By
From the * Department of Microbiology and Molecular Genetics, University of California Los Angeles,
Los Angeles, California 90095; the Department of Laboratory Medicine, University of California
San Francisco, San Francisco, California 94143; the § Howard Hughes Medical Institute, The
Children's Hospital, Boston, Massachusetts 02115; the
Unit of Applied Cell and Molecular Biology,
Umea University, S-901 87 Umea, Sweden; and the ¶ Howard Hughes Medical Institute, University
of California Los Angeles, Los Angeles, California 90095
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Abstract |
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Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine
kinase (Btk). Differences in the phenotypes of Btk/
and lyn
/
mice suggest that these kinases
may also have independent or opposing functions. B cell development and function were examined in Btk
/
lyn
/
mice to better understand the functional interaction of Btk and Lyn in
vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk
/
lyn
/
mice.
However, Btk
/
lyn
/
animals had a more severe immunodeficiency than Btk
/
mice. B cell
numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play
independent or partially redundant roles in the maintenance and function of peripheral B cells.
Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly
caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn
/
mice. A
transgene expressing Btk at ~25% of endogenous levels (Btklo) was crossed onto Btk
/
and
Btk
/
lyn
/
backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn
function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo
mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.
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Introduction |
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The development of a diverse repertoire of B cells and
the maintenance of self-tolerance depend on signals
transduced by the B cell antigen receptor (BCR).1 The
outcome of BCR engagement varies from proliferation and
differentiation to deletion depending on the developmental
stage of the B cell, concurrent signals, and the degree of
BCR cross-linking (for review see reference 1). A complex
signaling network translates BCR-mediated signals into the
appropriate response given the context in which they are
received. One of the initial biochemical consequences of
BCR engagement is the sequential activation of a cascade
of tyrosine kinases belonging to the Src, Btk/Tec, and Syk/
Zap70 families. The phosphorylation of multiple substrates
by these kinases leads to signaling events which include
stimulation of the Ras/mitogen-activated protein kinase
(MAPK) pathway, phosphoinositide hydrolysis, Ca2+ flux,
and the activation of PI3-kinase (for review see reference 2). B cell development is generally blocked at the proB to preB transition in the absence of preB receptor or BCR
subunits (3). syk
/
mice have a similar phenotype (7, 8),
but B lymphopoiesis is less severely affected in mice lacking
other molecules downstream of the BCR such as Bruton's
tyrosine kinase (Btk; references 9), Lyn (12), Fyn
(15, 16), PKC
(17), and Vav (18, 19). This suggests that,
although Syk plays a unique role early in B cell development, there may be a significant degree of redundancy among some components of BCR signaling pathways.
Src family kinases, including Lyn, Blk, Fyn, Lck, and
Fgr, are activated rapidly upon BCR cross-linking (2).
Among Src family kinases, only mutations in Lyn have been
described as affecting BCR signaling (12, 20). Intriguingly, Lyn appears to be involved in both the initiation of
BCR signals and their subsequent downregulation (14, 20).
Anti-IgM-mediated cross-linking of the BCR results in
slightly delayed and reduced tyrosine phosphorylation of Ig, Syk, shc, and several other substrates in B cells from lyn
/
mice (13, 14). The residual phosphorylation is probably catalyzed by other Src family kinases present in these cells.
Despite delayed signal initiation, lyn
/
murine B cells are
hypersensitive to anti-IgM stimulation (14, 20). This results
from impaired downregulation of BCR signaling via both
Fc
RIIb-dependent and -independent mechanisms (14).
Mutations in Lyn also affect B cell development. The
frequency of peripheral B cells is reduced approximately
twofold in lyn/
mice (12, 20). The remaining cells
have an immature cell surface phenotype and a shorter life
span than do wild-type B cells (14). Serum IgM and IgA
levels are increased (12, 13). Aged lyn
/
animals develop
autoantibodies and exhibit splenomegaly due to extramedullary hematopoiesis and the expansion of IgM-secreting B
lymphoblasts (12). The phenotype of lyn
/
mice is
strikingly similar to that of motheaten (me) mice (21), which are deficient in the negative regulator of BCR
signaling SH2-containing protein tyrosine phosphatase 1 (SHP1). This suggests that other Src kinases cannot compensate for Lyn in the termination of BCR signals.
