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* Departments of Pathology and
Microbiology and Immunology, Weill Medical College, Cornell University, New York, NY 10021; and
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. These results
provide the first evidence that primary plasma cells synthesize TRAIL
and are direct targets of TRAIL-mediated apoptosis, which may relate to
the inactivation of the NF-B survival
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expansion of activated B cells is tightly regulated by survival and
apoptotic signals mediated by CD40 and Fas, two members of the TNFR
family. Likewise, the proliferation and survival of germinal center B
cells in a T-dependent immune response critically depends on CD40
signaling through TNFR-associated factor
(TRAF)3 and NF-B
(5, 6). TRAIL/Apo-2 ligand (Apo2L) is a proapoptotic
member of the TNF family (7, 8), which appears to
predominantly induce apoptosis of tumor cells (9, 10),
including lymphoma and myeloma (malignant plasma) cell lines (7, 8, 10, 11, 12, 13). Whether TRAIL has a role in the control of B lineage
cells has not been defined.
TRAIL functions to either accelerate or attenuate apoptosis depending on its interaction with five distinct receptors (DRs). After ligand binding and receptor trimerization, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) recruit Fas-associated death domain-containing protein (FADD) to their cytoplasmic domains (14, 15, 16, 17). This newly formed complex initiates an apoptotic cascade through the recruitment and activation of caspase-8 (15, 16, 17) or by a caspase-independent mechanism involving the receptor-interacting protein (RIP) (18). The other known TRAIL receptors are decoy receptor (DcR)1 (TRAIL-R3) and DcR2 (TRAIL-R4) (14), which lack intact cytoplasmic death domains, and the soluble osteoprotegerin (19). These receptors sequester TRAIL from DR4 and DR5, thereby antagonizing TRAIL-mediated apoptosis (14).
The humoral immune response requires IL-6, given that IgG and IgA
responses are defective in the absence of IL-6 (20, 21).
In vitro, stimulation of EBV-immortalized, IgG-bearing human
lymphoblastoid cells with IL-6 recapitulates all major hallmarks of B
cell terminal differentiation (22, 23, 24, 25). These include
prominent increases in Ig synthesis and secretion, extinction of
surface MHC class II expression, and cell cycle arrest
(22, 23, 24, 25). Most importantly, the IL-6-differentiated plasma
cells no longer exhibit a transformed phenotype due to loss of
EBV-transforming gene expression and rapidly undergo apoptosis
(22), thereby mimicking short-lived plasma cells. The
human lymphoblastoid cells resemble CD40-activated B cells in that the
TRAF signaling pathway is constitutively activated by the EBV-encoded
latent membrane protein-1 (LMP1) (26, 27). The loss of
LMP1 expression in IL-6-differentiated plasma cells suggests that
apoptosis of plasma cells may relate to the loss of NF-B activation
and an altered balance between survival and death signals.
Here, we show that the IL-6-differentiated human plasma cells retain
the expression of TRAIL, DR4, and DR5 and rapidly undergo apoptosis
mediated by endogenous and exogenous TRAIL. Induction of apoptosis by
TRAIL extends to primary mouse plasma cells but not resting or
activated B cells. This preferential TRAIL-mediated killing correlates
with loss of CD40 expression and NF-B activation in plasma cells.
Thus, plasma cells synthesize TRAIL and are subject to TRAIL-mediated
apoptosis, which may relate to the inactivation of the CD40-NF-B
pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CESS is an EBV-immortalized, IgG-bearing human lymphoblastoid B cell line (22), Jurkat cells were obtained from the American Type Culture Collection, whereas the P11 human T cell line was provided by Dr. K. Elkon. CESS cells were terminally differentiated by treatment with baculovirally expressed recombinant human IL-6 and soluble IL-6 receptor (gp80) for 4 days as previously described (23). IL-6-differentiated (IgGhigh/surface MHC class IIlow) cells were separated from IL-6-refractory (IgGlow/surface MHC class IIhigh) cells using anti-MHC class II Abs conjugated to magnetic beads (Dynal, Lake Success, NY), also as previously described (23).
When indicated, cells were cultured with 20 µM
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD; Enzyme System
Products, Livermore, CA) and 1 µg/ml recombinant soluble TRAIL/Apo2L
(28), DR5-Fc (aa 52–180 of DR5 fused to the Fc portion of
human IgG) (29), both generously provided by A. Ashkenazi
(Genentech, South San Francisco, CA), or the Fc portion of human IgG
(Rockland, Bridgeport, NJ). Recombinant TRAIL and DR5-Fc provided by
Dr. Ashkenazi were used in Figs. 3
and 6A. Otherwise, cells
were incubated with recombinant soluble FLAG-tagged TRAIL/Apo2L (0.1
µg/ml) together with a FLAG enhancer Ab (1 µg/ml) and DR5-Fc (1
µg/ml) purchased from Alexis Biochemicals (San Diego, CA).
