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,
* Department of Molecular Oncology, Graduate School of Medicine,
Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; and
Laboratory for Cytokine Signaling, Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Kanagawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dextran plus IL-4 plus IL-5 showed augmented IgM and IgG1
production in vitro that was corrected by the retrovirus-mediated
transfection of the wild-type Gab1 gene, clearly demonstrating
the cell-autonomous, negative role of Gab1. Furthermore, we showed that
the negative role of Gab1 required its Src homology 2-containing
tyrosine phosphatase-2 binding sites. Cell fractionation analysis
revealed that nonfollicular B cells were responsible for the augmented
Ab production in vitro. Consistent with these results, the Gab1 gene
was expressed in marginal zone B cells but not follicular B cells.
These results indicated that Gab1 is a unique negative regulator
specific for TI-2 responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Grb2-associated binder-1 (Gab1) is a member of the Gab/daughter of sevenless family of adapter molecules (12, 13). Gab1 is phosphorylated on tyrosine upon the stimulation of various receptors for growth factors and cytokines (12, 13, 14, 15, 16, 17, 18, 19, 20). Gab1 is also activated upon ligation of B and T cell Ag receptors in cultured cell lines (19, 21). Upon stimulation, Gab1 has been shown to interact with Grb2, Src homology 2-containing tyrosine phosphatase-2 (SHP-2), the p85 phosphatidylinositol 3 (PI-3) kinase, and Shc. Recently it has been reported that Gab1 links the BCR signals to the PI-3 kinase/Akt pathway and to the SHP-2 phosphatase in a B cell line, WEHI231 (22). Targeted disruption of the mouse Gab1 gene revealed that Gab1 is essential for development of the heart, placenta, and skin and is required for activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway mediated by hepatocyte growth factor, epidermal growth factor, platelet-derived growth factor, and gp130 (23). Furthermore, Gab1 is essential for the migration of myogenic precursor cells through c-Met signaling (24). Although Gab1 has been suggested to play roles in hematopoiesis and in the immune response, the embryonic lethality of Gab1-/- mice has made this issue unresolved. To reveal functions of Gab1 proteins in hematopoiesis and in the immune response in vivo, we generated a Gab1-deficient immune system in mice (radiation chimeras) by transplanting fetal liver (FL) cells from Gab1-/- mice to x-ray-irradiated mice. Analyses of these chimeras revealed that Gab1 was dispensable in hematopoiesis. Most importantly, immunization of the chimera with TI-2 Ag induced an enhanced production of Abs of the IgM and IgG1 classes, whereas TI-1 and TD antigenic responses were normal. Among the splenic B cell subsets, the expression of the Gab1 gene was restricted to MZ B cells. These results indicated that Gab1 is a specific negative regulator of the TI-2 response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The establishment of Gab1 knockout mice was described previously, and animals with a mixed background of 129 and C57BL/6 were used in this study. B6C3F1 mice (H-2b/k) were irradiated with 1300 rad of x-rays and injected i.v. 1 day later with FL at 14.5 days post-coitum (1–2 x 106) from Gab1-/- or Gab1+/+ mice (H-2b). Eight to 9 wk after transfer, the reconstituted chimeras were subjected to a variety of analyses. Donor-derived hematopoietic cells in the lymphoid organs were detected as H-2Kk-negative cells by multicolor flow cytometry analysis. Cells originating from the donors usually repopulated >90% of the hematopoietic cells in the chimeras.
