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Cutting Edge: Recruitment of the CD19/CD21
Coreceptor to B Cell Antigen Receptor Is
Required for Antigen-Mediated Expression of
Bcl-2 by Resting and Cycling Hen Egg
Lysozyme Transgenic B Cells
Teresa Roberts and E. Charles Snow
J Immunol 1999; 162:4377-4380; ;
http://www.jimmunol.org/content/162/8/4377
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
●
Cutting Edge: Recruitment of the
CD19/CD21 Coreceptor to B Cell
Antigen Receptor Is Required for
Antigen-Mediated Expression of Bcl-2
by Resting and Cycling Hen Egg
Lysozyme Transgenic B Cells
Teresa Roberts and E. Charles Snow1
B
cl-2 is the prototype for the Bcl-2 family of survival/
death proteins and represents the first oncoprotein demonstrated to promote cell survival rather than cellular expansion. Members of the Bcl-2 family share homology at
functional domains responsible for the formation of homo- and
heterodimeric complexes between family members (1). Since
Bcl-2 can heterodimerize and thus interfere with the activity of
proapoptotic Bcl-2 family members such as Bax, the relative levels
of Bcl-2 in relationship to the other family members is important
in determining cell fate (2). Bcl-2 interferes with Bax-mediated
cell death, at least in part, by blocking Bax-induced release of
mitochondrial cytochrome c (3, 4). Once in the cytosol, cytochrome c serves as a required cofactor for the initiation of a
Department of Microbiology and Immunology, University of Kentucky Medical Center, Lexington, KY 40536
Received for publication October 9, 1998. Accepted for publication January 27, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. E. Charles Snow, Department of
Microbiology and Immunology, University of Kentucky Medical Center, Lexington,
KY 40536-0084.
Copyright © 1999 by The American Association of Immunologists
●
caspase cascade. In addition, Bcl-2 acts downstream of cytochrome c release (5), most probably by sequestering the cytochrome c receptor, Apaf-1 (6).
Liu et al. (7) originally showed that cross-linking B cell Ag
receptor (BCR)2 expressed by centroblasts/centrocytes recovered
from human germinal centers by anti-BCR immobilized onto
sheep RBC induces elevated expression of Bcl-2. We extended this
observation by showing that restimulation of cycling B cells with
soluble F(ab9)2 anti-BCR induces accumulation of Bcl-xL while
restimulation with immobilized anti-BCR induces both Bcl-xL and
Bcl-2 (8). In this study, hen egg lysozyme (HEL)-specific transgenic B cells (9) are used to define the parameters necessary for
specific Ag to regulate Bcl-2 expression in both resting and cycling
B cell populations. Our results show that although occupancy of
BCR by either soluble or immobilized Ag elicits cellular accumulation of Bcl-xL, the CD19/CD21 coreceptor must be recruited to
induce Bcl-2 expression. The accumulation of both survival proteins correlates with enhanced responsiveness of resting or cycling
B cells to Th cell-induced proliferation. This suggests, therefore,
that stimulation of either resting or cycling B cells with Ags derived from inflammatory loci provides a survival and functional
advantage to the responding B cells.
Materials and Methods
Mice
A colony of HEL transgenic mice (the MD4 line, a generous gift of Dr.
Noelle, Dartmouth Medical School and originally described by Goodnow
and colleagues (9)) was maintained by breeding transgenic mice to normal
C57BL/6J females. Transgenic male and female mice were used at 6 –12
wk of age. The handling and care of all mice were approved by the University of Kentucky Animal Care Committee.
Preparation of resting and cycling B cells
Resting B cells were isolated from spleens of HEL transgenic B cells as
described (10). Cycling B cells were prepared from resting B cell preparations as described (8, 10). Briefly, 2.5 3 106 resting B cells/ml in complete media (RPMI 1640 supplemented with 10% FCS (Sigma, St. Louis,
MO), penicillin, streptomycin, gentamicin, glutamine, and 50 mM 2-ME)
were stimulated with 25 mg/ml LPS (purified from Salmonella enteritidis,
Sigma) in 100-ml bulk cultures per 162-cm2 flasks (Costar, Cambridge,
MA) for 48 h. The recovered cells were separated on three-step Percoll
gradients (Amersham Pharmacia Biotech, Piscataway, NJ), and the cells
2
Abbreviations used in this paper: BCR, B cell Ag receptor; HEL, hen egg lysozyme.
