By
From the * Department of Oncology, Institute of Medical Science, The University of Tokyo, Tokyo
108-8639, Japan; and the Department of Biophysics and Biochemistry, Graduate School of Science,
The University of Tokyo, Tokyo 113-0033, Japan
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Abstract |
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B cells from young lyn/
mice are hyperresponsive to anti-IgM-induced proliferation, suggesting involvement of Lyn in negative regulation of B cell antigen receptor (BCR)-mediated
signaling. Here we show that tyrosine phosphorylation of Fc
RIIB and CD22 coreceptors,
which are important for feedback suppression of BCR-induced signaling, was severely impaired in lyn
/
B cells upon their coligation with the BCR. Hypophosphorylation on tyrosine
residues of these molecules resulted in failure of recruiting the tyrosine phosphatase SHP-1 and
inositol phosphatase SHIP, SH2-containing potent inhibitors of BCR-induced B cell activation, to the coreceptors. Consequently, lyn
/
B cells exhibited defects in suppressing BCR-induced Ca2+ influx and proliferation. Thus, Lyn is critically important in tyrosine phosphorylation of the coreceptors, which is required for feedback suppression of B cell activation.
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Introduction |
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The Src family protein-tyrosine kinase, Lyn, is highly
expressed in hematopoietic cells. Lyn physically associates with the BCR in B cells and with FcRI in mast
cells, and is rapidly activated upon cross-linking of the antigen receptors (1). Lyn interacts with and phosphorylates
a number of substrates, such as the Syk kinase (4, 5), HS1
protein (6, 7), and Cbl protooncogene product (8). Taken
together, Lyn is thought to play important roles in the antigen receptor-mediated positive signaling.
Recently, however, two groups reported that B cells
from young lyn/
mice were hyperresponsive to anti-IgM-induced proliferation due in part to the impairment
of Fc
RIIB-mediated feedback suppression of the B cell
antigen receptor (BCR) signaling (9, 10). Therefore, it is
suggested that Lyn may play some roles in the antigen receptor-mediated negative signaling, too. In this study, using splenic B cells or bone marrow-derived mast cells
(BMMCs) from lyn
/
mice, we addressed molecular
mechanisms by which the Lyn kinase would act as a key
regulator of antigen receptor signaling.
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Materials and Methods |
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Cells and Cell Culture.
All lynProliferative Responses.
For proliferation assay, B cells (105/ well) were cultured in 96-well flat-bottomed tissue culture plates either alone or in the presence of goat F(ab')2 anti-IgM (Cappel, Durham, NC) or intact anti-IgM (Southern Biotechnology Assoc., Inc., Birmingham, AL). Cultured cells were pulse-labeled and assayed as described (13). All assays were performed in triplicate with <20% variation among assays.Qualification of Total Ig by ELISA.
Amounts of each Ig isotype in sera and in culture supernatants were determined by ELISA with antibodies specific for each membrane-bound Ig (mIg) isotype (13).Immunoprecipitation and Immunoblotting.
For the activation of B cells, splenic B cells were treated with goat F(ab')2 anti-IgM (Cappel) or intact anti-IgM (Southern Biotechnology Assoc., Inc.) for 2 min at 37°C. For the activation of mast cells, BMMCs were sensitized for 1 h with antidinitrophenyl (anti-DNP) monoclonal IgE (10 µg/ml; Sigma Chemical Co., St. Louis, MO) followed by stimulation for 2 min at 37°C with 30 ng/ml DNP-conjugated human serum albumin (DNP-HSA; resulting in FcFcR Cross-linking and Degranulation Assay.
FcMeasurement of Internal Ca2+ Concentration.
Splenic B cells and IgE-sensitized BMMCs from lyn+/+ and lyn ![]() |
Results and Discussion |
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lyn/
mice, generated by gene targeting, exhibit splenomegaly as they age. The enlarged spleen accumulates unusual lymphoblast-like cells and plasma cells (13, 15). lyn
/
mice with splenomegaly contain high concentrations of serum IgM and IgA, and often develop glomerulonephritis
due to the production of autoreactive antibodies (13, 15).