Cell viability was determined by trypan blue exclusion.
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Resting splenic B cells were isolated from 5- to 8-wk-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) and cultured with CD40 ligand (CD40L)-expressing L cells in the presence of human IL-6 (40 U/ml) and the soluble gp80 subunit of the IL-6R (40 U/ml) as previously described (30). From day 9 onward, the B cells were cocultured with osteoblastic MC3T3 cells (Deutsche Sammlungion Mikroorganismen und Zell Kulturen, purchased from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
Isolation of plasma cells generated in the
4-hydroxy-3-nitrophenyl-chicken 
-globulin (NP-CGG) response and
induction of apoptosis by TRAIL
Splenic B cells were isolated from B6 mice (8-wk of age) 10 days after s.c. injection with 75 µg of NP-CGG as previously described (30). To enrich for syndecan-1-positive plasma cells, viable cells were isolated by Ficoll gradient centrifugation and incubated at a concentration of 107 cells/ml with a biotinylated rat anti-mouse syndecan-1 Ab (1/500 dilution; BD Biosciences, Franklin Lake, NJ). This was followed by a second incubation with streptavidin conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA). Syndecan-1-positive cells were either plated onto CD40L-expressing L cells in the absence or presence of DR5-Fc or Fc or cultured directly onto 2PK3 cells or a stable transfectant expressing membrane-bound murine TRAIL (mTRAIL) (31). Cell viability was determined by trypan blue exclusion after 5 h in culture. Resting and activated B cells present in the syndecan-1-negative fraction were separated by Percoll (Sigma-Aldrich, St. Louis, MO) gradient centrifugation, as previously described (30) and cultured onto mTRAIL or control 2PK3 cells for 24 h. Cell viability was determined by annexin V binding and trypan blue exclusion.
Immunofluorescence microscopy and flow cytometric analysis
Immunofluorescence microscopy was performed essentially as described (23). Unless indicated, all Abs were obtained from BD Biosciences. Fas expression in CESS cells was detected by incubation with an anti-Fas mouse mAb (1/100; PanVera, Madison, WI) followed by a second incubation with a FITC-conjugated rabbit anti-mouse Ab (1/200; DAKO, Carpinteria, CA). Intracellular IgG was detected with a Rhodamine-conjugated goat anti-human IgG Ab (1/400; Cappel, Bryan, OH). Cells were counterstained with 17 mM 4',6'-diamino-2-phenylindole dihydrochloride and visualized by fluorescence microscopy.
Flow cytometric analysis was performed as previously described
(30). CESS cells were stained with the above mentioned
anti-Fas, or CD40 Abs (1/100), and were revealed by a secondary
incubation with a FITC-conjugated rabbit anti-mouse Ab (1/200;
DAKO). In vitro-differentiated primary mouse B cells were stained with
a PE-conjugated hamster anti-mouse FasAb (1/100) or a
FITC-conjugated rat anti-mouse CD40 Ab (1/100). For three-color
staining shown in Fig. 7A, syndecan-negative and -positive
cells were stained with a PE-conjugated rat anti-mouse syndecan-1
Ab (1/200), a FITC-conjugated rat anti-mouse CD40 Ab (1/200) and a
Cy-Chrome conjugated rat anti-mouse B220 Ab (BD PharMingen, San
Diego, CA) (1/400). Cells were analyzed using a BD Biosciences
FACSCalibur.
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Detection of human IgG secreted by IL-6-differentiated CESS cells by ELISA was performed in 96-well plates coated with 1 µg of a rabbit anti-human IgG polyclonal Ab (Cappel) (30). To detect NP-specific IgG secreted by primary plasma cells, wells were coated with 2.5 µg 4-hydroxy-3-iodo-5-nitrophenylacetyl succinimide ester conjugated to BSA. Bound Ab was revealed by incubation with biotinylated goat anti-human IgG (1/20,000; Jackson ImmunoResearch Laboratories, West Grove, PA), or biotinylated goat anti-mouse IgG (1/30,000; Sigma-Aldrich) followed by a second incubation with HRP-conjugated streptavidin (1/2000; Vector, Burlingame, CA).
A secondary immune response was elicited 5 wk after the primary
response by injecting tail veins with 15 µg NP-CGG in PBS.
Splenic B cells were isolated 6 days later, cultured for 4 h in
medium alone, with parental 2PK3 cells or mTRAIL-expressing 2PK3 cells
at a 10:1 ratio. An ELISPOT was then performed in 96-well plates coated
with 1 µg of a goat anti-mouse Ab (Southern Biotechnology
Associates, Birmingham, AL) or 2.5 µg
4-hydroxy-3-iodo-5-nitrophenylacetyl succinimide ester conjugated to
BSA (30).
