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,
*
Division of Immunology, Department of Medicine, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021; Departments of
Microbiology and
Medicine, Boston University School of Medicine, Boston, MA 02118; and
Center for Immunology, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel transcription factor family has been shown to
protect many cell types from apoptotic signals. However, it is not
known whether NF-B is required for all survival pathways and whether
each NF-B member plays a unique or a redundant role. Here we
describe the results of studies on the role of c-Rel in survival.
Mature B cells from c-Rel-/- mice exhibit defects in
survival, including sensitivity to Ag receptor-mediated apoptosis as
well as increased sensitivity to ionizing radiation and
glucocorticoids. Transgene expression of Bcl-xL, a c-Rel
target gene, rescues c-Rel-/- B cells from their survival
defects. Thus, c-Rel-dependent survival pathways are crucial for
protection from apoptotic signals that target the mitochondrial
pathway. Despite a lack of Bcl-xL, c-Rel-/- B
cells can still be rescued from Fas-mediated apoptosis via B cell
receptor signaling. The Fas apoptosis inhibitor molecule and FLICE
inhibitory protein (c-FLIP) proteins are up-regulated normally in
c-Rel-/- B cells, and these two molecules may play a more
physiological role in the Fas pathway. Furthermore, unlike the TNF
sensitivity of RelA-/- fibroblasts, c-Rel-deficient
fibroblasts are refractory to TNF-mediated cell death. Thus, c-Rel is
dispensable for protection against death receptor-mediated apoptosis.
Taken together, our data suggest that distinct NF-B/Rel members are
required for protecting cells from different types of apoptotic
signals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B lymphocytes are subject to apoptotic regulation by many death signals, including the B cell Ag receptor, Fas, gamma irradiation, and glucocorticoids (5). For example, the B cell lymphoma lines WEHI231 and CH31 have been used extensively in the studies of anti-IgM-induced cell death of immature B cells (6, 7, 8). Alternatively, activation of mature B cells by CD40 up-regulates Fas expression and can sensitize them to Fas-mediated apoptosis. Such CD40-sensitized Fas-mediated death was found to be protected by Ag receptor signaling (reviewed in Ref. 9). Subsequently, several survival proteins, including Bcl-xL, FAIM, and c-FLIP, were shown to be responsible for anti-IgM-mediated protection mechanisms (4, 10, 11).
NF-B/Rel, a family of dimeric transcription factors, has been shown
to protect many cells from apoptotic signals (reviewed in Ref.
12). For example, the RelA knockout mouse fails to develop
beyond embryonic day 15 and is characterized by massive apoptosis in
the liver (13). This death was shown to be mediated mostly
by TNF-
, as RelA/TNF- double knockout mice are rescued from
embryonic lethality and have normal livers (14). There are
also examples where NF-B/Rel was found to be pro-apoptotic. For
example, NF-B/Rel was implicated in the apoptosis of
CD4+CD8+ double positive
thymocytes, as double positive thymocytes expressing the IB
super-repressor were protected from anti-CD3-mediated apoptosis
(15). In embryonic fibroblasts, RelA was shown to play a
role in Fas-induced death (16), and in NIH-3T3 cells it
was shown to be important for expression of Fas as induced by TNF-
(17). Thus, the contribution of NF-B/Rel in either
protecting or inducing apoptosis has to be considered in the context of
stimuli, cell types, and differentiation stages of the cells.
