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Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3 h, when compared with
wild-type cells. Likewise, in in vitro bone marrow cultures,
Btk-deficient large cycling pre-B cells showed increased IL-7 mediated
expansion and reduced developmental progression into noncycling
CD2+CD25+ surrogate L chain-negative small
pre-B cells and subsequently into Ig-positive B cells. Furthermore, the
absence of Btk resulted in increased proliferative responses to IL-7 in
recombination-activating gene-1-deficient pro-B cells. These findings
identify a novel role for Btk in the regulation of the differentiation
stage-specific modulation of IL-7 responsiveness in pro-B and pre-B
cells. Moreover, our results show that Btk is critical for an efficient
transit through the small pre-B cell compartment, thereby regulating
cell surface phenotype changes during the developmental progression of
cytoplasmic µ H chain expressing pre-B cells into immature
IgM+ B cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in the Btk gene lead to X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (xid) in the mouse (6, 7, 8, 9). XLA is characterized by recurrent bacterial infections, and very low serum Ig levels of all classes due to the lack of plasma cells in the secondary lymphoid organs. There is a severe deficiency of peripheral B cells, and those B cells that are present have an aberrant IgMhigh phenotype (10). In the bone marrow (BM) of XLA patients, the numbers of cytoplasmic µ H chain (cµ)-expressing pre-B cells are variable, but in most cases reduced; those pre-B cells present are mainly nonproliferating small cells (11, 12, 13). The absence of Btk apparently results in deficient expansion of the earliest cµ-expressing pre-B cells. Therefore, the XLA disease phenotype most likely reflects defective signaling through the pre-BCR, by which the expression of a functional µ H chain normally is monitored, together with surrogate L chain (SLC; Ref. 14). As most patients have substantial numbers of pro-B cells in their BM, XLA generally results in an increased ratio of pro-B to pre-B cells (11, 12, 13).
In contrast, Btk deficiency in the mouse is associated with an
impairment of peripheral B cell maturation, without a major early B
cell developmental block (15, 16, 17). In xid mice
the mature
IgMlowIgDhigh B cell
populations in spleen and lymph nodes are severely reduced, B-1 B cells
are absent, and serum IgM and IgG3 levels are low. Xid B
cells do not proliferate upon surface IgM (sIgM) stimulation. The most
obvious difference between the BM B cell compartment of wild-type (WT)
mice and Btk-deficient mice is the reduction in mature recirculating
cells (15, 16, 17). The absolute numbers of pre-B cells that
are generated in the BM of Btk-deficient mice are normal, and
Btk-deficient B cell precursors in the BM have the same kinetics of
turnover (18, 19). Nevertheless, several lines of evidence
point at a role for Btk at the transition of pre-B cells into immature
surface Ig+ B cells, thereby indicating the
involvement of Btk in pre-BCR signaling in the mouse. Using an in vivo
competition assay in heterozygous Btk+/- female
mice, we identified a small but significant selective disadvantage of
Btk-deficient cells to contribute to the
IgM+IgD- immature B cells
stage in the BM (17). Our recent finding of an intrinsic
reduction of Ig 
L chain usage in Btk-deficient B cells in the mouse
implicated Btk in the activation of gene rearrangements at the L
chain locus (20). Furthermore, while mice deficient for
another Tec kinase family member, Tec, showed normal B cell
development, an almost complete block at the
CD43+B220+ stage of B cell
development was observed in Btk/Tec double-deficient mice
(21). These results indicated that Btk is critically
involved in pre-BCR-mediated signaling in the mouse and that Tec is
able to compensate for the loss of Btk during early B cell development.
A role for Btk in pre-BCR signaling would be consistent with the recent
finding that Btk is able to function in CD79b (Ig
)-mediated
signaling in recombination-activating gene (RAG)-2-deficient pro-B
cells (22).
