mature splenic B lymphocytes renders them susceptible to anti-Ig-mediated negative selection
The relative inability of immature B cells to increase
PIP2 hydrolysis in response to BCR ligation, coupled with
the ability of PMA to rescue immature B cells from BCR-induced
apoptosis, suggested that BCR-induced activation of PKC may be
necessary to prevent anti-Ig-induced death. Therefore, we next
determined if PKC-depleted mature B cells are susceptible to
BCR-induced apoptosis. Chronic exposure to high doses (100 ng/ml) of
the phorbol ester PDBu results in a depletion of PKC activity, at least
in part, through an increased rate of degradation of the enzyme 45 .
Previous studies have demonstrated that primary splenic B cells are
susceptible to PKC depletion and that PKC-depleted B cells are no
longer able to proliferate in response to anti-Ig 46, 47 . PDBu
was utilized in these studies because it is effective in depleting PKC,
yet it is less hydrophobic than PMA and, thus, can be removed from the
cultures by washing 48 . PKC depletion in PDBu- or mock-treated
cultures were monitored by Western blot analysis (Fig. 3). The two conventional, phorbol
ester-responsive isoenzymes of PKC expressed in B cells, PKC- and
-ß, are efficiently depleted in PDBu-treated cells, while the two
novel isoenzymes, PKC- and -, are also depleted, although PKC-
is depleted somewhat less efficiently (Fig. 3). As predicted, the level
of expression of the lower m.w. phorbol ester-insensitive PKC- was
decreased only slightly 49 , while a higher m.w. PKC isoenzyme
detected by this particular anti-PKC- antiserum (previously
reported to be a partially characterized phorbol ester-sensitive
isoenzyme 50, 51) was also depleted by PDBu. As can also be seen, PKC
levels in unstimulated immature and mature cells were quite similar,
making it unlikely that differential regulation of PKC activity would
occur at the level of expression of the individual isoenzymes.
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-PDD.
After an 8-h incubation, the cells were harvested and subjected to a
modified TUNEL analysis. While mock-depleted B cells did not show
increases in the percentages of apoptotic cells in any of the culture
conditions, a strikingly different result was observed with the
PDBu-treated B cells. After PKC-depletion, mature B cells were
specifically susceptible to anti-Ig-mediated apoptosis (Fig. 4). These data suggest that an uncoupling
of PKC activation from BCR-induced signal transduction is sufficient to
render mature B cells sensitive to anti-Ig-induced apoptosis.
Interestingly, PMA was still capable of rescuing the cells from
anti-Ig-induced apoptosis, perhaps because of its ability to
activate the very low residual amounts of the conventional isoenzymes
(PKC- and -ß) or the more substantial amounts of the novel phorbol
ester-responsive isoenzymes, PKC- and - (Fig. 4). Thus, it
appears that the BCR-induced activation of PKC in PDBu-treated mature B
cells is insufficient to protect them from BCR-induced cell death.
However, activation of PKC by PMA, leading to either more efficient
activation of PKC or to the activation of a subset of PKC isoenzymes
that are not normally coupled to BCR-induced signal transduction
events, is sufficient to protect PKC-depleted mature B cells from
apoptosis. The hypothesis that only a subset of PMA-inducible PKC
isoenzymes (perhaps the conventional isoenzymes PKC- and -ß that
are efficiently depleted following PDBu treatment) are normally coupled
to BCR signal transduction is substantiated by the nonredundant nature
of the PKC-ß isoenzyme in mature B cell signal transduction observed
in PKC-ß-deficient mice 33 . Interestingly, mature B cells from
PKC-ß-deficient mice proliferate normally in response to phorbol
ester and calcium ionophore, suggesting that while other PKC isoenzymes
are capable of promoting a proliferative signal, only PKC-ß is
activated to induce proliferation following BCR ligation.
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While phorbol ester treatment depletes conventional and novel PKC
isoenzymes in mature B cells, this depletion is preceded by activation
of these phorbol ester-responsive isoenzymes. To assure that this prior
PKC activation was not rendering mature B cells susceptible to
BCR-induced apoptosis, we utilized two PKC inhibitors,
bisindolylmaleimide I, an inhibitor of conventional and novel PKC
isoenzymes 52 , and Ro-32-0432, an inhibitor with a greater
selectivity for the conventional isoenzymes, PKC- and -ß 53 .
Addition of these inhibitors to mature B cells 15 min before
stimulation with anti-Ig lead to the induction of apoptosis (Fig. 5A). This enhanced cell death
was not due to toxic effects because the frequency of apoptosis in
unstimulated or LPS-stimulated mature B cells was not affected
(Fig. 5A).