The lymphoblast-like cells are IgM+/
, IgD+/
, CD5
,
Mac-1+, CD23
, B220+/
, and IL-5R+/
, and are therefore B-1b-like cells. These cells produce spontaneously a
large amount of the antibodies. However, we found that
serum levels of IgM and IgA were extremely high in lyn
/
mice compared to wild-type mice even at their early ages
(<1 mo old) when the lymphoblast-like cells did not yet
accumulate (Fig. 1 A). This suggests that Lyn is involved in
negative regulation of antibody production by the normal
B cells.
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In vitro cross-linking of the BCR on resting B cells induces a proliferative response. The response is prominent
only when F(ab')2 anti-IgM is used as a stimulating antibody, whereas intact anti-IgM fails to fully activate B cells.
This is due to the inhibitory signals induced by coligation
of the BCR and FcRIIB, a low-affinity immunoglobulin
G receptor (16, 17). In contrast to wild-type B cells, lyn
/
B cells from young mice (<6 wk old) were highly responsive to the intact anti-IgM, exhibiting a proliferation level
similar to that with F(ab')2 anti-IgM. The data suggest that
the lyn
/
B cells are defective in Fc
RIIB-mediated suppression of BCR signaling (9, 10, and Fig. 1 B). Furthermore,
the disorders of lyn
/
mice, such as the hyperresponse to
proliferation after BCR ligation and high concentrations of
serum Igs, are found in mice lacking Fc
RIIB (18). Moreover, the phenotypes of mice deficient in SHP-1, an SH2-containing protein tyrosine phosphatase (19, 20), or CD22,
a B-lineage specific surface molecule (21), overlap those
of lyn
/
mice. Note that both SHP-1 and CD22 participate in the feedback suppression of BCR signaling, and that
tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs; reference 25) of CD22 and
Fc
RIIB is involved in the suppression (26). The expression levels of SHP-1, CD22, and Fc
RII in lyn
/
mice did not differ from those in wild-type mice (data not
shown). Therefore, the inhibitory signalings through SHP-1,
CD22, and Fc
RIIB are likely related to Lyn kinase activity.
Upon stimulation of the BCR, CD22 and SHP-1 become rapidly tyrosine-phosphorylated (29) and associate
with one another (30). To determine whether Lyn is
involved in the tyrosine phosphorylation of CD22 and
SHP-1, we immunoprecipitated these proteins from the lysates of BCR-cross-linked splenic B cells and probed the
immunoprecipitates with antiphosphotyrosine antibody. As
shown in Fig. 2 A, tyrosine phosphorylation of CD22 was
extremely impaired in lyn/
B cells (lanes 5 and 6), as
compared with that in wild-type B cells (lanes 2 and 3). A
level of tyrosine phosphorylation of SHP-1 was also low,
and association of CD22 with SHP-1 was poor in the absence of Lyn (Fig. 2 B and data not shown).
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Next, we examined the level of tyrosine phosphorylation of anti-FcRIIB immunoprecipitates prepared from
the lysates of wild-type and lyn
/
splenic B cells on which
Fc
RIIB and BCR had been cross-linked. Immunoblot
analysis of the precipitates with antiphosphotyrosine antibody (Fig. 3 A) showed a very low level of tyrosine phosphorylation of Fc
RIIB in the absence of Lyn. The recruitment of SHIP, an inositol phosphatase important for
the feedback suppression (26), from cytoplasm to membrane was also impaired in lyn
/
B cells (Fig. 3 B). Accordingly, the magnitude of BCR-induced calcium influx
was less affected by coligated Fc
RIIB in lyn
/
B cells than
in wild-type B cells (Fig. 3 C). Moreover, as mentioned above, the proliferative response to BCR cross-linking was
not suppressed by coligated Fc
RIIB in lyn
/
B cells (Fig.
1 B). Thus, in the case of B cells, Lyn is directly involved in
the feedback suppression mechanisms as well as in the
BCR positive signaling. The controversial observation that B cells from relatively older lyn
/
mice are hyporeactive
to BCR stimulation (13, 15) may suggest that negative signaling pathways are differently tuned with age.
