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CUTTING EDGE |
2 in B Cell Development and Function1
*
Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan;
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
First Department of Pathology, Kansai Medical University, Moriguchi, Japan; and
§
Department of Biomolecular Sciences, Fukushima Medical College, Fukushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2, which is involved in
the activation of the phosphatidylinositol pathway. To assess the
importance of PLC-2 in murine lymphopoiesis, the PLC-2 gene was
inducibly ablated by using IFN-regulated Cre recombinase. Mice with a
neonatally induced loss of PLC-2 function displayed reduced numbers
of mature conventional B cells and peritoneal B1 cells and defective
responses in vitro to BCR stimulation and in vivo to immunization with
thymus-independent type II Ags. In contrast, T cell development and
TCR-mediated proliferation were normal. Taken together, PLC-2 is a
critical component of BCR signaling pathways and is required to promote
B cell development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2, tyrosine phosphorylation of
which is mediated by Syk and Btk (5, 6, 7). PLC-2
hydrolyzes phosphatidylinositol-4,5-bisphosphate
(PIP2) to form inositol-1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG), and this
catalytic activity is thought to be increased upon its tyrosine
phosphorylation (8). It is now well
established that these two products, IP3 and DAG,
mediate the calcium release from intracellular stores and activation of
protein kinase C (PKC), respectively (9, 10).
To elucidate the in vivo function of PLC-2, we undertook a
gene-targeting approach in the mouse. Because
PLC-1-/- mice result in embryonic lethality
at approximately embryonic day 9.0 (11), we were concerned
that PLC-2-/- mice might suffer a similar
fate. Thus, to circumvent this potential problem, we made use of an
IFN-induced Cre-recombinase transgene (MxCre) (12) to
delete an essential loxP-flanked portion of the PLC-2 gene. Analysis
of MxCre/PLC-2flox/flox mutant mice revealed a
similar, though not identical, phenotype to defects observed in mice
lacking Btk (13, 14, 15), supporting the previous model that
PLC-2 and Btk lie along the common BCR signaling pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2flox/flox mice
A targeting vector to generate
PLC-2flox/flox was designed so that the
PIP2 binding site in the PLC-2 X domain could
be deleted by expression of Cre protein. The
PstI-BamHI-digested 1.7-kb fragment of the
PLC-2 genomic DNA was inserted between two loxP sites for the vector
pKSTKNEOLOXP, which has HSV thymidine kinase and loxP-flanked pGK-neo.
Then, the PstI-PstI 7.9-kb fragment 5' upstream
and the 1.0-kb fragment 3' downstream of the exon containing the
PIP2 binding site were inserted (see Fig. 1
A).
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A).
Cre protein was transiently expressed in the targeted ES clones to
delete the loxP-flanked neo gene. Two types of clones,
PLC-2 flox and PLC-2
, were obtained (see Fig. 1A).
The PLC-2 flox ES clones were used to generate chimeric mice, which
successfully contributed to the germline transmission. Mice homozygous
for the loxP-flanked PLC-2 gene
(PLC-2flox/flox) were born at the expected
Mendelian ratios and presented no obvious abnormalities.
PLC-2flox/+ mice were mated with MxCre mice to
generate MxCre/PLC-2flox/+ mice. These mice
were further mated with PLC-2flox/flox mice to
generate MxCre/PLC-2flox/flox mice.
For analysis of genotypes (see Fig. 1B), 
0.5 µg of DNA
was subjected to 35 cycles of amplification with each cycle consisting
of 0.5 min at 94°C, 1 min at 64°C, and 1 min at 74°C followed by
an extension of 10 min at 74°C on a thermal cycler.
Ca2+ measurement and protein analysis
Splenic non-B cells were depleted by mAbs against CD4, CD8,
Gr-1, and CD11b in combination with magnetic beads conjugated with
sheep anti-rat IgG Ab (Dynabeads M-450; Dynal, Lake Success, NY).