Several lines of evidence indicate that Btk is downstream of Src family kinases in a BCR signaling pathway. Coexpression of Btk and Lyn in fibroblasts leads to the transphosphorylation of Btk on Y551 and activation of Btk kinase activity (22, 23). Btk is also phosphorylated on Y551 in response to BCR cross-linking (22, 24). The ability of an activated form of Btk to transform fibroblasts is dependent on both the activity of Src family kinases and the presence of Y551 (25, 26). Mutation of Y551 also prevents Btk from mediating BCR-induced Ca2+ flux in B cells (27, 28). These combined observations indicate that transphosphorylation by Src kinases is critical for Btk function.
Mutations in Btk result in the B cell immunodeficiencies
X-linked agammaglobulinemia (XLA) in humans (29, 30)
and X-linked immunodeficiency (xid) in mice (31, 32).
XLA patients have a block at the preB stage of development, resulting in a severe deficit of circulating B cells and
serum Ig (for review see reference 33). Both xid and Btk/
(9) mice have a more subtle phenotype (for review see
reference 33). They have a 30-50% decrease in the number
of peripheral B cells, with the most profound reduction in
the mature IgMloIgDhi subset. xid mice have reduced levels
of serum IgM and IgG3 and do not respond to type II T
cell-independent antigens. They also lack B1 cells. Responses to the engagement of several cell surface receptors
including BCR, IL-5R, IL-10R, and CD38 are impaired in the absence of Btk. B cells expressing reduced levels of
Btk are hyposensitive to anti-IgM (34), suggesting that Btk
is limiting for the transmission of signals from the BCR.
Despite the biochemical evidence that Lyn and Btk operate sequentially in common signaling pathways, the different phenotypes of Btk/
and lyn
/
mice (low versus
high serum IgM, hypo- versus hypersensitivity to BCR
cross-linking) suggest that these kinases may also have opposing roles in BCR signaling. To clarify this issue, we examined B cell development in mice lacking both Btk and
Lyn. If Btk and Lyn oppose each other, Btk deficiency
might be expected to rescue the lyn
/
phenotype, analogous to the rescue of the me B cell phenotype by CD45 deficiency (35). If Lyn is the sole upstream activator of Btk,
then effects on B cell development should be no more severe in Btk
/
lyn
/
mice than in lyn
/
mice alone. Increased severity of phenotype would indicate that Btk and
Lyn are partially redundant components of one signaling pathway or participants in independent pathways. A combination of these possibilities was observed, indicating that Lyn
both opposes Btk-mediated signals and plays a positive signaling role independent of or partially redundant with Btk.
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Materials and Methods |
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Mice
Btk/
(10) and lyn
/
mice (14), each on a mixed C57B/6 × 129/Sv genetic background, were crossed to generate Btk+/
lyn+/
F1 progeny. These F1 animals were mated resulting in wild-type (wt), Btk-deficient, Lyn-deficient, and Btk/Lyn-deficient progeny. Genotypes were determined by Southern blot (10) or PCR
(14) analysis of tail biopsy DNA as described. For the analysis of limiting Btk dosage in Fig. 5, crosses were performed as above starting with Btk
/
mice carrying an Ig heavy chain enhancer/
promoter-driven Btk transgene expressing ~25% of endogenous
Btk levels in B cells (34). The resulting progeny were on a mixed
C57B/6 × 129/Sv × Balb/c background. The presence of the
Btk transgene was determined by Southern blot as previously described (34).
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Flow Cytometry
Phenotypic Analysis.
Single cell suspensions from spleen, bone marrow, or peritoneal wash were depleted of red blood cells and stained with combinations of the following antibodies (from PharMingen, San Diego, CA, unless otherwise noted): PE-conjugated (PE) anti-B220 (RA2-6B2); FITC-conjugated (FITC) anti-IgM (R6-60.2); anti-IgDa (AMS 9.1) PE; anti-IgDb (217-170) PE; anti-CD5 (53-7.3) FITC; anti-Thy-1.2 (53-2.1) FITC; anti-CD4 (H129.19) PE; anti-CD8 (53-6.7) FITC; anti-Ter119 (TER-119) PE; anti-Gr1 (RB6-8C5) PE; and anti-Mac1 (M1/ 70HL) FITC (Boehringer Mannheim Corp., Indianapolis, IN). Data was acquired on a FACScan® (Becton Dickinson, San Jose, CA) and analyzed using LYSIS II software. Live cells were gated based on forward and side scatter. A gate characteristic of lymphoid cells based on forward and side scatter was used for acquisition of peritoneal cells.Analysis of In Vivo Bromodeoxyuridine Incorporation.
