By
§
§
From the * Amgen Institute, Toronto, Ontario, Canada M5G 2C1; the Department of Medical
Biophysics and the § Department of Immunology, University of Toronto, Ontario, Canada M5G 2M9;
and the
Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9
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Abstract |
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Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has
been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fc receptor IIB (Fc
RIIB), a low affinity receptor for the Fc portion of
immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor
(BCR)-Fc
RIIB coligation. The function of Ship in lymphocytes was investigated in Ship
/
recombination-activating gene (Rag)
/
chimeric mice generated from gene-targeted Ship
/
embryonic stem cells. Ship
/
Rag
/
chimeras showed reduced numbers of B cells and an
overall increase in basal serum Ig. Ship
/
splenic B cells displayed prolonged Ca2+ influx,
increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR-Fc
RIIB coligation. These results demonstrate that Ship plays an
essential role in Fc
RIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca2+ flux and MAPK activation.
![]() |
Introduction |
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Ship is an inositol polyphosphate 5-phosphatase that hydrolyzes phosphatidylinositol-3,4,5-polyphosphate (PIP3)1 and inositol-1,3,4,5-polyphosphate (IP4; references 1 and 2). The catalytic domain of Ship has been shown to reduce the intracellular PIP3 levels and to inhibit the biological effects induced by phosphatidylinositol 3'-kinase activation in Xenopus oocytes (3). In addition to the catalytic domain, Ship contains an Src homology (SH)2 domain, three putative SH3 interacting motifs, and two potential phosphotyrosine binding (PTB) domain binding sites. Ship can interact with membrane receptors (4, 5), tyrosine kinases (6), and adapter proteins (7, 8). It has been suggested that Ship functions as a negative regulator of cell growth (2) and as a positive factor in cellular apoptosis (9).
Immune complexes consisting of antigen and IgG antibodies are potent inhibitors of humoral immune responses
(10). The immune complex-mediated inhibition of antibody production depends on the coligation of the antigen-specific B cell antigen receptor (BCR) and FcRIIB, a low
affinity receptor for the Fc portion of IgG (11). Engagement of the BCR in the absence of coligation induces
rapid activation of tyrosine kinases, generation of inositol phosphates, elevation of the cytoplasmic Ca2+ concentration, and mitogen-activated protein kinase (MAPK) activation (12). These events result in cellular activation and lead
to B cell proliferation, differentiation, and antibody secretion (13). In contrast, coligation of the BCR and Fc
RIIB
leads to inhibition of the extracellular Ca2+ influx (14), reduction of cell proliferation (15), and blockage of blastogenesis (16).
FcRIIB delivers the inhibitory signal to downstream
SH2-containing proteins through its immunoreceptor tyrosine-based inhibitory motif (ITIM), a 13-amino acid
sequence that is tyrosine phosphorylated in response to
BCR and Fc
RIIB coligation (17). Several SH2-containing molecules bind to the ITIM of Fc
RIIB (18), including the SH2-containing tyrosine phosphatase SHP-1 (19)
and the phosphatidylinositol phosphatase Ship (4). SHP-1
was thought to play a significant role in Fc
RIIB signaling
(15). However, recent studies have shown that SHP-1 is
dispensable for Fc
RIIB-mediated inhibition of mast cell
degranulation (4) and BCR-triggered Ca2+ influx (20),
suggesting that SHP-1 is not involved in the early signaling
events of Fc
RIIB inhibition. Another candidate for a key
role in Fc
RIIB-mediated inhibition is the Ship protein. Ship interacts with the ITIM of Fc
RIIB (4) and is rapidly tyrosine phosphorylated in response to BCR-Fc
RIIB
coligation (21, 22). Deletion of Ship in a chicken B cell
line rendered the cells resistant to Fc
RIIB-mediated inhibition of Ca2+ accumulation (23), suggesting a direct involvement of Ship in the Fc
RIIB pathway.
To determine the function of Ship in B and T lymphocytes in vivo, we generated embryonic stem (ES) cell lines
with a homozygous mutation in the Ship gene and Ship/
Rag
/
chimeric mice. Ship
/
Rag
/
mice had reduced
numbers of B cells, but increased basal serum Igs. Ship
/
B
lymphocytes exhibited prolonged Ca2+ influx and increased
proliferation upon BCR-Fc
RIIB coligation, demonstrating an essential requirement for Ship in Fc
RIIB-mediated negative signaling. Furthermore, MAPK activation in Ship
/
B cells was increased after BCR-Fc
RIIB coligation, suggesting that, once recruited to Fc
RIIB, Ship can act as a
negative regulator of MAPK signaling.
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Materials and Methods |
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Generation of Ship/
Rag-1
/
Mice.
Flow Cytometry.
The following FITC-conjugated, PE-conjugated, or biotinylated antibodies were used for flow cytometry: anti-FcIn Vitro B Cell Proliferation.