EMSAs
EMSA was performed to analyze the NF-B DNA binding activity
using 2.6 µg of whole cell lysate and a
[32P]dATP-labeled H2K site as
previously described (30), or to analyze the Oct-1 DNA
binding activity using a [32P]dATP-labeled
Oct-1 probe (H2B)
(5'-GATCCCAACTCTTCACCTTATTTGCATAAGCGATTCTATAG). In
competition assay, an unlabeled double-stranded oligonucleotide
containing the Oct site of the Eµ enhancer
(5'-AATTCACCCTGTCTCATGAATATGCAAATCAGGTGAGTCTATG-3') was
used at a 280-fold molar excess.
Immunoblotting
Whole cell lysates were prepared by incubating cells in lysis
buffer (250 mM NaCl, 50 mM HEPES, pH 7; 0.1% Nonidet P-40) on ice for
10 min, supernatants were clarified by centrifugation, and 10–30 µg
of total protein was resolved on SDS-PAGE and transferred onto
polyvinylidine difluoride membranes. Membranes were blocked in TBS-T
(10 mM Tris (pH 8), 150 mM NaCl, 0.1% Tween 20) containing 5%
powdered milk for 1 h followed by a 3-h incubation with one of the
following Abs: mouse anti-human caspase-7 (1-1-11; 1/1000), mouse
anti-human caspase-8 (1-1-40; (1/1000), rabbit anti-human
caspase-3 (585R; 1/1000) (32), all provided by Dr. Y.
Lazbenik; rabbit anti-human DR4 (66901N; BD Transduction
Laboratories, Lexington, KY; 1/1000), rabbit anti-human DR5
(AAP-430) (1/1000; Stressgen, Vancouver, Canada), rabbit anti-human
A1 (FL-175; 1/1000; Santa Cruz Biotechnology, Santa Cruz, CA), hamster
anti-mouse Bcl-2 (554218; 1/1000), or mouse anti-chick
-tubulin (T9026) (1/5000; Sigma-Aldrich). Membranes were rinsed in
TBS-T and incubated with biotinylated donkey anti-mouse, donkey
anti-hamster, or donkey anti-rabbit Abs (1/20,000; Jackson
ImmunoResearch Laboratories) for 45 min followed by a second 30-min
incubation in streptavidin-HRP (1/20,000; Jackson ImmunoResearch
Laboratories). To detect IgG, membranes were probed directly with a
biotinylated goat anti-human IgG Ab (1/20,000) or a biotinylated
goat anti-mouse IgG Ab (1/20,000), followed by a streptavidin-HRP
incubation. The immunoreactive proteins were detected by
chemiluminescence (ECL; Amersham, Arlington Heights, IL). For
sequential blotting, membranes were stripped in 2% SDS, 62.5 mM Tris,
pH 6.8, at 50°C for 20 min.
RT-PCR and RNase protection assay
CESS cell RNA was isolated by the guanidine isothiocyanate procedure as previously described (25). Total RNA from primary B lineage cells were isolated using Trizol (Life Technologies, Gaithersburg, MD). For RT-PCR analysis, reverse transcription was performed on total RNA from 3 x 105 CESS cells or 1 µg of primary cell RNA using 1 µg oligo(dT) primer and Superscript II reverse transcriptase (Life Technologies). For the PCR analysis, 1 µl of cDNA was used with 10 pmol of each of the following primers: Fas ligand (FasL), 5'-TCAGCTCTTCCACCTACAGAA-3' and 5'-TACAACATTCTCGGTGCCTG-3'; GAPDH, 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3'; mTRAIL, 5'-GGTCTCAAAGGACAAGGTG-3' and 5'-TTAGTTAATTAAAAAGGCTCC-3'; mouse DR5, 5'-GTCAAAGCCGAAACACTGG-3' and 5'-TCAAACGCACTGAGATCC-3'; mouse actin, 5'-AAGATCCTGACCGAGCGTGGC-3' and 5'-CTGGAAGGTGGACAGTGAGGC-3'. DNA was amplified using the following PCR conditions: FasL, 35 cycles (30 s at 94°C; 30 s at 55°C; 45 s at 72°C); GAPDH, 20 cycles (1 min at 94°C; 1 min at 62°C; 1 min at 72°C), mTRAIL: 40 cycles (1 min at 94°C; 1 min at 52°C; 2 min at 72°C); mDR5, 40 cycles (1 min at 94°C; 1 min at 52°C; 2 min at 72°C) and mouse actin, 30 cycles (1 min at 94°C; 1 min at 68°C; 2 min at 72°C) The PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide.