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B). Again, these
inhibitors were relatively nontoxic, as the frequency of apoptotic
cells in unstimulated or LPS-stimulated immature B cells was
relatively unaffected (Fig. 5B). The ability of PKC
inhibitors to reduce PMA-induced protection of immature B cells from
BCR-induced apoptosis and render mature B cells sensitive to
BCR-induced apoptosis greatly supports our hypothesis that PKC plays an
active role in preventing BCR-induced apoptosis and that PKC activation
is differentially coupled to BCR-induced signal transduction events
in immature and mature B cells. Phorbol ester-induced receptor desensitization does not totally account for the ability of PMA to rescue cells from BCR-induced death
It has previously been shown that phorbol ester treatment of
mature B cells can result in a decrease in calcium influx and
PIP2 hydrolysis in response to subsequent BCR ligation
54, 55, 56, 57 , an effect known as receptor desensitization. This suggested
that PMA may be rescuing anti-Ig-stimulated immature B cells by
reducing the early BCR-induced signal transduction events following BCR
ligation. Since calcium influx is one of the earliest of these events,
we monitored the effect of PMA on BCR-induced calcium influxes. Fura
2-AM-loaded immature B cells were prestimulated with medium, PMA, or
4-PDD for 5 min and then stimulated with 50 µg/ml of anti-Ig.
As shown in Fig. 6A, acute
stimulation with the active compound PMA could indeed reduce
BCR-induced increases in intracellular calcium levels in immature B
cells, whereas the inactive 4-PDD did not. This data confirmed
studies by others showing that PMA can desensitize the BCR with respect
to its coupling to calcium flux when it is present at the onset of BCR
stimulation.
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B), it did not alter the BCR-induced calcium
flux in PDBu-treated cells at all (Fig. 6C), indicating that
the ability of PMA to rescue B cells from BCR-induced death does not
result from receptor desensitization. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that mature B cells that have either been depleted of PKC
or are stimulated with anti-Ig in the presence of PKC inhibitors
are rendered sensitive to BCR-induced apoptosis strengthened our
hypothesis that an "imbalance" in BCR-induced signal transduction
events resulting from a relative inability to activate PKC play a role
in the induction of B cell apoptosis following receptor engagement.
Specifically, PDBu treatment of splenic B cells, which results in
markedly reduced levels of PKC- and -ß and PKC- expression,
renders them susceptible to anti-Ig-induced apoptosis, suggesting
that uncoupling of the conventional, and perhaps novel, PKC isoenzymes
from BCR-induced activation events plays a critical role in this
process. The ability of PMA to rescue PKC-- and PKC-ß-depleted
mature B cells from anti-Ig-induced death suggests that perhaps the
novel DAG-responsive isoenzyme of PKC (PKC-) is still efficiently
activated by phorbol ester and is capable of rescuing the cells from
cell death. The relative inability of the residual amounts of
DAG-responsive PKC isoenzymes found in PDBu-treated mature B cells to
rescue mature B cells from apoptosis following BCR engagement may
reflect either the inefficient coupling of a subset of these isoenzymes
to the BCR or to the more efficient activation of these isoenzymes by
PMA than by DAG (generated during BCR-induced signaling) 58 . Support
for the suggestion that anti-Ig may activate a subset of PKC
isoenzymes, while PMA may activate all conventional and novel PKC
isoenzymes, comes from the observation that the induction of the
immediate early gene, egr-1 is markedly higher following
stimulation with PMA relative to induction by cross-linking sIg 59, 60 . In addition, the dramatic effect on B cell function observed in
PKC-ß-deficient mice indicates that PKC-ß is directly coupled to
the BCR and that other PKC isoenzymes are not able to compensate for
its function following receptor engagement although they are capable of
doing so when activated pharmacologically 33 . The relative inability
of PKC-ß-deficient mice to mount a response to a T-independent type
II Ag or an efficient primary response to a T-dependent Ag in vivo or
to proliferate to anti-Ig in vitro suggests that the activation of
PKC-ß is specifically regulated by BCR engagement and is critical for
B cell proliferation. It remains to be tested whether this lack of
response is due to inefficient signal transduction, an inability to
proliferate, or to induced apoptosis, as our results might suggest.