Splenic lymphocytes from 8-14-wk-old mice were incubated in 0.155 M ammonium chloride, 0.1 mM sodium EDTA, 0.1% potassium bicarbonate, pH 7.3, for 5 min on ice to lyse red blood cells. Cells were washed with PBS and incubated with anti-Thy-1, anti-CD8, and anti-CD4 in combination with rabbit complement (Cedarlane Labs Ltd., Hornby, Ontario, Canada) for complement-mediated lysis of T cells. Lymphocytes were collected from the interface of a lympholyte purification gradient (Lympholyte-M; Cedarlane Labs Ltd.). The purity of each of the splenic B cell preparations was verified by flow cytometric analysis, and was typicallyIn Vitro T Cell Proliferation.
Freshly isolated lymphocytes from lymph nodes were placed into round-bottomed 96-well plates (Fisher Scientific, Nepean, Ontario, Canada) in RPMI medium supplemented with 5% FBS, 2 µM sodium pyruvate, 1 µM glutamine, 50 µMIntracellular Ca2+ Measurements.
Splenocytes (5 × 106/ml) were loaded with 3 µM Indo-1 (Molecular Probes, Inc., Eugene, OR) at 37°C for 1 h in IMDM supplemented with 2% FCS. After washing with medium, cells were labeled with PE-conjugated anti-TCR-Western Blot Analysis.
Purified splenic B cells (2 × 106 cells/ 100 µl PBS) were stimulated with PBS alone, goat anti-mouse IgM antibody (20 µg/ml), the F(ab')2 fragment of goat anti- mouse IgM (15 µg/ml), LPS (2 µg/ml), or anti-CD40 antibody (5 µg/ml) at 37°C for various time periods. At the end of the stimulation, cells were immediately diluted with 1 ml ice-cold PBS containing 1 mM sodium vanadate (Na3VO4), pelleted by centrifugation, and resuspended in 20 µl ice-cold lysis buffer consisting of 1% Triton X-100, 1% deoxycholate, 50 mM Hepes buffer, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM PMSF, and 1 mM Na3VO4. Cell debris was pelleted, and supernatants containing the whole cell lysates were analyzed on 15% SDS-polyacrylamide gels at an acrylamide to bis-acrylamide ratio of 120:1. Proteins were transferred to nitrocellulose membranes and immunoblotted with phospho-specific P44/P42 MAPK antibody (Thr202/Tyr204; New England Biolabs Inc., Beverly, MA) to reveal the presence of activated MAPK; phospho-specific stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) antibody (New England Biolabs Inc.) to reveal the presence of activated SAPK/ JNK; and phospho-specific IDetection of Ig Levels.
ELISA for Ig subclasses was performed on serially diluted serum samples using anti-mouse Ig (IgG plus IgA plus IgM) antibodies and alkaline phosphatase-conjugated anti-mouse Ig isotype antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) according to the manufacturer's directions.Serum Neutralization Test.
Vesicular stomatitis virus (VSV) Indiana (Mudd-Summers isolate) seeds were grown on BHK21 cells infected with a low multiplicity of infection and plaqued on Vero cells. Sera were collected from mice at defined time points after VSV infection. The sera were prediluted 40-fold in MEM containing 5% FCS and then heat-inactivated at 56°C for 30 min. Serial twofold dilutions were mixed with equal volume of VSV-containing medium (500 PFU/ml) and incubated in a 5% CO2 incubator at 37°C for 90 min. 100 µl of the mixture was transferred onto Vero cell monolayers in 96-well plates and incubated at 37°C for 1 h. The monolayers were then overlaid with 100 µl of DMEM containing 1% methylcellulose. After incubating at 37°C for 24 h, the monolayer was fixed and stained with 0.5% crystal violet. The highest dilution of serum that reduced the number of plaques by 50% was taken as titer. To determine IgG titers, undiluted serum was pretreated with an equal volume of 0.1 mMIn Vitro Th Cell Differentiation.
Splenocytes (2 × 106/ml) depleted of red blood cells were cultured in duplicate in RPMI medium supplemented with 5% FBS, 50 µM ![]() |
Results |
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Targeted inactivation of the Ship gene in ES cells was accomplished
by replacement of the first coding exon and part of the following intron with the LacZ gene from Escherichia coli and the gene encoding neomycin phosphotransferase (neo) (Fig.