For the RNase protection analysis, antisense RNA was generated from the human hAPO-3c multiprobe template set (BD PharMingen) as described (33). Total RNA (5 µg) was hybridized with 1 x 106 cpm of the radiolabeled probe, subjected to RNase protection analysis, and electrophoresed on 6% denaturing acrylamide gels containing urea (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-6-differentiated human IgG plasma cells rapidly undergo
apoptosis as evidenced by annexin V binding and poly(ADP)ribose
polymerase cleavage (22, 23). These plasma cells
(IgGhigh), which no longer possess a transformed
phenotype due to reversal of EBV immortalization (22), can
be enriched to >80% purity by negative selection for the loss of MHC
class II expression (23). To verify the
involvement of caspases in plasma cell death, the IL-6-differentiated
plasma cells and control CESS cells were cultured in the presence or
absence of the general caspase inhibitor, ZVAD (Fig. 1A). Although ZVAD did not
alter the viability of control cells, it reduced the death of
IL-6-differentiated plasma cells by 60% in 24 h. Plasma cell
apoptosis is therefore primarily caspase dependent.
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B). Confirming this
observation, immunofluorescent staining revealed a prominent reduction
of Fas on plasma cells expressing high levels of intracellular IgG
(Fig. 1C). Moreover, FasL mRNA was undetectable in CESS
cells before or after IL-6 differentiation by either RT-PCR or RNase
protection assays (Figs. 1D and 2A). Thus,
IL-6-differentiated plasma cells express reduced level of Fas and no
FasL. The Fas pathway is unlikely to be instrumental in the death of
these clonal plasma cells in vitro. Expression of TRAIL, DR4 and DR5 on IL-6-differentiated human plasma cells
Apoptosis of IL-6-differentiated plasma cells in the absence of
FasL prompted us to investigate the role of TRAIL in plasma cell death.
RNase protection assays revealed that mRNAs encoding TRAIL, DR4, and
DR5 were expressed in IL-6-differentiated plasma cells
(IgGhigh), at levels comparable with those
observed in IL-6-refractory (IgGlow) and
untreated CESS cells (Fig. 2A). The Fas mRNA levels were
similarly maintained in IL-6-differentiated plasma cells (Fig. 2A), implying that reduction of Fas protein (Fig. 1) might
occur at the posttranscriptional level. The maintenance of DR4 and DR5
expression was confirmed at the protein level by immunoblot analysis
(Fig. 2B), where detection of DR5 but not DR4 in Jurkat T
cells (15) served as a control for the Ab
specificity. The purity of the plasma cell population was confirmed
through the detection of high IgG levels, and tubulin expression
controlled for protein loading (Fig. 2B). There was no
evidence for the presence of DR3 or TNFR p55 mRNAs in plasma cells
(Fig. 2A). The expression of TRAIL, DR4, and DR5 is
therefore retained during IL-6 differentiation of human B
lymphoblastoid cells to plasma cells. The reduction of surface Fas
expression and the absence of FasL indicate that Fas is unlikely to be
instrumental in the apoptosis of IL-6-differentiated plasma cells. The
sustained expression of TRAIL and its receptors in plasma cells is
consistent with a potential role for TRAIL in inducing plasma cell
apoptosis in vitro.
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The function of TRAIL in plasma cell apoptosis was characterized,
first in IL-6-differentiated human IgG-secreting plasma cells due to
the relative ease in enriching this plasma cell population. To
determine whether endogenous TRAIL induces plasma cell death,
IL-6-differentiated plasma cells were incubated with DR5-Fc, an
inhibitory chimeric soluble TRAIL receptor comprised of the
extracellular domain of DR5 fused to the Fc portion of human IgG
(29) (Fig. 3). Indeed,
DR5-Fc inhibited plasma cell death by 40% within 24 h, as
assessed by trypan blue staining (Fig. 3, top). This
correlated with a 4-fold increase in the relative amounts of IgG
secreted into the medium during the 24-h period, as measured by ELISA
(Fig. 3, bottom). Inhibition of cell death by DR5-Fc was
specific to the DR5 extracellular domain, because the Fc portion of
human IgG had no effect (Fig. 3). Thus, endogenous TRAIL functions to
induce plasma cell death. The addition of soluble, trimerized
recombinant human TRAIL further enhanced plasma cell death, whereas the
presence of both DR5-Fc and TRAIL led to a marked reduction of plasma
cell death and a corresponding increase in IgG secretion (Fig. 3).
These results suggest that plasma cell death is induced by endogenous
TRAIL and augmented by exogenous TRAIL.
Induction of cell death by TRAIL, however, did not extend to the
control CESS lymphoblastoid cells (Fig. 3). They were intrinsically
less apoptotic than IL-6-differentiated plasma cells, and were
refractory to TRAIL-mediated killing at a concentration that enhanced
plasma cell death (Fig. 3). TRAIL therefore preferentially induces the
death of IL6-differentiated plasma cells.
TRAIL induces apoptosis of primary plasma cells generated in T-dependent immune responses
The susceptibility of IL-6-differentiated human plasma cells to
TRAIL killing prompted us to address whether primary plasma cells are
also direct targets of TRAIL-mediated apoptosis (Fig. 4). Primary mouse plasma cells were
generated in vivo by immunization with NP-CGG, a T cell-dependent Ag
and enriched to >70% homogeneity by selecting syndecan-1-positive
cells from splenic B cells isolated on day 10 of immunization. As
controls, resting and activated B cells present in the
syndecan-1-negative fraction were subsequently separated by Percoll
gradient centrifugation (30). TRAIL and DR5 (there is no
DR4 in mice) mRNAs were expressed at comparable levels in primary
resting, activated, and plasma cells, as indicated by RT-PCR analysis
(Fig. 4A).
