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From the
Received for publication, April 5, 2002, and in revised form, July 22, 2002
SHPS-1 is a receptor-type glycoprotein that binds
and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and
thereby negatively modulates intracellular signaling initiated by
various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems
through its association with CD47 (integrin-associated protein) on
adjacent cells. Furthermore, recent studies with fibroblasts derived
from mice expressing an SHPS-1 mutant that lacks most of the
cytoplasmic region suggested that the intact protein contributes to
cytoskeletal function. Mice homozygous for this SHPS-1 mutation have
now been shown to manifest thrombocytopenia. These animals did not
exhibit a defect in megakaryocytopoiesis or in platelet production.
However, platelets were cleared from the bloodstream more rapidly in
the mutant mice than in wild-type animals. Furthermore, peritoneal
macrophages from the mutant mice phagocytosed red blood cells more
effectively than did those from wild-type mice; in addition, they
exhibited an increase both in the rate of cell spreading and in the
formation of filopodia-like structures at the cell periphery. These
results indicate that SHPS-1 both contributes to the survival of
circulating platelets and down-regulates the macrophage phagocytic response.
SHPS-1 is a transmembrane glycoprotein that is abundant in neural
and myeloid tissues (1-6). This molecule is also known as SIRP Tyrosine phosphorylation of SHPS-1 is induced by soluble growth factors
(1, 7, 14, 15), integrin-mediated cell adhesion (16-18), or
cross-linking of Fc Recent studies have suggested that SHPS-1, through its association with
CD47, contributes to cellular functions that depend on intercellular
communication, including T cell activation (13), T cell arrest on
inflammatory vascular endothelium (22), B cell aggregation (23),
macrophage multinucleation (24), and phagocytosis of red blood cells
(RBCs)1 by splenic
macrophages (25, 26). SHPS-1-CD47 interaction also promotes the
adhesion of cerebellar neurons (12) and modifies synaptic activity in
the retina (27). Thus, SHPS-1 appears to interact with CD47 on adjacent
cells and thereby regulates various cellular responses in the neural
and immune systems. The biological consequences of this interaction
in vivo, however, remain to be clarified.
We recently generated mice that lack most of the cytoplasmic region of
SHPS-1. Characterization of immortalized fibroblasts from these mice
revealed important roles for SHPS-1 in both integrin-mediated cytoskeletal reorganization and the down-regulation of growth factor-induced activation of mitogen-activated protein kinase cascades
(28). To elucidate further the physiological roles of SHPS-1, we have
now characterized the phenotype of the SHPS-1 mutant mice. The mutant
animals were found to exhibit thrombocytopenia, which results from an
increased rate of clearance of circulating platelets. Furthermore,
peritoneal macrophages (PEMs) from these mice exhibit an enhanced
phagocytic response.
Animals--
The generation of mutant mice that lack most of the
cytoplasmic region of SHPS-1 has been described (28). The mice were bred and maintained by brother-sister mating on a mixed C57BL/6 x 129Sv
genetic background. Genotyping of the mice was performed by polymerase
chain reaction analysis as described (28). Mice were housed under
pathogen-free conditions and handled in accordance with the animal care
guidelines of Kobe University.
Immunoprecipitation and Immunoblot Analysis--
Mouse tissues
were homogenized on ice in lysis buffer (20 mM Tris-HCl (pH
7.6), 140 mM NaCl, 1 mM EDTA, 1% Nonidet P-40)
containing 5 mM NaF, 1 mM phenylmethylsulfonyl
fluoride, aprotinin (10 µg/ml), and 1 mM sodium vanadate.
The lysates were centrifuged at 10,000 × g for 15 min
at 4 °C, and the resulting supernatants were subjected to
immunoprecipitation and immunoblot analysis as described (28). A rat
monoclonal antibody (mAb) to p84 (SHPS-1) was kindly provided by C. Lagenaur; rabbit polyclonal antibodies to SHP-2 or to SHP-1 were
described previously (1); and normal rat IgG were obtained from Santa
Cruz Biotechnology.
Tissue Histology and Peripheral Blood Counts--
Tissues were
fixed in 3.7% formaldehyde in phosphate-buffered saline, embedded in
paraffin, sectioned, and stained with Mayer's hematoxylin-eosin.
Peripheral blood samples were obtained from the retro-orbital plexus
with 75-mm heparinized capillary tubes (Funakoshi, Tokyo, Japan).
