Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While
B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here
induction of the complete complement of cell cycle regulatory molecules, though the level of
induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig
stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of
apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle
progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell
cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly
regulate Bcl-xL.
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Introduction |
Bcell development proceeds as a series of sequential
check points designed to ensure the production and
coupling of functional heavy and light chains, and the integrity of the Ig signal transduction cascade (1). Mutations
which lead to disruption of the Ig receptor complex or
downstream signaling components can have dramatic effects on the generation and/or function of developing B
cells (2). The X chromosome-encoded cytoplasmic
Bruton's tyrosine kinase (Btk)1 is required for efficient signaling through the antigen receptor and B cell development in both mice and humans (5). In humans, mutations in Btk result in X-linked agammaglobulinemia (XLA), a
severe immunodeficiency that manifests as a block in B cell development at the pre-B cell stage, rendering affected
males virtually devoid of peripheral B cells (9). A spontaneous mutation in Btk in mice results in the milder X-linked
immunodeficiency, xid, which is characterized by reduced
numbers of splenic B cells and a failure of these B cells to
enter the long-lived B cell pool (12, 13). Additionally, xid
B cells have an unusual surface phenotype that is not characteristic of either immature or mature B cells (14, 15),
suggesting some defect in development after their generation in the bone marrow. Furthermore, peritoneal B1 cells
are absent (16) and there are specific Ig isotype deficiencies. However, despite these immunodeficient traits, the mice
are robust and have a normal life span. The variability in
severity of disease between xid and XLA reflects genuine
species-specific differences in the requirement for Btk during B cell development rather than differences in site-specific mutations in Btk (7, 8, 11).
Although Btk is thought to be a component of several
signal transduction pathways, it is almost certainly the disruption of the Ig signal transduction pathway that results in
the immunodeficiencies of XLA and xid. In vitro cross-linking of surface IgM on xid B cells fails to promote cell
cycle progression, consistent with the observation that
XLA and xid phenotypes include a failure to expand specific B cell populations (pre-B cells in humans and B1 cells
in mice) whose development is thought to require productive antigen receptor signaling (17, 18).
The molecular control of cell cycle progression involves
the sequential activation of a family of serine/threonine kinases known as cyclin-dependent kinases, or cdks, and their
subsequent phosphorylation of specific substrates (19, 20).
cdks are activated in part by physical coupling with cyclins,
a family of regulatory subunits that are induced at phase-specific stages during the cell cycle. The decision to enter S
phase occurs late in G1, at the restriction point, R, after
which the cells are committed to DNA replication and cellular division. A major component of the restriction point
is the phosphorylation and inactivation of the protein production of the retinoblastoma gene, Rb, during G1 by the
cyclin D-associated kinase activity (21). However, despite
the requirement for Rb phosphorylation for S phase entry, inactivation of Rb does not guarantee cell cycle progression since Rb phosphorylation can be detected in cell lines
undergoing apoptosis (22, 23). In such systems, ectopic expression of the antiapoptotic molecule, Bcl-2, permits orderly cell cycle progression. These and other experiments
led to the hypothesis that cellular division can only be
achieved with the engagement of the proliferative machinery in the presence of antiapoptotic proteins (22, 24, 25).
The Bcl-2-related antiapoptosis regulatory protein, Bcl-xL, is expressed at different stages during B cell ontogeny
(26). Intriguingly, Bcl-xL is expressed at stages of B cell
development arrested in XLA (pre-B cells) and xid (B1 B
cells) (29) and is known to be upregulated as a consequence
of antigen receptor cross-linking (27). The importance
of Bcl-xL expression during B cell development is demonstrated by the expansion of the pro-pre-B cell compartment in two independently generated bcl-xL transgenic mouse models (26, 27) and the dramatic loss of peripheral lymphoid system in chimeric mice homozygous for a targeted deletion in bcl-x (30).
Given that the defects in XLA and xid both affect expansion of B cells at developmental stages dependent on Ig
signaling, we explored the influence of defective Btk on
the induction of cell cycle regulatory proteins after anti-Ig
stimulation. Our results indicate that the Ig signal transduction cascade that uses mutant Btk does stimulate the induction of cyclins, cdks, and kinase activity; however, there is
a specific failure to normally upregulate Bcl-xL. The induction of cell cycle regulatory machinery via mutant Btk was
sufficient to promote proliferation in the presence of transgene-driven Bcl-xL expression. These results demonstrate a
specific requirement for the antiapoptotic protein Bcl-xL
during antigen receptor-induced B cell proliferation and links certain immunodeficient traits in xid with failure to
induce this viability-enhancing protein.
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Materials and Methods |
Mice.
Male and female CBA/N (xid) mice were obtained
from The Jackson Laboratory (Bar Harbor, ME) and used as
breeding pairs at DNAX Research Institute. CBA/Ca mice were
obtained from The Jackson Laboratory and used as control mice.
Males from two previously described and independently derived
strains of bcl-x transgenic mice (bcl-x-87; reference 26, and bcl-x-
81; reference 25) were used in breedings with female CBA/N
(xid).
Reagents.
Antibodies used in this study were monoclonal rat
anti-mouse cyclin D3 and D2, rabbit anti-mouse cyclin E, cyclin
A, cyclin B, cdk2, cdk4, and cdk6; these reagents were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse
anti-mouse cell division cycle (cdc)2, mouse anti-human Rb,
and anti-Bcl-2 antibodies used for Western blotting were purchased from PharMingen (San Diego, CA). The anti-CD43
FITC, anti-B220 PE, anti-CD5 PE, and anti-B220 FITC used in
flow cytometric analysis were purchased from PharMingen. Anti-Bcl-xL and anti-Bad were purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated anti-rat, rabbit, and mouse secondary reagents were purchased from
Amersham Corp. (Arlington Heights, IL). The anti-cdc2 antibody used for immunoprecipitations was purchased from GIBCO
BRL (Gaithersburg, MD). The substrate for the kinase assay, histone H1, was purchased from Boehringer Mannheim (Indianapolis, IN). The affinity purified F(ab')2 goat anti-mouse IgM used to
stimulate B cells was purchased from Cappel Labs. (Malvern, PA).