Much effort has been focused on characterizing the pro-survival genes
that are up-regulated by NF-B/Rel. Some of the genes identified
include TNFR-associated factor 1, TNFR-associated factor 2, the Bcl-2
family members Bcl-xL (18, 19) and
A1/Bfl1 (20, 21), IEX-1L, and the inhibitor of apoptosis
proteins (12). However, not all these molecules are
definitive candidates for anti-apoptotic NF-B targets. While
several studies have established a link among NF-B,
Bcl-xL, and survival (18, 19),
others have not (21, 22, 23). These findings are complicated
by the fact that the results were obtained in transformed cell lines of
different tissue types, using various means of activating or inhibiting
NF-B. Therefore, additional studies are necessary to sort out the
specific regulatory relationships among activating signals,
NF-B/Rel, and survival proteins in the context of primary B
lymphocytes.
c-Rel is a lymphoid-specific member of the NF-B/Rel family; thus,
c-Rel knockout (c-Rel-/-) mice provide a unique
system in which to study the requirements for NF-B/Rel in immune
cells. Previous studies have shown that c-Rel-deficient B lymphocytes
fail to receive anti-IgM-mediated activation, proliferation, and
survival signals (24, 25). Furthermore, Myc-transformed
c-Rel-deficient cell lines are still sensitive to apoptosis when
cross-linked with anti-IgM (21). In this case,
however, it is unclear whether the death sensitivity is due to the
transformation by Myc.
In the present study we address the following questions. 1) Is c-Rel
required for protection from all, or just selective, types of apoptosis
in B lymphocytes? 2) What are the survival genes regulated by c-Rel in
primary B cells that are responsible for this protection? Since
transformed B cell lines may not faithfully reflect the survival
requirements of their untransformed counterparts, we decided to examine
the role of c-Rel in the modulation of survival/apoptosis using primary
splenic B cells derived from c-Rel knockout mice. Here we show that
c-Rel-/- B cells, when compared with wild-type
cells, are more sensitive to anti-IgM-, 
irradiation-, and
dexamethasone-induced death even in the presence of survival signals
such as LPS or anti-CD40. We also confirm
Bcl-xL and A1/Bfl1 as bonafide c-Rel target genes
in mature splenic B cells. By introducing a
Bcl-xL transgene into the
c-Rel-/- background, we were able to rescue the
survival defect in B cells. Interestingly, we find that
c-Rel-/- B cells have no defect in their
ability to be rescued from CD40-sensitized Fas-mediated apoptosis.
These results indicate that while c-Rel and its target genes are
required for B cell protection against certain forms of programmed cell
death, c-Rel and Bcl-xL may not play as large a
protective role in the Fas pathway. The implications of these findings
in the context of the regulation of apoptosis are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-Rel-/- mice were generated in the C57BL/6 background as described previously. They were subsequently crossed with the B cell-restricted Bcl-xL transgenic mice (26) (from T. W. Behrens). c-Rel+/-;Bcl-xL transgenic (Tg) F1 mice were intercrossed to generate the c-Rel-/-;Bcl-xL Tg F2 progeny.
Cell culture and proliferation assays
B cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin, and 50 µM 2-ME. B cells were purified by complement-mediated lysis in the presence of J1J (anti-Thy1.2; a gift from Dr. J. Nikolic-Zugic, Sloan-Kettering Institute, New York, NY), GK1.5 (anti-L3T4), and 3.115 (anti-Ly2) to remove T cells. Enriched B cell populations were >95% B220+. For proliferation assays, B cells were plated at 105 cells in 96-well U-bottom plates in triplicate. Where indicated, cells were stimulated with 5 µg/ml LPS (Sigma-Aldrich, St. Louis, MO), 10 µg/ml goat anti-mouse IgM F(ab')2 (anti-IgM, Jackson ImmunoResearch Laboratories, West Grove, PA), or 10 µg/ml anti-CD40 (mAb 1C10 provided by Drs. M. Howard (Corixa, Seattle, WA) and A. Heath (University of Sheffield Medical School, Sheffield, U.K.)). Before the indicated time points, cultures were incubated with 0.5 µCi [3H]thymidine (Amersham, Arlington Heights, IL). Cells were then harvested, and incorporation into DNA was quantified by scintillation.
T cells
The T cell clone 2C2 was provided by Dr. A. Houghton. T cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin, 1 mM nonessential amino acids, and 50 µM 2-ME. They were maintained by stimulating with irradiated splenocytes pulsed with pigeon cytochrome c.