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The similarities between the BCR- and FcRI-mediated
signaling prompted us to examine whether tyrosine phosphorylation of Fc
RIIB coligated with Fc
RI was impaired in lyn
/
mast cells. As shown in Fig. 4 A, tyrosine
phosphorylation of coligated Fc
RIIB was suppressed in
the lyn
/
BMMCs. This likely results in the failure of recruitment of SHIP and/or SHP-1 to the ITIM of Fc
RIIB,
leading to the lack of suppression of Fc
RI signaling by the
inhibitory coreceptor. However, in both wild-type and
lyn
/
BMMCs, the calcium response after IgE cross-linking was similarly suppressed by coligation of Fc
RIIB with
Fc
RI-IgE complexes (data not shown). Furthermore,
coligation of Fc
RIIB with Fc
RI-IgE complexes with increasing amounts of the cross-linking antibody resulted in
increasing inhibition of degranulation, as measured by
-hexosaminidase release, in both wild-type and lyn
/
BMMCs (Fig. 4 B). Coligation of Fc
RIIB with Fc
RIII
on both wild-type and lyn
/
BMMCs also inhibited receptor-triggered degranulation (the pair columns at the
foremost right in Fig. 4 B). These results suggest that, unlike the case with the B cells, the negative signaling pathways appear to function similarly in wild-type and lyn
/
BMMCs.
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The apparent contradiction between B cells and mast
cells with regard to the FcRIIB feedback function may be
due to differences in their Fc
RIIB-mediated signalings.
Such differences could produce the cell-type specific balance between the positive and negative signalings. Present
data together with previous observations (9, 10, 12, 13, 15)
indicate that positive signaling is less affected than negative
signaling in lyn
/
B cells, and vice versa in lyn
/
mast
cells. The balance of the positive and negative signalings in
lyn
/
cells could well be influenced by some other Src-like kinases and Syk kinase expressed in the cells. In B cells,
the imbalance induced by lyn deficiency seems to severely
affect the immune responses. As a consequence, lyn
/
mice, which have lost the control of positive and negative
signalings, exhibit abnormal phenotypes such as hyperactivation of B cells (9, 10) and elevated serum levels of IgM
and IgA (13, 15), resulting in development of autoimmune
disease (13, 15).
Our data show that Lyn is critically involved in tyrosine
phosphorylation of particular proteins such as CD22 and
FcRIIB that are involved in the feedback regulation in B
cells and/or BMMCs (for review see references 33).
Lyn is also important in tyrosine phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) family
of proteins and the proteins involved in positive regulation
of the BCR and Fc
RI signaling events (4, 5, 12, 13, 15, 36).
The other membrane molecules with the putative ITIMs
have been described (37, 38). These include KIR on NK
cells, glycoprotein 49B and MAFA-1 on mast cells, and CD23 and CD72 on B cells. To understand the molecular
basis of the balance of the positive and negative signalings,
it is important to address what kinases preferentially tyrosine phosphorylate and regulate these molecules. The
candidates include the Src family kinases, JAK kinases, Tec
family kinases, and Syk/ZAP-70 kinases. The balance, of
course, could be affected by the absence of a critical kinase(s).
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Footnotes |
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Address correspondence to Tadashi Yamamoto, Department of Oncology, Institute of Medical Science, The University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5301; Fax: 81-3-5449-5413; E-mail: tyamamot{at}ims.u-tokyo.ac.jp
Received for publication 22 October 1997 and in revised form 28 January 1998.
We thank J.V. Ravetch (Rockefeller University, New York), M. Ono (Rockefeller University, New York), E.A. Clark (University of Washington, Seattle, WA), and M.N. Lioubin (Fred Hutchinson Cancer Research Center, Seattle, WA) for antibodies, and A. Tanaka and Y. Yamanashi for critical reading of the manuscript.
This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, Human Frontier Science Program Organization, and Japan Society for the Promotion of Science.
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