To examine the role of Btk at the pre-BCR checkpoint in vivo, we compared the expression of early B cell surface markers in the BM from Btk-deficient and WT control mice. In this report, we show that Btk is involved in the pre-BCR-dependent induction of cell surface phenotype changes during the progression of large cycling cµ+ pre-B cells into surface Ig+ immature B cells, thereby affecting the transit time through the small pre-B cell compartment. In addition, we performed IL-7-driven BM cultures, which point at a role for Btk in the differentiation stage-specific modulation of IL-7 responsiveness in early murine B cell development, even in stages preceding cµ expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Btk-/lacZ mice (17) were crossed onto a C57BL/6 background for >6 generations. Specific experiments were additionally performed with Btk mice on a mixed C57BL/6 x 129 or C57BL/6 x FVB background, but no significant influence of the genetic background was observed. RAG-1-/- mice (23) were on a 129/Sv background. All mice were bred and maintained in the animal care facility at the Erasmus Medical Center, Rotterdam (Rotterdam, The Netherlands). To determine the Btk genotype, tail DNA was analyzed by Southern blotting of BamHI digests, as described (17). The RAG-1 genotype was determined using a 1.9-kb partial RAG-1 probe (encoding amino acid positions 387-1008, kindly provided by D. van Gent, Erasmus Medical Center) to hybridize to BamHI-NcoI genomic digests (23).
Flow cytometric analysis
Preparations of single-cell suspensions, flow cytometry, and
determination of -galactosidase activity by loading cells with
fluorescein-di--D-galactopyranoside substrate (Molecular
Probes Europe, Leiden, The Netherlands), have previously been described
(17, 24). Events (1-3 x 105)
were scored using a FACSCalibur flow cytometer and analyzed by
CellQuest software (BD Biosciences, Mountain View, CA). The following
mAbs were obtained from BD PharMingen (San Diego, CA): FITC-conjugated
anti-BP-1/6C3, anti-B220/RA3-6B2, anti-
(R5-240),
and anti-IgM (II/41); PE-conjugated anti-CD2,
anti-(187.1), anti-CD25, and anti-CD43;
CyChrome-conjugated anti-B220/RA3-6B2 and biotinylated
anti-IL7R, anti-(187.1),
anti-1/2(R26-46),
and anti-IgM (II/41). PE-conjugated IgD was obtained from Southern
Biotechnology Associates (Birmingham, AL). Biotinylated anti-SLC Ab
LM34 (25) was kindly provided by A. Rolink (Basel
Institute for Immunology, Basel, Switzerland). Secondary Abs were PE-,
tricolor-, or APC-conjugated streptavidin, purchased from Caltag
Laboratories (Burlingame, CA).
For intracellular flow cytometric detection of cytoplasmic Ig H or L
chain or SLC, the following Abs were applied: FITC-conjugated
anti-Ig (R5-240, BD PharMingen) or polyclonal anti-Ig µ H
chain (Jackson Immunoresearch Laboratories, West Grove, PA) and
biotinylated
anti-1/2 (BD
PharMingen), or anti-SLC (LM34). Cells were first stained for cell
surface markers and subsequently fixed in 2% paraformaldehyde and
permeabilized using 0.5% saponin.
Cell cycle analysis
To analyze cell cycle status of cultured cell suspensions, cells were incubated in ice-cold ethanol for >2 h. Subsequently, cells were incubated at room temperature for 30 min in PBS containing 0.02 mg/ml propidium iodide, 0.1% v/v Triton X-100, and 0.2 mg/ml RNase, left overnight at 4°C, and analyzed with a FACSCalibur flow cytometer. Doublet cells were excluded from the analysis by measuring peak area and width. For simultaneous analysis of cell surface markers, cµ expression, and DNA content, we used a method described by Constantinescu and Schlissel (26). Cells were sequentially stained for surface CD2 expression using PE-labeled anti-CD2, for cµ using polyclonal anti-µ H chain, and 7-aminoactinomycin-D (7-AAD, Molecular Probes Europe).
IL-7-driven BM cultures
Primary pre-B cell cultures were essentially performed as described previously (20), using erythrocyte-depleted total BM cell suspensions. For specific experiments, BM cell suspensions were depleted of sIgM+ cells, using biotinylated anti-IgM and streptavidin-coated microbeads, as described (20). Recombinant murine IL-7 was from R&D Systems (Minneapolis, MN). To measure IL-7-dependent proliferative responses, cells were cultured in flat-bottom 96-well plates for 5 days with various amounts of IL-7 at different cell concentrations (0.25–1 x 105 cells/well). Cultures were pulsed for 24 h with 0.5 µCi/well of [3H]thymidine, harvested on glass-fiber filters, and the incorporated radioactivity was determined using a beta counter, according to standard procedures.
