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*
Department of Internal Medicine and Molecular Science, Graduate School of Medicine B5, Osaka University, Osaka, Japan; and
Pharmaceutical Research Laboratories, Kirin Brewery Company, Gunma, Japan
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4
7 via mucosal
addressin cell adhesion molecule-1 (MAdCAM-1; Refs. 3 and
4). These interactions mediate the tethering and rolling
of lymphocytes on endothelial venule, followed by
1 and 2
integrin-mediated adhesion and transmigration (7).
CD47, also known as integrin-associated protein, is an ubiquitously
expressed 50-kDa cell surface glycoprotein with an extracellular Ig
domain and five putative transmembrane domains (8, 9). It
physically and functionally associates with 3
integrins and modulates a variety of cell functions including cell
activation, adhesion, migration, and phagocytosis
(10, 11, 12, 13). Treatment of leukocytes with anti-CD47 mAbs
inhibits 3 integrin-mediated ligand binding,
activation of an oxidative burst, and FcR-mediated phagocytosis
(8, 12, 14). Neutrophils require CD47 to migrate across
the endothelial and colonic epithelial cells after firm adhesion
(15, 16, 17). Regarding lymphocytes which express little
3 integrins, anti-CD47 mAbs costimulate
the proliferation of human T cells and the production of IL-2
(18, 19, 20). Concerning B lymphocytes, we have recently
demonstrated that soluble form of an anti-CD47 mAb B6H12 stimulates
the migratory activity on fibronectin via integrin
41 in some human B
cell lines. In addition, the immobilized B6H12 induced polarization in
these B cell lines via the activation of Cdc42, a member of Rho family
small GTPase, in an integrin-independent manner (21).
These findings suggest that CD47 itself may transduce polarization
signals into B lymphocytes.
Thrombospondin (TSP) is known to be a ligand for CD47 and involved in modulating the differentiation, proliferation, and migration of various cells (22, 23). The interaction between TSP and CD47 induces the activation of autoreactive T lymphocytes and the spreading of platelets and melanoma cells on fibrinogen (24, 25, 26). Recently, Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) has been shown to be a ligand for CD47 (27, 28). SHPS-1 is a glycosylated receptor-like protein with three Ig-like domains in its extracellular region and has two immunoreceptors with tyrosine-based inhibition motifs in its intracellular region (29). The intracellular region of SHPS-1 seems to act as a site for the recruitment of Src homology 2 domain containing phosphatases, leading to inhibition of signals evoked by cytokines and integrins that promote tyrosine kinase activity (30, 31). However, the biological roles of the extracellular region of SHPS-1 remain obscure. In this study, we have demonstrated the expression of SHPS-1 even on nonactivated endothelial cells including HEV in tonsils, and suggested a novel role of SHPS-1/CD47 interaction on B lymphocyte biology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAbs specific for CD47 (B6H12 and 2D3, IgG2b) were generously
provided by Dr. E. Brown (Washington University, St. Louis, MO; Ref.
32). B6H12 has been reported to modulate the
3 integrin-mediated cell functions and inhibit
the transmigration of neutrophils (8, 12, 14). In
contrast, 2D3 has not been reported to modulate the cell functions. An
anti-CD47 mAb, SE5A5, which can specifically block the binding of
SHPS-1 to CD47 was generously provided by Dr. H.-J. Bühring
(EberhardKarls Universität, Tübingen, Germany; Ref.
28). MOPC195 (IgG2b) was purchased from Cappel
Laboratories (Cochranville, PA). Wortmannin was purchased from Sigma
Aldrich (St. Louis, MO). Rabbit anti-SHPS-1 polyclonal Ab was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human
rIL-1 was purchased from PeproTech (London, U.K.). Pertussis toxin
(PTX) was generously provided by Chemo-Sero Therapeutic Research
Institute (Kumamoto, Japan).
Cell culture
A human B cell leukemia cell line (Nalm6 and BALL) and human myeloma cell line (RPMI 8226) were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Establishment and characterization of a human lymphoma cell line (ONHL-1) originated from pleural effusion, a human mature B cell line (OPM-3) harboring EBV, and a human myeloma cell line (OPM-2) derived from peripheral blood were described previously (33, 34, 35). These cell lines were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Flow Laboratories, Rockwell, MD) at 37°C in the humidified atmosphere of 5% CO2. HUVECs were obtained as previously reported (36). HUVECs were cultured in MCDB131 medium (Sigma Aldrich) supplemented with 10% FCS and human basic fibroblast growth factor (10 ng/ml; PeproTech). HUVECs, which were conducted through the passage two or three times, were used for the experiments. A human embryonic kidney cell line, 293T, was generously provided by Dr. T. Hirano (Osaka University, Osaka, Japan). 293T cells were cultured in DMEM medium (Nikken Biomedical Laboratory, Kyoto, Japan) supplemented with 10% FCS.