Complete blood cell counts were performed with the Sysmex automatic
microcell counter F-800 (Toa Medical Electronics, Kobe, Japan). To
determine the percentage of circulating leukocyte subsets, we stained
blood smears for differential counts with May-Grünwald Giemsa
solution. At least 200 randomly chosen leukocytes were classified. For
determination of platelet turnover in myelosuppressed animals, female
mice (10-12 weeks of age) were injected intravenously with
5-fluorouracil (5-FU) at a dose of 150 mg/kilogram of body mass, and
complete blood counts were performed every 5 days.
Determination of the Number and Ploidy Distribution of
Megakaryocytes--
Marrow cells obtained from femoral bone were
plated at a density of 1.0 × 105 cells per well in
96-well plates, fixed in 100 µl of 100 mM sodium phosphate buffer (pH 6.0) containing 2.5% glutaraldehyde (Wako, Osaka,
Japan), and stained with acetylcholinesterase (AChE). The number of
megakaryocytes, determined as AChE-positive cells, was counted with an
inverted light microscope. The ploidy distribution of megakaryocytes in
bone marrow was analyzed by flow cytometry as described (29).
Colony-forming Unit-Megakaryocyte (CFU-MK) Assay--
The
CFU-MK assay was performed essentially as described (30). In brief,
bone marrow or spleen cells (3.0 × 105 cells per
35-mm culture dish) were cultured under a humidified atmosphere
containing 5% CO2 at 37 °C in Iscove's modified
Dulbecco's medium supplemented with pegylated recombinant human
megakaryocyte growth and development factor (PEG-rHuMGDF; 100 ng/ml),
0.3% Noble agar (Difco), 10% fetal bovine serum (Hyclone, Logan, UT),
2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol (Merck, Darmstadt, Germany). After 7 days of culture, agar disks were removed from the culture dishes,
placed onto glass slides, and stained with AChE. CFU-MK-derived
colonies were defined as colonies with at least three AChE-positive cells.
In Vivo Turnover of Biotinylated Platelets--
Platelet
turnover in vivo was measured as described (31) with minor
modifications. Female mice (9 weeks of age) were injected intravenously
with 1.5 mg of sulfo-NHS-LC-biotin (Pierce). Blood samples subsequently
obtained were fixed for 30 min with modified Tyrode's-Hepes buffer (10 mM Hepes-NaOH (pH 7.4), 129 mM NaCl, 8.9 mM NaHCO3, 2.8 mM KCl, 0.8 mM KH2PO4, 0.8 mM
MgCl2, 5.6 mM glucose) containing 2%
formaldehyde, washed with modified Tyrode's-Hepes buffer containing
0.35% bovine serum albumin (Sigma), and incubated for 20 min at room
temperature with a fluorescein isothiocyanate-conjugated hamster mAb to
mouse platelets (2 µg/ml) (Seikagaku, Tokyo, Japan). The cells were
then washed before incubation for 20 min with
streptavidin-phycoerythrin (6.25 µg/ml) (Pharmingen, San Diego, CA).
Biotinylated platelets were detected by phycoerythrin fluorescence with
a FACSort instrument (BD Biosciences) after gating for both
fluorescein isothiocyanate positivity and the characteristic light
scatter of these cells.
Phagocytosis Assays--
PEMs were prepared essentially as
described (32). In brief, the peritoneum was flushed with ice-cold
phosphate-buffered saline containing 0.2% bovine serum albumin 4 days
after intraperitoneal injection of mice with 3 ml of 3% thioglycolate
broth (Nissui, Japan). The exuded cells were centrifuged at
400 × g for 5 min at 4 °C, washed with ice-cold
Dulbecco's modified Eagle's medium, and then resuspended in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Cells were transferred at a density of 1.0 × 106 cells/ml to glass coverslips coated with fibronectin
(10 µg/ml) (Sigma) that had been placed in 35-mm culture dishes.
After incubation for 24 h at 37 °C in a humidified incubator
containing 5% CO2, nonadherent cells, which include
neutrophils, B cells, and T cells, were washed away. RBCs from
wild-type donor mice were labeled with a PKH26 red fluorescent cell
linker kit (Sigma) and then overlaid at a density of 1.0 × 108 cells per dish on the adherent cells, 99% of which
were identified as macrophages (32). After incubation for 2 h, the
cells were washed with phosphate-buffered saline and then incubated for
5 min at room temperature with hemolysis buffer (154 mM
NH4Cl (pH 7.3), 10 mM KHCO3, 0.1 mM EDTA) to remove attached, but not phagocytosed, RBCs.