Mice were fed 0.25 mg/ml bromodeoxyuridine (BrdU) and 2.5% glucose in their drinking water continuously for up to 15 d. Single cell suspensions of spleens and peripheral blood were depleted of red blood cells, stained with anti-BrdU FITC (Becton Dickinson) and anti-B220 (RA3-6B2) PE (PharMingen) as previously described (36), and analyzed as above.Proliferation Assays
BrdU Labeling.
Total splenocytes were depleted of red blood cells and plated in RPMI with 10% heat-inactivated FCS at 106/ml. Where indicated, goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch Labs., West Grove, PA) were added at either 2 or 20 µg/ml. At 24 h, BrdU (Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 10 µM. Cells were harvested at 48 h and FACS® analysis was performed as above.[3H]Thymidine Labeling.
B220+ spleen cells were isolated using the Minimacs magnetic bead system (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions. Single cell suspensions were depleted of red blood cells before incubation with magnetic beads. B cell-enriched populations were >90% B220+ by FACS® analysis. B220+ splenic B cells were seeded into 96-well plates at 5 × 105/ml in RPMI with 10% heat-inactivated FCS. Where indicated, cells were incubated for 60 h with 2 or 20 µg/ml goat anti-mouse IgM F(ab')2 fragments (Jackson ImmunoResearch Labs.). 1 µCi [3H]thymidine (NEN Life Science Products, Boston, MA) was added per well for the final 12-18 h. Cells were harvested and counted on a scintillation counter.ELISA
Serum Ig.
Plates were coated with 2 µg/ml goat anti-mouse Ig (Southern Biotechnology Associates, Huntington, AL) and blocked with 1% BSA in borate-buffered saline (BBS). Serum or Ig standards (mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA; Sigma Chemical Co.) were diluted serially into BBS and added to wells in duplicate. Plates were washed, incubated with secondary antibody (goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA-alkaline phosphatase [ALPH], Southern Biotechnology Associates) diluted 1:500 in BBS/0.05% Tween 20/1% BSA and developed with an ALPH substrate kit (Bio-Rad, Hercules, CA). OD405 was read on a Vmax kinetic microplate reader (Molecular Devices Corp., Sunnyvale, CA).Keyhole Limpet Hemocyanin.
Mice were immunized intraperitoneally with 100 µg of keyhole limpet hemocyanine (KLH) (Sigma Chemical Co.) in incomplete Freund's adjuvant (GIBCO BRL, Gaithersburg, MD), boosted on day 21 with 50 µg of KLH in PBS, and bled on day 28. ELISAs were performed as above with the following modifications: plates were coated with 8 µg/ ml of KLH, and secondary antibodies were goat anti-mouse IgM-ALPH and goat anti-mouse IgG1-ALPH.2,4,6-Trinitrophenyl-Ficoll.
Mice were immunized intraperitoneally with 10 µg of 2,4,6-trinitrophenyl (TNP)-Ficoll (a gift of Dr. John Inman, National Institutes of Health, Bethesda, MD) and bled 6 d later. ELISA was performed as above with the following modifications: plates were coated with 25 µg/ml of TNP-BSA in PBS, and serum and secondary antibody (goat anti-mouse IgM-ALPH) were diluted into PBS/0.1% BSA, and 0.05% Tween 20.dsDNA.
Unimmunized mice were bled at 16-20 wk of age. Serum antibodies to dsDNA were measured in triplicate by ELISA as previously described (14).Immunofluorescence
Unimmunized mice were bled at 16-20 wk of age. Serum antibodies to nuclear antigens were measured by immunofluorescence as previously described (14).
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Results |
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We bred Btk+/lyn+/
mice to generate wild-type, Btk
/
, lyn
/
, and Btk
/
lyn
/
progeny in order to examine the interaction of these kinases in B cell development and function. All genotypes
occurred in the predicted Mendelian frequencies, indicating that embryonic development occurs normally in the
absence of both Btk and Lyn. Btk
/
lyn
/
mice were
healthy and fertile. lyn+/+ and lyn+/
animals were indistinguishable in the assays described below and were therefore
used interchangeably. All mice were on a mixed C57B/6 × 129/Sv background. Although the phenotype of Btk deficiency has been described to differ according to genetic
background (10, 37, 38), little variation was observed
within each group of mice analyzed here.