Flow cytometry
Flow cytometry was performed and analyzed as described
previously (25). Anti-mouse mAbs, FITC-anti-BP-1
(6C3), anti-CD23 (B3B4), PE-anti-CD4 (RM4-4), anti-CD45R
(RA3-6B2), anti-CD23 (B3B4), anti-CD3
(145-2C11), and
allophycocyanin-anti-CD11b (M1/70) were purchased from BD
PharMingen (San Diego, CA). PE-F4/80 and biotinylated
anti-H-2Kk(CTKk)
were purchased from Caltag Laboratories (Burlingame, CA). Quantum
Red-anti-CD8a (53-6.7) and PE-anti-IgD (11-26c) were purchased
from Sigma-Aldrich (St. Louis, MO) and Southern Biotechnology
Associates (Birmingham, AL), respectively. Anti-IgM (AM/3),
anti-heat-stable Ag (M1/69), anti-CD11b (M1/70), anti-Gr-1
(RB6-8C5), and anti-erythroid cells (TER-119) were purified from
the supernatant of hybridomas using protein G-Sepharose 4B (Pharmacia,
Peapack, NJ) and were labeled with FITC or Cy5 (Amersham, Arlington
Heights, IL) using standard procedures. Purified anti-CD43 (S7) and
anti-CD21 (7G6) were kindly provided by P. W. Kincade
(Oklahoma Medical Research Foundation, Oklahoma City, OK) and T.
Kinoshita (Osaka University, Osaka, Japan), respectively, and were
labeled with FITC. Biotinylated Abs were detected with FITC-, PE-, or
PerCP-labeled streptavidin. Data from stained cells were acquired using
FACSCalibur and CellQuest software (BD Biosciences, Mountain View,
CA).
Immunohistochemistry and 
-galactosidase staining
For immunofluorescence analysis, the spleens were isolated from Gab1+/+ and Gab1-/- FL chimeras and frozen in ornithine carbamoyltransferase compound with liquid nitrogen. Six-micrometer-thick sections were cut and fixed with ethanol. The sections were sequentially incubated with rat anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) Ab (MECA-367; BD PharMingen) and biotinylated F(ab')2 donkey anti-rat IgG. After blocking with 5% heat-inactivated rat serum in PBS, rhodamine-streptavidin and FITC-anti-IgM (AM/3) were added. For the staining of metallophilic macrophages and MZ macrophages, the hybridoma supernatants of MOMA-1 and ERTR-9 (both from Serotec, Oxford, U.K.), respectively, were used as the first Ab. The bound Abs were visualized with biotinylated F(ab')2 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).
For the analysis of the localization of Gab1+
cells, 35- and 6-µm-thick serial frozen sections were prepared from
the spleens of Gab1+/- mice and fixed with
ethanol. To detect -galactosidase activity, the 35-µm sections
were stained with 0.1%
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)
in PBS containing 2 mM MgCl2, 5 mM
K3Fe(CN)6, and 5 mM
K4Fe(CN)6 at room
temperature. The marginal lining cells were detected by staining the
6-µm sections with anti-MAdCAM-1, biotinylated
F(ab')2 donkey anti-rat IgG, and a Vectastain
Elite ABC kit.
Analysis of Gab1 gene expression in the sorted B cell subsets by reverse transcription PCR
Spleen cells of wild-type mice were stained with FITC-anti-CD21, PE-anti-CD23, and Quantam Red-anti-CD45R Abs, and MZ B cells (CD45R+CD21++CD23-) and FO B cells (CD45R+CD21+CD23+) were sorted using a FACSVantage cell sorter (BD Biosciences). The purity of both the sorted MZ B cells and the FO B cells was 95%. RNA was isolated from sorted cells lysed with Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan) and reverse transcribed. RT-PCR was conducted as described previously (19). Primer sequences for Gab1 are 5'-GCGTGGAAGAGAAGGTGGTTTGTGT-3' and 5'-CGCGACTGAAGAAGCTTCCATCTGA-3'.
Immunization
To examine the TI responses, 8–9 wk after reconstitution, the chimeric mice were injected i.p. with 50 µg of 2,4,6-trinitrophenyl (TNP)-LPS (prepared in this laboratory) or 20 µg TNP-Ficoll (Biosearch Technologies, Novato, CA). Blood was taken through the retro-orbital plexus every 7 days. For the TD Ag responses, the mice were i.p. injected with 100 µg of DNP-keyhole limpet hemocyanin (KLH) (LSL, Tokyo, Japan) precipitated with 4 mg of alum (LSL) on day 0 and were boosted with 20 µg of DNP-KLH in saline on day 21.