In vivo 5-bromo-2'-deoxyuridine (BrdU) labeling
BrdU (Sigma Aldrich, St. Louis, MO) was dissolved in PBS at 2 mg/ml. Mice were injected i.p. with 200 µl, and sacrificed at various time points. Total BM cell suspensions were analyzed by flow cytometry for BrdU incorporation, using the BrdU flow kit (BD PharMingen) in conjunction with cell surface marker expression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the involvement of Btk in pre-BCR signaling, we
compared the pre-B cell compartment in the BM of
Btk+ and Btk- mice.
Consistent with published findings (16, 17, 21), we did
not observe a major early B cell developmental block in
Btk- mice, but only a small increase in the
fraction of CD43+ cells within the population of
sIgM-B220+ pro-/pre-B
cells: 20–25% in Btk+ and 50–55% in
Btk- mice (Fig. 1
A).
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L chain in the cytoplasm, compared
with 23 ± 2% in Btk+ mice, while the
frequency of cytoplasmic expression pre-B cells was reduced by a
factor 3 (Fig. 1
B). These findings suggest reduced or
delayed Ig L chain rearrangement.
Signaling through the pre-BCR initiates proliferative expansion of
those cells with a productively rearranged Ig H chain. Subsequently,
these cells progress into small resting pre-B cells that have changed
their cell surface phenotype and initiate and L chain
rearrangement (14). Therefore, we analyzed
IgM-B220+ B cell
precursors for the expression of several cell surface markers in
four-color flow cytometric experiments (Fig. 1C). In
Btk- mice, the
IgM-B220+ compartment
contained a unique subpopulation of CD43low cells
which manifested increased expression of the BP-1 molecule, a
metallopeptidase that is selectively induced coincident with
IL-7-driven pre-B cell proliferation (28). As soon as a
pre-BCR is expressed, the synthesis of SLC is turned off
(29). However, Btk- cells had an
increase in the fraction of
IgM-B220+ cells that
expressed SLC in their cytoplasm, as compared with
Btk+ cells (Fig. 1C). In a specific
analysis of cµ+ pre-B cells,
Btk- mice showed significantly reduced
expression of the IL-2R CD25 (30 ± 6%), and the CD2 adhesion
molecule (32 ± 4%), as compared with Btk+
mice (66 ± 7% and 70 ± 6%, respectively). These molecules
were previously reported to be up-regulated upon pre-BCR signaling
(30, 31).
Collectively, these findings indicated that the absence of Btk resulted
in a defective progression of
SLC+CD43+CD2-CD25-
into
SLC-CD43-CD2+CD25+cµ+
pre-B cells. Analysis of the absolute numbers of the various B-lineage
subpopulations in the BM (Table I)
revealed that in Btk- mice the size of the total
population of CD2- cµ+
early pre-B cells was increased with a factor of 2.4, whereas the
total population of CD2+
cµ+ late pre-B cells was decreased with a
similar factor.
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45% of CD2- and 10% of
CD2+cµ+ pre-B cells in S,
M, or G2 phase, using 7-AAD staining for DNA
content (Fig. 1C). In contrast, the
CD2-cµ+ pre-B cell
population in Btk- mice contained only 23%
of cycling cells, i.e., about half the number found in
Btk+ mice. However, as the size of the total
population of CD2-cµ+
pre-B cells was increased with a factor 2.4 in
Btk- mice, we conclude that
Btk+ and Btk- mice contain
comparable numbers of large cycling cµ+ cells
(0.8 x 106 cells per hind leg). In summary, the absence of Btk in pre-BCR signaling resulted in defective down-regulation of CD43 and SLC, defective up-regulation of CD2 and CD25, augmented expression of BP-1, whereas in vivo proliferative expansion of pre-B cells appeared to be unaffected.
Btk- cells show a developmental delay within the small pre-B cell compartment in vivo
The inefficient induction of cell surface phenotype changes in
Btk- mice at the pre-B cell stage was still
noticeable at the next developmental stage of
IgM+ immature B cells. As shown by flow
cytometric analysis (Fig. 2A),
IgM+B220low immature B
cells in Btk- mice manifested lower expression
of CD2 (66 ± 5%, n = 9) and CD25 (19 ±
1%, n = 12), compared with immature B cells in
Btk+ animals (CD2+: 90
± 3%, n = 8, and CD25+: 36
± 1%, n = 12). In addition, considerable fractions of
Btk- immature B cells expressed surface BP-1
(45 ± 3%, n = 6) or cytoplasmic SLC (13 ±
3%, n = 8), which were not detected in
Btk+ immature B cells (<3%; Fig. 2A).