To understand whether Lyn and Btk are components of a common signaling pathway or function independently in B cells, we examined B cell development in the
absence of Btk alone, Lyn alone, or both Btk and Lyn.
Both the frequency and number of splenic B cells in
8-wk-old Btk/
lyn
/
mice was reduced two- to fourfold
relative to either Btk
/
or lyn
/
animals and four- to sixfold compared with wild-type controls (Fig. 1 A and Table
1). The remaining B cells had an immature IgMhiIgDlo
phenotype similar to Btk
/
B cells (Fig. 1 B). This decrease was specific to the B lineage. No significant difference in myeloid cell numbers was observed in the spleens
of young mice, and T cell numbers were reduced less than
twofold compared with wild-type controls (Table 1). The
frequency of conventional (B220+CD5
) B cells in the
peritoneum was also diminished in Btk
/
lyn
/
mice
compared with single knockouts alone (Fig. 1 D, Table 1).
|
|
Either a block in the bone marrow phase of B cell development or reduced half life of peripheral B cells could be
responsible for the small number of splenic B cells in Btk/
lyn
/
mice. Both the B220+IgM
population, consisting
of proB and preB cells, and B220intIgM+ immature B cells
were present in similar frequencies in bone marrow from
wild-type, Btk
/
, lyn
/
, and Btk
/
lyn
/
mice (Fig. 1 C
and Table 1). Therefore, the antigen-independent phase of
B cell development proceeds normally in the absence of both Btk and Lyn. B220hiIgM+ recirculating B cells were
three- to fourfold less frequent in both lyn
/
(as previously
reported in references 12 and 13) and Btk
/
lyn
/
mice
than in wild-type controls (Fig. 1 C and Table 1). This is
consistent with the significant reduction in mature B cell numbers in the periphery.
The turnover rate of the peripheral B cell population was
assessed with in vivo BrdU labeling since an early developmental block did not account for the reduced number of
splenic B cells in Btk/
lyn
/
mice. Short-lived B cells in
the periphery are replaced by newly generated cells that
have incorporated BrdU, whereas long-lived resting cells
remain unlabeled. Both splenic and peripheral blood B cells
from Btk
/
lyn
/
mice turned over faster than wild-type,
Btk
/
, or lyn
/
B cells (Fig. 2). Greater than 70% of Btk
/
lyn
/
B220+ cells were labeled with BrdU after 8 d, compared with ~35% in wild-type mice and 50% in mice lacking either Btk or Lyn alone (Fig. 2). These observations indicate that the reduced number of B cells in mice lacking both
Btk and Lyn is due to poor survival in the periphery rather
than decreased production of B cells in the bone marrow.
|
To determine whether the reduced
number of peripheral B cells in Btk/
lyn
/
mice was accompanied by alterations in serum Ig levels, the amount of
each Ig isotype present in the serum of 6-8-wk-old wild-type, Btk
/
, lyn
/
, and Btk
/
lyn
/
mice was measured
(Fig. 3). Btk
/
mice had a >10-fold reduction in IgM and
IgG3 levels, and a 2-4-fold decrease in IgG2a and IgG2b.
IgM and IgA levels were slightly elevated in lyn
/
mice
compared with wild-type controls, although the 5-10-fold increase in these isotypes described in lyn
/
mice in two
previous studies (12, 13) was not observed. This may be
due to differences in the age of the animals at the time of
analysis, since serum IgM levels in lyn
/
mice increase
with time (12, 13). Btk
/
lyn
/
mice resembled Btk
/
mice with respect to IgM, IgG3, and IgG2a levels, and had
increased IgA similar to lyn
/
animals. The amount of
IgG2b and IgG1 was reduced in Btk
/
lyn
/
mice compared with all other genotypes, consistent with the low numbers of peripheral B cells in these animals.