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B, left panel).
Conversely, it was reduced by 30% by incubation with DR5-Fc
(p = 0.02), but not with Fc (Fig. 4B, middle panel). The reduction of plasma cell
death by DR5-Fc was corroborated by a 2-fold increase
(p = 0.03) in the amount of NP-specific IgG Ab
secreted in the medium (Fig. 4B, right panel).
Thus, endogenous and exogenous TRAIL cooperate to induce the death of
primary plasma cells generated in a primary T cell-dependent
response.
To further confirm that TRAIL induces apoptosis of primary plasma cells
generated in vivo, the loss of plasma cells secreting NP-specific IgG
in response to TRAIL was assayed functionally by ELISPOT (Fig. 4C). NP-specific plasma cells were first amplified in vivo
in a secondary response elicited 5 wk after primary NP-CGG
immunization. Splenic B cells were isolated at the peak of the
secondary Ab response (day 6) and directly cultured, without selection
for syndecan-1-positive cells, for 4 h in medium, alone or
together with mTRAIL or the control 2PK3 cells (Fig. 4C).
mTRAIL, but not the control cells, induced a 2-fold reduction of the
number of NP-specific IgG plasma cells (Ab-forming cell)
(p = 0.006) as well as the polyclonal
IgG-secreting plasma cells (p = 0.03). Thus,
TRAIL directly induces the death of Ag-specific and polyclonal,
class-switched, primary plasma cells generated in a T-dependent immune
response.
Incubation with mTRAIL-expressing cells ex vivo, however, did not
enhance apoptosis of resting or activated splenic B cells isolated from
a primary NP response, as determined by either the annexin V-binding
activity of early apoptotic cells or trypan blue staining of dead cells
(Fig. 4D). Primary resting and activated mouse B cells are
therefore refractory to TRAIL-mediated killing ex vivo, in agreement
with our observation during IL-6 differentiation of human
lymphoblastoid cells (Fig. 3). TRAIL therefore preferentially induces
the death of primary plasma cells but not resting or activated B cells
in a T-dependent immune response.
TRAIL-mediated apoptosis of IL-6-differentiated human plasma cells is primarily caspase dependent
TRAIL has been shown to initiate the caspase cascade by
recruitment and activation of caspase-8 (15, 16, 17).
Spontaneous and TRAIL-induced death of IL-6-differentiated plasma cells
are inhibited by ZVAD (Figs. 1A and 5A),
suggesting that the TRAIL death signals are mediated by caspases.
Consistent with this possibility, both isoforms of procaspase-8 (55 and
53 kDa) as well as procaspases-3 and -7 were abundantly expressed in
IL-6-differentiated IgG plasma cells as determined by immunoblotting
(Fig. 5B). The initiator
caspase-8 was activated within 1 h of TRAIL stimulation, as
indicated by the emergence of its cleavage products. This was rapidly
followed by the activation of the effector caspases-7 and -3, also in
agreement with observations in other cell types (15, 16, 17, 34). Although the cleavage of all three caspases in response to
TRAIL was inefficient in IL-6-differentiated plasma cells (Fig. 5B), the levels of cleavage were comparable with those
observed by others in a subset of cell lines including human B cell
lines and colon and lung carcinomas (16, 17). Moreover,
activation of these three caspases was also very modest after prolonged
(7 h) Fas cross-linking, in striking contrast to their efficient
activation in the control lymphoblastoid cells under the same Fas
cross-linking conditions (Fig. 5C and data not shown).
Activation of caspases-8, -3, and -7 is therefore inefficient in plasma
cells by either TRAIL or Fas.
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).
However, protection from apoptosis by ZVAD was incomplete, implying
that a caspase-independent mechanism may function in concert with the
caspase-dependent pathway to induce plasma cell apoptosis in response
to TRAIL.
Decreased CD40 expression and inactivation of NF-B in
IL-6-differentiated human plasma cells
Our data point to preferential killing of plasma cells, but not B
cells before terminal differentiation, by TRAIL despite comparable
levels of expression of both the ligand and receptors. This raises the
possibility that either functional TRAIL/DR complexes cannot form on
activated (lymphoblastoid) cells, or that plasma cells have
specifically lost the ability to respond to one or more survival
signals. To address the latter possibility, we investigated the
regulation of the TRAF/NF-B survival pathway, which is constitutive
in EBV-transformed lymphoblastoid cells (26, 27) but may
no longer operate in IL-6-differentiated plasma cells (22)
due to loss of LMP1 expression.