Representational difference analysis (RDA)
RDA was performed essentially as described by Hubank and Schatz (27). Splenocytes from wild-type and c-Rel-/- mice were stimulated with anti-CD40 for 4 h in the presence of 5 µg/ml cycloheximide. Total RNA was isolated using STAT-60 (Tel-Test, Friendswood, TX), and poly(A)+ RNA (isolated by Oligotex, Qiagen, Chatsworth, CA) from these populations was then used to generate cDNA. Three rounds of RDA were performed using wild-type representations as the tester population. Difference product 3 was cloned into pBSKII+. Dot blots yielded 18 independent clones, which were sequenced and compared with the nucleotide and dbEST databases. Inducibility of message was verified by RNase protection.
RNase protection assay
Bcl-xL and A1/Bfl-1 message induction were initially assayed with the mApo-2 RNase protection assay kit (PharMingen, San Diego, CA), according to the manufacturer’s protocol. RNA used was isolated from purified B cells stimulated for 4 h with the indicated reagent in the presence of 10 µg/ml cycloheximide. Five micrograms of total RNA from each condition were used as samples in the assay. Relative induction of Bcl-xL and A1/Bfl1 was determined by phosphorimager analysis, normalized to L32 and GAPDH.
Western blot
Whole cell lysates were generated by lysing cells in 1x RIPA
buffer and sonicating four times with five pulses of a Branson 250
microtip sonicator (VWR International, West Chester, PA) at 20%
duty cycle. Supernatants were separated from pellets and stored at
-80°C. Protein concentrations were determined by Bradford assay.
Twenty to 30 µg whole cell lysate were loaded onto SDS-polyacrylamide
gels and transferred to polyvinylidene difluoride membrane (Millipore,
Bedford, MA) using a semidry method. Blots were probed with the
following Abs diluted into 1% nonfat milk and Tris-buffered saline
(TBS) containing 0.05% Tween 20: rabbit anti-human/mouse
Bcl-xL/S (S-18, sc-634, Santa Cruz Biotechnology,
Santa Cruz, CA), rat anti-human/mouse c-FLIP (Dave-2; Alexis
Biochemicals, San Diego, CA), rabbit polyclonal raised against
full-length FAIM, or anti-mouse actin (Sigma). HRP-conjugated
anti-rat or anti-rabbit secondary Ab was purchased from
Amersham. The ECL Plus chemiluminescence detection system was used to
visualize Western blots (RPN 2132, Amersham). In all experiments equal
protein loading was controlled for by stripping blots in 63 mM Tris-HCl
(pH 6.8), 2% SDS (w/v), and 100 mM 
-ME at 50°C for 30 min,
followed by three washes with TBS containing 0.05% Tween 20, and
reprobing blots with mouse anti-actin Ab (A4700) from Sigma or was
verified using nonspecific signals on the immunoblot for
comparison.
Cell cycle and apoptosis analysis
B cells were cultured at 5 x 105 cells/well in 96-well flat-bottom plates for 2–3 days. Where indicated, cells were treated with 5 µg/ml LPS, 10 µg/ml anti-IgM, or 10 µg/ml anti-CD40. For the irradiation experiments, cells were prestimulated for 18 h before treatment. 137Cs (Gammacell 1000; MDS Nordion, Kanata, Ontario, Canada) was used as the source of gamma irradiation. Dexamethasone stocks were dissolved in ethanol. The final ethanol concentration in culture was 0.004%. At the indicated time points, cells were collected and stained with a solution containing 50 µg/ml propidium iodide (PI), 20 µg/ml RNase A, 0.1% Triton X-100, and 0.1% sodium citrate. Apoptosis was assessed by flow cytometry to quantify the percentage of cells with <2 N DNA content.