Isolation of human B cells
Preparation of normal tonsilar B cells was conducted as previously described (34). Briefly, cells were separated from tonsils of patients with chronic tonsillitis, who received tonsillectomy, by centrifugation over Ficoll-Hypaque density gradient (Nycomed, Oslo, Norway) and washed three times with RPMI 1640 medium. T cells were depleted by rosetting with neuraminidase-treated sheep erythrocytes. Monocytes were partially depleted by adhering to plastic dishes. More than 95% of the cells were CD19-positive in every sample. Informed consent was given from each patient before surgery, and the investigation was approved by the research ethical committee of Osaka University Medical School (Osaka, Japan).
Preparation of fusion proteins
The full-length cDNA encoding human SHPS-1 ligated into pTracer CMV vector was generously provided from Dr. T. Sudoh (Kirin Brewery Company, Gunma, Japan) and Dr. T. Matozaki (Osaka University; Ref. 37). A construct of Ig fusion protein of human SHPS-1 (SHPS-1-Ig/pEFBOS) was produced as previously described (38). The extracellular domain of SHPS-1 was amplified from cDNA using sense primer (5'-GGATGGTACCTACAACTGG-3') and antisense primer (5'-GCAGCTCGAGTTAGATCCAG-3') under the following conditions: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles. The amplified fragments digested with KpnI and XhoI and the 5' fragments of SHPS-1/pTracer CMV vector digested with NotI and KpnI were ligated into the Ig/pEFBOS vector (SHPS-1-Ig/pEFBOS). The sequence of this vector was confirmed by using the DNA Sequencing Kit (PerkinElmer, Warrington, U.K.). SHPS-1-Ig and CD44-Ig (38) were purified by Immunopure protein A-Sepharose column (Pierce, Rockford, IL) from culture supernatants of 293T cells transfected with SHPS-1-Ig/pEFBOS and CD44-Ig/pEFBOS, respectively.
Flow cytometry
The surface phenotypes of cells were examined with indirect immunofluorescent method as previously described (21). Briefly, the aliquots (100 µl) of cell suspension (1 x 103 cells/µl) were first incubated with an appropriate mAb (1 µg/ml) or rabbit polyclonal Ab (1 µg/ml) at 4°C for 30 min, and then rinsed and incubated with secondary FITC-conjugated goat anti-mouse IgG (BD Biosciences, Mountain View, CA) or goat anti-rabbit Ig (DAKO, Copenhagen, Denmark). To measure the binding of SHPS-1-Ig or CD44-Ig, the aliquots (100 µl) of cell suspension (1 x 103/µl) were incubated with each fusion protein (1 µg/ml) in HBSS at 4°C for 30 min, and then rinsed and incubated with FITC-conjugated goat anti-human IgG (Southern Biotechnology Associates, Birmingham, AL). The analysis was performed using FACSort (BD Biosciences).
Immobilization of fusion proteins, peptides, or mAbs
Immobilization of fusion proteins or mAbs was performed as previously described (21). In brief, 96-well microtiter plates were coated with 50 µl of SHPS-1-Ig, CD44-Ig (10 µg/ml), or 1% BSA at 37°C for 30 min, and washed with PBS. For blocking nonspecific-binding sites, 96-well microtiter plates were further incubated with 50 µl of 1% BSA at 37°C for 30 min. The wells were washed with PBS three times before cell culture. In some experiments, 96-well microtiter plates were coated with various mAbs (10 µg/ml) by the same procedure described above. ELISA using anti-human Ig or anti-mouse-Ig showed that an equal amount of each fusion protein or mAb was coated on each well.
Polarization assay
The polarization assay was performed as described previously (21). The cells were washed with chemically defined medium (AIM-V medium, Life Technologies) and resuspended in AIM-V medium with 0.1% BSA. The aliquots (100 µl) of cell suspension (5 x 102/µl) were applied into the 96 microtiter wells coated with SHPS-1-Ig, CD44-Ig, or mAb as described above. After a 2-h incubation, the number of a polarization-like morphology was counted in the indicated area under the phase-contrast microscopy. At least 300 cells/well were counted blindly. The polarization was scored according to the criteria by Wilkinson (39). The proportion of polarized cells was calculated using the following formula: the percentage of polarization = (polarized cell number/total cell number) x 100.