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or
L chain, and analyzed by flow cytometry. Twelve hours after injection,
30–35% of Ig- B cell precursors
incorporated BrdU, both in Btk+ and
Btk- mice. At various time points after BrdU
injection, we analyzed BrdU incorporation within
+ and +
IgD- immature B cells. As shown for the 48-h
time point in Fig. 2B, the fractions of
BrdU+ cells within the surface
+ or + immature B
cells were decreased in Btk- mice when compared
with Btk+ mice. As small pre-B cells are
noncycling cells, the first appearance of BrdU+
cells within the surface Ig+ immature B cells
reflects the minimum transit time through the small pre-B compartment.
When for + immature B cells, the data from six
time points were subjected to linear regression analysis
(33), it was found that
BrdU++ immature B cells
emerged 8 h after BrdU injection in Btk+ mice
and only after 11 h in Btk- mice (Fig. 2C). In agreement with reported experiments (33, 34), BrdU++
immature B cells only emerged 30 h after BrdU injection. Likewise,
linear regression analysis revealed a delay of 3.5 h for
Btk-deficient + immature B cells (32.5 h),
as compared with Btk+ cells (29 h).
In summary, we conclude that in Btk- mice the
inefficient induction of cell surface phenotype changes in
µ+ pre-B cells was accompanied by a specific
developmental delay within the small pre-B cell compartment of 3 h.
In addition, the immature B cells generated manifest an aberrant
surface phenotype.
Btk- pre-B cells show deficient developmental progression in vitro
To further investigate the role of Btk in the developmental
progression of cµ+ pre-B cells and the
initiation of Ig L chain expression, IL-7-driven BM culture experiments
were performed, as described (35, 36). Pre-B cells that
have a productive Ig H chain rearrangement undergo rapid cell division
in response to IL-7. When IgM- BM cell
suspensions from Btk+ mice were cultured in the
presence of 100 U/ml IL-7 for 5 days, the majority of cells consisted
of B220+IgM- cells, while
a significant fraction matured to
sIgM+IgD- (10%) or
IgM+IgD+ B cell stages
(5%) (Fig. 3). This differentiation
into IgM+ cells did not result from an IL-7
insufficiency in culture, as it was also present when higher IL-7
concentrations were used. In contrast, only <5% and <0.5% of the
Btk- cells exhibited an
IgM+IgD- or
IgM+IgD+ profile in
culture, respectively. Therefore, these findings indicated that Btk
signaling supported the progression from pre-B cell to surface
Ig+ B cell in vitro in the presence of IL-7.
Similar differences were observed, when cells were subsequently
cultured for 48 h on S17 stroma cells in the presence of IL-7:
30% of Btk+B220+ cells
were surface Ig+, but only 5% of
Btk-B220+ cells (Fig. 3).
After removal of IL-7, which strongly induces the cells to exit from
cell cycle and to further differentiate (36), significant
numbers of sIgM+ B cells were generated in the
Btk- BM cultures. Nevertheless, when compared
with Btk+ B cells, the maturation of
Btk- B cells to the
IgM+IgD+ stage was reduced
(Fig. 3). Therefore, the analyses of IL-7-driven BM cultures showed
that Btk signaling promotes the maturation of pre-B cells into surface
Ig+ B cells in the presence of IL-7 in vitro.
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Next, we analyzed the kinetics of in vitro B cell development in IL-7-driven BM cultures, using total BM cells from Btk+, Btk-, and Btk+/- mice, without S17 stroma. Similar results were obtained in IL-7-driven BM cultures in the presence of S17 stromal cells, either throughout the 7-day culture period or from day 5 onwards (data not shown).
In these experiments, we used biotinylated Abs to detect sIgM
expression, which resulted in higher fluorescence intensity values (up
to 2 x 103), as compared with the
experiments described in Fig. 3 (up to 5 x
102). We observed that most
Btk-B220+ cells stained
with the anti-µ H chain Ab after culture with IL-7 (fluorescence
intensities ranging from 20 to 100; Fig. 4A). These cells were cµ
positive, surface, or cytoplasmic or negative, and expressed
SLC both in their cytoplasma and on the cell surface (see
Results; Fig. 5). Therefore,
we concluded that these cells were pre-B cells, and that the
anti-µ H chain Ab staining in Btk- cells
reflected high surface pre-BCR expression. In contrast, the
Btk+ cells with anti-IgM fluorescence
intensities ranging from 20 to 100 were surface +
cytoplasmic SLC- immature B cells (see Results;
Fig. 5).