Chromium release assays
B cells were stimulated with anti-CD40 for 2 days, with or without overnight stimulation with IgM on the last day. Cells were loaded with 100 µCi sodium chromate (51Cr) for 1 h at 37°C, washed three times, and counted. B cells were distributed in 96-well plates at 5000 cells/well, and 2C2 T cells were added at E:T ratios of 20:1, 10:1, 5:1, and 2.5:1. Con A (Sigma-Aldrich) was added at 2.5 µg/ml. Plates were incubated for 4 h in a 37°C incubator, then spun for 5 min at 1000 rpm. Supernatants were collected and then assayed with a gamma counter. The percent specific lysis was calculated as: (Cr release - minimum release)/(maximum release - minimum release).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced cell death
The importance of NF-B in mediating survival signals was
highlighted by studies in the RelA knockout (28).
RelA-deficient EFs are exquisitely sensitive to TNF-induced cell death.
In contrast, cells from other tissues of the RelA-deficient mice, such
as spleen or heart, are resistant to TNF cytotoxicity despite the
expression of receptors for TNF, TNFR1, and TNFR2
(14).
To test whether all NF-B/Rel members play equal roles in apoptosis
protection, EFs from wild-type and c-Rel-/-
cells were isolated and treated with various doses of TNF- for 3
days. Viability was compared with that of L929 cells, which are very
sensitive to TNF- treatment. Interestingly, unlike
RelA-/- EF cells, both wild-type and
c-Rel-/- EFs are highly resistant to
TNF-induced cell death. L929 cells were sensitive at the lowest dose
used (Fig. 1
a). Thus, c-Rel is
not specifically required to protect fibroblasts from the effects of
TNF-. One possible explanation is that RelA, which is absolutely
required for TNF resistance, could compensate for the lack of c-Rel in
EF cells. These data thus suggest that each NF-B/Rel member plays a
distinct death protection role depending upon its expression levels in
certain cell types and its response to specific stimuli.
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The predominant expression of c-Rel in the lymphoid and myeloid
lineages would predict that it may play a significant role in
lymphocytes. We therefore chose to focus our apoptosis studies on B
cells. Previous studies indicated that c-Rel-/-
B cells fail to be rescued from spontaneous apoptosis by B cell
receptor (anti-IgM) signals (25, 29). Our results
confirm this finding and show that, compared with wild-type cells,
primary B cells from c-Rel-/- mice are
extremely sensitive to anti-IgM-induced death (Fig. 1
b).
In fact, they exhibit more apoptosis than cells cultured in medium
alone, indicating a key role for c-Rel in maintaining the survival of
anti-IgM-stimulated cells.
c-Rel-deficient B cells are more sensitive to
irradiation-induced apoptosis.
Resting B cells are exquisitely sensitive to low doses of
irradiation, and they are protected only after several hours of
activation by stimuli such as LPS (30). To further explore
the role of c-Rel in survival signaling, B cells from wild-type and
c-Rel-/- mice were cultured with medium alone,
LPS, anti-IgM, or anti-CD40 overnight and then exposed to 0,
250, or 500 rad. Unstimulated wild-type and
c-Rel-/- B cells are equally sensitive to gamma
irradiation. Two days after exposure to 250 rad, the viability of the
cultures is <20% (Fig. 2a).
Overnight stimulation with LPS, anti-IgM, or anti-CD40 is able
to significantly rescue wild-type B cells from this induced apoptosis
(Fig. 2, b–d; compare with Fig. 2a). The ability
of these stimuli to rescue is radiation dose-dependent (data not
shown). c-Rel-/- B cells, however, are greatly
impaired in their response to these survival signals even at 250 rad,
the lowest dose tested. Importantly, c-Rel is required for
anti-CD40- or LPS-mediated protection from gamma irradiation (Fig. 2, b and d). As expected, anti-IgM treatment
cannot rescue these c-Rel-/- B cells, and
irradiation promotes even greater levels of apoptosis (Fig. 2c). We hypothesize that LPS, anti-IgM, and
anti-CD40 exert their protective effects by inducing c-Rel to
activate the transcription of anti-apoptotic genes, as all three
stimuli have been shown to induce c-Rel nuclear translocation and DNA
binding (31, 32).