They were then fixed with 3.7% formaldehyde in phosphate-buffered saline, after which phagocytosed RBCs were examined with a
laser-scanning confocal microscope (Bio-Rad, model MRC-1024) and random
fields were photographed. Clearance of labeled RBCs in vivo
was determined essentially as described (25).
Cell Spreading Assay--
Macrophages were prepared from the
peritoneum of mice that had not been injected with thioglycolate, and
were transferred at a density of 2.5 × 105 cells/ml
to glass coverslips that had been coated with fibronectin (10 µg/ml)
and placed in 35-mm culture dishes. After incubation in serum-free
Dulbecco's modified Eagle's medium for 0.5-4 h at 37 °C under a
humidified atmosphere containing 5% CO2, the cells were
fixed, stained with rhodamine-labeled phalloidin (Sigma), and examined
by confocal microscopy as described above.
Statistical Analysis--
Data are presented as mean ± S.E. The significance of differences between independent means was
assessed by Student's t test. A p value of
<0.05 was considered statistically significant.
Histological Changes in the Spleen of SHPS-1 Mutant Mice--
We
previously generated mutant mice that lack most of the cytoplasmic
region of SHPS-1 (28). Crossing of mice heterozygous for this mutation
revealed that, among the resulting 44 offspring, the ratio of genotypes
was consistent with that predicted by Mendelian inheritance (wild-type,
1.73 ± 0.22; heterozygous, 3.34 ± 0.28; homozygous mutant,
1.91 ± 0.23; these values are the number of mice per litter).
Mice homozygous for the SHPS-1 mutation appeared healthy and remained
viable for >18 months, although their mean body mass was reduced by
~10% compared with that of wild-type littermate controls. Expression
of SHPS-1 was examined in lysates of the brain and PEMs by
immunoprecipitation and immunoblot analysis with a mAb to SHPS-1 that
recognizes the extracellular region of the protein and thus reacts with
both the wild-type and mutant forms. Only the truncated form of SHPS-1
was detected in both the brain (Fig.
1A) and PEMs (Fig.
1B) of the homozygous mutant mice, although the abundance of
this protein was markedly reduced compared with that of the full-length
protein in wild-type animals. Unlike wild-type SHPS-1, the mutant
neither underwent tyrosine phosphorylation nor associated with SHP-1 in
response to exposure of PEMs to pervanadate, which greatly increases
the extent of tyrosine phosphorylation of cellular proteins (Fig.
1B; data not shown). These results indicate that the
truncated SHPS-1 is unable to recruit and activate SHP-1, and are
consistent with our previous observations with embryonic fibroblasts
from these mice (28).
The tissue histology of the homozygous mutant mice and wild-type
littermate controls was examined at 3 weeks, 7 weeks, 11 weeks, and 1 year of age. No marked differences were apparent in most tissues,
including the brain, heart, lung, kidney, thymus, and bone marrow,
between the two types of mice, although the liver of the mutant animals
exhibited a markedly increased fat content (data not shown). However,
at 11 weeks of age, a large number of megakaryocytes were observed in
the spleen of the homozygous mutant mice (Fig.
2, right panels) but not in
that of wild-type animals (Fig. 2, left panels). In the
spleen of wild-type mice, megakaryocytes were apparent at 3 weeks of
age, began to disappear by 7 weeks, and were no longer detectable at 11 weeks after birth (data not shown). At 1 year of age, megakaryocytes
were absent from the spleen of both types of mice (data not shown). The
amount of white pulp in the spleen appeared smaller in the mutant mice than in wild-type controls (Fig. 2, upper panels). However,
flow cytometric analysis of spleen cells did not reveal a substantial decrease in the proportion of B220-positive B lymphocytes, which are a
major component of white pulp, in the mutant mice (wild-type, 45.2%;
homozygous mutant, 54.4%). In contrast, the proportion of CD4- and
CD8-positive T lymphocytes in the mutant mice was significantly smaller
than that in wild-type mice (15.8 versus 22.1%,
respectively, for CD4-positive cells; 9.9 versus 14.2%, respectively, for CD8-positive cells).