Adhesion assays
To measure the activity to adhere to SHPS-1-Ig, the adhesion assays using a crystal violet method were performed as previously reported (21). Briefly, 1 x 105 cells treated with or without various mAbs in AIM-V medium were incubated for 1 h on the 96-well microtiter plates coated with SHPS-1-Ig or CD44-Ig. After washing the wells with the medium three times, adherent cells were fixed with 1% glutaraldehyde in PBS for 15 min and stained with 0.1% crystal violet solution for 30 min. Following the destaining by distilled water for 15 min, the dyes were solubilized with 200 µl of distilled water containing 0.2% Triton X-100, and then the absorbance at 620 nm was measured by microphotometry. In some experiments, human tonsilar B cells (5 x 105 cells/well) were incubated on HUVEC monolayer in 96 microtiter wells in the presence or absence of anti-CD47 mAb.
Phosphatidylinositol-3 kinase (PI3K) activity assay
PI3K activity assay was performed as previously described
(40). After the incubation on immobilized SHPS-1-Ig or
CD44-Ig for 15 min, Nalm6 cells were lysed in a lysis buffer (1%
Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 5 mM EDTA, 10%
glycerol, and 1% Nonidet P-40) containing protease inhibitors, and
insoluble material was removed by centrifugation at 10,000 x
g at 4°C for 10 min. The cell lysates were subjected to
immunoprecipitation for 2 h at 4°C with anti-PI3K p85
subunit mAb (U5; Wako Junyaku, Osaka, Japan). Immunocomplexes were
collected by protein G-Sepharose, subsequently washed twice with lysis
buffer, twice with LiCl buffer (HEPES buffer containing 0.5 M LiCl and
0.2% Nonidet P-40), and finally with 10 mM HEPES and 0.15 M NaCl.
Following the addition of 30 µl of 10 mM phenyl phosphate, the
immunocomplexes were incubated with 0.2 mg/ml
L--phosphatidylinositol, 40 mM ATP, 30 mM
MgCl2, and 20 µCi of
[
-32P]ATP for 15 min. The reactions were
terminated with 200 µl of 1 N HCl, and lipids were exracted with 200
µl of chloroform/methanol (1:1, v/v). After washing with methanol/1 N
HCl (1:1, v/v), phosphorylated lipids were then extracted and resolved
by TLC using
chloroform/methanol/H2O/NH4OH
(43:38:7:5, v/v) as solvent. Radioactive spots were detected by
autoradiography.
Immunohistochemistry
The 4% paraformaldehyde-fixed cryostat sections (8 µm) of the tonsils were incubated with rabbit anti-SHPS-1 polyclonal Ab, followed by the incubation of biotinylated goat anti-rabbit Ig and alkaline phosphatase-conjugated streptavidin (DAKO). Reactions were visualized using the 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium kit (Promega, Madison, WI). Following that, the sections were incubated with mouse anti-PNAd mAb, MECA-79 (IgM; BD PharMingen, San Diego, CA), and then incubated with FITC-conjugated anti-mouse Ig mAb (DAKO). As a negative control, the primary Ab was replaced with normal rabbit IgG (DAKO). The sections were counterstained with methyl green, dehydrated, and mounted. Tonsilar B cells, which were adhered to immobilized SHPS-1 on cell-culture slide (four-chamber, noncoating; BD Biosciences), were fixed with 4% paraformaldehyde for 15 min. Following the incubation with PBS containing 0.1% goat serum, the cells were incubated with mouse anti-human CD19 mAb (B4, Coulter, Fullerton, CA) in PBS containing 0.2% BSA (5 µg/ml) for 12 h at 4°C. After washing with PBS containing 0.2% BSA, the cells were incubated with PE-conjugated anti-mouse Ig mAb (DAKO) for 1 h. Finally, for visualization of nucleus, the cells were incubated with 4'6-diamino-2-phenylindole dihydrochloride solution and mounted. A total of 50 fields in each section were examined under the fluorescence microscopy and then photographed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The binding of soluble SHPS-1-Ig to human B cell lines was
evaluated using flow cytometry. All B cell lines examined in this study
show a similar expression level of CD47 irrespective of their
maturation stage, and Nalm6 cells show the highest expression of CD47
(Table I
). Although SHPS-1-Ig bound to