B cell purification
Spleen cells were obtained from mice 8–9 wk after injection and
were treated with 0.165 M NH4Cl to lyse the
erythrocytes. B cells were purified by negative sorting using
anti-Thy1, anti-CD43, anti-CD11b, and anti-CD11c
Ab-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) and a
MiniMACS column (Miltenyi Biotec). The purity of the splenic B cell
preparation was verified by flow cytometry analysis and was typically
>95%. From the purified B cells, FO B and non-FO B cells were further
purified by positive and negative sorting using biotin-anti-CD23 Ab
(BD Pharmingen) and streptavidin-conjugated magnetic beads (Miltenyi
Biotec) and MiniMACS column (Miltenyi Biotec). Non-FO B cell
preparation consists of MZ B and newly formed B cells with a ratio of
1:1.
B cell proliferation assay
Purified splenic B cells (2 x
105/200 µl) were cultured in triplicate in RPMI
1640 supplemented with 10% FBS, 1 mM glutamine, 50 µM 2-ME,
and antibiotics. The F(ab')2 of goat
anti-mouse IgM (Jackson ImmunoResearch Laboratories),
LPS (Escherichia coli 0111:B4; Sigma-Aldrich),
anti--dextran (kindly provided by C. M. Snapper, Uniformed
Services University of the Health Sciences, Bethesda, MD), or
anti-CD40 Ab (HM40-3; BD PharMingen) was added to the culture
medium at various concentrations. Triplicate samples of cells were
pulsed with 0.5 µCi/well [3H]thymidine (NEN
Life Science Products, Boston, MA) for the final 6 h of a
72-h-long culture. The amount of [3H]thymidine
incorporation was determined using a micro beta scintillation counter
(Wallac, Gaithersburg, MD).
Ig production of B cells in vitro
Purified splenic B, FO B, and non-FO B cells (5 x
104/200 µl) were cultured in the presence or
absence of anti--dextran (3 ng/ml), anti--dextran plus
IL-5 (150 U/ml; a generous gift from K. Takatsu, University of Tokyo,
Tokyo, Japan), or anti--dextran plus IL-5 plus IL-4 (1000 U/ml;
Life Technologies, Rockville, MD). Supernatants from the cultures were
harvested at day 6 and Ab production was measured by ELISA.
Generation of viral supernatants and transduction of primary B lymphocytes
To construct the Gab1 mutant
Gab1F447/472/589, tyrosines 447, 472, and 589 of
human Gab1, corresponding to the YXXM motifs, were mutated to
phenylalanines by PCR-based mutagenesis using pcDNA3-hemagglutinin
(HA)-Gab1 (12, 17). The Gab1 mutant
Gab1F627/659 was constructed by replacing
tyrosines 627 and 659, corresponding to the YLDL and YVVV motifs, with
phenylalanines. The cDNA fragments of HA-tagged wild-type and mutant
Gab1 were excised from pcDNA3 and subcloned into the HindIII
site of pBluescript SK+ (Stratagene, La Jolla,
CA). Primers used for the mutagenesis are available on request. To
generate the internal ribosomal entry site (IRES)-enhanced green
fluorescent protein (EGFP) fragment, the EGFP cDNA from pC1EGFP
(Clontech Laboratories, Palo Alto, CA) was inserted into the
NcoI and BamHI sites of pBlKS-CITE (a gift
from H. Niwa, Osaka University). To construct the retroviral vectors,
the BglII-SalI fragment of IRES-EGFP was
subcloned into the BamHI and SalI sites of
pMX (a gift from T. Kitamura, University of Tokyo), then the
HA-Gab1 fragments from pBluescript SK+ were