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A, day 3), and a
reduced appearance of newly formed Ig+ B cells
(Fig. 4A, days 5 and 7). From the total cell counts and the
flow cytometric analyses of the cells, the expansion rates of
sIgM- and
sIgM+B220+ cells during
culture were calculated. As shown in Fig. 4B, pre-B cell
expansion was significantly higher in the cultures from
Btk- mice (30x) when compared with
Btk+ mice (<10x). Only for
Btk+ BM cells, the further differentiation of
pre-B cells generated in the cultures in the presence of IL-7 resulted
in an expansion of mature sIgM+ B cells
(4x).
Increased proliferation of Btk- cells was
confirmed by analysis of cell cycle and cell size (Fig. 4C).
After 5 days of culture, 53 ± 3% of the
Btk- cells (n = 6) and only
25 ± 3% of the Btk+ (n =
6) were in the S, M, or G2 phase of the cell
cycle. In these experiments, the Btk- BM
cultures contained higher proportions (77 ± 6%) of large cells
with high forward scatter (FSC) values than the
Btk+ BM cultures (42 ± 7%). To
further quantify cell proliferation in the presence of various
concentrations of IL-7, we performed
[3H]thymidine incorporation experiments. After
5 days of culture, Btk- cells showed
significantly higher proliferative responses to IL-7 when compared with
Btk+ cells (Fig. 4D). These findings
in Btk+ and Btk- BM
cultures demonstrated that in the absence of Btk, IL-7-driven pre-B
cell proliferation in vitro is increased.
Enhanced IL-7-driven expansion and decreased developmental progression is intrinsic to Btk- pre-B cells
The observed differences in IL-7-driven proliferation between
Btk+ and Btk- pre-B cells
cannot easily be explained by increased surface expression of IL-7R on
these cells in Btk- mice. In flow cytometric
experiments, the proportions of IL-7R
chain positive cells within
the large pre-B cell population in Btk+ and
Btk- mice were 77 ± 4% and 87 ±
2%, respectively, while the mean fluorescence intensities of
IL-7R+ cells were comparable.
To answer the question of whether the phenotype of
Btk- cells in IL-7-driven BM cultures is an
intrinsic feature of Btk- pre-B cells, or
alternatively, originates from a defective BM microenvironment in
Btk- mice, we also investigated BM cultures from
Btk+/- mice. Due to random X chromosome
inactivation, the pre-B cell compartment in these mice consists of two
functionally different populations, i.e., Btk+
and Btk- (17, 37, 38). In
Btk+/- heterozygous mice, IL-7-driven pre-B cell
proliferation was intermediate between Btk+ and
Btk- mice, as measured by pre-B cell expansion,
cell cycle status, and [3H]thymidine
incorporation (Fig. 4, B–D). At days 5 and 7, the surface
Ig expression profiles and the FSC values in
Btk+/- cultures were similar to those of
Btk- cells (Fig. 4, A and
C), indicating a proliferative advantage of
Btk- over Btk+ cells. The
presence of a lacZ reporter in the
Btk- allele enabled a separate evaluation
of the expansion of
Btk+/lacZ- and
Btk-/lacZ+ cells in
Btk+/- cultures. At days 5 and 7, the
proportions of lacZ+ cells in
Btk+/- and Btk- cultures
were comparable, indicating an intrinsic selective advantage of
Btk-deficient cells in culture. In these cultures, the majority of
IgM+ cells were
Btk+/lacZ-. Thus,
the increased expansion and reduced differentiation in IL-7-driven
pre-B cell cultures is an intrinsic feature of
Btk- cells, which is independent of the
xid BM microenvironment in vivo, or coculture of
Btk-deficient stromal cells in the BM cultures.
Aberrant phenotype of Btk- cells in IL-7-driven BM cultures
Next, we investigated whether the inefficient pre-BCR-mediated
induction of cell phenotype changes in Btk-
pre-B and immature B cells in vivo were paralleled in the BM cultures
in vitro. Large and small IgM- cells were
obtained from total BM cultures in the presence of IL-7 for 7 days.