all B cell lines tested, the amounts of the bound SHPS-1-Ig did not
correlate to the expression levels of CD47; larger amounts of SHPS-1-Ig
bound to Nalm6, BALL, RPMI8226, and OPM-2 than to ONHL-1 and
OPM-3. CD44-Ig failed to bind to B cell lines and was used as a
negative control in the subsequent experiments. In Nalm6, BALL, and
RPMI8226 cells, the binding of SHPS-1-Ig was increased in a
dose-dependent manner and reached maximal level at 1.0 µg of SHPS-1
Ig per 1 x 106 cells (Fig. 1A). To determine whether the
binding of SHPS-1-Ig is mediated via CD47, we performed blocking
experiments. B6H12 and SE5A5 blocked the binding of SHPS-1-Ig to Nalm6
cells, while 2D3, a nonfunctional mAb, did not block its binding (Fig. 1B). B6H12 completely blocked the binding of SHPS-1-Ig to
the other cell lines as well as Nalm6 cells (data not shown). The
complete blockade of SHPS-1-Ig binding by B6H12 suggests that the
binding of SHPS-1-Ig requires CD47. In addition, preincubation of Nalm6
cells with SHPS-1-Ig significantly inhibited the binding of B6H12 (data
not shown). Essentially the same results were obtained in other B cell
lines (ONHL-1 and RPMI8226; data not shown). These data suggest that
SHPS-1 binds to human B cells via CD47 and there are some differences
in the affinity/avidity of CD47 for SHPS-1 binding between the types of
human B lymphocytes.
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We next examined the adhesion of human B cell lines to immobilized
SHPS-1-Ig. Among B cell lines, pre-B cell lines (Nalm6 and BALL) and a
lymphoma B cell line (ONHL-1) markedly adhered to immobilized SHPS-1-Ig
in a dose-dependent manner (Fig. 2A). In contrast, two myeloma
cell lines (RPMI8226 and OPM-2) showed the dose-dependent but modest
adhesion to immobilized SHPS-1-Ig. A mature B cell line (OPM-3)
scarcely adhered in any doses of immobilized SHPS-1-Ig performed in
this study. Again, B6H12 completely inhibited the adhesion of Nalm6 to
immobilized SHPS-1-Ig, while 2D3 did not (Fig. 2B). These
findings suggest that SHPS-1 acts as an adhesive substrate via CD47 in
certain human B cell lines.
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We have reported that immobilized B6H12 induces an elongated and
polarized shape change (polarization) in pre-B cell lines (Nalm6 and
BALL) and mature B cell lines (ONHL-1 and OPM-3), but not in myeloma
cell lines (RPMI8226 and OPM-2; Ref. 21). To extend our
findings, we examined the morphological changes induced by immobilized
SHPS-1-Ig. Among B cell lines tested, SHPS-1-Ig induced polarization
only in Nalm6 cells, and the percentage of the polarized cells induced
by immobilized SHPS-1-Ig was smaller than immobilized B6H12 (Fig. 3). Immobilized CD44-Ig did not
significantly induce the polarization in Nalm6 cells. These data
suggest that SHPS-1 provokes the signals to induce the cytoskeletal
reorganization via CD47, at least in some pre-B cell lines.
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PTX-sensitive heterotrimeric guanosine triphosphate-binding
protein (G protein) has been reported to be involved in the signaling
via the TSP/CD47 interaction (41). Although the activity
of PTX was confirmed by Chinese hamster ovary cell cyotoxicity assay
(42), PTX could not significantly inhibit the immobilized
SHPS-1-Ig-induced polarization of Nalm6 cells. In contrast, a specific
PI3K inhibitor, wortmannin, completely inhibited the polarization of
Nalm6 cells (97% decrease of polarization; Fig. 4A). Wortmannin also
significantly inhibited the adhesion to immobilized SHPS-1-Ig (38%
decrease of adhesion), while PTX did not affect the adhesion activity
(Fig. 4B). To investigate whether PI3K is activated by
SHPS-1/CD47 interaction, we performed PI3K activity assay. PI3K is
constitutively activated. However, immobilized SHPS-1-Ig further
activated the PI3K activity (Fig. 4C). Therefore, PI3K, but
not PTX-sensitive G protein, is mainly involved in the signals through
the interaction between SHPS-1 and CD47, at least in Nalm6 cells.