inserted into the ClaI and EcoRI sites in
IRES-EGFP (located at the 5' end of the IRES sequence). To generate the
retroviruses, the 293T-based packaging cell line SBR432 (a
gift from G. Nolan, Stanford University, Stanford, CA) was transfected
with the retroviral vectors by a standard calcium phosphate
precipitation method. At 24 h after the transfection, the medium
was changed to RPMI 1640 containing 10% FBS and antibiotics. The
supernatants were harvested after additional 24- and 48-h incubations,
with fresh RPMI 1640 medium being added after the first harvest of
viral supernatant (26). Purified splenic B cells from
Gab1-/- FL chimeras were activated with
anti--dextran (3 ng/ml). At 24 h after stimulation, the
cells were infected by incubation with the viral supernatants in the
presence of 8 µg/ml polybrene (Sigma-Aldrich) and
anti--dextran (3 ng/ml). The infected B cells were stimulated
with anti--dextran (3 ng/ml), IL-4 (1000 U/ml), and IL-5 (150
U/ml) in 96-well plates at a concentration of 5 x
104 cells/well. After a 6-day incubation, the
supernatants were harvested and used to determine the production of
Igs. The efficiency of infection was verified by flow cytometry
analysis of the green fluorescent protein-positive cells and was
50% for each infection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gab1 is tyrosine-phosphorylated by the stimulation of
stem cell factor, IL-3, erythropoietin, and GM-CSF or by ligation of Ag
receptors, suggesting that Gab1 plays a role in hematopoiesis or immune
responses in vivo. Because Gab1-/- mice were
lethal to embryos, we made radiation chimeras with embryonic day
14.5 FL cells (FL chimeras) to explore the roles of the Gab1-mediated
signals in hematopoietic cell differentiation. Transplantation of
Gab1-/- FL cells could rescue the lethally
irradiated recipient mice, indicating that Gab1 is dispensable, at
least cell autonomously, for hematopoiesis. There were no differences
in the total cell numbers of the bone marrow, spleen, lymph nodes, and
peritoneum between Gab1-/- and
Gab1+/+ FL chimeras. Flow cytometry analyses of
Gab1-/- FL chimeras revealed that, in the bone
marrow, the frequencies of myeloid
(Gr-1+CD11b+, Gab1+/+: 26 ±
3%; Gab1-/-: 42 ± 7%) and erythroid
(TER-119+, Gab1+/+: 27 ± 3%;
Gab1-/-: 34 ± 2%) cells increased and those of
pre-B cells (CD45R+sIgM-),
but not pro-B and B cells, decreased
(Gab1+/+: 16.3 ± 2%;
Gab1-/-: 6.8 ± 2.9%) (Table I
and Fig. 1). Total cell numbers of thymus
decreased (Gab1+/+: 6.5 ± 1.9 x
106; Gab1-/-: 3.6 ±
1.6 x 106; p < 0.005) due
to the reduction of
CD4+CD8+ thymocytes
(Gab1+/+: 78.5 ± 3.7%;
Gab1-/-: 47.6 ± 32.3%). In the spleen,
cell numbers of CD4+ or
CD8+ T cells, B cell subsets defined by IgM/IgD
or CD21/CD23 expressions, and macrophages were normal. In the
peritoneum, mature macrophages
(F4/80+CD11b+) increased
(Gab1+/+: 28 ± 9%;
Gab1-/-: 44 ± 11%). These results
indicate that the Gab1 signaling pathway was not essential for the
generation of mature immunocompetent cells in the peripheral lymphoid
organs, although it played some regulatory roles in the early
development of erythroid, myeloid, and B lymphoid cells.