Electrophoretic mobility shift assays showed that NF-B was highly
activated in control and IL-6-refractory CESS cells, as evidenced by
the formation of NF-B-DNA complexes consisting of p50/p65, p50/RelB,
and p50/p50 (Fig. 6A,
lanes 1 and 2). However, the NF-B DNA-binding
activity was drastically reduced in IL-6-differentiated plasma cells
(Fig. 6A, lane 3). Although TRAIL stimulation can
activate NF-B in other cells (35, 36, 37, 38), it did not
enhance the NF-B DNA-binding activity in either IL-6-differentiated
plasma cells or refractory cells (Fig. 6A, lanes 4 and
5). The loss of NF-B activity could not be attributed to
an overall reduction in DNA-binding activity of cellular transcription
factors, because the Oct-1 DNA binding activity was comparable in each
lysate (Fig. 6B). Moreover, coordinated with the loss of
LMP1 expression (22), the expression of CD40, a potent
activator of NF-B in B lineage cells (5, 6), was also
profoundly reduced in IL-6-differentiated plasma cells (Fig. 6C). This correlated with the failure of CD40L to delay or
rescue apoptosis of these plasma cells (data not shown). Thus, terminal
differentiation of lymphoblastoid cells by IL-6 correlated with
coordinated loss of CD40 expression and NF-B activation.
Reduction of CD40 and Fas expression in primary mouse plasma cells
Spontaneous apoptosis ex vivo of primary plasma cells generated in
the NP response also could not be delayed or prevented by stimulation
with CD40L (Fig. 4B), suggesting that the expression of CD40
might be similarly reduced in primary plasma cells. Indeed, the
expression of CD40 on primary plasma cells
(Synhigh/B220low) was
significantly lower than that on B cells
(Synlow/B220high) from the
same immune response (Fig. 7A). To address the
possibility that the expression of CD40 might be temporally regulated
during B cell terminal differentiation, primary mouse plasma cells were
generated in vitro by sequential coculture of resting splenic B cells
with CD40L-expressing L cells and then MC3T3 osteoblastic cells in the
presence of IL-6. Under these conditions, the majority of B cells
(85%) entered the cell cycle by day 3 based on 5-bromo-2'-deoxyuridine
uptake and continued to proliferate until around day 7, when cell cycle
arrest began. From day 9 onward, coculture with MC3T3 cells facilitated
terminal differentiation, leading to plasma cells that had lost B220
and MHC class II, expressed syndecan-1 and secreted IgM (W. Zhang and
S. Chen-Kiang, unpublished observations). The expression of CD40 was
maintained on activated B cells (day 4), reduced as cells withdrew from
the cell cycle (day 7), and further decreased in plasma cells (day 11)
to levels substantially below that of resting B cells (Fig. 7B). Together, these in vivo and in vitro results
demonstrate that CD40 expression is progressively reduced during
terminal differentiation of primary B cells.
The reduction of CD40 expression suggests that the NF-B survival
pathway may be coordinately inactivated in primary plasma cells. To
address this possibility, we determined the expression of A1 in
activated B cells and plasma cells as a functional readout for NF-B
activity because A1 is a target for both NF-B and CD40 (39, 40). The A1 protein level was markedly lower in plasma cells
(Syn+) compared with activated B cells
(Syn-) generated in the primary NP response,
whereas Bcl-2 and tubulin levels remained unchanged (Fig. 7C). These results confirmed that CD40 and a NF-B target
gene, A1, are selectively and coordinately reduced in
primary plasma cells.
Surface Fas expression was prominently elevated on CD40L-activated B
cells (Fig. 7B), in agreement with previous reports
(41). Of interest, Fas expression was also drastically
reduced on plasma cells (day 11), to levels characteristic of resting B
cells (Fig. 7B). Fas and CD40 are therefore coordinately
regulated during terminal differentiation of primary B cells initiated
by CD40 signaling. Together with the reduction of Fas on
IL-6-differentiated human plasma cells (Fig. 1B), the loss
of surface Fas expression appears to be common in plasma cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B survival
signals. Induction of plasma cell apoptosis by TRAIL
The death of plasma cells is tightly coordinated with cell cycle
arrest and cellular differentiation to ensure their rapid elimination
at the end of a humoral immune response (2, 3, 22, 23).
Here, we provide the first direct evidence that TRAIL mediates
spontaneous and accelerated plasma cell death in two independent model
systems: primary plasma cells generated in a T-dependent immune
response; and human IgG plasma cells differentiated in vitro by IL-6.
First, we showed that the expression of TRAIL and the DR death
receptors were maintained in Ag-specific and polyclonal mouse plasma
cells generated in the 4-hydroxy-3-nitrophenyl (NP) response (Fig. 4A) as well as IL-6-differentiated human plasma cells (Fig. 2). Next, we demonstrated that both plasma cells were susceptible to
killing mediated by endogenous and exogenous TRAIL ex vivo (Figs. 3 and 4). In light of the lack of understanding of TRAIL function in primary
cells, lymphocytes in particular, these findings have significant
implications for the mechanisms that control primary plasma cell
apoptosis.