The rat anti-mouse monoclonal anti-CD40 antibody has been
described previously (31).
Purification of Splenic B Cells.
To isolate splenic B cells, single
cell suspensions were prepared from spleens of unprimed mice.
Red blood cells were lysed using red blood cell lysis buffer (Sigma
Chemical Co., St. Louis, MO) according to the manufacturer's
instructions. To deplete T cells, splenocytes were stained using
anti-Thy-1 (Dupont-NEN, Boston, MA) and anti-CD4 (RL172),
followed by incubation in rabbit complement (Cedarlane Labs.
Ltd., Hornby, Ontario, Canada) at 37°C for 45 min. These cells
were consistently >90% B2201/IgM1 and included both the low
and high density B cells.
In Vitro Stimulations and Whole Cell Lysate Preparation.
The ex-
pression of cell cycle regulatory proteins in anti-Ig-stimulated B
cell populations was evaluated using procedures previously described (32). In brief, 50-ml cultures were set up at 106 B cells/ml
in 75 cm2 flasks (Becton Dickinson Labware, Lincoln Park, NJ)
and stimulated with 25 µg/ml F(ab')2 anti-IgM. Cells were collected at 6, 12, 24, 36, 48, and 72 h after stimulation. All stimulations were carried out in supplemented RPMI containing 10%
FCS (JR Scientific, Woodland, CA), 5 × 105 M 2-ME (Polysciences, Inc., Warrington, PA), 2 mM glutamate (JR Scientific),
10 mM Hepes buffer (Irvine Scientific, Santa Ana, CA), 100 U/ml
penicillin, and 100 µg/ml streptomycin (Irvine Scientific). After
stimulation, cells were recovered, washed three times in cold
PBS, divided into three pellets, and stored at 
80°C for later use
in Western blotting and in vitro kinase assays. When all time
points were collected, one pellet from each time point was lysed
in NP-40 lysis buffer (1% NP-40, 250 mM NaCl, 1 mM Hepes, pH 7.5, and 1 mM dithiothreotol [DTT; United States Biochemical Corp., Cleveland, OH] with protease inhibitors added;
final concentration: 5 µg/ml aprotinin [Sigma Chemical Co.],
125 µg/ml Pefabloc [Boehringer Mannheim], 5 µg/ml pepstatin
[Sigma Chemical Co.]). Protein concentrations were calculated
using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's procedures. An equal volume of
2× SDS sample buffer (Novex, San Diego, CA) with 2-ME was
then added to the total cell lysates. 125 µg of lysate was run per time point on 12% Novex reducing gels according to manufacturer's suggested protocol (6% gels were used to detect the Rb protein), and transferred onto Immobilon PVDF membranes (Millipore,
Bedford, MA) at 30 V overnight in 4°C. The mouse plasmacytoma line P3X was obtained from the American Type Culture
Collection (Rockville, MD) and used as a positive control on all gels.
In Vitro Kinase Assay.
cdk2- and cdc2-dependent kinase assays were performed using the histone substrate according to procedures described elsewhere (33). In brief, frozen pellets were
lysed in 1 ml 0.1% NP-40 lysis buffer containing 50 mM Hepes,
pH 7.0, and 250 mM NaCl in the presence of the above protease
inhibitors. Protein concentration was calculated as described
above, and 200 µg of total protein was used for each kinase assay.
In brief, lysates were precleared with normal rabbit serum followed by Zysorbin (Zymed, South San Francisco, CA). 1 µg of
anti-cdk2, anti-cdc2, or normal rabbit sera was added to the lysate
and incubated at 4°C for 4 h with rocking. Protein G beads
(Pharmacia Biotech, Inc., Piscataway, NJ) were added and the lysates were rotated in the cold for another hour. Immunoprecipitates were washed 4 times in lysis buffer followed by two washes
in kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM
DTT). The kinase reactions were carried out in a 50 µl volume containing 2.5 µg histone H1 and 5 µCi [32P]
ATP (Amersham
Corp.) per reaction. Reactions proceeded for 30 min at room
temperature with rocking. Samples were run on 12% Novex precast gels, fixed, dried, and exposed to phosphor screen (Molecular
Dynamics, Sunnyvale, CA). The results were read on a Storm
860 phosphorimager (Molecular Dynamics).
Cell Cycle Analysis.
Cell cycle analysis was performed using a
modified method of Carayon and Bord (34). Purified B cells were
cultured at 106/ml in 200-µl aliquots in the presence of 50 µM
bromodeoxyuridine (BrdU; Sigma Chemical Co.) and an equimolar
concentration of cytidine (Sigma Chemical Co.) plus 25 µg/ml
anti-IgM for times indicated. Cells were harvested and stored in
PBS with 0.5% paraformaldehyde overnight. PBS with 5%
Tween 20 was added to permeabilize the nuclear membrane. The
cells were then washed and resuspended in a solution of 10 mg/ml
DNAse I (Sigma Chemical Co.) plus anti-BrdU (Becton Dickinson, Mountain View, CA). Finally propidium iodide (PI) is added
and then analysis is performed on FACScalibur® using Cellquest
software (Becton Dickinson).
Thymidine Incorporation.
200-µl aliquots (2 × 105 total cells)
of stimulated cells were placed in 96-well flat-bottomed plates at
times indicated and pulsed for 45 min in 1 µCi [3H]thymidine
(Amersham Corp.). Cells were harvested and incorporated cpms
were counted using a PHD cell harvester (Cambridge Technology Corp., Cambridge, MA).