Thrombocytopenia in SHPS-1 Mutant Mice--
We next compared
peripheral blood cell counts among the homozygous mutant mice,
heterozygous mutant mice, and their wild-type littermates at various
ages. No significant differences in the numbers of circulating RBCs or
reticulocytes or in the percentages of leukocyte subsets were apparent
among the three types of mice (Table I;
data not shown). In addition, mean corpuscular volume, mean corpuscular
hemoglobin concentration, and mean platelet cell volume were similar in
these animals. In contrast, the circulating platelet count of the
homozygous SHPS-1 mutant mice was significantly reduced (by ~25%)
compared with that of gender- and age-matched wild-type littermates
(Table I); the number of platelets in female mice was 15-20% smaller
than that in males regardless of genotype. The difference in platelet
number between the homozygous mutant and wild-type mice was detected as
early as 4 weeks after birth and remained apparent at 13 weeks of age
(Fig. 3). Thus, targeted deletion of the
cytoplasmic region of SHPS-1 in mice resulted in moderate
thrombocytopenia. The platelet count of the heterozygous mutant mice
was variably reduced (by ~20%) compared with wild-type littermates;
however, this reduction was not statistically significant (data not
shown). We therefore utilized the homozygous mutant mice for the
subsequent analyses with wild-type mice as controls.
Megakaryocytopoiesis in SHPS-1 Mutant Mice--
The
thrombocytopenia apparent in SHPS-1 mutant mice might have resulted
either from reduced proliferation or maturation of the
megakaryocyte-platelet lineage or from an increased rate of clearance
of circulating platelets. To test the former possibility, we counted
the number of megakaryocytes in femoral bone marrow and examined their
ploidy distribution by flow cytometry. Neither of these parameters
differed significantly between wild-type and homozygous mutant mice
(Table II). Given that platelets contain SHPS-1 mRNA (data not shown), megakaryocytes might also express the
SHPS-1; we therefore examined the ability of bone marrow or spleen
cells from the mutant mice to undergo megakaryocytopoiesis with a
CFU-MK assay in vitro. The CFU-MK from either the bone marrow or spleen of SHPS-1 mutant mice yielded statistically similar numbers of colonies in response to incubation with PEG-rHuMGDF as did
those of wild-type mice (Table II). The serum concentrations of
thrombopoietin were also similar in wild-type and mutant mice (320 ± 17.4 versus 337 ± 19.5 pg/ml, respectively),
indicating that the generation of endogenous thrombopoietin was not
affected by the SHPS-1 mutation. Together, these results demonstrate
that neither the generation of megakaryocyte progenitors nor the
maturation of these cells is affected by the absence of the cytoplasmic
region of SHPS-1, and that suppression of megakaryocytopoiesis
therefore is not responsible for the thrombocytopenia of the mutant
animals.
Shortened Life Span of Circulating Platelets in SHPS-1 Mutant
Mice--
We next examined the turnover rate of circulating platelets
in the homozygous mutant and wild-type mice after myelosuppression with
5-FU. Administration of a single high dose (150 mg/kg) of 5-FU resulted
in a decrease in the platelet count of peripheral blood at 5 days and
subsequent platelet recovery by 10 days in both types of mice (Fig.
4). Although the initial platelet number was smaller in the mutant mice than in the wild-type animals, the
increase in the number of circulating platelets apparent from 5 to 10 days after 5-FU administration did not differ significantly between the
two genotypes. At 15 days after 5-FU injection, while the platelet
count of wild-type mice remained unchanged compared with the value at
10 days, that of the mutant mice had decreased by ~23%. The number
of circulating platelets in the mutant mice remained smaller than that
in wild-type mice at all times after 5-FU treatment, with the most
marked difference being observed at 15 days (117 (±41.2) × 104 versus 182 (±13.7) × 104
platelets/µl, respectively). These results indicate that platelet production is not affected in SHPS-1 mutant mice, but that the life
span of circulating platelets is shortened by the lack of the SHPS-1
cytoplasmic region.
To characterize further the life span of circulating platelets, we
biotinylated platelets in vivo and then counted the number of labeled cells remaining in peripheral blood at various times. The
time course of the reduction in the percentage of biotinylated platelets in peripheral blood revealed that the life span of platelets in the mutant mice was significantly shorter than the corresponding value for wild-type mice (Fig. 5).