0022-1767/99/$02.00
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Recruitment of the CD19/CD21 coreceptor is thought to lower
the threshold for effective signaling through the B cell Ag receptor. We provide evidence supporting a second role for coreceptor recruitment, and that is to enhance the survival/proliferative potential of the responding B cells. We show that B
cell Ag receptor signaling in the absence of coreceptor recruitment induces cellular accumulation of the anti-apoptotic protein Bcl-xL, whereas CD19-mediated signals are required for
Bcl-2 accumulation. The expression of both anti-apoptotic proteins correlates with the enhanced responsiveness of both resting and cycling B cells to growth-promoting signals delivered
through CD40. These results provide further evidence for the
necessity of coreceptor recruitment during Ag-dependent B
cell activation and indicate that Ags derived from inflammatory sites function as better thymus-dependent Ags than their
counterparts not coated with complement fragments. The
Journal of Immunology, 1999, 162: 4377– 4380.
4378
CUTTING EDGE
banding to the 50%– 0% interface were collected and used as cycling B
cells.
Analysis of Ag-induced resting or cycling HEL transgenic B cell
proliferation
Both resting and cycling HEL transgenic B cells were cultured at 1 3 105
cells/well in 200 ml of complete media in 96-well flat-bottom plates
(Costar), and the cells were stimulated as described in the figure legends.
HEL (Sigma) and anti-CD19 (clone 1D, a rat IgG anti-mouse CD19, was
a generous gift of Dr. Bondada, University of Kentucky Medical Center)
were attached to cyanogen bromide-activated Sepharose 4B as suggested
by the vendor, Sigma. CD40 ligand-baculovirus-infected Sf9 cells were a
generous gift from Dr. Berton, University of Texas Health Science Center
at San Antonio, San Antonio, TX, and were fixed with paraformaldehyde
before use. Each well was pulsed with 1 mCi of [3H]thymidine (ICN, Irvine
CA, 67 Ci/mmol) for the final 6 h of culture.
Western analysis
Results and Discussion
Using normal B cells, we previously demonstrated that the restimulation of cycling B cells with either soluble F(ab9)2 or immobilized anti-m elicited the transient continuation of B cell proliferation (8). Although both forms of anti-BCR stimulation
induced the accumulation of the anti-apoptotic protein, Bcl-xL,
only restimulation with immobilized anti-m induced expression of
a second anti-apoptotic protein, Bcl-2. Thus, restimulation of cycling B cells with immobilized anti-m provided a potentially stronger survival/proliferative signal than that seen by cells restimulated
with a soluble anti-BCR reagent. Interestingly, the accumulation of
both anti-apoptotic proteins correlated with a heightened ability of
the cycling B cells to respond to CD40-mediated proliferative
signals.
Initial attempts at reproducing a similar pattern using cycling
HEL transgenic B cells restimulated with either soluble (HELs) or
immobilized (HEL:B) HEL failed. Two possibilities were considered to account for this discrepancy. First, immobilized anti-m provided a much stronger and/or longer lasting cross-linking of BCR
than similarly immobilized HEL. Second, delivering an equivalent
signal using HEL:B required the colocalization of the CD19/CD21
coreceptor to sites of cross-linked BCR. The present study addressed the second possibility by stimulating cycling or resting
HEL transgenic B cells with HEL coimmobilized with a monoclonal anti-CD19 Ab onto Sepharose beads (H:19:B).
Cycling HEL transgenic B cells recultured in the absence of
stimuli displayed a rapid reduction in their proliferation (media in
Fig. 1). As we have previously reported (10), restimulation of the
cycling cells with HELs, HEL-B, or H:19:B elicited transient increases in radionucleotide incorporation that was always less than
seen in cultures restimulated with CD40L alone (Fig. 1A). The
coaddition of either HELs or HEL-B with CD40L did not appreciably enhance proliferation beyond levels detected in cultures receiving only CD40L (Fig. 1B). In contrast, restimulation with
H:19:B both enhanced and extended CD40L-induced cycling B
cell proliferation (Fig. 1B). This result is reminiscent of our previous finding that immobilized anti-m extended and enhanced cycling B cell responsiveness to CD40-derived proliferative
signals (8).