Testing of Serum IgM and IgG3 Levels by ELISA.
Sera was col-
lected from the experimental mice. Samples were serially diluted
and compared with known standards of purified IgM and IgG3 as
previously described (35).
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Results |
Induction of Complete Complement of Cell Cycle Regulatory
Proteins in Anti-Ig-stimulated xid B Cells.
Consistent with
a previous report (36), purified B cells from xid mice
showed little [3H]thymidine incorporation in response to
stimulation with 25 µg/ml anti-IgM (Fig. 1 A). In contrast,
the level of [3H]thymidine incorporation in xid B cells after
stimulation with 25 µg/ml anti-CD40 was comparable to
normal B cells (Fig. 1 B). Since B cells from xid mice have
a selective inability to proliferate in response to anti-IgM
stimulation, it was possible that the xid mutation in Btk affected induction of proteins that control cell cycle progression in response to antigen receptor cross-linking. To test
this, lysates were prepared from anti-IgM stimulated xid and control B cells at different times after stimulation
through 72 h and Western blot analysis of cell cycle regulatory proteins was performed. Fig. 2 shows the induction of
regulatory proteins that control the entry into G1 (i.e., cyclin D2, cyclin D3, cdk4, cdk6, and Rb). Cyclins D2 and
D3 were induced after anti-Ig stimulation of xid B cells
with the same kinetics as in the control CBA/Ca B cells,
the only difference being the slightly greater induction of
the faster migrating form of cyclin D3 in the control B
cells, the significance of which is not known (Fig. 2, A and
B). Similarly, both cdk4 and cdk6 were present to comparable extents in both the anti-Ig-stimulated control and xid
B cells (Fig. 2, C and D). Most notable was the comparable
induction and hyperphosphorylation of Rb in both xid and
control B cells after anti-Ig stimulation (Fig. 2 E). Surprisingly, despite the inability of xid B cells to proliferate, the
induction of cyclins and cdks involved in S phase progression were also detected. Specifically, both cyclins E and A
(Fig. 3, A and B), along with their kinase partners cdk2 and
cdc2 (Fig. 3, C and D) were detected at about half the levels seen in control B cells. The only significant difference in
these two populations was found at the level of expression
of cyclin B, the regulatory protein that governs the progression through mitosis. Although cyclin B was only
weakly induced in anti-Ig-stimulated xid B cells, it was easily detected in control B cells (Fig. 3 C).
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Fig. 1.
Defective anti-IgM-triggered proliferation in xid B cells. B
cells from xid (black bar) and CBA/Ca (gray bar) were stimulated with
mAbs against CD40 (A) or F(ab')2 anti-IgM (B) and pulsed with [3H]thymidine for 1 h at the time indicated. Standard errors are shown and results
are representative of three independent experiments.
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Since both the cyclins and cdk partners required for cell
cycle progression were induced in anti-Ig-stimulated xid B
cells despite the inability of these cells to proliferate, we
performed cdk2- and cdc2-associated kinase assays to determine if cyclin/cdk complexes were assembled correctly
and that activating posttranslational modifications had occurred. Fig. 4 shows that both cdk2- and cdc2-associated kinase activities are clearly present in anti-IgM-stimulated
xid B cells at 48 and 72 h after stimulation, albeit at approximately half the levels detected in anti-Ig-treated control B
cells. Collectively, these data indicate that anti-Ig-stimulated xid B cells induce substantial levels of active cell cycle
regulatory proteins despite their inability to complete the
cell cycle in response to this stimulus.
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Fig. 4.
In vitro kinase assays from anti-IgM-stimulated B cells. Stimulated B cells were collected at the time points indicated and used to measure (A) cdk2- and (B) cdc2-associated specific kinase activity using Histone H1 as the substrate in the presence of [32P]gATP. Kinase activity was
quantitated by comparing the amount of 32P incorporated into the substrate by phosphorimager analysis. Kinase activity detected in xid (black
bars) and CBA/Ca (gray bars) are shown as arbitrary units. Similar results
were obtained in three independent experiments.
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Anti-Ig-stimulated xid B Cells Successfully Pass the G1 Restriction Point, then Enter an Aborted S Phase.
Since cell cycle regulatory protein induction and kinase activity were
detectable in anti-Ig-stimulated xid B cells, we carefully examined the various stages of cell cycle progression using
BrdU and PI labeling to distinguish the discrete steps of cell
cycle progression. As previously described (34, 37), the
combination of long-term BrdU incorporation plus PI
staining allows the discrimination of cells that are in (a) the
original G0/G1 phase; (b) the S phase; (c) the G0/G1 phase
of the second cell cycle; and finally (d) apoptotic cells (as illustrated diagrammatically in Fig. 5, top). Anti-IgM-stimulated control B cells clearly revealed a characteristic S phase
entry that began as early as 24 h after stimulation (Fig. 5,
center, arrow) (for review see reference 38). By 36 h, cells were beginning to successfully accumulate in the second
round of the cell cycle and could still be detected in S phase
at 48 and 72 h after stimulation. Such detailed analyses revealed that anti-IgM-stimulated xid B cells also successfully
passed the restriction point and entered S phase at 36 h
(Fig. 5, bottom, arrow), albeit some 12 h later than in the
control B cells. These data presumably explain the small
amounts of thymidine incorporation that can be detected at
48 and 72 h after stimulation of xid B cells with anti-Ig
(Fig. 1). However, BrdU and PI costaining convincingly established that there was no accumulation of anti-Ig-stimulated cells in the second round of the cell cycle (Fig. 5,
bottom), indicating that the cells do not successfully complete S phase. Indeed, by 48 and 72 h after stimulation,
>85% of the anti-Ig-stimulated xid B cells are apoptotic
(Fig. 5, bottom).