Enhanced Phagocytic Activity of PEMs from SHPS-1 Mutant
Mice--
SHPS-1 expressed in splenic macrophages contributes to the
survival of RBCs by negatively regulating phagocytosis (25, 26). This
effect requires the interaction of SHPS-1 with CD47 expressed on the
surface of RBCs and appears to be mediated by SHP-1 (19, 25, 26). Given
that the truncated form of SHPS-1 lacks the immunoreceptor
tyrosine-based inhibitory motifs that bind SHP-1 (Fig. 1B;
Ref. 28), it might be expected to have lost the ability to suppress
phagocytosis by macrophages. We tested this hypothesis with an ex
vivo phagocytosis assay in which thioglycolate-elicited PEMs were
incubated with PKH26-labeled RBCs from wild-type donor mice; RBCs were
used instead of platelets because of technical limitations. Whereas
PEMs derived from wild-type mice phagocytosed few RBCs, a substantial
proportion of PEMs from the homozygous mutant mice exhibited
phagocytosis of RBCs (Fig. 6). The
phagocytic activity of PEMs was thus markedly enhanced as a result of
the absence of the cytoplasmic region of SHPS-1.
Morphological Changes in PEMs from SHPS-1 Mutant
Mice--
Proteins of the Rho family of GTPases, including Rac1 and
Cdc42, regulate phagocytosis by macrophages (33, 34). These proteins
might thus also play a role in the enhanced phagocytosis apparent in
macrophages from SHPS-1 mutant mice. Because biochemical assays failed
to detect a substantial amount of activated Rac1 and Cdc42 in PEMs
(data not shown), we examined macrophage spreading on the extracellular
matrix as a more sensitive indicator for the activities of these
GTPases. PEMs from the mutant mice spread more extensively than did
wild-type PEMs on fibronectin-coated coverslips for up to 1 h
after attachment; they also exhibited a marked increase in the number
of filopodia-like structures at the cell periphery (Fig.
7, A-D), indicative of
enhanced activities of Rac1 and Cdc42. After 4 h, however, no
marked difference in the extent of spreading was apparent between
macrophages from the two types of mice, although PEMs from the mutant
mice appeared less polarized than did those from wild-type animals
(Fig. 7, E and F).
We recently generated mutant mice that express a truncated form of
SHPS-1 lacking most of the cytoplasmic region of this protein. We have
now characterized these mutant mice and demonstrated that the absence
of the cytoplasmic region of SHPS-1 results in thrombocytopenia. This
condition is attributable to an increased metabolism of circulating platelets, with platelet production being unaffected. Our results have
also revealed an important role for the cytoplasmic region of SHPS-1 in
down-regulation of the macrophage phagocytic response. However, we need
to be cautious in interpreting these results given that the mutant mice
would possess functional extracellular regions of SHPS-1 and thus
represent a hypomorphic or neomorphic mutation as opposed to a
true loss-of-function.
The mice homozygous for the SHPS-1 mutation exhibited mild
thrombocytopenia. Both the generation of megakaryocyte progenitors in
bone marrow and the ability of these progenitors to grow and differentiate into mature polyploid megakaryocytes in this tissue appeared unaffected in the mutant mice. Furthermore, these animals exhibited no significant defect in the recovery of platelet counts after transient myelosuppression. In contrast, platelets were cleared
from the bloodstream more rapidly in the mutant mice than in wild-type
mice. Thus, the thrombocytopenia of the SHPS-1 mutant mice appears to
be attributable, at least in part, to an increased rate of consumption
of circulating platelets, and not to a defect in the production of
these cells.
At least two possible mechanisms could account for the
accelerated clearance of circulating platelets in SHPS-1 mutant mice. Reverse transcription-polymerase chain reaction analysis has revealed the presence of SHPS-1 mRNA, albeit in small amounts, in platelets (data not shown). The deletion of the cytoplasmic region of SHPS-1 expressed in platelets might therefore increase the fragility of these
cells and thereby render them more susceptible to destruction, although
platelet functions such as aggregation in response to collagen or to
ADP are not affected by the SHPS-1
mutation.2 The second, and
more likely, possibility is that the enhanced rate of platelet
clearance in the mutant mice results from expression of the mutant
SHPS-1 protein in other cell types. The sequestration of aged platelets
is mediated by phagocytosis within the reticuloendothelial system,
especially by splenic and hepatic macrophages (35). In addition,
thrombocytopenia with abnormal platelet sequestration has been observed
in various diseases including sepsis syndrome (36) and chronic immune
thrombocytopenic purpura (37). Administration of cytokines such as
granulocyte-macrophage colony stimulating factor and macrophage colony
stimulating factor also results in either the clearance of circulating
platelets or the stimulation of megakaryocytopoiesis, presumably by
activating the monocyte-macrophage system (38-40). Given the fact that
SHPS-1 is abundant in monocytes-macrophages and that this protein
negatively regulates cytokine synthesis (21), enhanced phagocytosis or
cytokine production by monocytes-macrophages is likely responsible for
the thrombocytopenia in SHPS-1 mutant mice.