FIGURE 1. Impact of CD19-derived signals on BCR- and CD40-induced maintenance of cycling B cell proliferation. The cycling B cells were
restimulated with: media, no stimulant; CD40L, a 1:50 ratio of fixed
CD40L/Sf9 cells to input B cells; HELs, 10 mg/ml soluble HEL; HEL:B,
10 mg/ml immobilized HEL; H:19:B, 10 mg/ml HEL coimmobilized with
anti-CD19; CD19s, 10 mg/ml soluble anti-CD19; or CD19:B, 10 mg/ml
immobilized anti-CD19. All cultures were pulsed with [3H]thymidine for
the final 6 h of culture. This experiment is representative of at least 10
independent experiments.
Since the ability of immobilized anti-m to potentiate CD40-mediated cycling B cell proliferation correlated with a concomitant
increase in both Bcl-2 and Bcl-xL, cycling HEL transgenic B cells
restimulated as described in Fig. 1 were examined by Western
analysis for expression of the two survival proteins (Fig. 2). As
seen when cycling normal B cells were challenged with soluble
F(ab9)2 anti-m (9), restimulation of cycling HEL transgenic B cells
with either soluble (results not shown) or immobilized (Fig. 2, lane
D) HEL induced expression of only Bcl-xL. In contrast, restimulation with HEL coimmobilized with anti-CD19 induced expression of both Bcl-2 and Bcl-xL (Fig. 2, lane E). Restimulation with
immobilized anti-CD19 induced accumulation of Bcl-2 but not
Bcl-xL (Fig. 2, lane C). This indicates that BCR-derived signals
were responsible for regulating Bcl-xL expression, while CD19mediated signals regulated Bcl-2 expression. Interestingly, restimulation with soluble anti-CD19 did not induce accumulation of
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Resting or cycling HEL transgenic B cells were cultured at 1 3 107 cells
in 1.0 ml of complete media in 12-well Costar plates and stimulated as
described in the figure legends. The Western analyses were done as described (11). The following Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): rat anti-mouse Bcl-2 (mAb 4C11), rabbit antihuman/mouse Bcl-xL/S (S-18), goat anti-human actin (C-11), anti-goat IgG
conjugated with horseradish peroxidase (sc-2020), and goat anti-rat IgG
conjugated with horseradish peroxidase. A goat anti-rabbit IgG conjugated
with horseradish peroxidase was purchased from Sigma.
The Journal of Immunology
4379
Bcl-2 (Fig. 2, lane F). Thus, simply cross-linking the CD19/CD21
coreceptor was not sufficient to elicit Bcl-2 expression, suggesting
that the CD19/CD21 coreceptors must aggregate to some minimal
density in the plane of the membrane to impact upon cellular accumulation of Bcl-2. Restimulation of the cycling cells with LPS
mimicked restimulation with CD40L (8) by inducing only the expression of Bcl-xL (Fig. 2, lane A).
Next, we determined the ability of BCR-derived signals, with or
without colocalization of the CD19/CD21 coreceptor, to regulate
Bcl-2 and Bcl-xL expression by resting HEL transgenic B cells. As
expected (12), HEL in soluble, immobilized, or coimmobilized
with anti-CD19 delivered signals insufficient to elicit resting B cell
cycle progression (Fig. 3A). Unexpectedly, however, neither immobilized nor soluble HEL was found to substantially augment the
level of resting B cell radionucleotide incorporation induced by
CD40L (Fig. 3B). However, when HEL was coimmobilized with
anti-CD19, there was a dramatic increase in CD40L-induced B cell
proliferation (Fig. 3B). Although immobilized anti-CD19 modestly
increased CD40-mediated resting B cell proliferation, soluble antiCD19 always decreased CD40-mediated B cell proliferation (Fig.