Small IgM+ cells were obtained from total BM cell
suspensions that were cultured in the presence of IL-7 for 5 days and
subsequently in the absence of IL-7 on S17 stromal cells for 2 days. In
agreement with our findings in vivo (Fig. 1), we found by flow
cytometric analysis that in Btk- cultures
down-regulation of CD43 and up-regulation of CD2 and CD25 at the large
to small pre-B cell transition was impaired (Fig. 5). The expression of
BP-1 was elevated in all three stages analyzed. Most importantly, we
noticed that in the absence of Btk, SLC synthesis is not efficiently
turned off, resulting in significantly enhanced cytoplasmic and surface
SLC expression in Btk- large pre-B cells
(96 ± 1% and 29 ± 2%, n = 3,
respectively), compared with 67 ± 5% and 2.9 ± 0.9%
(n = 3) in Btk+ cells. Also in
small pre-B cells, cytoplasmic SLC expression was enhanced in
Btk- cultures (62 ± 5%), compared with
Btk+ cultures (17 ± 3%; Fig. 5). A small
but reproducible increase in the expression level of cµ was present
in Btk-deficient large and small pre-B cells. In addition, significant
fractions of cytoplasmic L chain-positive pre-B cells were only
present in Btk+ cultures (20% of small
sIgM- pre-B cells, compared with <2% in
Btk-deficient pre-B cells).
Taken together, these findings in IL-7-driven BM cultures confirm the involvement of Btk signaling in the pre-BCR-mediated down-regulation of CD43 and SLC and the induction of CD2 and CD25 expression, and the initiation of Ig L chain rearrangements.
Btk- pro-B cells show significantly increased IL-7-driven proliferation in vitro
To investigate whether Btk-dependent modulation of IL-7
responsiveness in the mouse is specific for the pre-B cell stage, or
alternatively, also occurs in earlier stages of B cell development, we
crossed the Btk-deficient mice onto a RAG-1-/-
background. In these mice, B cell development is arrested at the
CD43+ pro-B cell stage, in which the BCR-linked
CD79/CD79 heterodimers are expressed on the cell surface without
associated µ H chains (39). Btk+
and Btk-RAG-1-/- mice
contained similar numbers of
CD43+B220+ pro-B cells in
vivo (data not shown), but the expression of IL-7R was slightly
different (62% in Btk+ and 79% in
Btk-RAG-1-/- mice). In
[3H]thymidine incorporation experiments,
IL-7-induced proliferation was significantly enhanced in
Btk-RAG-1-/- pro-B
cells, when compared with control
Btk+RAG-1-/- pro-B cells
(Fig. 6A). Whereas we did not
detect significant differences between the two groups of mice in the in
vivo expression of the BP-1, the expression was elevated in the
Btk-RAG-1-/-BM cultures
(Fig. 6B). Collectively, these results indicate that
Btk-signaling can modulate the responsiveness to IL-7 of early B cell
precursors, even before pre-BCR expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Signaling through the pre-BCR complex mediates the checkpoint
function of µ H chain by inducing cell cycle entry and rapid
down-regulation of the rearrangement machinery, thereby ensuring
allelic exclusion (14, 29). Subsequently, the
proliferating pre-B cells exit the cell cycle, change the expression
profile of various cell surface markers, and perform Ig L chain
rearrangements with sequential activation of the and the loci
(14). In humans, mutations in the Btk gene
result in XLA, which reflects an almost complete block in B cell
development at the pre-B cell stage (12, 13, 40). Similar
to findings in mice deficient for the SLC component 5 or the
membrane form of the Ig µ H chain (41, 42), most XLA
patients lack the compartment of large cycling pre-B cells
(13). Therefore, the XLA disease phenotype implicates Btk
in the pre-BCR signaling-mediated expansion of those pre-B cells with a
productive µ H chain rearrangement in humans.