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To further investigate the role of CD47/SHPS-1 interaction in B
lymphocyte biology, we examined the effect of immobilized SHPS-1-Ig on
the adhesion of normal B lymphocytes isolated from tonsils. As shown in
Fig. 5A, enriched tonsilar B
lymphocytes significantly adhered to immobilized SHPS-1-Ig (percentage
of adhesion of applied cells was 43.5 ± 0.7%; n
= 3). The adhesion of human tonsilar B lymphocytes to SHPS-1-Ig was
again completely inhibited by B6H12. Moreover, after 1–2 h of
incubation on immobilized SHPS-1-Ig, 
20% of tonsilar B lymphocytes
showed the polarization-like or dendritic shape change (Fig. 5, B and C). To confirm that the adhered cells were
really B lymphocytes, we performed the immunofluorescence staining
using anti-CD19 mAb. As shown in Fig. 5D, CD19
immunoreactivity was detected in most of the cells adhered to
immobilized SHPS-1-Ig. In addition, the immunoreactivity for CD19 was
also observed in the polarized cells. These findings indicate that
interaction between SHPS-1 and CD47 may mediate normal B lymphocyte
adhesion and subsequent their morphological change.
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To further clarify the physiological roles of SHPS-1, we examined
the expression and localization of SHPS-1 in human tonsil by
immunohistochemical staining. SHPS-1 immunoreactivity was detected in
macrophages in interfollicular space and was weakly detected in
tingible macrophages (data not shown). In contrast, there was no
detectable stain of SHPS-1 in the lymphocytes in tonsils. These data
are consistent with previous reports, in which the SHPS-1 expression
was examined in rat and cattle lymph nodes (43, 44). In
addition to the expression on macrophages, we found that SHPS-1
immunoreactivity was detected on endothelial cells in tonsils (Fig. 6A). It is noteworthy that
immunoreactivity of PNAd, a specific marker of HEV, was detected in the
endothelial cells (Fig. 6B). These results indicate that
SHPS-1 is expressed on HEV.
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To investigate whether the interaction between SHPS-1 and CD47 may
be involved in the adhesion of human tonsilar B cells to endothelial
cells, we used HUVECs as a source of SHPS-1. As shown in Fig. 7A, HUVECs constitutively
express SHPS-1 on their surface (mean fluorescence intensity (MFI) 6.8)
as well as HEV cells under nonactivated conditions. Although the
expression of ICAM-1 was significantly up-regulated (MFI 14.3 and
128.2, before and after 36 h treatment, respectively), the
expression of SHPS-1 on HUVECs was scarcely changed by IL-1, a
proinflammatory cytokine, for up to 36 h (MFI 6.5, after
treatment; Fig. 7A). We then performed the adhesion assay
using enriched human tonsilar B cells and nonactivated HUVECs in static
conditions in the presence or absence of anti-CD47 mAb (SE5A5).
Human tonsilar B cells apparently adhered to nonactivated HUVECs, and
SE5A5, which is a blocking mAb for the binding between SHPS-1 to CD47,
but has no functional activity, significantly inhibited the adhesion of
human tonsilar B cells to nonactivated HUVECs (45% decrease of
adhesion; Fig. 7B). To exclude the possibility that the
contaminated cells such as monocytes adhered to HUVECs, we performed
flow cytometric analyses. Flow cytometric analyses showed that 90%
of the adhered cells to HUVECs were CD19 positive (data not shown).
These data suggest that the interaction between SHPS-1 and CD47 may be
involved in the adhesion of human B lymphocytes to nonactivated
endothelial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD47 has been reported to play important roles in the activation,
adhesion, and migration of leukocytes in concert with integrins.