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Fig. 5.
BrdU incorporation and PI analysis of anti-IgM-stimulated
B cells from CBA/Ca and xid B mice. The diagram at the top indicates
the cell cycle position of cells at the time the cells were harvested. This
analysis identifies cells in G0 (G0), S phase (S), and those which have
completed S phase and are now in the second cell cycle G0 phase (2nd
G0), and apoptotic cells (Apo). B cells from CBA/Ca (top row) and xid
(bottom row) mice were stimulated with 25 µg/ml of anti-IgM in the presence of BrdU. Cells were harvested at time points indicated and fixed to
permit intranuclear staining of DNA with PI. The initial entry of B cells
from xid and CBA/Ca is indicated with an arrow. Analysis was performed
on FACSCalibur® using CellQuest software.
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These analyses are also consistent with previous reports
showing that xid B cells are more prone to undergo apoptosis than control B cells (29, 39). The increased susceptibility of xid B cells to apoptosis was evident as early as 12 h
after stimulation with anti-Ig, when 20% of the cells were
found to be apoptotic by PI staining compared with 5% of
the normal B cells (Fig. 5). The increased susceptibility to
apoptosis was unrelated to anti-Ig stimulation, since xid B
cells cultured in media alone died at a significantly faster
rate than did their non-xid counterparts (data not shown).
Even in the presence of the strong mitogen, anti-CD40,
that clearly stimulates xid B cells to proliferate (see Fig. 1),
there is a higher proportion of apoptotic cells in the cultures than in the anti-CD40-stimulated control cells (data
not shown). Thus, in addition to the selective inability to
proliferate to anti-IgM stimulation, xid B cells are more apoptosis-susceptible than are their normal B cell counterparts.
Diminished Induction of Bcl-xL in Anti-Ig-stimulated xid B
Cells.
To explore the mechanism underlying the increased apoptosis susceptibility of xid B cells, we evaluated
expression of the apoptotic regulatory proteins Bcl-2, Bax,
and Bcl-xL in this population. As shown in Fig. 6, A and B,
the levels of Bcl-2 and Bax are comparable in anti-Ig-stimulated xid and control B cells. In contrast, there was a striking and consistent decrease in Bcl-xL induction in anti-Ig-
stimulated xid B cells compared with normal B cells (Fig. 6
C). The induction of Bcl-xL in anti-Ig-stimulated normal
B cells was found to begin as early as 12 h after stimulation,
presumably exerting its antiapoptotic effects long before
the cells enter S phase.
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Fig. 6.
Analysis of Bcl-2 and related proteins. B cells were stimulated
as described in the legend to Fig. 2 and were collected at the time points
indicated. Western blots were prepared and screened with antibodies specific for Bcl-2 (A), Bax (B) or Bcl-xL (C). Horseradish peroxidase conjugates were used as secondary reagents followed by visualization with enhanced chemiluminescence.
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Bcl-xL Transgene Permits Anti-Ig-induced xid B Cell Proliferation.
To determine whether the diminished induction of
Bcl-xL in anti-Ig-stimulated xid B cells contributed to their
failure to proliferate, we evaluated the consequence of introducing two independently generated bcl-x transgenes
into these animals. CBA/N (xid) female mice, homozygous
for the mutation in the X-linked gene Btk, were bred to
male mice that were heterozygous for a bcl-x transgene (bcl-x-87 or bcl-x-81). These crosses provided offspring in which all of the males were xid while the females were non-xid.
Additionally, the offspring were either heterozygous for the
bcl-x transgene or were wild-type. The presence of the bcl-x
transgene permitted B cells from F1 male (xid.bcl-x-87)
mice to proliferate to anti-IgM stimulation; these cells successfully completed the cell cycle and could be detected in
the G0/G1 stage of second cell cycle (Fig. 7), confirming
our observations that mutations in Btk do not block cell
cycle machinery induction. However, it should be noted
that there was still a delay in entry into S phase that was not
detected in the female F1 (xid.bcl-x-87) mice, suggesting that mutant Btk continued to effect the rate of entry into
cell cycle. Indeed, although splenic B cells from transgene
positive male F1 (xid.bcl-x-87) mice did proliferate to anti-Ig, the proportion of responding cells was only about half
of that detected in the female F1 (xid. bcl-x-87) mice. Consistent with this, we detected only about half the amount of
[3H]thymidine incorporation in the male F1 (xid.bcl-x-87)
mice compared to that in the female F1 (xid.bcl-x-87) mice
(Table 1).
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Fig. 7.
BrdU and PI analysis of stimulated B cells from xid and F1
(xid.bcl-x-87) offspring. B cells from xid, female F1 (xid.bcl-x-87), and
male F1 (xid.bcl-x-87) mice were stimulated with 25 µg/ml anti-IgM in
the presence of BrdU and collected at the indicated time points. Cells
were prepared and analyzed as described in the legend to Fig. 5. Arrows
indicate the initial entry of cells into S phase.
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Partial Correction of Other xid Traits.
Since the Bcl-xL trans-
gene restored the proliferative response in xid B cells, we
examined the male F1 (xid. bcl-x-87) mice to evaluate the
overall ability of the Bcl-xL transgene to complement other
immunodeficient traits. The male F1 (xid.bcl-x-87) mice, all
of which expressed the bcl-x transgene along with defective Btk (F1 male Nos. 1, 2, 3, and 4, Table 2) had ~3 times
the number of splenic B cells as were detected in normal
CBA/Ca mice and 15 times the number of splenic B cells
found in the CBA/N (xid) mice. The effect of the bcl-x
transgene was also detected in the female F1 (xid.bcl-x-87)
mice carrying the bcl-x transgene (F1 female Nos. 2 and 4, Table 2) increasing the splenic B cells numbers to 2-3
times that detected in the normal CBA/Ca controls or female F1 (xid.bcl-x-87) mice that did not express the transgene. Consistent with these findings, the presence of the
bcl-x transgene in the male F1 (xid.bcl-x-87) mice increased
the total number of peritoneal B cells to that detected in
the normal CBA/Ca control mice, which is >10 times the
number of B cells detected in the peritoneal cavity of xid
mice (Table 3).