The susceptibility of plasma cells to TRAIL-mediated killing is in part
determined by temporal changes in the composition of death receptors
during B cell terminal differentiation. Contrasting the sustained
expression of DR, Fas protein expression was drastically reduced in
IL-6-differentiated human plasma cells, and in primary mouse plasma
cells (Figs. 1 and 7B). FasL expression was absent in the
former (Figs. 1D and 2A). Although cross-linking
of Fas can be facilitated by an extracellular domain in the absence of
ligand (42), the low level of Fas expressed on plasma
cells would likely preclude efficient ligand-independent Fas
oligomerization. Fas signaling is therefore not responsible for the
rapid apoptosis of IL-6-differentiated plasma cells in vitro (Figs. 3 and 4) and unlikely to be the primary mechanism that controls the death
of primary plasma cells.
The death of plasma cells, either spontaneous or induced by exogenous
TRAIL, is caspase dependent based on its inhibition by ZVAD (Fig. 5).
The extent to which TRAIL activates the caspase machinery has been
shown to vary greatly according to cell types (15, 16, 17). In
some cell lines, including B cell lines, the levels of caspase cleavage
(15, 16, 17) are comparable with our findings in plasma cells
(Fig. 5). Despite the inefficiency of caspase cleavage by TRAIL, which
may be inherent to specific cell types, the ability of ZVAD to block
the majority of TRAIL-induced killing suggests that plasma cells
respond to TRAIL death signals primarily through caspase-dependent
mechanisms. However, this does not preclude the involvement of a
caspase-independent pathway that may function in concert with
activation of the caspase-8 pathway to promote plasma cell death. This
possibility is consistent with the finding that TRAIL can also activate
a caspase-independent pathway through the RIP serine/threonine kinase
to promote cell death (18). In addition, plasma death is
unlikely to be induced exclusively by TRAIL, because neutralization of
TRAIL killing by DR5-Fc significantly reduced, but did not block,
plasma cell death (Figs. 3 and 4). Further studies are required to
determine the relative contributions of TRAIL and additional death
signals to plasma cell death and the intracellular pathways that
mediate TRAIL-mediated plasma cell death.
TRAIL-mediated apoptosis is coincident with inactivation of the CD40-NF-kB signaling pathway
TRAIL induces apoptosis of terminally differentiated plasma cells,
but not resting or activated primary mouse B cells or human
lymphoblastoid cells (Figs. 3 and 4). Because the expression of key
components of the TRAIL death pathway (TRAIL, DR4, and DR5) does not
vary during B cell terminal differentiation (Figs. 2 and 4), this
differential sensitivity of TRAIL-mediated killing must be determined
by other factors. The most likely possibilities are regulated assembly
of the TRAIL death-inducing signaling complex and altered balance
between intracellular survival and apoptotic signals.
Inactivation of the CD40-NF-B pathway during B cell terminal
differentiation may contribute to the selective killing of plasma cells
by TRAIL (Figs. 6 and 7). Although CD40 expression is believed to be
constitutive in mature B cells, our results revealed that it is in fact
markedly reduced in primary mouse plasma cells generated in vivo and in
vitro as well as IL-6 differentiated human plasma cells (Figs. 6 and 7). This correlates with the failure of CD40L to protect plasma cells
from apoptosis mediated by endogenous and exogenous TRAIL (Fig. 4B; J. Ursini-Siegel, W. Zhang and S. Chen-Kiang,
unpublished observations). In the case of human lymphoblastoid cells,
the extinction of LMP1 expression during terminal differentiation by
IL-6 further ensures that the NF-B pathway no longer functions in
plasma cells (22). Confirming this prediction, the
p50/p50, p50/RelB, and p50/p65 NF-B DNA-binding activities were
drastically reduced in IL-6-differentiated plasma cells and could not
be restored by TRAIL (Fig. 6).
p50 and p65 are essential for the survival of primary lymphocytes
(43, 44) and highly activated in freshly isolated primary
resting and activated B cells (30). NF-B may attenuate
TRAIL-mediated killing through activation of specific Bcl-2 family
proteins, based on the inverse correlation between sensitivity to TRAIL
killing and expression of a specific NF-B target gene A1
in primary plasma cells and activated B cells (Fig. 7). In addition to
modulating the intracellular balance between survival and death
signals, NF-B may also impair the assembly and signaling of an
active TRAIL/death-inducing signaling complex through induction of DcR1
and c-FLIP expression (45, 46, 47). It would be of
interest to determine the expression of DcR1 and c-FLIP in plasma cells
and their roles in TRAIL-mediated apoptosis.