The purity of the resultant splenic B cells was 85–90%. Splenic B
and T cells (1 x 106) were loaded with 3
µM fura-2/AM at 37°C for 45 min. Cells were washed twice and
stimulated with anti-IgM F(ab')2 Ab (The
Jackson Laboratory, Bar Harbor, ME) or anti-CD3 Ab (PharMingen, San
Diego, CA). Continuous monitoring of fluorescence from the cell
suspension was performed using Hitachi F-2000 fluorescence
spectrophotometer (Hitachi, Tokyo, Japan). Calibration and calculation
of Ca2+ levels were performed as described
(16).
Splenic B cells (2 x 107) were solubilized
and immunoprecipitated with anti-PLC-2 Ab against its C-terminal
epitope (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PLC-1
Ab (Santa Cruz Biotechnology), and anti-BLNK Ab as described
previously (5). Immunoprecipitates were separated by 7.5%
SDS-PAGE, blotted, and detected by the same Abs using the enhanced
chemiluminescence system (Amersham, Arlington Heights, IL).
Flow cytometric analysis
Cells were stained with optimal amounts of FITC-, PE-, and biotin-conjugated mAbs and analyzed by a FACScan (Becton Dickinson, Mountain View, CA). Biotin-conjugated mAbs were revealed with streptavidin-CyChrome (PharMingen). The following mAbs used in the flow cytometric analyses were purchased from PharMingen: anti-IgM (R6-60.2), anti-CD8 (53-6.72), anti-CD4 (GK1,5), anti-B220 (RA3-6B2), anti-CD5 (53-7.3), and anti-CD43 (S7). Anti-IgD (11-26) was obtained from Southern Biotechnology Associates (Birmingham, AL).
Immunization and assay for Ab-forming cells
Mice were immunized with 20 µg thymus-independent Ag trinitrophenyl (TNP)-Ficoll in PBS i.p. To determine the thymus-dependent responses, mice were immunized with 20 µg TNP-keyhole limpet hemocyanin (KLH) in a 1:1 homogenate with CFA (Difco, Detroit, MI), and, at day 21, a second immunization with 20 µg TNP-KLH was performed. Ab-forming cells were measured by the modified Jerne’s plaque-forming cell (PFC) assay using TNP-SRBC as described previously (17).
In vitro PFC responses were also conducted after the culture of spleen cells (4 x 106) with the same number of SRBC or 1 µg/ml of TNP-Ficoll for 5 days.
Proliferation assay
Splenic B cells (2 x 105) were
cultured with 2.5 µg/ml goat anti-mouse IgM
F(ab')2 (Tago Scientific, Burlingame, CA), 25
µg/ml LPS (Calbiochem, La Jolla, CA), 10 U/ml IL-4 (Genzyme,
Cambridge, MA), and anti-CD40 mAb (PharMingen), singly or in
combinations as indicated in Fig. 4C. B cells were
stimulated for 72 h and pulsed with 0.5 µCi of
[3H]thymidine for the last 16 h of the culturing
period. They were then harvested and counted using a Matrix 96
(Packard, Meriden, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 gene, we crossed two types of
mice: one is a MxCre mouse that bears the Cre transgene under the
control of type I IFN-inducible Mx promoter (12), and the
other is a mouse in which an exon encoding the
PIP2 binding site of the PLC-2 gene is flanked
by two loxP sites (PLC-2flox/flox) (Fig. 1A; see Materials and
Methods for a detailed description in making this mouse). To
assess the efficacy of the PLC-2 inactivation, newborn mice were
injected i.p. with the IFN-inducer poly(I-C) (300 µg) at 2-day
intervals after birth without knowledge of their genotype. Genotyping
was performed 21 days after birth. Genomic DNA was extracted from
various tissues and subjected to PCR analysis using three primers as
indicated in Fig. 1A. In the spleen, bone marrow, lymph
nodes, peritoneum, and thymus of
MxCre/PLC-2flox/flox mice, Cre-mediated
deletion resulted in the appearance of a novel 308-bp band
corresponding to the PLC-2 gene. Despite the appearance of the
PLC-2-specific band, a 365-bp band corresponding to the floxed
PLC-2 gene was still detectable in these cells, indicating that
Cre-mediated deletion did not occur completely at the DNA level (Fig. 1B). In contrast to the high deletion efficiency in the
spleen, bone marrow, lymph nodes, and thymus, this efficiency was
relatively low in the peritoneum. Western blot analysis of splenic B
cells also detected a small amount of residual wild-type PLC-2
protein in poly(I-C)-treated
MxCre/PLC-2flox/flox mice (Fig. 1C). Poly(I-C)-treated
MxCre/PLC-2flox/flox (hereafter
iPLC-2flox/flox) mice showed no gross
abnormalities compared with iPLC-2flox/+. The
expression level of PLC-1 in the
iPLC-2flox/flox splenic B cells was almost the
same as that in iPLC-2flox/+ (Fig. 1C).