In contrast, Btk is not essential for the selective expansion of cµ+ pre-B cells in the mouse, as in Btk-deficient mice the pre-B cell population is normal in size and contains normal proportions of cycling cells (16, 17, 18, 19). In this report, we demonstrate that the mouse Btk is involved in later phases of pre-B cell differentiation. We compared the pre-B cell compartment in Btk+ mice and Btk- mice both in vivo and in vitro in IL-7-driven BM cultures. The absence of Btk resulted in 1) defective down-regulation of CD43 and SLC expression, 2) impaired up-regulation of CD2 and CD25, 3) aberrant expression of BP-1, 4) a 3-h delay in B cell development in vivo at the small pre-B cell stage and reduced progression to surface Ig+ B cells in vitro, and 5) a significant increase in IL-7-driven proliferative expansion of pro-B and pre-B cells in vitro. Because Btk has been reported to be in the pre-BCR signaling pathway in the mouse (5, 22), our findings implicate pre-BCR signaling in the control of CD2, CD25, CD43, BP-1, and SLC expression, IL-7 responsiveness, and the transit time through the small pre-B cell stage.
As pre-B cell production in Btk- mice is normal, we conclude that the inefficient down-regulation of BP-1, CD43, and SLC expression and up-regulation of CD2 and CD25 do not affect the proliferative capacities of pre-B cells in vivo. Nevertheless, Btk- large pre-B cells manifested increased proliferative expansion in vitro, when compared with Btk+ cells. The finding of significantly increased surface SLC expression on these Btk- large pre-B cells (when compared with Btk+ cells) would support the hypothesis by Melchers et al. (14) that large cµ+ pre-B cells stop cycling when they run out of a sufficient number of assembled pre-BCR molecules per cell. This is not necessarily in conflict with our finding that the increased cytoplasmic SLC expression in Btk- pre-B cells did not result in increased proliferation in vivo. It is very possible that only the level of membrane SLC expression may correlate with the proliferative capacity of pre-B cells, and we were unable to detect differences between Btk+ and Btk- large pre-B cells in vivo in membrane SLC expression levels.
Btk and L chain rearrangement
We have previously found that Btk- mice
show reduced L chain usage, which could either result from a defect
in receptor editing or alternatively from a decreased rate or
efficiency of L chain rearrangement (20). Although by
crossing 3-83 µ
autoantibody transgenic mice into
Btk- mice we have shown that Btk is not
essential for receptor editing (20), a possibility
remained that the extent of L chain replacement events is reduced in
Btk- mice. However, our BrdU-labeling
experiments, in which we identified a 3-h delay at the small pre-B cell
stage in Btk- mice, clearly argue against this
possibility. Reduction of the level of receptor editing in
Btk- mice would have the opposite effect of a
more rapid transit through the small pre-B cell compartment, as B cells
targeted for receptor editing were recently shown to be specifically
delayed in this compartment for at least 2 h (33).
Therefore, the finding of a 3-h delay supports a role for Btk in the
regulation of the initiation of L chain rearrangement events. As both
+ and +
Btk- cells are equally delayed, this cannot
explain the observed reduced usage in Btk-
mice (20). However, we conclude that the absence of Btk
specifically affects the production rate of +
immature B cells, as our linear regression analysis (Fig. 2C) revealed a lower slope value for
+ cells in Btk- mice
(1.3) than in Btk+ mice (1.7), while the values
for + cells were similar (1.2 and
1.3).
In this context, it is unknown whether Btk signaling directly or
indirectly regulates the activation of the L chain loci, or
alternatively, whether Btk acts by mediating developmental progression
to a pre-B cell stage in which L chain rearrangements are initiated.
The latter possibility would be consistent with the delayed modulation
of the expression of SLC, BP-1, CD43, CD2, and CD25. The finding that
the expression of germline transcripts is first detected in vivo in
large cycling CD25+ pre-B cells would be
consistent with a role for pre-BCR signaling the opening of the L chain
loci for rearrangement (29, 43). However, additional
experiments would be needed to address this issue, as it has been shown
that productive L chain rearrangement can also be induced by IL-7
withdrawal of pre-BCR-deficient 5-/- or
JH-/- pre-B cells in vitro
(14, 44).
The size of the immature B cell pool and the turnover of immature B
cells were reported to be essentially normal in xid or
Btk- mice (16, 17, 18, 45).
Nevertheless, we previously observed a selective disadvantage of
Btk- cells at the transition from pre-B cells to
immature B cells in the BM, which we determined in an in vivo
competition analysis in Btk+/- heterozygous
female mice (17). In the immature B cell subset, we found
lacZ expression values of 30%, whereas in the absence of
a competitive disadvantage,
Btk-/lacZ+ cells
would be expected to represent 50% of any B cell subpopulation. The
finding of a 3-h delay in production of immature B cells in
Btk- mice would explain the observed selective
disadvantage of Btk-deficient cells in Btk+/-
female mice.