|
The dramatic effect of double Btk and Lyn deficiency on
B cell numbers suggested that B cell function may also be
more severely inhibited in the absence of both kinases. The
ability of Btk/
lyn
/
mice to mount an immune response
to the T cell-independent antigen TNP-Ficoll and the T
cell-dependent antigen KLH was assessed. Btk
/
lyn
/
mice failed to produce antibodies against TNP-Ficoll (Fig. 4 A), as expected, since this response requires Btk. Mice lacking either Btk or Lyn alone have relatively normal secondary
responses to T cell-dependent antigens such as KLH (Fig. 4,
B and C). Strikingly, anti-KLH IgM was not detectable in
Btk
/
lyn
/
mice (Fig. 4 B). IgG1 titers against KLH were
measurable but lower on average than in mice of all other
genotypes (Fig. 4 C). Although the impaired IgG1 response
could be attributed to low B cell numbers, the reduction in
anti-KLH IgM was significant even when corrected for cell
number. This indicates that Btk and Lyn are redundant for
the production of IgM against T-dependent antigens.
|
Several aspects of B cell function differ significantly between lyn/
and Btk
/
mice despite the similar reduction
in conventional B cell numbers. Most strikingly, lyn
/
B
cells are hyperresponsive to BCR cross-linking (14, 20), whereas Btk
/
cells fail to proliferate in response to anti-IgM (10). A transgenic model, in which varying doses of a
Btk transgene driven by the Ig heavy chain enhancer and
promoter are expressed on an xid or Btk
/
background,
has been used to demonstrate that Btk dosage is limiting for
response to BCR cross-linking (34). B cells expressing low levels of transgenic Btk and no endogenous Btk (Btklo) develop normally but have reduced sensitivity to BCR cross-linking (34). This system was used to determine whether
the net effect of Lyn is to activate or inhibit Btk function.
Low doses of Btk should be less effective in mediating mitogenic response to BCR engagement on a lyn
/
background if Lyn is an essential upstream activator of Btk during signal initiation. Alternatively, the ability of limiting
amounts of Btk to transmit BCR signals should be enhanced in the absence of Lyn if the negative role of Lyn
predominates.
A Btk transgene expressing ~25% of endogenous Btk
levels in splenic B cells (34) was crossed onto both Btk/
(Btklo) and Btk
/
lyn
/
(Btklo lyn
/
) backgrounds. The
frequency and absolute number of B220+ and IgMloIgDhi
cells were increased two- to threefold in Btklo spleens relative to Btk
/
spleens in both the presence and absence of
Lyn (reference 34 and data not shown), indicating that the
transgene restored Btk-dependent signals for maintenance
of B cell numbers. Btklo B cells were less sensitive to BCR
engagement than were wild-type B cells (reference 34 and
Fig. 5 A). In contrast, the BCR-induced proliferative response of Btklo lyn
/
B cells was indistinguishable from
that of lyn
/
B cells even at low doses of anti-IgM (Fig. 5
A). Btk
/
lyn
/
B cells failed to proliferate upon anti-IgM
stimulation (Fig. 5 A). This was not due to altered T or
myeloid cell function, as the same result was obtained with
purified B cells (Fig. 5 B) and total splenocytes (Fig. 5 A).
Lyn deficiency therefore enhances Btk-dependent signaling
by the BCR but cannot bypass a requirement for Btk.
These observations suggest that Lyn has a net inhibitory effect on Btk-dependent BCR signaling pathways.
The development of autoimmunity in aged lyn/
mice is another
feature that distinguishes them from Btk
/
and xid mice
(12). The xid mutation has been shown to prevent autoantibody production in both NZB×NZW mice (39,
40) and me mice (41). Although six out of six lyn
/
mice
older than 16 wk developed IgM and IgG antibodies
against both dsDNA and nuclear antigens, no Btk
/
lyn
/
animals of similar age displayed signs of autoimmunity (Table 2). Peritoneal B1 cells are believed to be a major source
of anti-self antibodies in autoimmune strains of mice (42,
43). Btk
/
lyn
/
mice, like Btk
/
mice, have a reduced
frequency of B220+CD5+ cells in the peritoneum relative
to wild-type or lyn
/
animals (Fig. 1 D, Table 1).
|
Splenomegaly resulting from extramedullary hematopoiesis occurs after 14 wk of age in the
absence of Lyn (12). No myeloid phenotype has been
described in xid or Btk/
mice or in XLA patients, although a partial defect in Fc
RI-mediated cytokine induction has been reported in xid and Btk
/
mast cells (44).
Consistent with these observations, the increased frequency
of myeloid and erythroid cells characteristic of lyn
/
spleens was also observed in old Btk
/
lyn
/
mice (Fig. 6 B).