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Because the Gab1-/- FL chimeras had an
almost normal population of immunocompetent cells, we examined the Ab
production of the chimeras to further clarify the roles of Gab1 in the
immune regulation in vivo. The serum Ig levels were normal in the
Gab1-/- FL chimeras 9 wk after reconstitution
(data not shown). When the Gab1-/- FL chimeras
were immunized with TD Ag (DNP-KLH) and
TI-1 Ag (TNP-LPS), Ab responses were comparable to those of the
controls (Figs. 2 and 3A). In
contrast, their Ab productions in response to TI-2 Ag, TNP-Ficoll,
showed a higher production of Ag-specific IgM (136 ± 30 µg/ml
at day 7) and IgG1 (2.8 ± 0.7 µg/ml at day 14) than did their
Gab1+/+ counterparts (IgM: 88 ± 7 µg/ml
at day7; IgG1: 1.3 ± 0.3 µg/ml at day 14) (Fig. 3B),
indicating that Gab1 negatively regulates the TI-2 response in
vivo.
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-dextran
To elucidate the role for Gab1 in B cells, we examined the
proliferation of Gab1-/- B cells stimulated
with anti-IgM, LPS, and anti-CD40 (Fig. 4A). In each case, the
proliferation was similar for purified B cells from the spleens of
Gab1-/- and Gab1+/+ FL
chimeras. Anti--dextran, which is dextran conjugated with
anti-IgD mAb, provides stimuli similar to TI-2 Ags and is used as a
model system for studying immune response to TI-2 Ag in vitro
(27). First we examined the growth responses to
anti--dextran alone or in combination with IL-5 or IL-5 + IL-4
(Fig. 4B). There were no differences between
Gab1-/- and Gab1+/+ B
cells. To evaluate the role of Gab1 in B cell differentiation in vitro,
we stimulated B cells with anti--dextran, anti--dextran
plus IL-5, and anti--dextran plus IL-5 plus IL-4 and measured
the level of Abs in the supernatant. Only the stimulation with
anti--dextran plus IL-5 plus IL-4, which induces a class switch
to IgG1, caused the higher production of IgM (28 ± 4 µg/ml) and
IgG1 (7.6 ± 0.3 µg/ml) by Gab1-/- B
cells compared with Gab1+/+ B cells (IgM: 14
± 1 µg/ml; IgG1: 4.2 ± 0.1 µg/ml) (Fig. 5A). Although MZ B cells are
known to be responsible for TI-2 response, there is a possibility that
augmented Ab production is due to FO B, a major population in splenic B
cells. To eliminate this possibility, we examined the Ab
production of FO B and non-FO B fractions. The amounts of Abs produced
by Gab1-/- FO B and those produced by
Gab1+/+ FO B were similar, indicating that
Gab1-/- FO B is not responsible for the
enhanced Ab production in vitro. In contrast,
Gab1-/- non-FO B showed higher Ab production
(IgM: 27 ± 2 µg/ml; IgG1: 17.6 ± 1.5 µg/ml) than did
Gab1+/+ non-FO B (IgM: 22 ± 1 µg/ml;
IgG1: 9.7 ± 0.3 µg/ml) (Fig. 5B). Because non-FO B
fraction consists of mature MZ B cells and immature, newly formed B
cells that die by BCR ligation, Gab1-/- MZ B
cells are most likely to be responsible for the enhanced Ab production
in vitro. These results consistent with the in vivo data show that Gab1
is a negative regulator specific to the TI-2 response and further
suggest its cell-autonomous negative role in B cells.
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To confirm the negative regulatory roles for Gab1 in B cell
differentiation and to examine which molecular complex with Gab1 is
responsible for its negative effect, we made a series of retroviral
vectors expressing either wild-type or mutant Gab1, as illustrated in
Fig. 6A. Splenic B cells from
Gab1-/- FL chimeras were infected with the
retrovirus (the frequencies of infected cells were usually 50% and
there was no difference in the infection using each construct) and
cultured for 6 days in the presence of anti--dextran plus IL-5
plus IL-4. The elevated production of IgM and IgG1 was inhibited in the
cultured B cells that had been transfected with either wild-type Gab1
or Gab1F447/472/589, a mutant with a defect in
PI-3 kinase binding motifs. However, the expression of
Gab1F627/659, which has mutations at the SHP-2
binding motifs, did not rescue the inhibitory effect of Gab1 (Fig. 6B). These data indicate that Gab1 plays negative regulatory
roles in B cell differentiation through its SHP-2 binding motifs.