1 A). Ship/
ES cells were isolated by selecting Ship+/
ES cells in elevated levels of G418. Homozygous mutation
of the Ship gene was confirmed by Southern blot analysis of
genomic DNA (Fig. 1 B). Three different Ship
/
clones
and a parental Ship+/
ES cell clone were injected into
blastocysts from Rag-1
/
mice. Since Rag-deficient mice
do not produce any mature lymphocytes due to a block in
the initiation of V(D)J recombination (27), mature lymphocytes in the chimeric mice must be derived from the injected ES cells. Chimeric mice were characterized by
flow cytometric analysis of CD4+ and CD8+ T cells and
IgD+ B cells in circulating blood. Genetic chimerism was
further substantiated by Southern blot analysis of DNA
obtained from tail biopsies (data not shown). Western blot
analysis of lysates prepared from thymocytes (data not
shown) and splenocytes (Fig. 1 C) of the Ship
/
Rag
/
chimeras showed the absence of Ship protein, indicating
that the engineered Ship mutation was a null mutation. All
chimeric mice appeared healthy and had no apparent abnormalities.
|
The thymus and lymph
nodes were of normal size in Ship/
Rag
/
chimeric
mice, but the spleen was significantly enlarged. The total number of thymocytes and the percentages of CD4
CD8
pre-T cells and CD4+CD8+ immature T cells in Ship
/
Rag
/
mice were similar to those found in Ship+/
Rag
/
mice (Table 1), showing that early thymic development
was normal. However, the ratio of mature CD4+ to CD8+
T cells was higher in Ship
/
Rag
/
compared with Ship+/
Rag
/
chimeric mice, suggesting an effect of the mutation
on the progression of immature CD4+CD8+ thymocytes
to mature CD4+ and CD8+ T cells and/or CD4/CD8
homeostasis in peripheral lymphoid organs (Table 1). No
abnormalities were found in the surface expression levels of
TCR-
/
, CD3, CD28, or CD95 on either CD4+ or
CD8+ single positive thymocytes, CD4+CD8+ double
positive thymocytes, or peripheral T cells (data not shown).
|
To test the role of Ship in T cell proliferation, lymph
node T cells were stimulated in vitro with either anti-CD3 antibody, anti-CD3
plus anti-CD28 antibodies,
Con A, or PMA plus Ca2+ ionophore. No significant differences in the extent or kinetics of proliferation or IL-2
production were observed between the Ship+/
and Ship
/
T cells (data not shown). Similarly, no obvious differences
were observed in the levels of phosphorylated I
B, MAPK,
or SAPK between Ship+/
and Ship
/
T cells after anti-CD3
or anti-CD3
plus anti-CD28 stimulation (data not
shown). The extent and duration of Ca2+ mobilization also
appeared to be normal in Ship
/
thymocytes activated
with anti-CD3
or anti-CD3
plus anti-CD28 (data not
shown).
To
examine the effect of the Ship mutation on B cell development, single cell suspensions from spleen and bone marrow
of Ship+/Rag
/
and Ship
/
Rag
/
chimeras were
stained with mAbs against B lineage-specific markers. The
bone marrow of Ship
/
Rag
/
chimeric mice had normal
numbers of B220+CD43+ pro-B cells but significantly reduced numbers of B220+sIgM+ immature and B220+sIgD+
mature B cells (Fig. 2, and Table 1), suggesting a partial
maturational defect of Ship
/
B cells. We found that B
cell numbers were also reduced in the B220+sIgM
population that expresses the IL-2R
chain (CD25; Table 1), an
early B cell maturation marker that appears in the small
pre-B stage before sIgM expression (28). Consistent with
this finding, Ship
/
Rag
/
mice showed normal percentages of B220loHSAhi large pre-B cells, but significantly
reduced percentages of the more mature B220+HSAlo population (Table 1). These results suggest that B cell production is normal in Ship
/
Rag
/
chimeric mice until the
B220+CD43+ large pre-B stage, but fewer B cells were
present in the small pre-B and more mature populations.
|
Peripheral B cells from Ship/
Rag
/
chimeras expressed normal cell surface levels of CD19, CD40, CD44,
ICAM-1, and CD95, but reduced levels of CD23 (data not
shown). Closer examination of splenic B cell subpopulations revealed a decrease in the sIgMhisIgDhi population
with a shift towards the more mature sIgMlosIgDhi phenotype (Fig. 2). This shift is probably not a consequence of
lowered sIgM expression due to the Ship mutation, since
the level of sIgM expression is normal in Ship
/
B cells in
the bone marrow (Fig. 2). We favor the hypothesis that this
shift reflects an augmented maturational event occurring in
Ship
/
B lymphocytes.
To assess the functional consequences of Ship deficiency, we analyzed the production of
Igs in Ship/
Rag
/
chimeric mice. Sera from unimmunized Ship
/
Rag
/
chimeras showed an overall increase
in Ig levels despite a reduction in the number of peripheral
B cells. In particular, IgM, IgA, IgG2a, and IgG2b levels
were elevated, whereas IgG1 levels were reduced (Fig. 3 A).
|
To further characterize the functional significance of the
Ship deficiency in vivo, chimeric mice were immunized
with VSV. Unexpectedly, antivirus-specific antibody production occurred at a normal level and with similar kinetics
in the T help-independent neutralizing IgM response as
well as in T cell-dependent class switching from IgM to
IgG (29; Fig. 3 B). Moreover, similar titers of neutralizing
IgG were detected for both Ship+/Rag
/
and Ship
/
Rag
/
mice 80 d after immunization (Fig. 3 B, and data
not shown), although the levels of nonneutralizing Ig were
significantly higher in Ship
/
Rag
/
chimeras (data not
shown). These results show that Ship is not essential to
maintain the homeostasis of VSV-neutralizing IgM and
IgG responses, and suggest that multiple negative signaling molecules regulate in vivo B cell responses.