T and B cell development can be assessed by flow cytometric analysis of
lymphocyte populations stained with Abs to various surface Ags.
Staining of thymocytes with Abs to CD4 and CD8 revealed that normal T
cell development occurred in both iPLC-2flox/+
and iPLC-2flox/flox (Fig. 2E). The absolute numbers of
thymocytes and splenic T cells of
iPLC-2flox/flox were comparable to
iPLC-2flox/+ (Table I). In contrast, the numbers of
B220+IgM+ mature B cells in
the spleen, bone marrow, and lymph nodes of the
iPLC-2flox/flox mice showed a 2- to 3-fold
reduction compared with those in control littermates (Fig. 2, A–C). Slight reduction of the population of
B220+CD43-IgM-
pre-B cells and concomitant increase of the
B220+CD43+IgM-
pro-B cells were observed in the bone marrow of the
iPLC-2flox/flox mice (Fig. 2A and
Table I). In both spleen and lymph nodes (Fig. 2, B and
C), the population of the mature B cells characterized as
IgMlowIgDhigh was reduced
in the iPLC-2flox/flox mice, whereas the
population of immature B cells
(IgMhighIgDlow) was
considerably increased. In addition, the number of the CD5-expressing
B1 B cells in the iPLC-2flox/flox mice,
usually found in the peritoneum, were 2-fold less than those of control
littermates (Fig. 2D).
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2 on the immune response was analyzed for both
T-independent (TNP-Ficoll) and T-dependent Ags (TNP-KLH). As shown in
Fig. 3A, in contrast to the
control littermates, the iPLC-2flox/flox mice
were found to have reduced anti-TNP IgM responses to the T
cell-independent Ag, when examined by PFC assays (17). In
the T-dependent responses, anti-TNP IgM responses were also
reduced, whereas anti-TNP IgG responses were comparable to those of
the control littermates. Consistent with these data in the
iPLC-2flox/flox mice, these mutant mice
exhibited reduced in vitro T-independent and -dependent IgM responses
(Fig. 3B).
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2flox/flox mice mobilized
Ca2+ to a much lower extent than control B cells
(Fig. 4, A and B).
In contrast, CD3-mediated Ca2+ mobilization from
the iPLC-2flox/flox splenic T cells was
essentially the same as that from the control T cells (Fig. 4A). The proliferative responses were also perturbed; the
iPLC-2flox/flox splenic B cells stimulated via
the BCR by treatment with anti-IgM or anti-IgM+IL-4 showed
about a 10-fold reduction in proliferative responses. Stimulation with
anti-CD40 Ab was also reduced in the mutant cells, while
LPS-dependent proliferation was indistinguishable between
iPLC-2flox/flox and
iPLC-2flox/+ (Fig. 4B). In contrast
to abnormalities in B cells, anti-CD3-induced T cell proliferative
responses in the iPLC-2flox/flox mice were the
same as those in the iPLC-2flox/+ mice (data
not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 in an inducible fashion. Consistent with the
predominant expression of PLC-1 in T lineage cells (8),
T cell number, development, and function were apparently normal in
iPLC-2flox/flox mice. Thus, as reported
previously, poly(I-C) treatment by itself appears to affect no gross
abnormalities of lymphocyte development and function (12, 18).