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Although the analyses in vitro suggested that Gab1 negatively
regulates the differentiation of MZ B cells, it is not clear why
targeting Gab1 resulted in abnormality selective to TI-2 Ag responses.
Because the frequency of
CD21++CD23- MZ B cells in
the spleen of Gab1-/- FL chimeras was normal (Fig. 1
and
Table I), we analyzed the structure of the spleen of the
Gab1-/- FL chimera by immunohistochemistry.
Dual staining with anti-IgM and anti-MAdCAM-1 Abs, which stain
B cells and MZ lining cells, respectively, showed similar localization
of these cells in Gab1+/+ and
Gab1-/- FL chimeras (Fig. 7A). Staining with the Abs,
MOMA-1 (for metallophilic macrophages), and ERTR-9 (for MZ
macrophages) showed normal localization of these cells as well (data
not shown). These results indicated that the structure around the MZ of
the Gab1-/- FL chimera was intact. Then we
examined the localization of the cells expressing Gab1 in the spleen.
The targeting vector for Gab1 was designed to include an IRES -geo
cassette, which enabled us to detect cells expressing the Gab1 gene by
X-gal staining (23). Staining of serial frozen sections
showed that Gab1-expressing cells localized to areas overlapping or
outside the MAdCAM-1-positive cells, but not in the FO area (Fig. 7B), suggesting that the expression of Gab1 is restricted to
the MZ B cells. To test whether MZ B cells express Gab1, we purified
the splenic B cell subsets of wild-type mice using a cell sorter and
examined their expression of the Gab1 gene. RT-PCR analysis clearly
demonstrated that MZ B cells, but not FO B cells, expressed Gab1 mRNA
(Fig. 7C). These results indicated that Gab1 is an adapter
molecule specifically expressed in MZ B cells that negatively regulates
TI-2 response in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the immunocompetent cells in the peripheral lymphoid organs of
the Gab1-/- FL chimera developed normally, we
were able to analyze the roles for Gab1 in the immune response in vivo.
Analysis of the humoral immune responses in the
Gab1-/- FL chimera revealed an enhanced
production of IgM and IgG1 in response to TI-2 Ag and normal Ig
production in response to TI-1 and TD Ag. We concluded that the
enhanced TI-2 response was primarily due to a loss of cell-autonomous
negative regulation by Gab1 in MZ B cells because 1) the
Gab1 gene was specifically expressed in the MZ B cells of
normal mice, 2) MZ B cell development and organization of the MZ were
normal in Gab1-/- FL chimeras, 3) a higher
production of IgM and IgG1 was also induced in vitro using purified
Gab1-/- non-FO B cells stimulated with a
combination of cytokines and anti--dextran that provides a
stimulus similar to that of TI-2 Ag, and 4) Ab production of
Gab1-/- B cells induced by cytokines plus
anti--dextran was inhibited by the retrovirus-mediated
expression of wild-type Gab1. Furthermore, we demonstrated that the
negative regulatory effect of Gab1 was mediated through its binding
sites for SHP-2 but not for PI-3 kinase, suggesting the involvement of
SHP-2 in the Gab1-mediated negative regulation. In contrast, Gab2
negatively regulates TCR-evoked activation of the IL-2 promoter in T
cells, which is mediated through PI-3 kinase (31). These
findings raise the possibility that Gab family proteins regulate B cell
and T cell functions in different ways.