The reduced level of serum IgG1, an IL-4-driven
Ig isotype, and the increased level of IgG2a, which depends
on IFN- for Ig class switching, suggested that Th cell differentiation might be affected in Ship
/
T cells. Therefore, we examined the response of Ship
/
T cells to two
different stimuli known to induce the differentiation of
Th1 and Th2 cells. Ship
/
splenocytes stimulated with
anti-CD3
in the presence of IL-4, which induces Th2 differentiation, showed normal levels of IL-4 and IL-6 production. Similarly, when Ship
/
T cells were stimulated
with anti-CD3
in the presence of IL-12, which induces
Th1 differentiation, there was no significant difference between Ship
/
and Ship+/
cells in the production of Th1-type cytokines IFN-
and TNF-
(data not shown). Although these results do not preclude a role of Ship in Th1
and Th2 cytokine production in vivo, our in vitro data imply that Ship has no essential role in Th1 and Th2 lineage differentiation.
To test the hypothesis that Ship downregulates B cell activation (23), B cell proliferation in Ship/
Rag
/
chimeric mice was examined. BCR signaling can
be activated by the F(ab')2 fragment of anti-IgM (or anti-IgG) antibodies that cause cross-linking of sIgM (or sIgG;
reference 11). Intact antibodies fail to stimulate BCR-
mediated cellular activation because they coligate the BCR
and Fc
RIIB (11), resulting in activation of the Fc
RIIB
inhibitory pathway. When sIgM was cross-linked on purified Ship
/
and Ship+/
B cells using the F(ab')2 fragment
of anti-IgM, comparable proliferative responses were induced, suggesting that BCR signaling is normal in Ship
/
B cells (Fig. 4). However, whereas coligation of sIgM and
Fc
RIIB with intact anti-IgM did not significantly stimulate proliferation in Ship+/
cells, Ship
/
B cells proliferated just as strongly in response to intact antibody as they
had in response to anti-IgM F(ab')2 stimulation (Fig. 4).
Similar, but less dramatic, results were obtained using anti- mouse IgG (data not shown). No differences in proliferation were detected when Ship
/
and Ship+/
B cells were
stimulated with LPS or anti-CD40, agents that do not use
the Fc
RIIB pathway (30, 31; Fig. 4 B). These results show that Ship is required for the delivery of a negative
regulatory signal in response to BCR-Fc
RIIB coligation.
|
A well-documented effect of
BCR and FcRIIB coligation is the inhibition of extracellular Ca2+ influx (14, 20, 23). To determine whether Ship
acts by downregulating the Ca2+ influx associated with
BCR stimulation, we compared Ca2+ mobilization in
Ship+/
and Ship
/
B lymphocytes after sIg activation or
sIg-Fc
RIIB coligation. Ship+/
B cells activated with the
F(ab')2 fragment of anti-IgG exhibited a rapid increase in
intracellular free Ca2+ (Fig. 5 B), a response that was reduced in Ship+/
B cells stimulated with intact anti-IgG
antibody. In contrast, an increased and prolonged Ca2+ response was observed in Ship
/
B cells stimulated with intact anti-IgG antibody (Fig. 5 B), despite normal Fc
RIIB
expression on the cell surface (Fig. 5 A). This increased
Ca2+ response to intact anti-IgG could be normalized to
the level observed in Ship+/
B cells by the addition of the
Ca2+ chelator EGTA, which exhausts the extracellular
Ca2+ store (data not shown). These data suggest that Ship
acts as negative regulator in the Fc
RIIB pathway by controlling the Ca2+ influx.
|
BCR signaling has also been
shown to activate the ERK2 isoform of MAPKs (32, 33).
This activation is accompanied by an increase in phosphorylation of ERK2 (34). To test whether Ship is involved in
the MAPK pathway, we examined ERK2 phosphorylation after BCR activation. As shown in Fig. 6, ERK2 was activated equally in Ship+/ and Ship
/
B cells in response to
anti-IgM F(ab')2 stimulation. As expected, ERK2 phosphorylation was reduced in Ship+/
B cells when the BCR
and Fc
RIIB were coligated by intact anti-IgM. In contrast, Ship
/
B cells showed no reduction in ERK2 phosphorylation after intact anti-IgM stimulation, suggesting
that Ship plays a role in the downregulation of the MAPK
pathway, and that the MAPK pathway is involved in the
delivery of the Fc
RIIB inhibitory signal.
|
![]() |
Discussion |
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Ship is an inositol phosphatase that plays important roles
in signal transduction (5, 20, 23). To investigate Ship's function in lymphocytes, we generated Ship-deficient ES
cell lines through homologous recombination, and created
Ship/
Rag
/
chimeric mice. Our analyses of Ship
/
Rag
/
chimeras show that Ship is required for immune
complex-mediated inhibition of B cell proliferation and
the regulation of antibody production. In addition, although the primary function of Ship appears to be one of
negative regulation of BCR signaling, our studies suggest
that Ship is also involved in pre-B cell maturation and homeostasis of T cell subsets.