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We next studied the effect of the glucocorticoid dexamethasone on
the survival of c-Rel-/- B cells.
Glucocorticoids are widely used as immunosuppressive agents, and their
mechanism of action has been proposed to be via modulation of
transcription and/or promotion of apoptosis in lymphocytes
(33). Wild-type and c-Rel-/- B
cells were cultured with medium alone, LPS, or anti-CD40, and
dexamethasone (100, 200, or 400 nM) was added to the cultures
immediately after addition of the indicated stimuli. Although
dexamethasone does not further increase spontaneous apoptosis (Fig. 3c), it decreases the survival
response of wild-type B cells to LPS and anti-CD40 (Fig. 3, a and b). Significantly,
c-Rel-/- B cells were more sensitive to the
suppressive effect of dexamethasone on survival. Thus, c-Rel is also
critical for maintaining viability in the presence of
dexamethasone.
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To elucidate the molecular mechanisms responsible for c-Rel’s role in survival, we decided to identify c-Rel-regulated target genes. RDA (27) of cDNA from wild-type and c-Rel-/- cells was employed. Three rounds of subtractive hybridization and amplification yielded several independent clones. One of the clones was identical to mouse Bcl-x, and this particular representation corresponded to the 3'-untranslated region (34, 35). From this experiment it was not possible to determine whether Bcl-xL or Bcl-xS message was the source of this representation. To clarify this point and to test expression levels, we performed RNase protection assays and Western blots.
In wild-type cells, Bcl-xL message is highly
induced within 4 h by anti-IgM and anti-CD40 (Fig. 4a). Anti-IgM induces
Bcl-xL message 2- to 3-fold over the medium
control, while anti-CD40 induces the message 9- to 10-fold.
c-Rel-deficient B cells have a lower basal level of message, and
anti-IgM and anti-CD40 fail to induce
Bcl-xL to normal levels. As determined by
migration distance, the form induced is clearly the anti-apoptotic
long form, or Bcl-xL. Other experiments confirmed
that Bcl-xL message is also induced by LPS, but not in
c-Rel-/- B cells (data not shown).
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a). This is in agreement with a previous report
(21). Bax expression remains relatively constant and is
not affected by loss of c-Rel. Bcl-2 message is below background in
this assay.
We then assessed the level of Bcl-xL protein that
is induced in B cells by LPS, anti-IgM, or anti-CD40 (Fig. 4b). Unstimulated B cells show low amounts of Bcl-xL
protein. In agreement with the RNase protection data,
Bcl-xL is strongly induced in wild-type B cells
by all three stimuli (Fig. 4b, lanes 3,
5, and 7). c-Rel-/- B
cells, on the other hand, are severely deficient in induction of
Bcl-xL (Fig. 4b, lanes 4,
6, and 8). The pro-apoptotic short form of Bcl-x,
Bcl-xS, was not detected under any conditions
(data not shown). These data thus identify Bcl-xL
as a bonafide c-Rel target gene in primary B lymphocytes.
Transgene expression of Bcl-xL can rescue the c-Rel-/- survival defects
To assess the role of Bcl-xL in B cell
survival, we crossed Bcl-xL transgenic mice into
the c-Rel-/- background. These mice (-/- Tg)
appeared normal, although spleen cellularity for both +/+ Tg and -/-
Tg mice was approximately double that of wild-type mice (data not
shown). The Bcl-xL transgene was able to rescue
the c-Rel-deficient B cells from anti-IgM-induced death (Fig. 5a). However, it did not
correct the proliferation defects described previously (data not
shown).
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b) or dexamethasone-induced death (Fig. 5c), in collaboration with LPS or anti-CD40. For
example, when irradiated at 250 rad, anti-CD40-stimulated wild-type
B cells are still 73% viable, while c-Rel-/-
cells are only 50% viable (Fig. 5b, middle
panel). -/- Tg B cells are 68% viable, similar to wild-type
cells, and +/+ Tg cells are 78% viable, slightly more than wild-type
cells.