Flow cytometric analysis of iPLC-2flox/flox
mice exhibits defects in B cell development at the pre-B cell stage.
The transition of
B220+CD43- pre-B cells
from B220+CD43+ pro-B cells
in the bone marrow is perturbed, which, in turn, limits their
differentiation into
B220highIgM+ mature B cells
in the bone marrow. Moreover, more immature B cells are present in the
spleen of mutant mice. Hence, in the absence of PLC-2, the signals
from the pre-BCR and BCR is less efficient in promoting further
maturation stages. These developmental deficits in the
iPLC-2flox/flox mice are similar to those in
BLNK-deficient mice (19, 20, 21, 22), strengthening the previous
model that PLC-2 and BLNK are components of a common BCR signaling
pathway (23, 24). However, the more severe
developmental block from the
B220+CD43+ to
B220+CD43- stage observed
in the BLNK-/- mice may suggest the requirement
for additional downstream targets of BLNK in pre-BCR signals.
The iPLC-2flox/flox mice have a phenotype that
is also similar to that in mice lacking Btk (13, 14, 15). Both
mutant mice show reduced levels of mature B cells in the bone marrow
and periphery; a poor humoral response to T-independent Ags; and a poor
proliferative response upon BCR stimulation. Thus, these data strongly
suggest that the functional roles of Btk and PLC-2 lie along the
common signaling pathways initiated by BCR. In contrast to
Btk-/- mice, the incomplete blockade of B1 B
cell differentiation (Fig. 2D) could be accounted for by the
low efficacy of Cre-mediated deletion in the peritoneum (Fig. 1B) and/or the residual B1 B cells before inducible
targeting. Another marked difference between
iPLC-2flox/flox and
Btk-/- is the in vitro proliferative response
via LPS (Fig. 4C); in contrast to the reduced response in
Btk-/- mice, this responses is not
significantly affected in the iPLC-2flox/flox
mice. The straightforward explanation of these results is that both Btk
and PLC-2 are associated with BCR signaling, while the signal
through LPS uses Btk, but not PLC-2. Because a small amount of
PLC-2 still remains in the iPLC-2flox/flox
splenic B cells (Fig. 1C), we cannot exclude the alternative
possibility that this residual B cell fraction expressing PLC-2
could compensatorily proliferate in response to LPS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tomohiro Kurosaki, Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan.
3 Abbreviations used in this paper: BCR, B cell Ag receptor; PTK, protein tyrosine kinase; PLC, phospholipase C; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; PFC, plaque-forming cell; TNP, trinitrophenyl; KLH, keyhole limpet hemocyanin; iPLC-2, poly(I-C)-treated MxCre/PLC-2.
Received for publication April 10, 2000. Accepted for publication June 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. J. Exp. Med. 184:31.1 in mammalian growth and development. Proc. Natl. Acad. Sci. USA 94:2999.2 and Rac1-JNK in B cells. Immunity. 10:117.[Medline]
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)
and open triangles (
) represent exons and loxP sites,
respectively. The restriction endonuclease cleavage sites are
abbreviated as P (PstI) and B (BamHI).
B, PCR analysis of genomic DNA using primers a, b,
and c as indicated in A. A novel 308-bp band was
observed in iPLC-
, respectively.
B, In vitro PFC responses were analyzed 5 days after the
culture with TNP-Ficoll and SRBC; symbols are the same as
A.