In splenic macrophages, SHPS-1 generates an intracellular signal that
inhibits phagocytosis of RBCs (25, 26) in a manner that depends both on
the binding of its extracellular IgV-like domain to CD47 expressed by
the RBCs and on the recruitment of SHP-1 to the immunoreceptor
tyrosine-based inhibitory motifs of SHPS-1 (19, 25, 26). CD47
engagement of macrophage SHPS-1 has also been shown to prevent the
clearance of lymphohematopoietic cells (41). Given that the truncated
SHPS-1 expressed in macrophages of the homozygous mutant mice is not
able to bind SHP-1, these cells would not be expected to transmit the
inhibitory signal initiated by the interaction with CD47. CD47 is
abundant in platelets (42, 43). If the functional consequences of the
interaction of macrophage SHPS-1 with CD47 expressed on platelets are
similar to those of the interaction of macrophage SHPS-1 with CD47 on RBCs or lymphocytes, then platelets would be expected to be
phagocytosed by splenic macrophages more effectively in SHPS-1 mutant mice.
In agreement with the lack of a signal that prevents phagocytosis, the
number of CD4- and CD8-positive T lymphocytes in spleen was reduced in
SHPS-1 mutant mice. In these mice, however, we failed to detect any
sign of accelerated RBC clearance such as anemia and reticulocytosis.
Also, no marked reduction in the survival ratio of transfused RBCs was
observed in the mutant mice.3
These results appear inconsistent with what might have been predicted from the previous reports (25, 26). This apparent discrepancy presumably results from the difference in the experimental approach adopted; whereas we mutated SHPS-1 to interrupt the inhibitory signal
by CD47, Oldenborg et al. (25, 26) followed the fate of RBCs
deficient in CD47. Redundant pathways may also explain the difference
in phenotype between the SHPS-1 and CD47 mutant mice. Nevertheless, our
ex vivo analyses have demonstrated that PEMs from SHPS-1
mutant mice phagocytosed RBCs more effectively than did wild-type
cells, supporting the notion that the cytoplasmic region of SHPS-1
negatively regulates the macrophage phagocytic response.
PEMs from SHPS-1 mutant mice are reminiscent of macrophages (44),
lymphocytes (45), or neutrophils (46) from SHP-1-deficient mice, in
that they manifest an increase in the actin polymerization. Although
this similarity suggests that the inability of the truncated SHPS-1 to
recruit SHP-1 might partly account for the phenotype of the mutant
PEMs, the mechanism by which SHPS-1 truncation stimulates phagocytosis
remains unclear. Rac1 and Cdc42 positively regulate Fc
receptor-mediated phagocytosis by macrophages (33, 34), indicating that
up-regulation of the small GTPases might play a causal role. We could
not, however, provide direct evidence for this hypothesis presumably
because of the low stoichiometry and transient nature of the activation
of these GTPases in PEMs. This idea nevertheless appears consistent
with the observation that cell spreading and filopodia formation, which
correlate well with the activation of Rac1 and Cdc42, were enhanced in
SHPS-1-deficient macrophages. Phagocytosis and Rac-mediated
cytoskeletal reorganization also require the participation of another
low molecular weight GTPase, ARF6 (47, 48). Thus, it is possible that
Rac1 and/or Cdc42 activity is unaffected by the SHPS-1 mutation and
that it may instead be regulating ARF6.