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Table 2
Comparison of Splenic B Cell Numbers and Phenotype in CBA/Ca, xid, Female F1 (xid.bcl-x-87), and Male F1
(xid.bcl-x-87) Mice
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In addition to expanding the peripheral B cell pool, the
bcl-x-87 transgene provided some correction of the unusually high levels of sIgM expressed on xid splenic B cells
(Table 2). Transgene positive splenic B cells from male F1
(xid.bcl-x-87) mice expressed less IgM than the B cells from
the xid mice, whereas the levels of IgD were comparable
between these two groups (Table 2). However, the levels
of IgM on B cells from the male F1 (xid.bcl-x-87) was still
higher than that detected on either the CBA/Ca mice or
the female F1 (xid.bcl-x-87). Additionally, though the peritoneal compartment of the male F1 (xid.bcl-x-87) mice was
expanded, most of the cells did not express the typical cell
surface phenotype of CD5 B cells, which represent the major B cell subset in a normal peritoneal cavity (Table 3).
The transgene did increase the number of CD5 B cells to
fourfold that detected in the xid mice. However, this still
reflected a 90% reduction when compared with wild type animals.
To determine the effect of the bcl-x transgene on viability, splenic B cells from xid, CBA/Ca controls, male and
female F1 (xid.bcl-x-87) mice that carried the bcl-x transgene were cultured in vitro and viability compared. As expected, viability of xid B cells was less than B cells from
normal control CBA/Ca (Fig. 8), however, the bcl-x-87
transgene substantially enhances the viability of the B cells
in both the male and female F1(xid.bcl-x-87) mice.
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Fig. 8.
Comparison of in
vitro viability. Splenic B cells
from CBA/Ca, xid, male F1
(xid.bcl-x-87), and female F1
(xid.bcl-x-87) mice were cultured
in 96-well flat-bottomed wells at
106 cells/ml in RPMI with 10%
FCS for times indicated. From
days 1 to 6, viability of spleen B
cells was assessed by Trypan blue
exclusion. Results are representative of three independent experiments.
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Although the bcl-x-87 transgene increased the number
and viability of B cells from xid F1 male mice, Bcl-x was
incapable of correcting the reduced levels of serum IgM
and IgG3 levels in the male F1 (xid.bcl-x-87) mice compared with xid mice (Fig. 9). Curiously, the levels of IgM
in the F1 male (xid.bcl-x-87) mice were consistently lower
than levels detected in the male xid mice. Serum IgM and
IgG3 levels in the female F1 (xid.bcl-x-87) mice were indistinguishable from that detected in the parent C57BL/6 strain.
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Fig. 9.
Serum immunoglobulin levels of IgM and IgG3. Sera
was collected from 8-wk-old xid
(n = 5), CBA/Ca (n = 4), F1 female (n = 5), and F1 male (n = 6) mice. Biotin-conjugated anti-IgG3 and anti-IgM reagents
were used in combination with
horseradish peroxidase-conjugated streptavidin in ELISAs to
quantitate serum Ig levels. Serial
dilutions of each sample were
made and compared with standard curves generated by IgM or
IgG3 preparations of known
concentrations. Asterisks indicate
mice carrying the bcl-x transgene.
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Interestingly, only the F1 offspring from the bcl-x-87
transgenic subline showed any correction of the xid phenotype. Table 1 indicates that anti-Ig-stimulated B cells from
male F1 (xid.bcl-x-81) mice showed little difference in
[3H]thymidine incorporation when compared with the xid
mice. The bcl-x-81 transgene expresses at high levels in developing B cells (26), but at low levels in peripheral B cells
(Behrens, T., et al., unpublished data). Additionally, there
was minimal increase in the peripheral B cell pool, little enhanced viability, and no difference in serum Ig levels between the F1 male (xid.bcl-x-81) in comparison with the
xid males (data not shown).
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Discussion |
Our initial experiments focused on the hypothesis that
the observed inability of xid B cells to proliferate to antigen
receptor cross-linking stemmed from a failure to induce
components of the cell cycle machinery. However, although there was an ~50% reduction in cyclin, cdk, and
kinase activity in anti-Ig-stimulated xid B cells compared
with normal controls, the complete complement of cyclins
and cdks were induced in anti-Ig-stimulated xid B cells. Additionally, the kinetics of protein induction and active
kinase induction in stimulated xid B cells was comparable
to that detected in anti-Ig-treated control B cells. In data
not shown, we confirm that the destruction of the cdk inhibitor, p27, after stimulation with anti-Ig antibodies was
also indistinguishable in xid and control B cells, as was the
appearance of the INK4 inhibitors, p18 and p19. Together
these results suggest that the signals inducing cell cycle machinery after antigen receptor cross-linking in B cells from
xid mice are intact although insufficiently amplified due to
mutations in Btk.
The decision to undergo cellular replication occurs late
in G1 at the restriction point, R, after which the cell is
committed to undergo DNA replication and cellular division. Despite the diminished induction of cell cycle regulatory proteins in anti-Ig-stimulated xid B cells, these cells
passed the restriction point and began entry into S phase,
albeit some 12 h after control B cells. The ability of anti-Ig-stimulated xid B cells to pass the restriction point suggests that the insufficient amplification of the Ig-generated signal simply resulted in a delay in passage through R,
rather than an inability to pass R. Typically, unless severe
damage to DNA is encountered, the entry into S phase
guarantees completion of cell cycle progression. Indeed,
there was significant cdk2- and cdc2-associated kinase activity present at time points during which the S and G2/M
phases should have occurred; however, at this time at least
85% of the cells were apoptotic, indicating that the cell cycle machinery was still engaged despite the fact that the
cells were dying.