The inactivation of Ship in B lymphocytes resulted in
enhanced proliferation in response to BCR and FcRIIB
coligation by intact anti-Ig, indicating that Fc
RIIB-mediated inhibition of BCR signaling is Ship dependent. Ship
/
B cells did not show any defects in other signaling pathways that bypass Fc
RIIB, such as stimulation by F(ab')2,
anti-CD40, or LPS; therefore, the predominant role of
Ship in resting B cells appears to be confined to the
Fc
RIIB pathway. We have also noticed that the proliferative responses of Ship
/
B cells to intact anti-Ig were very
similar to that of F(ab')2 stimulation, whereas B cells from
me/me mice (which are SHP-1-deficient) had a proliferative response to intact anti-Ig stimulation at 40% of their
response to F(ab')2 activation (15). Thus, although SHP-1
may be involved, Ship is the predominant signaling molecule downstream of Fc
RIIB.
In B cells, BCR-FcRIIB coligation triggers molecular
events that lead to the inhibition of the Ca2+ influx normally initiated by BCR activation, resulting in reduction of
BCR signaling (23). In this study, we have shown that the deletion of Ship abrogates Fc
RIIB-mediated inhibition of
Ca2+ influx in B cells. Interestingly, the extent and duration of Ca2+ mobilization that occurred in response to
BCR-Fc
RIIB coligation in the absence of Ship were significantly increased over the Ca2+ influx observed in response to BCR activation. Since the prolonged Ca2+
mobilization was clearly associated with BCR-Fc
RIIB
coligation, we speculate that other signaling molecules may
be interacting with the phosphorylated ITIM of Fc
RIIB
in the absence of Ship, and that these interactions generate
signals leading to a delayed closing of the membrane Ca2+
channels. For example, the phosphorylated ITIM of Fc
RIIB
has been shown to be an ideal docking site for several SH2-containing proteins, some of which might indirectly modulate Ca2+ mobilization (15, 18, 19).
An interesting question is whether modulation of Ca2+
mobilization is the sole function of Ship in FcRIIB signaling. It has been shown that the catalytic domain alone of
Ship is capable of delivering the inhibitory effect mediated
by Fc
RIIB, suggesting direct involvement of the Ship
substrates IP4 and/or PIP3 in this signaling process (23).
Since IP4 is able to activate the cytoplasmic membrane
Ca2+ channels (35), it has been postulated that the ITIM of
Fc
RIIB, once phosphorylated after BCR-Fc
RIIB coligation, recruits Ship to the membrane, where it hydrolyzes
IP4 and brings the membrane Ca2+ channels to a closed
state (23, 36). Our results indicate that this is probably not
the only function of Ship in delivering Fc
RIIB signal,
since the MAPK ERK2 was found to be hyperphosphorylated in Ship
/
B cells after BCR-Fc
RIIB coligation.
We speculate that, once recruited to the membrane and tyrosine phosphorylated, Ship may also modulate the extent
of BCR-triggered MAPK signaling through interaction
with molecules involved in the BCR pathway. A well-documented interacting partner for Ship is the adapter protein Shc (22, 37). BCR signaling induces the tyrosine
phosphorylation of Shc and the subsequent formation of
Shc-growth factor receptor-bound protein (Grb)2-Sos
complexes, which mediate Ras and MAPK activation (38,
39). However, BCR-Fc
RIIB coligation appear to enhance the formation of Ship-Shc complexes and to reduce
Shc-Grb2 interaction (37), suggesting that Ship may downregulate the MAPK pathway by competing with Grb2 for
Shc binding (40).
Another candidate molecule that may link Ship to BCR
signaling is the BCR coreceptor CD19. CD19 becomes
rapidly tyrosine phosphorylated after engagement of the
BCR (41), but is dephosphorylated upon BCR-FcRIIB
coligation (42, 43). Furthermore, CD19-deficient B cells
were unable to respond to Fc
RIIB-mediated inhibition (44). Although it is not clear at this point how an inositol phosphatase like Ship could regulate the dephosphorylation
of tyrosines in CD19, it is conceivable that Ship is instrumental in the formation of a multiprotein signal transducing complex. It is possible that one component protein of
the complex could be a tyrosine phosphatase; for example,
Ship has been shown to associate with SHP-2 in hematopoietic cell lines (45).
Although Ship was found to interact with the immunoreceptor tyrosine activation motifs (ITAMs) from the CD3
complex and TCR chain in vitro (46), normal proliferation, IL-2 production, MAPK and SAPK phosphorylation,
and Ca2+ mobilization were detected in Ship
/
T cells after
TCR activation. These findings indicate that Ship is probably
not involved in TCR signaling. However, the elevation in
the ratio of CD4+ to CD8+ single positive cells in Ship
/
Rag
/
mice, in conjunction with the upregulation of Ship
expression in single positive thymocytes after positive selection (47), suggests that Ship plays a role in mature T cells.