3C). The reason for this is unclear. The same pattern of Bcl-2 and
Bcl-xL accumulation seen after restimulation of cycling HEL
transgenic B cells was seen when resting HEL transgenic B cells
were stimulated with the various forms of Ag (Fig. 4). Both soluble (results not shown) and immobilized HEL induced Bcl-xL
accumulation in the absence of Bcl-2 expression. Immobilized, but
not soluble, anti-CD19, induced Bcl-2 expression with or without
FIGURE 3. Impact of CD19-derived signals on BCR- and CD40-induced resting B cell proliferation. Resting B cells were stimulated as indicated (notations and concentration of reagents shown in the legend to Fig.
1). All cultures were pulsed with [3H]thymidine for the final 6 h of culture.
This experiment is representative of at least five independent experiments.
being coimmobilized with HEL. Immobilized anti-CD19 in the
absence of HEL, however, failed to elicit Bcl-xL accumulation.
Therefore, BCR-derived signals regulated Bcl-xL expression while
signals through CD19 regulated Bcl-2 expression.
CD19 is recruited to BCR aggregated by complement-coated
Ags because it is noncovalently associated within the plane of the
membrane with CD21, the C3d receptor (13). The importance of
this recruitment is highlighted by studies showing that both CD21
(14, 15) and CD19 (16, 17) knockout mice are impaired in their
ability to respond to thymus-dependent Ags. One explanation for
this dramatic codependence is that CD19/CD21 recruitment effectively lowers the threshold of Ag required to induce B cell cycle
progression because CD19 amplifies BCR-mediated activation signals (18). Our results support a previously proposed second alternative (19), that CD19 recruitment to sites of active BCR signaling
provides B cells with a survival advantage. Furthermore, the data
suggest that this is accomplished by a CD19-mediated induction of
Bcl-2 accumulation to complement Bcl-xL induced by virtue of
BCR signaling.
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FIGURE 2. Impact of BCR- and CD19-derived signals on the expression of Bcl-2 and Bcl-xL by cycling B cells. The cycling B cells were
restimulated with: no stimulant (A), 25 mg/ml LPS (B), 10 mg/ml immobilized anti-CD19 (C), 10 mg/ml immobilized HEL (D), 10 mg/ml HEL
coimmobilized with anti-CD19 (E), and 10 mg/ml soluble anti-CD19 (F).
In this experiment, the same blot was probed on three separate occasions
with anti-Bcl-2, anti-Bcl-xL, and anti-actin as described in Materials and
Methods. The bands were identified based on their migration (Bcl-2 migrated even with the 29-kDa standard, Bcl-xL migrated between the 29- and
33-kDa standards, and actin migrated just below the 51-kDa standard). The
migratory patterns were verified by exposure of the primary Abs to blocking peptides before assay (results not shown). This experiment is representative of five independent experiments.
4380
CUTTING EDGE
not recruit the CD19/CD21 coreceptors, induces Bcl-2 accumulation (8). It is possible that multimeric thymus-independent Ags that
are capable of inducing extensive and long lasting BCR aggregation can elicit the same pattern of survival protein expression as
seen when thymus-dependent Ags are derived from inflammatory
sites and coaggregate BCR and CD19/CD21 coreceptors. This possibility will require further attention.
Acknowledgments
We thank Dr. Michael Berton for the generous gift of the CD40L/Sf9 cells
and Drs. Subbarao Bondada and Thomas Roszman for their critical comments on the manuscript.
References
Both resting and cycling B cells require BCR-derived signals to
either be recruited into or remain part of an ongoing humoral immune response. Our results suggest that whether or not the Ag is
derived from inflammatory foci impacts upon the ultimate survival/proliferative potential of the responding B cells. Soluble or immobilized Ags not derived from inflammatory foci only induce the
accumulation of Bcl-xL, and elicit only minimal enhancement of
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The one caveat to the above hypothesis is raised by our demonstration that immobilized, but not soluble, anti-BCR, which does
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FIGURE 4. The impact of BCR- and CD19-derived signals on the expression of Bcl-2 and Bcl-xL by resting B cells. The experimental groups
were identical with those described in the legend for Fig. 2 except that
resting HEL transgenic B cells were used, a sample of which was assayed
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