Stimulation with PMA did not result in BCR down-regulation on immature B cells (data not shown), ruling out one trivial explanation for why PMA might be able to rescue immature B cells from BCR-induced apoptosis. However, short-term stimulation with PMA is known to desensitize the Ag receptor of mature B cells and prevent the induction of both calcium flux and PIP2 hydrolysis 54, 55, 56, 57 through an, as yet, unknown mechanism. While this phenomenon could potentially explain the ability of PMA to rescue immature B cells from BCR-induced death, it appears to only partially account for the ability of PKC activation to inhibit apoptosis. First, PMA can reverse the apoptosis-inducing effects of both ionomycin and thapsigargin, agents that increase intracellular calcium levels, but do so by bypassing the BCR (data not shown). Second, mature B cells that have been treated overnight with PDBu are susceptible to BCR-induced death and can be rescued by PMA, but PMA does not desensitize the BCR in these cells. Thus, PKC activation appears to exert its effects at points downstream of the BCR as well as at the level of receptor desensitization. It is also possible that PKC activation may engage cell survival pathways. For instance, both Bcl-2 and Bad, a molecule that can heterodimerize with Bcl-x and promote cell death, can be regulated at the level of phosphorylation 61, 62 .
The mechanisms by which primary immature B cells undergo apoptosis
stand in apparent contrast to those observed in cell lines such as
WEHI-231 and DT40 that are thought to serve as model systems for the
negative selection of immature B cells. In these systems, death is
associated with the activation of phospholipase C , leading
to an increase in both PKC activity and intracellular calcium levels
34, 54, 63 . It is not clear why the two systems differ, but it may
reflect differences between primary cells that have not yet entered the
cell cycle compared with actively proliferating tumor cells.
Briefly summarized, the data presented here and in previous reports 35 have demonstrated that cross-linking the Ag receptor of immature B cells initiates intracellular biochemical changes. Thus, the induction of apoptosis is not associated with the absence of signal transduction in immature B cells, but rather it is an active process that is initiated by BCR cross-linking and subsequent signaling events. Immature B cells have a specific Ag-receptor-associated signaling phenotype; cross-linking the BCR leads to increases in intracellular calcium concentrations in the absence of PIP2 hydrolysis 35 . The ability of PMA treatment to inhibit the anti-IgM mediated apoptosis, as well the sensitivity of PKC-depleted mature splenic B cells to anti-Ig-induced apoptosis, implicates the activation of PKC with protection from anti-Ig-induced apoptosis. Future efforts will focus on the identification of the specific PKC-isoenzymes activated by stimulation through the Ag receptor and characterizing the specific downstream actions of PKC that mediate the inhibition of anti-Ig-mediated apoptosis and, thus, regulate the process of negative selection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leslie B. King, University of Pennsylvania School of Medicine, Room 538A, Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: BCR, B cell Ag receptor; PIP2, polyphosphoinositide bis phosphate; PKC, protein kinase C; s, surface; DAG, diacylglycerol; PI, propidium iodide; PDBu, phorbol dibutyrate; PDD, phorbol didecanoate; DOPPA, 12-deoxyphorbol 13-phenylacetate 20-acetate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling; HSA, heat-stable Ag.
Received for publication September 1, 1998. Accepted for publication November 20, 1998.
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References |
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2 activation in surface immunoglobulin M-induced B cell apoptosis. J. Exp. Med. 182:907.This article has been cited by other articles:
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 P. A. Welch, V. P. Sinha, A. L. Cleverly, C. Darstein, S. D. Flanagan, and L. C. Musib Safety, Tolerability, QTc Evaluation, and Pharmacokinetics of Single and Multiple Doses of Enzastaurin HCl (LY317615), a Protein Kinase C-{beta} Inhibitor, in Healthy Subjects J. Clin. Pharmacol., September 1, 2007; 47(9): 1138 - 1151. [Abstract] [Full Text] [PDF]
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A. Brauweiler, K. Merrell, S. B. Gauld, and J. C. Cambier Cutting Edge: Acute and Chronic Exposure of Immature B Cells to Antigen Leads to Impaired Homing and SHIP1-Dependent Reduction in Stromal Cell-Derived Factor-1 Responsiveness J. Immunol., March 15, 2007; 178(6): 3353 - 3357. [Abstract] [Full Text] [PDF]