The effect of Ship deficiency appeared to be more dramatic in B cells. We observed a significant reduction of the
percentage of B220+CD25+sIgM small pre-B cells in
Ship
/
Rag
/
chimeric mice. These early B cell populations do not yet express sIgM, suggesting a role of Ship besides inhibition of BCR signaling. The role of Ship in early
B cell maturation and the receptor(s) for signaling molecules on which Ship acts during pre-B cell differentiation need to be determined. The percentages of premature
IgM+ and mature IgD+ B cells were also reduced in bone
marrow and peripheral immune organs. This reduction is
not caused by a defect in cell proliferation, because Ship
/
B cells showed enhanced proliferative response toward intact antibody stimulation and normal responses toward LPS
and anti-CD40 activation (Fig. 4). Since B cells go through
negative selection during maturation to assure immunological tolerance to self-antigens (48, 49), and since this selection depends on the threshold of intracellular signals (48,
50), deletion of the inhibitory regulator Ship may produce
a stronger signal, which exceeds the signaling threshold for
negative selection and leads to a greater reduction of bone
marrow B cells. Consistent with this hypothesis, we have
also observed a shift from IgMhiIgDhi to the more mature
IgMloIgDhi population in Ship
/
B cells, a phenotype normally correlating with B cell hyperresponsiveness caused by
the deletion of negative regulators (51).
The enhanced proliferative response of Ship/
B cells
after anti-Ig stimulation and increased basal serum levels of
different Ig subclasses suggest a possible deregulation of antibody production in vivo due to the disruption of an immune complex-mediated inhibition. Surprisingly, the Ig
levels of neutralizing IgM and IgG after VSV infection
were comparable among Ship+/
Rag
/
and Ship
/
Rag
/
chimeric mice, suggesting that additional pathways for
maintaining antibody homeostasis are operating in VSV-specific responses. Whether Ship
/
Rag
/
chimeric mice
would respond differently to pathogenic stimulation other
than VSV remains to be determined.
![]() |
Footnotes |
---|
Address correspondence to Qiurong Liu, Amgen Institute, 620 University Avenue, Suite 706, Toronto, Canada, M5G 2C1. Phone: 416-204-2264; Fax: 416-204-2277; E-mail: qliu{at}amgen.com
Received for publication 20 April 1998 and in revised form 23 June 1998.
We thank Kurt Bachmaier, Anne Hakem, Takehiko Sasaki, Yun Kong, and Connie Krawczyk for comments, and Mary Saunders and Marissa Luchico for assistance during preparation of the manuscript. We also give special thanks to Andrew Wakeham, Wilson Khoo, Annick Itie, Arda Shahinian for technical help, and Dr. Tak W. Mak for support.
Abbreviations used in this paper BCR, B cell antigen receptor; ERK, extracellular signal-regulated protein kinase; ES, embryonic stem; FBS, fetal bovine serum; Grb, growth factor receptor-bound protein; HSA, heat stable antigen; ICAM, intracellular adhesion molecule; IP4, inositol-1,3,4,5-polyphosphate; ITIM, immunoreceptor tyrosine-based inhibitory motif; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PIP3, phosphatidylinositol-3,4,5-polyphosphate; Rag, recombination-activating gene; SAPK, stress-activated protein kinase; SH, Src homology domain; Ship, SH2-containing inositol polyphosphate 5-phosphatase; SHP, SH2-containing protein tyrosine phosphatase; VSV, vesicular stomatitis virus.
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References |
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1. |
Damen, E.D.,
L. Liu,
P. Rosten,
R.K. Humphries,
A.B. Jefferson,
P.W. Majerus, and
G. Krystal.
1996.
The 145-kDa protein
induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate
5-phosphatase.
Proc. Natl. Acad. Sci. USA.
93:
1689-1693
|
2. | Lioubin, M.N., P.A. Algate, S. Tsai, K. Carlberg, R. Aebersold, and L.R. Rohrschneider. 1996. p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev 10: 1084-1095 [Abstract]. |
3. | Deuter-Reinhard, M., G. Apell, D. Pot, A. Klippel, L.T. Williams, and W.M. Kavanaugh. 1997. SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase. Mol. Cell. Biol. 10: 2559-2565 . |
4. |
Ono, M.,
S. Bolland,
P. Tempst, and
J.V. Ravetch.
1996.
Role of the inositol phosphatase SHIP in negative regulation
of the immune system by the receptor Fc![]() |
5. |
Kimura, T.,
H. Sakamoto,
E. Appella, and
R.P. Siraganian.
1997.
The negative signaling molecule SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) binds to the
tyrosine-phosphorylated ![]() |
6. |
Crowley, M.T.,
S.L. Harmer, and
A.L. DeFranco.
1996.