Recently, we have demonstrated that CD47 transduces the signals
involved in the cell migration through Cdc42, a member of Rho family
GTPases (21). However, these results were obtained using
ligand-mimic anti-CD47 mAb, B6H12. In this study, using a fusion
protein composed of the extracellular domain of SHPS-1 and the Fc
portion of human IgG1 we have investigated the role of
SHPS-1 as a physiological ligand for CD47. We revealed that SHPS-1-Ig
acts as an adhesive substrate in human B lymphocytes. In addition,
SHPS-1-Ig showed similar effects on certain human B lymphocytes to
B6H12. B6H12 completely blocked the binding of SHPS-1-Ig to CD47, and
SHPS-1-Ig partially blocked the B6H12 binding to CD47. These findings
strongly suggest that the binding sites of SHPS-1 and B6H12 in CD47 are
very close to each other, which may account for similar effects of
these ligands on human B cells. Seiffert et al. (28)
reported that extracellular signal-regulatory protein, which is
identical to SHPS-1, cannot bind to the CD47-deficient human ovarian
carcinoma cell line OV10, but to human CD47-transfected OV10. Taken
together, it is strongly suggested that SHPS-1 exclusively bound to
CD47 on human B lymphocytes. Although all B cell lines examined in this
study expressed CD47, the binding capacity of soluble SHPS-1-Ig varied
between B cell lines: pre-B cell lines (Nalm6 and BALL) and myeloma
cell lines (RPMI8226 and OPM-2) bound larger amounts of SHPS-1-Ig than
mature B cell lines (ONHL-1 and OPM-3). Of particular interest was that
pre-B cell lines, but not myeloma cell lines, well-adhered to
immobilized SHPS-1-Ig irrespective of the similar binding capacity for
soluble SHPS-1-Ig. Our findings suggest that the affinity/avidity state
of CD47 for SHPS-1 may change during B cell maturation. In addition,
the intercellular signaling evoked by the CD47/SHPS-1 interaction may
also change during B cell maturation. Because SHPS-1-Ig contains the Fc
portion of human IgG1, there exists the possibility that
FcR would be involved in the binding and the adhesion to SHPS-1-Ig.
Flow cytometric analysis showed that the expression of FcRII (CD32)
was modestly expressed on Nalm6 and RPMI8226 cells and abundantly on
OPM-2 (data not shown). However, there is no relationship between the
SHPS-1-Ig function and the expression of FcR. In addition, CD44-Ig,
which also contains the Fc portion of IgG, did not bind to any human B
cell lines in this study. These results suggest that SHPS-1-Ig binds to
human B cell lines via CD47.
In this study, we investigated the signaling via SHPS-1/CD47 interaction in Nalm6 cells. Wortmannin, but not PTX, affected the adhesion to immobilized SHPS-1-Ig and the morphological change induced by SHPS-1 in Nalm6 cells. In contrast, it has been shown that PTX-sensitive heterotrimeric GTP-binding protein (G protein) is involved in the signals evoked by TSP/CD47 interaction (10, 41). These results suggested that SHPS-1 and TSP may play differential roles in human B lymphocyte biology. In addition to marked inhibition of the polarized shape change and cell adhesion by wortmannin, SHPS-1/CD47 interaction activates PI3K activity in Nalm6 cells. Although the intracellular signaling cascade evoked by SHPS-1/CD47 interaction in B lymphocyte remains to be clarified, PI3K may be involved in a downstream effector of Cdc42 and Rac1 and regulate integrin-mediated cell motility, as suggested in mammary epithelial cells (45). Experiments of this regard are in progress.
The recruitment of lymphocytes is essential to the development and
function of the immune system. Circulating lymphocytes continuously
enter the peripheral lymphoid tissue by traversing endothelial cells
under steady state conditions. After the differentiation and the
maturation, memory and effector lymphocytes migrate from lymphoid
tissue to the effector sites. The interaction between endothelial cells
and lymphocytes is an essential step in this recruitment. It has been
demonstrated that adhesion molecules play central roles in the
extravasation of lymphocytes. Naive lymphocytes interact with PNAd and
MAdCAM-1 on HEV via L-selectin and integrin
47, respectively
(3, 4, 7). Memory and effector lymphocytes, which express
little L-selectin but highly express
47, mainly interact
with MAdCAM-1 via integrin
47 for the
extravasation at effector sites, although the homing mechanism of
memory/effector lymphocytes is heterogeneous (3, 4, 46).
In addition, the existence of another mechanism for the extravasation
of memory/effector lymphocytes has been speculated, especially
IgA-producing mucosal B lymphocytes (47). In this study,
we demonstrated that SHPS-1 is expressed on human endothelial cells and
HEV, an entrance of lymphocytes from blood stream, in human tonsil.