Our finding that Bcl-x induction was diminished in
anti-Ig-stimulated xid B cells is consistent with reports
from Anderson et al. (29) and Choi et al. (28). We extend
these observations by demonstrating that transgene driven
expression of Bcl-xL substantially corrects the inability of
xid B cells to proliferate in response to stimulation with
anti-Ig antibodies in vitro. It is noteworthy that we could
detect some Bcl-xL induction in anti-Ig-stimulated B cells
from xid mice, indicating again that the signaling pathway leading to Bcl-xL induction is not entirely disrupted. It
seems likely that some critical level of Bcl-xL protein is
needed to permit anti-Ig-induced proliferation since reconstitution of the proliferative response was only detected
in xid crosses with the bcl-x-87 transgenic mouse, which
expresses high levels of Bcl-xL in the peripheral B cell compartment. Despite the difference in phenotype, both the
bcl-x-87 and bcl-x-81 transgenics are carried on the C57BL/6
background. Thus, it is unlikely that the correction in the
xid phenotype observed with the male F1 (xid.bcl-x-87) mice is due to C57BL/6 background genes.
It is significant that the proportion of anti-Ig-responsive
B cells in the male F1 (xid.bcl-x-87) mice and the rate of
entry into S phase was still reduced compared with female
F1 (xid.bcl-x-87) mice. We suspect this results from the still
incompletely amplified signal through Btk leading to half-maximal induction of cell cycle regulatory proteins. These
results suggest that the crippling effect of mutant Btk on the
proliferative response is the failure to properly amplify signals generated by the antigen receptor. This is supported by
our data showing the induction of both cell cycle regulatory proteins plus Bcl-xL although at levels far below those
detected in wild-type mice. Thus, one would predict that if
signals generated at the antigen receptor could be significantly enhanced that perhaps B cells from xid mice could
be stimulated to proliferate. Indeed, reports from Lindsberg
et al. (40) showed that xid B cells can proliferate in the
presence of anti-Ig reagents, which induces extensive and
prolonged antigen receptor cross-linking.
The physiological significance of defective Bcl-xL induction in relation to other immunodeficient traits is established in our male F1 (xid.bcl-x-87) mice, where there was a
dramatic increase in the number of splenic and peritoneal B
cells plus enhanced in vitro viability. The hyperreconstitution in splenic B cell numbers we observed is comparable
to that reported in the original description of the bcl-x-87
transgenic line (27). We suspect that the expansion of the
splenic B cell pool stems from the demonstrated enhancement of B cell viability coupled with inhibition of cell death rather than from enhancing B cell proliferation. The
transgene, however, provided only minimal correction of
the unusual phenotype of splenic B cells in the xid mice.
Splenic B cells expressed IgM at levels intermediate between the xid and the CBA/Ca controls suggesting, at
most, a partial correction of the atypical xid splenic B cell
phenotype.
The number of B lymphocytes in the peritoneal cavity
of male F1 (xid.bcl-x-87) mice was also expanded to numbers comparable to normal control mice. However, there
was only a modest increase in the total number of CD5-
expressing B cells compared with control mice. Furthermore, the expanded peritoneal B cell population in the
male F1 (xid.bcl-x-87) mice was not the CD5 "sister" population since the B cells did not express Mac-1 nor the
characteristic ratio of IgMhi/IgDlo (data not shown). The
inability to reconstitute CD5 B cells is difficult to interpret
since it has not been established that the bcl-x transgene is
expressed in the CD5 B cell compartment; normal CD5 B
cells express Bcl-xL constitutively (29), making the efficiency of transgene-driven Bcl-xL expression difficult to establish in these cells. Alternatively, we cannot rule out the
possibility that there are additional defects in the Btk signal-transduction cascades that effect development of this particular B cell subset. Regardless of the mechanism, the poor
reconstitution of the CD5 B cell population may partially
explain the failure to correct the serum IgM and IgG3 deficiencies since CD5 B cells have been shown to be a major
source of these isotypes in vivo (41).
Our demonstration that anti-Ig-stimulated xid B cells
can induce the complete complement of cell cycle regulatory proteins while the cells continue to undergo apoptosis
indicates that induction of cell cycle machinery alone is insufficient to ensure cellular replication. At least for B lymphocytes, induction of cell cycle machinery must be coupled
with antiapoptotic proteins for proliferation. Furthermore,
our results suggest a specific requirement for the antiapoptotic protein Bcl-x, since Woodland et al. has shown that transgene driven expression of Bcl-2 in xid mice could not
support anti-Ig induced proliferation (39). Therefore, Bcl-xL may have a unique and nonredundant role in maintaining viability during antigen-driven B cell expansion.
Address correspondence to Nanette Solvason at her current address, Anergen, Inc., 301 Penobscot Dr.,
Redwood City, CA. 94063. Phone: 650-361-8901; Fax: 650-361-8958; E-mail: nsolvason{at}anergen.com
| 1.
|
Billups, L.G.,
K. Lassoued,
C. Nunez,
J. Wang,
H. Kubagawa,
G.L. Gartland,
P.D. Burrows, and
M.D. Cooper.
1995.
Human B-cell development.
Ann. N.Y. Acad. Sci.
764:
1-8
[Medline].
|
| 2.
| Satterthwaite, A., and O.N. Witte. 1996. Genetic analysis of
tyrosine kinase function in B cell development. Annu. Rev.