Activation-induced association of a 145-kDa tyrosine-phosphorylated protein with Shc and Syk in B lymphocytes and macrophages.
J. Biol. Chem.
271:
1145-1152
|
7. |
Lamkin, T.D.,
S.F. Walk,
L. Liu,
J.E. Damen,
G. Krystal, and
K.S. Ravichandran.
1997.
Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires
the Shc-phosphotyrosine binding domain and two specific
phosphotyrosines on SHIP.
J. Biol. Chem.
272:
10396-10401
|
8. | Damen, J.E., L. Liu, R.L. Cutler, and G. Krystal. 1993. Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-kd tyrosine phosphorylated protein. Blood. 82: 2296-2303 [Abstract]. |
9. |
Liu, L.,
J.E. Damen,
M.E. Hughes,
I. Babic,
F.R. Jirik, and
G. Krystal.
1997.
The Src homology (SH2) domain of SH2-containing inositol phosphatase (SHIP) is essential for tyrosine phosphorylation of SHIP, its association with Shc, and
its induction of apoptosis.
J. Biol. Chem.
272:
8983-8988
|
10. | Köhler, H., B.C. Richardson, D.A. Rowley, and S. Smyk. 1997. Immune response to phosphorylcholine. III. Requirement of the Fc portion and equal effectiveness of IgG subclasses in anti-receptor antibody-induced suppression. J. Immunol. 119: 1979-1986 [Abstract]. |
11. |
Phillips, N.E., and
D.C. Parker.
1983.
Fc-dependent inhibition of mouse B cell activation of whole anti-µ antibodies.
J.
Immunol.
130:
602-606
|
12. | DeFranco, A.L.. 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9: 296-308 [Medline]. |
13. | Pleiman, C.M., D. D'Ambrosio, and J.C. Cambier. 1994. The B-cell antigen receptor complex: structure and signal transduction. Immunol. Today. 15: 393-397 [Medline]. |
14. |
Diegel, M.L.,
B.M. Rankin,
J.B. Bolen,
P.M. Dubois, and
P.A. Kiener.
1994.
Cross-linking of Fc![]() |
15. | Pani, G., M. Kozlowski, J.C. Cambier, G.B. Mills, and K.A. Siminovitch. 1995. Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling. J. Exp. Med. 181: 2077-2084 [Abstract]. |
16. |
Phillips, N.E., and
D.C. Parker.
1984.
Cross-linking of B
lymphocyte Fc-![]() |
17. |
Muta, T.,
T. Kurosaki,
Z. Misulovin,
M. Sanchez,
M.C. Nussenzweig, and
J.V. Ravetch.
1994.
A 13-amino-acid motif in the cytoplasmic domain of Fc![]() |
18. | Vély, F., S. Olivero, L. Olcese, A. Moretta, J.E. Damen, L. Liu, G. Krystal, J.C. Cambier, M. Daëron, and E. Vivier. 1994. Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs. Eur. J. Immunol. 27: 1994-2000 . |
19. |
D'Ambrosio, D.,
K.L. Hippen,
S.A. Minskoff,
I. Mellman,
G.I. Pani,
K.A. Siminovitch, and
J.C. Cambier.
1995.
Recruitment, and activation of PTP1C in negative regulation of
antigen receptor signaling by Fc![]() |
20. |
Nadler, M.J.S.,
B. Chen,
J.S. Anderson,
H.H. Wortis, and
B.G. Neel.
1997.
Protein-tyrosine phosphatase SHP-1 is dispensable for Fc![]() |
21. |
D'Ambrosio, D.,
D.C. Fong, and
J.C. Cambier.
1996.
The
SHIP phosphatase becomes associated with Fc![]() |
22. | Chacko, G.W., S. Tridandapani, J.E. Damen, L. Liu, G. Krystal, and K.M. Coggeshall. 1996. Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP. J. Immunol. 157: 2234-2238 [Abstract]. |
23. | Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, and J.V. Ravetch. 1997. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell. 90: 293-301 [Medline]. |
24. | Tybulewicz, V.L.J., C.E. Crawford, P.K. Jackson, R.T. Bronson, and R.C. Mulligan. 1991. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell. 65: 1153-1163 [Medline]. |
25. | Wurst, W., and A.L. Joyner. 1993. Gene Targeting. Oxford University Press, New York. |
26. | Wallace, V.A., W.-P. Fung-Leung, E. Timms, D. Gray, K. Kishihara, D.Y. Loh, J. Penninger, and T.W. Mak. 1992. CD45RA and CD45RBhigh expression induced by thymic selection events. J. Exp. Med. 176: 1657-1663 [Abstract]. |
27. | Mombaertz, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and V.E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 68: 869-877 [Medline]. |
28. | Osmond, D.G., A. Rolink, and F. Melchers. 1998. Murine B lymphopoiesis: towards a unified model. Immunol. Today. 19: 65-68 [Medline]. |
29. |
Leist, T.P.,
S.P. Cobbold,
H. Waldmann,
M. Aguet, and
R.M. Zinkernagle.