At the same time, wild-type B cells stimulated with LPS and treated
with 200 nM dexamethasone are 65% viable, and
c-Rel-/- cells are only 37% viable (Fig. 5c, top panel). However, -/-Tg B cells are 67%
viable. In the presence of the transgene, the viability of
c-Rel-/- B cells approaches that of wild-type
cells. Thus, replacement of the c-Rel target gene
Bcl-xL can compensate for the loss of c-Rel.
c-Rel and Bcl-xL are not required for protection against Fas-mediated apoptosis
The Fas molecule is a key cell surface receptor involved in lymphocyte apoptosis (36). Previous studies have shown that Ag receptor engagement can protect B cells from cell death induced by anti-Fas Ab or by Fas ligand-bearing Th1 T cells (37, 38). This protection is thought to be mediated partly by Bcl-xL, because the protein is strongly up-regulated by anti-IgM stimulation with kinetics similar to those of Fas resistance, and because overexpression of Bcl-xL can also confer resistance. Thus, in our c-Rel-deficient system, we sought to address the role of c-Rel and Bcl-xL in resistance to Fas-mediated cell death.
For this study we sought to reproduce earlier work (37) performed with BALB/c cells (H-2d) and to verify it in the C57BL/6 background (H-2b). In this system stimulation of B cells with anti-CD40 causes up-regulation of surface Fas, which then sensitizes them to Fas-mediated apoptosis via Fas ligand-bearing Th1 T cells. We used the 2C2 T cell clone, which has previously been used to specifically kill Fas-bearing targets of H-2b background (39, 40).
CD40 stimulation up-regulated Fas surface expression equivalently in
wild-type and c-Rel-/- B cells (data not shown)
and sensitized them to Fas-mediated killing in a dose-dependent manner
(Fig. 6a). Surprisingly,
anti-IgM signaling was able to rescue both types of B cells from
this apoptosis despite the fact that Bcl-xL was
still deficient in c-Rel-/- B cells (Fig. 6b, lanes 7 and 8). Thus, other
molecules that are independent of c-Rel regulation must be mediating
the protection.
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d, lane 4). Interestingly,
c-Rel-/- B cells also up-regulated c-FLIP as
well as the wild-type cells (Fig. 6d, lane 8).
There also appears to be some induction with CD40 alone in the
c-Rel-/- cells (Fig. 6d, lane
7).
FAIM was also up-regulated at comparable levels in both wild-type and
c-Rel-/- B cells (Fig. 6c). The
induction of both c-FLIP and FAIM correlates kinetically with
protection from Fas-mediated cell death. These results suggest that the
presence of these two molecules in c-Rel-/- B
cells could account for this protection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We show that c-Rel is indeed crucial
for protecting B cells from Ag receptor-mediated death signals. It is
also an important component of CD40- and LPS-mediated survival pathways
for preventing death induced by ionizing irradiation and
glucocorticoids. Furthermore, we show that c-Rel-mediated protection is
partly achieved through the induction of Bcl-xL
and A1/Bfl1, as supported by the rescuing effect of a
Bcl-xL transgene. Interestingly, however,
c-Rel-deficient B lymphocytes, which lack Bcl-xL
expression, are still able to receive anti-IgM/anti-CD40
costimulatory survival signals in the protection from CD40-sensitized
Fas-mediated apoptosis. Taken together, our studies reveal the
significance of c-Rel in protecting from some, but not all, types of
apoptosis. This emphasizes that the role of a given NF-B/Rel member
in death protection needs to be tested and verified in the context of
cell types and specific inducing signals.
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b). This finding implies that
anti-IgM ligation can simultaneously trigger both survival and
apoptotic signals in mature B lymphocytes. In wild-type B cells the
anti-IgM survival signals are mediated in part by c-Rel and its
downstream survival proteins, thus protecting them from apoptotic
signals. In contrast, in the absence of c-Rel and its survival
proteins, the anti-IgM-mediated pro-apoptotic pathway becomes
predominant. Therefore, c-Rel is one of the critical components in
mature B cells to ensure the proper response to antigenic signals.