The number of splenic megakaryocytes in SHPS-1 mutant mice ~11 weeks
after birth was markedly increased compared with that in wild-type
controls, suggesting that megakaryocytopoiesis in the spleen might be
transiently activated as a consequence of the deletion of the SHPS-1
cytoplasmic region. This observation appears inconsistent with our
in vitro data showing that the growth rate of spleen-derived
megakaryocyte progenitors from SHPS-1 mutant mice is increased only
slightly. A likely explanation is that the relative contribution of
SHPS-1 to megakaryocytopoiesis is greater in vivo than
in vitro. Under the former conditions, megakaryocytopoiesis might be supported more extensively by cytokines secreted by leukocytes into the microenvironment surrounding megakaryocyte progenitors. Given
that SHPS-1 inhibits cytokine synthesis by leukocytes (21), its
cytoplasmic truncation might reverse this inhibition and thus facilitate megakaryocytopoiesis in the spleen. In conclusion, our results demonstrate that SHPS-1 inhibits the clearance of circulating platelets as well as down-regulates the macrophage phagocytic response, thereby possibly contributing to hemostasis and
host defense.
We thank C. Lagenaur for providing the rat
mAb to p84 (SHPS-1), as well as H. Miyazaki and T. Kuwaki for critical
discussions throughout the study.
*
This work was supported by a grant-in-aid for cancer
research and a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by a grant-in-aid from the Research for the Future Program of the Japan Society for the Promotion of Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Division of Diabetes,
Digestive, and Kidney Diseases, Dept. of Clinical Molecular Medicine,
Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: 81-78-382-5861; Fax:
81-78-382-2080; E-mail: noguchi@med.kobe-u.ac.jp.
Published, JBC Papers in Press, August 7, 2002, DOI 10.1074/jbc.M203287200
2
T. Hagiwara, unpublished data.
3
T. Yamao, unpublished data.
The abbreviations used are:
RBCs, red blood
cells;
PEMs, peritoneal macrophages;
mAb, monoclonal antibody;
5-FU, 5-fluorouracil;
AChE, acetylcholinesterase;
CFU-MK, colony-forming
unit-megakaryocyte;
PEG-rHuMGDF, pegylated recombinant human
megakaryocyte growth and development factor.
Negative Regulation of Platelet Clearance and of the Macrophage
Phagocytic Response by the Transmembrane Glycoprotein SHPS-1*
,§,
,,,,,,
Division of Diabetes, Digestive, and Kidney
Diseases, Department of Clinical Molecular Medicine, and
§§ Division of Surgical Pathology, Department of
Biomedical Informatics, Kobe University Graduate School of Medicine,
7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan, the
¶ Department of Host Defense, Research Institute for Microbial
Diseases, Osaka University, Suita 565-0871, Japan,
Pharmaceutical Development Laboratories and
** Pharmaceutical Research Laboratories, Kirin Brewery Co.,
Ltd., Takasaki 370-1295, Japan, and the
Biosignal Research Center, Institute for
Molecular and Cellular Regulation, Gunma University,
Maebashi 371-8512, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
(7), BIT (8), MFR (9), and p84 neural adhesion molecule (10). The
cytoplasmic region of SHPS-1 contains two immunoreceptor tyrosine-based
inhibitory motifs, which recruit and activate the Src homology 2 domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2 in a
phosphorylation-dependent manner (1, 7, 11). The putative
extracellular region of this protein comprises three immunoglobulin
(Ig)-like domains, of which the most amino-terminal, IgV-like domain
associates with the ligand CD47, also known as integrin-associated
protein (6, 12, 13).
receptors (19). Overexpression of SHPS-1
inhibits the activation of extracellular signal-regulated kinases
induced by growth factors such as insulin, epidermal growth factor, and
platelet-derived growth factor (7); it also inhibits promotion of the
motility and survival of glioblastoma cells by epidermal growth factor
(20). Furthermore, SHPS-1 inhibits IgE-induced mediator secretion and
cytokine synthesis by mast cells (21). These observations suggest that
SHPS-1, presumably by recruiting SHP-1 or SHP-2, negatively modulates a
wide range of cellular activation signals initiated by tyrosine
kinase-coupled receptors. However, the physiological significance of
these observations remains unclear.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
Expression of a truncated form of SHPS-1 in
the brain and macrophages of mutant mice. A, brain lysates
derived from wild-type (+/+) and homozygous SHPS-1 mutant ( 
/) mice
were subjected to immunoblot analysis either with a mAb to SHPS-1
(upper panel) or with polyclonal antibodies to SHP-2
(lower panel). The positions of wild-type and mutant
(
SHPS-1) SHPS-1, SHP-2, and molecular size standards (in
kilodaltons) are indicated. B, thioglycolate-elicited PEMs
were incubated for 5 min at room temperature in the absence () or
presence (+) of 100 µM pervanadate
(NaVO4), after which cell lysates were subjected
to immunoprecipitation (IP) with a mAb to SHPS-1
(SHPS-1) or with normal rat IgG (NRG). The
resulting precipitates were subjected to immunoblot analysis either
with the mAb to SHPS-1 (upper panel) or with polyclonal
antibodies to SHP-1 (lower panel). Data in both panels are
representative of three independent experiments.