1987.
Functional analysis of T lymphocyte
subsets in antiviral host defense.
J. Immunol.
138:
2278-2281
|
30. | Noelle, R.J., M. Roy, D.M. Shepherd, I. Stamenkovic, J.A. Ledbetter, and A. Aruffo. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal of cognate activation of B cells. Proc. Natl. Acad. Sci. USA. 89: 6550-6554 [Abstract]. |
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. | Kashiwada, M., Y. Kaneko, H. Yagita, K. Okumura, and T. Takemori. 1996. Activation of mitogen-activated protein kinases via CD40 is distinct from that stimulated by surface IgM on B cells. Eur. J. Immunol. 26: 1451-1458 [Medline]. |
33. | Cyster, J.G., J.I. Healy, K. Kishihara, T.W. Mak, M.L. Thomas, and C.C. Goodnow. 1996. Regulation of B-lymphocyte negative and positive selection by tyrosine phosphatase CD45. Nature. 381: 325-328 [Medline]. |
34. | Chan, V.W.F., F. Meng, P. Soriano, A.L. DeFranco, and C.A. Lowell. 1997. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity. 7: 69-81 [Medline]. |
35. | Lückhoff, A., and D. Clapham. 1992. Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca2+-permeable channel. Nature. 355: 356-358 [Medline]. |
36. | Ravetch, J.V.. 1997. Fc receptors. Curr. Opin. Immunol. 9: 121-125 [Medline]. |
37. | Tridandapani, S., G.W. Chacko, J.R. Van Brocklyn, and K.M. Coggeshall. 1997. Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interaction. J. Immunol 158: 1125-1132 [Abstract]. |
38. |
Saxton, T.M.,
I. van Oostveen,
D. Bowtell,
R. Aebersold, and
M.R. Gold.
1994.
B cell antigen receptor cross-linking
induces phosphorylation of the p21ras oncoprotein activators
SHC and mSOS1 as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein.
J. Immunol.
153:
623-636
|
39. | Kumar, G., S. Wang, S. Gupta, and A. Nel. 1995. The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line. Biochem. J. 307: 215-223 [Medline]. |
40. | Tridandapani, S., T. Kelley, D. Cooney, M. Pradhan, and K.M. Coggeshall. 1997. Negative signaling in B cells: SHIP Grb2 Shc. Immunol. Today. 18: 424-427 [Medline]. |
41. | Tedder, T.F., M. Inaoki, and S. Sato. 1997. The CD19-CD21 complex regulates signal transduction threshold governing humoral immunity and autoimmunity. Immunity. 6: 107-118 [Medline]. |
42. | Sato, S., D.A. Steeber, P.J. Jansen, and T.F. Tedder. 1997. CD19 expression levels regulate B lymphocyte development. J. Immunol. 158: 4662-4669 [Abstract]. |
43. |
Kiener, P.A.,
M.N. Lioubin,
L.R. Rohrschneider,
J.A. Ledbetter,
S.G. Nadler, and
M.L. Diegel.
1997.
Co-ligation of the antigen and Fc receptors gives rise to the selective modulation of
intracellular signaling in B cells.
J. Biol. Chem.
272:
3838-3844
|
44. |
Hippen, K.L.,
A.M. Buhl,
D. D'Ambrosio,
K. Nakamura,
C. Persin, and
J.C. Cambier.
1997.
Fc![]() |
45. | Sattler, M., R. Salgia, G. Shrikhande, S. Verman, J.-L. Choi, L.R. Rohrschneider, and J.D. Griffin. 1997. The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors. Oncogene. 15: 2370-2384 . |
46. | Osborne, A., G. Zenner, M. Lubinus, X. Zhang, Z. Songyang, L.C. Cantley, P. Majerus, P. Burn, and J.P. Kochan. 1996. The inositol 5'-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation. J. Biol. Chem. 217: 29271-29278 . |
47. |
Liu, Q.,
F. Shalaby,
J. Jones,
D. Bouchard, and
D.J. Dumont.
1998.
The SH2-containing inositol polyphosphate 5-phosphatase, Ship, is expressed during haematopoiesis and spermatogenesis.
Blood.
91:
1-8
|
48. |
Goodnow, C.C..
1996.
Balancing immunity and tolerance:
deleting and tuning lymphocyte repertoires.
Proc. Natl. Acad.
Sci. USA.
93:
2264-2271
|
49. | Hartley, S.B., M.P. Cooke, D.A. Fulcher, A.W. Harris, S. Cory, A. Basten, and C.C. Goodnow. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell. 72: 325-335 [Medline]. |
50. | Klinman, N.R.. 1996. The "clonal selection hypothesis" and current concepts of B cell tolerance. Immunity. 5: 189-195 [Medline]. |
51. |
O'Keefe, T.L.,
G.T. Williams,
S.L. Davies, and
M.S. Neuberger.
1996.
Hyperresponsive B cells in CD22-deficient
mice.
Science.
274:
798-801
|