In addition to the importance of c-Rel in anti-IgM-mediated
responses, our experiments with irradiation and
dexamethasone-induced death have also uncovered a further requirement
for c-Rel in survival responses induced by LPS or anti-CD40. Thus,
c-Rel activation can counteract environmental as well as regulatory
death signals. We have identified Bcl-xL as a
c-Rel target gene and demonstrated its ability to rescue
c-Rel-/- B cells from programmed cell death
mediated by irradiation, dexamethasone, and anti-IgM (Fig. 5).
The protective effect of Bcl-xL correlates well
with data showing that these forms of apoptosis are integrated at the
level of mitochondria, which controls the release of cytochrome
c and the ultimate activation of pro-caspase 9. In support
of this view, caspase-9-deficient thymocytes were shown to be resistant
to dexamethasone-, irradiation-, and etoposide-induced cell death
(41).
It is interesting that c-Rel and Bcl-xL are both dispensable for protection of B cells against Fas-mediated cell death. Previously, we and others have found that Bcl-xL, when overexpressed, can protect B cells from Fas-mediated apoptosis (10, 38). Because anti-IgM can induce high levels of Bcl-xL, this led to the hypothesis that anti-IgM can confer protection, because it induces a more prolonged and higher level of Bcl-xL expression than anti-CD40 alone. Our current results suggest that in primary B lymphocytes, Bcl-xL is not necessary for anti-IgM-mediated protection of CD40-activated cells, at least in the time period tested. This raises the possibility that other molecules, such as FAIM and c-FLIP, may play a more significant role in vivo. Indeed, we find that both proteins are up-regulated normally in c-Rel-/- cells, and the timing of induction correlates well with protection. These results are in agreement with earlier work on FAIM and c-FLIP indicating that these molecules, when expressed in B cell lines, can prevent Fas-mediated apoptosis (4, 11). Other candidate protective molecules, such as A1/Bfl1, Bcl-2, and the inhibitor of apoptosis proteins, do not have expression kinetics that match the onset of Fas resistance (B. R. Schram and T. L. Rothstein, unpublished observations; reviewed in Ref. 9).
The differential requirements for c-Rel in protecting from different
types of programmed cell death (e.g., irradiation vs Fas) can be
reconciled by the fact that there are two major apoptotic pathways in
lymphocytes (reviewed in Ref. 42). One is initiated by
death receptors and is mediated by caspase-8, and the other is
initiated by signals that directly affect the mitochondria, resulting
in the activation of caspase-9. Both pathways eventually converge
downstream at caspase-3. Work in various caspase knockout mice as well
as Apaf1 knockouts has revealed that the Fas apoptotic pathway can
still function in the absence of caspase-9 and Apaf1, suggesting the
involvement of a nonmitochondrial pathway in Fas-mediated cell
death (41, 43). On the other hand, caspase-9-deficient
thymocytes are resistant to etoposide, dexamethasone, and
irradiation; these data correlate well with the requirement for
Bcl-xL induction in our B cell model. In
addition, the fact that Bcl-xL can rescue
c-Rel-/- B cells from IgM-mediated apoptosis is
in agreement with recent work illustrating that this death pathway
involves the mitochondria, and subsequently caspase-9 activation
(44, 45).