Immunol. 14:131-154.
|
| 3.
|
Yel, L.,
Y. Minegishi,
E. Coustan-Smith,
R.H. Buckley,
H. Trubel,
L. Pachman,
G.T. Kitchingman,
D. Campana,
J. Rohrer, and
M.E. Conley.
1996.
Mutations in the mu
heavy-chain gene in patients with agammaglobulinemia.
N.
Engl. J. Med.
335:
1486-1493
[Abstract/Free Full Text].
|
| 4.
| Kitamura, D., J. Roes, R. Kurn, and K. Rajewsky. 1991. A B
cell deficient mouse by targeted disruption of the membrane
exon of the immunoglobulin mu chain gene. Nature. 350:
423-426.
|
| 5.
| Thomas, J.D., P. Sideras, C.I.E. Smith, I. Vorechovsk, V. Chapman, and W.E. Paul. 1993. Colocalization of X-linked
agammaglobulinemia and X-linked immunodeficiency genes.
Science. 261:355-358.
|
| 6.
|
Rawlings, D.J.,
D. Saffron,
D. Tsukada,
D. Largaespada,
J.C. Grimaldi,
L. Cohen,
R.N. Morh,
F.J. Bazan,
M. Howard,
N.G. Copeland,
N.A. Jenkings, and
O.N. Witte.
1993.
Mutations in unique region of Btk in immunodeficient XID
mice.
Science.
261:
358-361
[Abstract/Free Full Text].
|
| 7.
|
Khan, W.N.,
F.W. Alt,
R.M. Gerstein,
B.A. Malynn,
I. Larsson,
G. Rathbun,
L. Davidson,
S. Muller,
A.B. Kantor,
L.A. Herzenberg,
R.S. Rosen, and
P. Sideras.
1995.
Defective B
cell development and function in Btk-deficient mice.
Immunity.
3:
283-299
[Medline].
|
| 8.
| Lerner, J.D., M.W. Appleby, R.N. Morh, S. Chien, D.J.
Rawlings, C.R. Maliszewski, O.N. Witte, and R.M. Perlmutter. 1995. Impaired expansion of mouse B cell progenitors lacking Btk. Immunity. 3:301-312.
|
| 9.
|
Tsukada, S.,
D.C. Saffran,
D.J. Rawlings,
O. Parolini,
R.C. Allen,
I. Klisak,
R.S. Sparkes,
H. Kubagawa,
T. Mohandas,
S. Quan,
J.W. Belmont,
M.D. Cooper,
M.E. Conley, and
O.N. Witte.
1993.
Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia.
Cell.
72:
279-284
[Medline].
|
| 10.
|
Vetrie, D.,
I. Vorechovsky,
P. Sideras,
P. Holand,
A. Davies,
A. Flinter,
A.L. Hammarstrom,
C. Kinnon,
R. Levinsky,
M. Bobrow, et al
.
1993.
The gene involved in X-linked agammaglobulinemia is a member of the src family of protein-
tyrosine kinases.
Nature.
361:
226-229
[Medline].
|
| 11.
|
Conley, M.E.,
O. Parolini,
J. Rohrer, and
D. Campana.
1994.
X-linked agammaglobulinemia: new approaches to old
questions based on the identification of the defective gene.
Immunol. Rev.
138:
5-21
[Medline].
|
| 12.
|
Scher, I..
1982.
The CBA/n mouse strain; an experimental
model illustrating the influence of the X-chromosome on
immunity.
Adv. Immunol
33:
1-71
[Medline].
|
| 13.
|
Oka, Y.,
A.G. Rolink,
J. Andersson,
M. Kamanaka,
J. Uchida,
T. Yasui,
H. Kishimoto,
H. Kikutani, and
F. Melchers.
1996.
Profound reduction of mature B cell numbers, reactivities and serum Ig levels in mice which simultaneously carry the XID and CD40 deficiency genes.
Int. Immunol.
8:
1675-1685
[Abstract/Free Full Text].
|
| 14.
|
Hardy, R.R.,
K. Hayakawa,
D.R. Parks, and
L.A. Herzenberg.
1983.
Demonstration of B cell maturation in X-linked
immunodeficient mice by simultaneous 3-color immunofluorescence.
Nature.
306:
270-272
[Medline].
|
| 15.
|
Mosier, D..
1985.
Are xid B lymphocytes representative of
any normal B cell population?
J. Mol. Cell. Immunol
2:
70-78
[Medline].
|
| 16.
|
Hayakawa, K.,
R.R. Hardy, and
L.A. Herzenberg.
1986.
Peritoneal Ly1 B cells: genetic control and autoantibody production.
Eur. J. Immunol.
16:
450-465
[Medline].
|
| 17.
|
Melchers, F.,
D. Haasner,
U. Grawunder,
C. Kalberer,
H. Karasuyama,
T. Winkler, and
A.G. Rolink.
1994.
Roles of
IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage.
Annu. Rev.
Immunol.
12:
209-225
[Medline].
|
| 18.
|
Wortis, H.H..
1992.
Surface markers, heavy chain sequences
and B cell lineages.
Int. Rev. Immunol.
8:
235-246
[Medline].
|
| 19.
|
Sherr, C.J..
1996.
Cancer cell cycle.
Science.
274:
1672-1677
[Abstract/Free Full Text].
|
| 20.
|
Lees, E..
1995.
Cyclin dependent kinase regulation.
Curr.
Opin. Cell Biol.
7:
773-780
[Medline].
|
| 21.
|
Weinberg, R.A..
1995.
The retinoblastoma protein and cell
cycle control.
Cell.
81:
323-330
[Medline].
|
| 22.
|
Evan, G.I.,
L. Brown,
M. Whyte, and
E. Harrington.
1995.
Apoptosis and the cell cycle.
Curr. Opin. Cell Biol.
7:
825-834
[Medline].
|
| 23.
|
Pandey, S., and
E. Wang.
1995.