Surprisingly, splenomegaly did not occur in Btk
/
lyn
/
mice. Spleens of 10-11-mo-old Btk
/
lyn
/
mice were
four- to fivefold smaller by both weight (0.208 ± 0.042 g
vs. 1.1 ± 0.4 g, n = 2) and cell count (3.74 × 107 ± 2.5 × 107, n = 5, vs. 1.6 × 108 ± 0.72 × 108, n = 6, nucleated
cells) than those of age-matched lyn
/
mice (Fig. 6 A).
These results suggest that although extramedullary hematopoiesis does not require Btk, the subsequent expansion of myeloid and erythroid elements in lyn
/
mice is Btk dependent.
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![]() |
Discussion |
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A large body of evidence derived
from biochemical and tissue culture-based experiments
demonstrates that activation of Btk requires Src family kinases (22). However, the increased severity of the B cell
phenotype in Btk/
lyn
/
mice compared with lyn
/
mice alone indicates that Btk can still transmit signals in the absence of Lyn. In fact, the ability of limiting doses of Btk to signal is enhanced in lyn
/
B cells. This suggests either
that Lyn is redundant with other Src family kinases for the
activation of Btk in mature B cells or that Btk can be activated by Src family-independent mechanisms. Although
activation of Btk is critically dependent on Src kinases in a
fibroblast model (22, 23, 26), additional regulatory mechanisms cannot be ruled out in the B lineage.
Although Lyn may contribute to the activation of Btk in
concert with other Src kinases, it also plays a Btk-independent role in the maintenance of the peripheral B cell population. lyn/
mice have fewer B cells than did wild-type
littermates (12). This has been suggested to result from
increased negative selection of B cells that are hypersensitive to BCR cross-linking (14). Diminished B cell numbers
in Btk
/
lyn
/
mice could be explained by the additive effect of reduced positive selection in the absence of Btk and
increased negative selection in the absence of Lyn. However, this is unlikely since lyn
/
B cells do not respond to
BCR engagement when they also lack Btk. Lyn is therefore likely to transmit a positive signal for B cell survival
that is independent of Btk.
B cells from both Btk/
and lyn
/
mice have an
increased turnover rate relative to wild-type B cells. The
number of long-lived B cells is even further reduced in the
absence of both Btk and Lyn. This could be secondary to
a block in maturation as the long-lived B cell pool consists
predominantly of mature B cells (45). However, this possibility is unlikely as Btk
/
and Btk
/
lyn
/
mice have a similar developmental block at the IgMhiIgDhi to IgMloIgDhi
transition. The reduced half-life of Btk
/
lyn
/
B cells
could also be a result of impaired positive BCR signaling due to the combined effects of Lyn deficiency on signal initiation and Btk deficiency on signal transmission. The BCR
is required for survival of B cells in the periphery (46), and
deletion of the cytoplasmic tail of Ig
in mice results in a B
cell phenotype (4) similar to that of Btk
/
lyn
/
mice. Independent roles for Btk and Lyn in BCR signaling are also
supported by the recent demonstration that the Btk/Tec
family kinase Itk and the Src family kinase Fyn have independent functions in TCR signaling (47). Alternatively,
Btk and Lyn may be redundant for CD40 signaling. Btk
/
lyn
/
mice resemble mice deficient in both Btk and CD40
(48, 49). The impaired response to T cell-dependent antigens would also be explained by failure to transmit CD40
signals (50). Finally, defects in homing of Btk
/
lyn
/
B cells to the proper compartments in secondary lymphoid
organs could contribute to their poor survival (53).
Mice lacking the three Src
family kinases Lyn, Blk, and Fyn have a block in development at the proB to preB transition (Tarakhovsky, A., personal communication) similar to that observed in Ig heavy chain- (5) or surrogate light chain-deficient (6) mice. These combined results imply that Src family kinases are
redundant for the transmission of preB receptor signals. B
lymphopoiesis is also blocked at the preB stage in XLA patients (33), suggesting that Btk is an essential substrate of
Src family kinases in human preB receptor signaling. In
contrast, the antigen-independent phase of B cell development is normal in Btk/
mice even in the absence of Lyn.
The critical target of Src family kinases in murine preB cells
is probably Syk rather than Btk since the proB to preB
transition is impaired in syk
/
mice (7, 8).