Work by Scaffidi et al. (46) has identified two types of Fas-expressing cells that differ in their sensitivity to Fas ligation. Type I cells activate caspase-8 rapidly, whereas type II cells have a delayed response. Fas-mediated apoptosis was blocked by Bcl-2 and Bcl-xL in type II cells only. The density of Fas and the amount of endogenous caspase-8 determined whether the apoptosis would proceed independently of mitochondrial factors. The authors were able to convert MCF7, a type II breast carcinoma cell previously shown to be protected by Bcl-xL (47), into a type I cell by overexpressing caspase-3. Thus, mouse primary B lymphocytes may be type I cells, which have high levels of Fas and endogenous caspase-8. Indeed, it has been found in both murine and human B cells that caspase-8 is recruited to the death-inducing signaling complex, but only after signaling through Fas (11, 48). Stimulation of murine B cells with anti-IgM inhibits this recruitment, presumably due to c-FLIP up-regulation (11).
Physiologically, it is reasonable to assume that inhibition of the Fas pathway would occur more effectively at the level of the receptor. Thus, c-FLIP and FAIM may play a more important role in vivo. However, our results do not preclude the possibility that Bcl-xL plays an important role in other contexts of Fas-mediated apoptosis, such as higher E:T cell ratios. Previously, we have shown that overexpression of Bcl-xL in transgenic mice diminished the Fas sensitivity of CD40 ligand-stimulated primary B cells (10). Thus, all three molecules may play a role physiologically, but in our in vitro system, FAIM and c-FLIP can fully compensate for the loss of Bcl-xL. Another possibility is that the residual amount of Bcl-xL expression in the c-Rel-/- mouse is sufficient to cooperate with FAIM and c-FLIP. Alternatively, there may be other molecules (perhaps pro-apoptotic) that are affected by the loss of c-Rel. Overall, however, our current results are consistent with the idea that the absence of c-Rel and Bcl-xL does not have a major impact on the protection of B cells from Fas-mediated death.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hsiou-Chi Liou, Weill Medical College, Cornell University, 515 East 71st Street, Room S-210, New York, NY 10021. E-mail address: hcliou{at}med.cornell.edu
3 Abbreviations used in this paper: c-FLIP, FLICE inhibitory protein; EF, embryonic fibroblast; FAIM, Fas apoptosis inhibitor molecule; PI, propidium iodide; RDA, representational difference analysis; Tg, transgenic.
Received for publication May 8, 2001. Accepted for publication August 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in promoting double positive thymocyte apoptosis. J. Exp. Med. 189:145.B in regulation of multiple immune-response genes and in Fas-induced cell death. J. Exp. Med. 189:999.B rela (p65) is essential for tnf--induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death. J. Immunol. 166:4949.B family directly activates expression of the apoptosis inhibitor Bcl-x(L). Mol. Cell. Biol. 20:2687.B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc. Natl. Acad. Sci. USA 96:9136.B that blocks TNF-induced apoptosis. Genes Dev. 13:382.B-mediated cell survival. Science 281:998.B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680.-induced apoptosis by NF-B. Science 274:787.B1 (NF-B1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells. J. Exp. Med. 187:663.B/Rel family proteins during B-cell terminal differentiation. Mol. Cell. Biol. 14:5349.B/Rel are at the divergence of CD40-mediated proliferation and survival pathways. J. Immunol. 165:3860.This article has been cited by other articles:
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C. S. Schmidt, J. Liu, T. Zhang, H. Y. Song, G. Sandusky, K. Mintze, R. J. Bensch |
) for 2 days with or without
overnight (o/n) stimulation with anti-IgM on the last day (
).
Chromium release assay was performed as described with the indicated
E:T cell ratios. Supernatants were collected and then assayed with a
gamma counter. Error bars indicate the SD for triplicate samples.
b, Bcl-xL is still deficient in c-Rel-/- B
cells. Lysates from wild-type or c-Rel-/- cells were
generated at the indicated time points after stimulation with
anti-CD40, with or without anti-IgM. Western blotting for Bcl-x
was performed as described. c and d,
Expression of FAIM (c) and c-FLIP (d)
protein. The same lysates were used as in B, Western
blotted with a rabbit polyclonal Ab raised against full-length FAIM
(c) or rat anti-human/mouse c-FLIP
(d). FAIMS runs at 20 kDa.
c-FLIPL runs at approximately 55 kDa.