View larger version (168K):
[in a new window]
Fig. 2.
Histological changes in the spleen of SHPS-1
mutant mice. The spleen was removed from 11-week-old
wild-type (left panels) or homozygous SHPS-1 mutant
(right panels) mice, fixed, and stained with Mayer's
hematoxylin-eosin. Sections were examined with a light microscope
equipped with phase-contrast optics, and megakaryocytes are indicated
by arrowheads. Data are representative of three independent
experiments. Original magnification: ×100 (upper panels) or
×400 (lower panels).
Peripheral blood cell counts in SHPS-1 mutant (/) and wild-type
(+/+) mice at 4 weeks of age
View larger version (14K):
[in a new window]
Fig. 3.
Circulating platelet counts in SHPS-1 mutant
and wild-type mice at 4-13 weeks of age. The number of platelets
in peripheral blood was determined for male and female wild-type and
homozygous mutant mice at the indicated ages. Data are means of
triplicate determinations for five animals per group. The platelet
count differed significantly (p < 0.05) between
wild-type and mutant mice of each gender at all ages.
Megakaryocytopoiesis in SHPS-1 mutant (/) and wild-type (+/+) mice
at 10 to 12 weeks of age
View larger version (14K):
[in a new window]
Fig. 4.
Platelet turnover in SHPS-1 mutant and
wild-type mice after 5-FU administration. Female mice (10-12
weeks of age) were injected intravenously with 5-FU (150 mg/kg), and
the number of platelets in peripheral blood was counted at the
indicated times thereafter. Data are mean ± S.E. of triplicate
determinations for five mice of each genotype. The platelet count
differed significantly (p < 0.05) between wild-type
and mutant mice at 15 days.
View larger version (12K):
[in a new window]
Fig. 5.
Platelet survival in SHPS-1 mutant and
wild-type mice. Female mice at 9 weeks of age were injected
intravenously with sulfo-NHS-LC-biotin (1.5 mg), and the number of
biotinylated platelets in peripheral blood was determined as a
percentage of the total number of circulating platelets at the
indicated times thereafter (A); the total number of
circulating platelets is shown in B. Data are mean ± S.E. of triplicate determinations for five mice per group.
View larger version (29K):
[in a new window]
Fig. 6.
Phagocytosis of RBCs by PEMs from SHPS-1
mutant and wild-type mice in vitro. PKH26-labeled
RBCs from wild-type donor mice were overlaid on thioglycolate-elicited
PEMs that were derived from wild-type (+/+) or homozygous SHPS-1 mutant
( /) mice and adherent to fibronectin-coated coverslips.
After incubation for 2 h, nonattached RBCs were washed away and
attached (but not ingested) RBCs were removed by hemolysis. The cells
were examined by confocal microscopy (upper panels), and the
number of RBCs phagocytosed by PEMs was counted in at least six fields.
Quantitative data are expressed as the number of RBCs ingested per 100 macrophages (phagocytosis index) (lower panel) and are
mean ± S.E. of values from three independent experiments.
Arrowheads in the upper panel indicate
phagocytosed RBCs; original magnification, ×630.
View larger version (77K):
[in a new window]
Fig. 7.
Spreading of PEMs from SHPS-1 mutant and
wild-type mice. Residual (non-thioglycolate-elicited) PEMs from
wild-type (A, C, and E) or homozygous
SHPS-1 mutant (B, D, and F) mice were
plated in serum-free Dulbecco's modified Eagle's medium on coverslips
coated with fibronectin. After incubation for 0.5 (A and
B), 1 (C and D), or 4 h
(E and F), cells were fixed, stained with
rhodamine-labeled phalloidin, and examined by confocal microscopy. Data
are representative of three independent experiments. Original
magnification, ×630.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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