Hypersensitivity to BCR cross-linking in lyn/
B cells indicates that Lyn plays a critical role in the negative regulation of BCR signaling. Both the failure of Btk
/
lyn
/
B
cells to proliferate in response to anti-IgM and the observation that Btk is no longer limiting for response to BCR engagement in the absence of Lyn suggest that Lyn downregulates Btk-dependent signaling pathways. The ability of Btk
to promote depletion of intracellular calcium stores in response to BCR cross-linking is prevented by Fc
RIIb signaling (27). This inhibition may be mediated by Lyn since
Fc
RIIb function is partially impaired in lyn
/
B cells (14).
The negative regulatory role of Lyn is not limited to Btk-dependent pathways, as BCR-induced activation of the
classical mitogen-activated protein kinase (MAPK) pathway does not require Btk (54, 55) but is enhanced in lyn
/
B cells (14).
The transgenic mice expressing low levels of Btk (34) are shown here (Fig. 5 A) to be a useful sensitized system with which to identify negative regulatory components of Btk signaling pathways. Similarly, molecules that contribute positively to Btk signaling could be defined by mutations that further impair BCR signaling in Btklo mice. It will be interesting to determine the effect of mutations in the remaining Src family kinases, BCR signal threshold modulators, and other B cell signaling molecules on the transmission of BCR signals by limiting amounts of Btk.
A Role For Btk in Myeloid Expansion.A distinguishing
characteristic of older lyn/
mice is the development of
splenomegaly due to extramedullary hematopoiesis (12- 14). Surprisingly, although splenomegaly did not occur in
old Btk
/
lyn
/
mice, these animals had a similar increase
in the frequency of myeloid and erythroid cells as lyn
/
mice. This suggests that the splenomegaly in lyn
/
mice is
caused by two separate defects. The first, in which the frequency of splenic myeloid and erythroid cells is increased, is independent of Btk. This phase could result from either a
shift in the site of hematopoiesis to the spleen or simply
"space filling" (56) secondary to a reduction in the number
of lymphoid cells. Myeloid and erythroid elements that are
present in lyn
/
spleens then expand in a Btk-dependent
manner. Lyn deficiency may render myeloid cells hypersensitive to cytokines, analogous to the reduction of BCR
signaling thresholds in B cells. This enhanced response may
be attenuated in the absence of Btk. Btk has been implicated as a component of the IL-5 (25, 57, 58), IL-6 (59),
and IL-10 (60) cytokine pathways in B cells. However, no
alterations in myeloid or erythroid cell development have
been reported in xid mice, Btk
/
mice, or XLA patients
except for some defects in Fc
RI signaling in mast cells
(44).
Btk may serve in a general capacity to regulate mitogenic
responses and cell survival. These functions would normally be observed only in B cells because of redundant signaling pathways in other lineages. Variation in genetic context or the mutation of other signaling molecules on a
Btk/
background may reveal additional roles for Btk in
the development and function of hematopoietic cells.
![]() |
Footnotes |
---|
Address correspondence to Owen N. Witte, 5-748 MacDonald Research Laboratories, 675 Circle Dr. South, Los Angeles, CA 90095-1662. Phone: 310-206-6411; Fax: 310-206-8822; E-mail: owenw{at}microbio.ucla.edu
Received for publication 6 March 1998 and in revised form 22 April 1998.
Anne Satterthwaite was supported by a postdoctoral fellowship from the Irvington Institute for Immunological Research. Paschalis Sideras is supported by the Swedish Medical Research Council (MFR). Owen Witte and Frederick Alt are Investigators of the Howard Hughes Medical Institute. This work was partially funded by a US Public Health Service grant (CA-12800; Principal Investigator Randy Wall).We thank Kim Palmer, Fiona Willis, and Prim Kanchanastit for excellent technical assistance and Vivien Chan for technical advice. We are grateful to Dr. John Inman for his gift of TNP-Ficoll. We also thank Julia Shimaoka and Jamie White for assistance with manuscript preparation.
Abbreviations used in this paper ALPH, alkaline phosphatase; BBS, borate-buffered saline; BCR, B cell antigen receptor; BrdU, bromodeoxyuridine; Btk, Bruton's tyrosine kinase; Btklo, mice lacking the endogenous Btk gene and expressing 25% of endogenous levels of Btk in B cells from an Ig heavy chain enhancer/promoter-driven transgene; KLH, keyhole limpet hemocyanin; TNP, 2,4,6-trinitrophenyl; me, motheaten; wt, wild-type; xid, X-linked immunodeficiency; XLA, X-linked agammaglobulinemia.
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