MZ B cells comprise a minor subset of B cells that play an important
role in the first line of defense against bacterial infection by
producing Abs against TI Ags, such as components of the bacterial cell
wall (1, 2, 4, 5). MZ B cells localize to the MZ extending
outside of the marginal sinus, where microorganisms are carried by the
circulation (32). Defective development of MZ B cells
has been found in the gene targeting of CD19 (33),
Pyk-2 (11), NF-
Bp50 (34), and Aiolos
(35, 36). Because the targeting of Gab1 did not affect the
development of MZ B cells, Gab1 seems to be dispensable for MZ B cell
development. Several reports indicate that MZ B cells have unique
functional characteristics compared with FO B cells. MZ B cells show
higher growth responses and Ab production after LPS stimulation or BCR
ligation than do FO B cells (8, 9, 37). Such
hyperresponsiveness may be advantageous for the role of MZ B cells as a
front-line defender against bacterial infection (38).In
contrast, MZ B cells show poorer responses in anti-Ig-induced
survival and growth (10). Several negative regulators of
MZ B cells have been reported. Analysis of knockout mice of another
negative regulatory molecule, Src homology 2 domain-containing
inositol-5-phosphatase (SHIP), showed a phenotype partly similar to
that of the Gab1-/- FL chimera. SHIP-deficient
mice have lower numbers of pre-B cells in the bone marrow, higher
numbers of myeloid cells, and enhanced IgG1, IgG2a, and IgG3 Ab
responses to TI-2 Ags, but this enhancement is not cell autonomous
(39). These phenotypes are partly explained by the
increased expression of SHIP in MZ B cells (37). It is
interesting to speculate that there may be a link between the signaling
pathways of SHIP and Gab1 in B cells. Targeting of programmed death-1,
a member of the Ig superfamily that contains an immunoreceptor
tyrosine-based inhibitory motif in its cytoplasmic region, demonstrated
that a lack of negative regulation by programmed death-1 resulted in
higher TI-2 responses (40) and autoantibody-mediated
cardiomyopathy (41). The report indicated that impairment
of negative regulation of TI-2 response could result in the development
of an autoimmune disease (42). Thus, it is possible that
the negative regulation of a very narrow range of humoral immune
responses by Gab1 could also be important for preventing the
development of autoimmune disease.
Collectively, the present results indicate that Gab1 is a unique adapter molecule that is specifically expressed in MZ B cells and negatively regulates their differentiation in response to TI-2 Ags in vivo.
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Acknowledgments |
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Footnotes |
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2 Current address: Unit on Vertebrate Neural Development, Lab of Molecular Genetics, National Institute of Child Health and Human Development/National Institutes of Health, Building 6B, Room 3B-315, 9000 Rockville Pike, Bethesda, MD 20892.
3 Current address: Laboratory for Vertebrate Axis Formation, Institute of Physical and Chemical Research, Center for Developmental Biology, Kobe, Japan.
4 Address correspondence and reprint requests to Dr. Toshio Hirano, Yamada-oka Suita, Osaka 565-0871, Japan, Department of Molecular Oncology (C-7), Graduate School of Medicine, Osaka University, Osaka, Japan. E-mail address: hirano{at}molonc.med.osaka-u.ac.jp
5 Abbreviations used in this paper: TD, thymus dependent; TI, thymus independent; BCR, B cell Ag receptor; MZ, marginal zone; FO, follicular; Gab1, Grb2-associated binder-1; SHP-2, Src homology 2-containing tyrosine phosphatase-2; PI-3, phosphatidylinositol 3; FL, fetal liver; MAdCAM-1, mucosal addressin cell adhesion molecule-1; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; TNP, 2,4,6-trinitrophenyl; KLH, keyhole limpet hemocyanin; HA, hemagglutinin; IRES, internal ribosomal entry site; EGFP, enhanced green fluorescent protein; SHIP, Src homology 2 domain-containing inositol-5-phosphatase.
Received for publication December 31, 2001. Accepted for publication March 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is required for the development of marginal zone B lymphocytes. J. Exp. Med. 192:1175.This article has been cited by other articles:
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) and Gab1-/- (•) FL chimeras
(n = 5), respectively, are shown.