Btk and IL-7 responsiveness
An inhibitory effect of Btk on proliferation of B-lineage cells in long-term Whitlock-Witte BM cultures has previously been reported (46). However, analysis of growth kinetics of cultures established from mixtures of WT and xid cells indicated that the observed differences resulted from changes in the BM microenvironment associated with the xid mutation. In contrast, our separate analyses of lacZ+ and lacZ- cells in the BM cultures from Btk+/- mice showed that increased IL-7 responsiveness is an intrinsic feature of Btk- pre-B cells. This would be consistent with the finding of an increased frequency of CFU responsive to IL-7 by limiting dilution analysis of day 15 fetal liver cells from CBA/N xid mice (47).
Finally, crosses of Btk-deficient and RAG-1-deficient mice showed that
IL-7-dependent expansion in vitro is constrained by an inhibitory
signal mediated by Btk, even before the expression of µ H chain.
Cross-linking of the Ig signaling component of the (pre-)BCR, which
is expressed on the cell surface in cµ- pro-B
cells in association with calnexin induced a rapid phosphorylation of
several intracellular signaling molecules, including Btk (22, 48). Therefore, it is possible that Btk-mediated Ig/calnexin
signaling would serve to stop pro-B cell proliferation to facilitate
V(D)J recombination, which is exclusively initiated in the
G0/G1 cell cycle phase
(49). This hypothesis may be supported by a significant
reduction of the level of VH to
DJH rearrangements observed in Ig-deficient
mice (50), which would then be explained by a reduced
signal to make pro-B cells competent to undergo
VH-DJH rearrangement
(48). In this context, it has been shown previously that
in later stages of B cell development, modulation of the IL-7
responsiveness is dependent on pre-BCR and BCR signaling (51, 52). However, it is very possible that in pro-B cells, Btk may
function in a pathway that is unrelated to the Ig/calnexin complex,
as Btk has been implicated in signaling of various other receptors,
including IL-5R, CD38, IL-10R, and Fc
RI (1, 2).
Taken together, our findings implicate Btk in two different pathways in
early B cell development. First, Btk is required for the efficient
developmental progression of cytoplasmic µ+
pre-B cells, as Btk was shown to be involved in BP-1, CD43, and SLC
down-regulation, CD2 and CD25 up-regulation, and the rate of transit
through the small pre-B cell compartment. Additional experiments are
required to show whether Btk-mediated pre-BCR signaling directly or
indirectly influences the expression of SLC, germline , or
transcripts or the RAG genes. Second, the IL-7-driven BM culture
experiments indicate a role for Btk as a pro- and pre-BCR associated
negative regulator of IL-7 expansion. Although XLA patients may
occasionally have a significantly increased pro-B cell compartment
(12, 53), in general there is no absolute pro-B expansion
in XLA. Obviously, both in the mouse and in humans, other signaling
molecules or pathways must be able to compensate for the loss of Btk in
pro-B cells in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rudolf W. Hendriks, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Room Ee853, Dr. Molewaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail address: hendriks{at}immu.fgg.eur.nl
3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BCR, B cell receptor; BM, bone marrow; FSC, forward scatter; WT, wild type; xid, X-linked immunodeficiency; XLA, X-linked agammaglobulinemia; SLC, surrogate L chain; RAG, recombination-activating gene; sIgM, surface IgM; BrdU, 5-bromo-2'deoxyuridine; cµ, cytoplasmic µ H chain; 7-AAD, 7-aminoactinomycin-D.
Received for publication October 11, 2001. Accepted for publication January 10, 2002.
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light chain locus in precursor B cells in the mouse. J. Exp. Med. 193:1169.5 surrogate light chain on the surface of µ heavy chain-negative early precursor B cell lines. J. Exp. Med. 178:469. transgene, but not VJ gene segment targeted into the Ig locus, can rescue B cell development in 5-deficient mice. Immunity 5:229.[Medline]
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light chain genes can occur without µ heavy chain expression during differentiation of pre B cells. Int. Immunol. 5:1609./Ig heterodimer on µ-negative proB cells is competent for transducing signals to induce early B cell differentiation. Immunity 7:559.[Medline]
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