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J. Ke, M. Gururajan, A. Kumar, A. Simmons, L. Turcios, R. L. Chelvarajan, D. M. Cohen, D. L. Wiest, J. G. Monroe, and S. Bondada The Role of MAPKs in B Cell Receptor-induced Down-regulation of Egr-1 in Immature B Lymphoma Cells J. Biol. Chem., December 29, 2006; 281(52): 39806 - 39818. [Abstract] [Full Text] [PDF]
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K. L. Hoek, P. Antony, J. Lowe, N. Shinners, B. Sarmah, S. R. Wente, D. Wang, R. M. Gerstein, and W. N. Khan Transitional B Cell Fate Is Associated with Developmental Stage-Specific Regulation of Diacylglycerol and Calcium Signaling upon B Cell Receptor Engagement J. Immunol., October 15, 2006; 177(8): 5405 - 5413. [Abstract] [Full Text] [PDF]
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D. H. Li, J. W. Tung, I. H. Tarner, A. L. Snow, T. Yukinari, R. Ngernmaneepothong, O. M. Martinez, and J. R. Parnes CD72 Down-Modulates BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes J. Immunol., May 1, 2006; 176(9): 5321 - 5328. [Abstract] [Full Text] [PDF]
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 R. H. Carter B Cells in Health and Disease Mayo Clin. Proc., March 1, 2006; 81(3): 377 - 384. [Abstract] [Full Text] [PDF]
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V. Roy, N.-H. Chang, Y. Cai, G. Bonventi, and J. Wither Aberrant IgM Signaling Promotes Survival of Transitional T1 B Cells and Prevents Tolerance Induction in Lupus-Prone New Zealand Black Mice J. Immunol., December 1, 2005; 175(11): 7363 - 7371. [Abstract] [Full Text] [PDF]
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 A. Bredberg, G. Henriksson, A. Larsson, R. Manthorpe, and A. Sallmyr Sjogren's syndrome and the danger model Rheumatology, August 1, 2005; 44(8): 965 - 970. [Full Text] [PDF]
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F. G. Karnell, R. J. Brezski, L. B. King, M. A. Silverman, and J. G. Monroe Membrane Cholesterol Content Accounts for Developmental Differences in Surface B Cell Receptor Compartmentalization and Signaling J. Biol. Chem., July 8, 2005; 280(27): 25621 - 25628. [Abstract] [Full Text] [PDF]
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D. Mielenz, C. Vettermann, M. Hampel, C. Lang, A. Avramidou, M. Karas, and H.-M. Jack Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition J. Immunol., March 15, 2005; 174(6): 3508 - 3517. [Abstract] [Full Text] [PDF]
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 S. Hussain, K. V. Salojin, and T. L. Delovitch Hyperresponsiveness, Resistance to B-Cell Receptor--Dependent Activation-Induced Cell Death, and Accumulation of Hyperactivated B-Cells in Islets Is Associated With the Onset of Insulitis but not Type 1 Diabetes Diabetes, August 1, 2004; 53(8): 2003 - 2011. [Abstract] [Full Text] [PDF]
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S. L. Christian, R. L. Lee, S. J. McLeod, A. E. Burgess, A. H. Y. Li, M. Dang-Lawson, K. B. L. Lin, and M. R. Gold Activation of the Rap GTPases in B Lymphocytes Modulates B Cell Antigen Receptor-induced Activation of Akt but Has No Effect on MAPK Activation J. Biol. Chem., October 24, 2003; 278(43): 41756 - 41767. [Abstract] [Full Text] [PDF]
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J. B. Chung, A. D. Wells, S. Adler, A. Jacob, L. A. Turka, and J. G. Monroe Incomplete Activation of CD4 T Cells by Antigen-Presenting Transitional Immature B Cells: Implications for Peripheral B and T Cell Responsiveness J. Immunol., August 15, 2003; 171(4): 1758 - 1767. [Abstract] [Full Text] [PDF]
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R. J. Benschop, E. Brandl, A. C. Chan, and J. C. Cambier Unique Signaling Properties of B Cell Antigen Receptor in Mature and Immature B Cells: Implications for Tolerance and Activation J. Immunol., October 15, 2001; 167(8): 4172 - 4179. [Abstract] [Full Text] [PDF]
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X. Li, F. Martin, A. M. Oliver, J. F. Kearney, and R. H. Carter Antigen Receptor Proximal Signaling in Splenic B-2 Cell Subsets J. Immunol., March 1, 2001; 166(5): 3122 - 3129. [Abstract] [Full Text] [PDF]
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M.-Y. Cao, F. Shinjo, S. Heinrichs, J.-W. Soh, J. Jongstra-Bilen, and J. Jongstra Inhibition of Anti-IgM-induced Translocation of Protein Kinase C beta I Inhibits ERK2 Activation and Increases Apoptosis J. Biol. Chem., June 29, 2001; 276(27): 24506 - 24510. [Abstract] [Full Text] [PDF]
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