Cells en route to apoptosis
are characterized by the upregulation of c-fos, c-myc, c-jun,
cdc2, and RB phosphorylation, resembling events of early
cell-cycle traverse.
J. Cell. Biochem
58:
135-150
[Medline].
|
| 24.
|
Fanidi, A.,
E. Harrington, and
G.I. Evan.
1992.
Cooperative
interaction between c-myc and Bcl-2 proto-oncogenes.
Nature.
359:
554-556
[Medline].
|
| 25.
|
Fukada, T.,
M. Hibi,
Y. Tamanaka,
M. Takahashi-Texuka,
Y. Fujitani,
T. Tamaguchi,
K. Nakajima, and
T. Hirano.
1996.
Two signals are necessary for cell proliferation induced
by a cytokine receptor gp130: involvement of STAT 3 in
anti-apoptosis.
Immunity.
5:
449-460
[Medline].
|
| 26.
|
Fang, W.,
D.L. Mueller,
C.A. Pennel,
J.J. Rivard,
Y.S. Li,
R.R. Hardy,
M.S. Schlissel, and
T.W. Behrens.
1996.
Frequent aberrant immunoglobulin gene rearrangements in
pro-B cells revealed by a bcl-xL transgene.
Immunity.
4:
291-299
[Medline].
|
| 27.
|
Grillot, D.A.M.,
R. Merino,
J.C. Pena,
W.C. Fanslow,
F.D. Finkelman,
C.B. Thompson, and
G. Nunez.
1996.
bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic
mice.
J. Exp. Med.
183:
381-391
[Abstract/Free Full Text].
|
| 28.
|
Choi, M.S.K.,
M. Holman,
C.J. Atkins, and
G.G.B. Klaus.
1996.
Expression of bcl-x during mouse B cell differentiation
and following activation by various stimuli.
Eur. J. Immunol.
26:
676-682
[Medline].
|
| 29.
|
Anderson, J.S.,
M. Teutsch,
Z. Dong, and
H.H. Wortis.
1996.
An essential role for Bruton's tyrosine kinase in the
regulation of B-cell apoptosis.
Proc. Natl. Acad. Sci. USA.
93:
10966-10971
[Abstract/Free Full Text].
|
| 30.
|
Motoyama, N.,
F. Want,
K.A. Roth,
H. Sawa,
K. Nakayama,
K. Nakayama,
I. Negishi,
S. Senju,
Q. Zhang,
S. Fujii, and
D.Y. Loh.
1995.
Massive cell death of immature
hematopoietic cells and neurons in bcl-x-deficient mice.
Science.
267:
1506-1510
[Abstract/Free Full Text].
|
| 31.
|
Heath, A.W.,
W.W. Wu, and
M.C. Howard.
1994.
Monoclonal antibodies to murine CD40 define two distinct functional epitopes.
Eur. J. Immunol.
24:
1828-1834
[Medline].
|
| 32.
|
Solvason, N.,
W.W. Wu,
N. Kabra,
X. Wu,
E. Lees, and
M.C. Howard.
1996.
Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphocytes.
J. Exp. Med.
184:
407-417
[Abstract/Free Full Text].
|
| 33.
|
Draetta, G., and
D. Beach.
1988.
Activation of cdc2 protein
kinase during mitosis in human cells: cell cycle-dependent
phosphorylation and subunit rearrangement.
Cell.
54:
17-26
[Medline].
|
| 34.
|
Carayon, P., and
A. Bord.
1992.
Identification of DNA-replicating lymphocyte subsets using a new method to label the
bromo-deoxyuridine incorporated into the DNA.
J. Immunol. Methods.
147:
225-230
[Medline].
|
| 35.
|
Ferlin, W.G.,
E. Severinson,
L. Strom,
A.W. Heath,
R.L. Coffman,
D.A. Ferrick, and
M.C. Howard.
1996.
CD40 signaling induces interleukin-4-independent IgE switching in
vivo.
Eur. J. Immunol.
26:
2911-2915
[Medline].
|
| 36.
|
Sieckmann, D.G.,
I. Scher,
R. Asofsky,
D.E. Mosier, and
W.E. Paul.
1978.
Activation of mouse lymphocytes by anti-IgM. II. A thymus-independent response by a mature subset
of B lymphocytes.
J. Exp. Med
148:
1628-1643
[Abstract/Free Full Text].
|
| 37.
|
Lund-Johansen, F.,
T. Frey,
J.A. Ledbetter, and
P.A. Thompson.
1996.
Apoptosis in hematopoietic cells is associated with
an extensive decrease in cellular phosphotyrosine content that
can be inhibited by the tyrosine phosphatase antagonist pervanadate.
Cytometry
25:
182-190
[Medline].
|
| 38.
|
DeFranco, A.L.,
J.T. Kung, and
W.E. Paul.
1982.
Regulation
of growth and proliferation in B cell subpopulations.
Immunol. Rev.
64:
161-182
[Medline].
|
| 39.
|
Woodland, R.T.,
M.R. Schmidt,
S.J. Korsmeyer, and
K.A. Gravel.
1996.
Regulation of B cell survival in xid mice by the
proto-oncogene bcl-2.
J. Immunol.
156:
2143-2154
[Abstract].
|
| 40.
|
Lindsberg, M.-L.,
M. Brunswick,
H. Yamada,
A. Lees,
J. Inman,
C.H. June, and
J.J. Mond.
1991.
Biochemical analysis
of the immune B cells defect in xid mice.
J. Immunol.
147:
3774-3779
[Abstract].
|
| 41.
|
Solvason, N.,
A. Lehuen, and
J.F. Kearney.
1991.
An embryonic source of Ly1 but not conventional B cells.
Inter. Immunol.
3:
543-550
[Abstract/Free Full Text].
|
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