In the context of a humoral immune response, CD40 has dual functions in
B cell activation and survival, and NF-B is essential for the
transcription of Ig genes (48). Moreover, CD40 engagement
of germinal center B cells induces memory B cell formation but inhibits
their terminal differentiation into Ab-secreting plasma cells (1, 49, 50), and removal of CD40 signals allowed germinal center B
cells to undergo proper terminal differentiation (49).
Thus, reduction of CD40 expression and inactivation of NF-B during B
cell terminal differentiation must be exquisitely regulated. Consistent
with this possibility, we show that reduction of CD40 expression on
activated B cells is coincidental with the onset of cell cycle arrest
and continues progressively during subsequent differentiation to plasma
cells in vitro (Fig. 7). On this basis, we suggest that the reduction
of CD40 expression promotes not only B cell terminal differentiation
but also the decline of CD40-activated NF-B activity, at a point
when plasma cells have accumulated and secreted sufficient amounts of
Ig, may in fact signal the end of their life span.
Conditional induction of apoptosis of tumor and primary cells by TRAIL
TRAIL is thought to preferentially kill transformed cells of
various lineages in vitro, including B and T cell lymphomas and
multiple myeloma cells (7, 8, 10, 11, 12). Induction of
apoptosis by the administration of TRAIL in vivo has been shown to
reduce tumor incidence in experimental mouse models of colon carcinoma
(9) and mammary adenocarcinoma (10).
Conversely, neutralization of endogenous TRAIL signals led to
accelerated liver metastases (51). The protection of HL60
cells from TRAIL-mediated killing by p65 activation (52)
and sensitization of myeloma cells to TRAIL killing by inactivation of
NF-B (13, 53), however, suggest that susceptibility of
tumor cells to TRAIL-mediated apoptosis may also be determined by the
balance between apoptosis and survival signals mediated by NF-B.
With the exception of human astrocytes and prostate epithelial cells, most human primary cells are highly resistant to TRAIL-mediated apoptosis (9, 10, 54). However, they can be sensitized to TRAIL-mediated killing by CD3 ligation in the case of primary human thymocytes (55) and by inhibition of protein synthesis of primary human thyroid follicular cells and keratinocytes (56, 57). Our finding that primary resting and activated B cells are refractory to TRAIL killing does not preclude the possibility that additional signals may render them sensitive to TRAIL killing. Elucidating the pathways that mediate and modulate TRAIL-induced apoptosis in primary B lineage cells should help to better understand the control of TRAIL-mediated apoptosis of both normal and malignant plasma cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Selina Chen-Kiang, Department of Pathology C-338, Weill Medical College, Cornell University, 1300 York Avenue, New York, NY 10021. E-mail address: sckiang{at}mail.med.cornell.edu
3 Abbreviations used in this paper: TRAF, TNFR-associated factor; Apo2L, Apo-2 ligand; DR, death receptor; FADD, Fas-associated death domain-containing protein; DcR, decoy receptor; LMP1, latent membrane protein-1; CD40L, CD40 ligand; mTRAIL, membrane-bound murine TRAIL; NP, 4-hydroxy-3-nitrophenyl; NP-CGG, NP-chicken -globulin; RIP, receptor-interacting protein; ZVAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; FasL, Fas ligand.
Received for publication May 31, 2002. Accepted for publication September 16, 2002.
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B and JNK activation and apoptosis through distinct pathways. J. Biol. Chem. 274:30603.B kinase and c-Jun N-terminal kinase. Mol. Cell Biol. 20:6638.B. Immunity 7:831.[Medline]
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.B1 (NF-B1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells. J. Exp. Med. 187:663.B transcription factors protect from TRAIL-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1. J. Biol. Chem. 276:27322.B inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol. Cell Biol. 21:3964.B Signals Induce the Expression of c-FLIP. Mol. Cell Biol. 21:5299.B. Nat. Cell Biol. 3:409.[Medline]
B blockade in multiple myeloma: therapeutic applications. Blood 99:4079.This article has been cited by other articles:
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) or
presence (
) of ZVAD. The percent of cell death was determined by
trypan blue staining; values represent three independent experiments.
B, Flow cytometric analysis (open histogram) of surface
Fas expression on control CESS cells or IL-6-differentiated plasma
cells. The shaded areas represent signals obtained from an isotype
control Ab. C, Immunofluorescence analysis of Fas
(green) and intracellular IgG (red) expression in control CESS cells
and IL-6-differentiated plasma cells. All cells were counterstained
with 4',6'-diamidino-2-phenylindole (DAPI; blue) to detect nuclei.
D, RT-PCR analysis of FasL mRNA expression in control
(Cont), IL-6-differentiated (IgGhigh), and IL-6-refractory
(IgGlow) CESS cells and the positive control P11 cells. The
analysis of GAPDH mRNA serves as a control for RNA loading.
) cells at a 10:1 ratio
for 5 h before determination of cell death by trypan blue staining
(left panel). In vivo-generated plasma cells were plated
onto CD40L-expressing L cells at a 10:1 ratio in the absence (