Flow cytometric analysis also showed that primary cultured HUVECs
constitutively express SHPS-1 and that the expression of SHPS-1 on
HUVECs was not changed by the addition of IL-1. In addition,
anti-CD47 mAb SE5A5 significantly inhibited the adhesion of human
tonsilar B lymphocytes to nonactivated HUVECs. From these findings, we
suggest that SHPS-1/CD47 interaction may be involved in the
extravasation of B lymphocytes under the conditions without
inflammatory stimuli. Recently, Ticchioni et al. (48)
demonstrated that CD47 contributes to the arrest of Jurkat cells (human
T leukemia cell line) on HUVECs activated by TNF- under
flow.
In immune response, the contact between lymphocytes and APCs such as dendritic cells is the first step to initiate the immune response (49). Several reports have suggested that CD47 is involved in the activation and the differentiation of T lymphocyte (18, 19, 20, 50). Brooke et al. (44) demonstrated that SHPS-1 expressed on monocytes and dendritic cells mediates the binding to CD4-positive T lymphocytes in cattle. In B lymphocyte activation, cocultivation of dendritic cells with B lymphocytes induces the cell-cell interaction and enhances the Ig production (51). Although LFA-1/ICAM-1 interaction is partially involved in the cell-cell contact between dendritic cells and B lymphocytes (52), other mechanisms should exist. The morphological change, like polarization, may also facilitate the cell-cell contact between lymphocyte and APC. Because macrophages expressed SHPS-1 in human tonsils, it is also possible that SHPS-1/CD47 interaction may be involved in the cell-cell contact between B lymphocytes and macrophages or dendritic cells in human tonsils for the differentiation and the maturation of B lymphocytes.
In summary, we demonstrate that SHPS-1 acts as an adhesive substrate of human B lymphocyte via CD47, and suggest that the affinity/avidity of CD47 may be regulated during the maturation of human B lymphocyte. We also show that SHPS-1/CD47 interaction may be involved in human B lymphocyte adhesion to nonactivated HUVEC. Our results suggest that SHPS-1/CD47 interaction may play important roles in the immune system. The investigations concerning the roles and signals through SHPS-1/CD47 interaction in human B lymphocytes would provide further understanding of the immune system.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshiaki Tomiyama, Department of Internal Medicine and Molecular Science, Graduate School of Medicine B5, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: yoshi{at}hp-blood.med.osaka-u.ac.jp
3 Abbreviations used in this paper: HEV, high endothelial venule; PNAd, peripheral node addressin; MAdCAM-1, mucosal addressin cell adhesion molecule-1; SHPS-1, Src-homology 2 domain bearing protein tyrosine phosphatase substrate-1; PI3K, phosphatidylinositol-3 kinase; TSP, thrombospondin; PTX, pertussis toxin; MFI, mean fluorescence intensity.
Received for publication August 3, 2001. Accepted for publication February 1, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v3-dependent ligand binding. J. Cell Biol. 123:485.IIb3. J. Biol. Chem. 272:1470.
21 integrin in vascular smooth muscle cells. Mol. Biol. Cell 9:865.21-mediated platelet activation. Blood 94:642.v3 function through integrin-associated protein. J. Cell Biol. 135:533.v3 antibodies in chronic immune throbocytopenic purpura. Thromb. Haemost. 85:36.[Medline]
family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells. Eur. J. Immunol. 28:1.[Medline]
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|
 T. Ishikawa-Sekigami, Y. Kaneko, H. Okazawa, T. Tomizawa, J. Okajo, Y. Saito, C. Okuzawa, M. Sugawara-Yokoo, U. Nishiyama, H. Ohnishi, et al. SHPS-1 promotes the survival of circulating erythrocytes through inhibition of phagocytosis by splenic macrophages Blood, January 1, 2006; 107(1): 341 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Miyashita, H. Ohnishi, H. Okazawa, H. Tomonaga, A. Hayashi, T.-T. Fujimoto, N. Furuya, and T. Matozaki Promotion of Neurite and Filopodium Formation by CD47: Roles of Integrins, Rac, and Cdc42 Mol. Biol. Cell, August 1, 2004; 15(8): 3950 - 3963. [Abstract] [Full Text] [PDF]
|
|
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|
 A. Fukunaga, H. Nagai, T. Noguchi, H. Okazawa, T. Matozaki, X. Yu, C. F. Lagenaur, N. Honma, M. Ichihashi, M. Kasuga, et al. Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate 1 Regulates the Migration of Langerhans Cells from the Epidermis to Draining Lymph Nodes J. Immunol., April 1, 2004; 172(7): 4091 - 4099. [Abstract] [Full Text] [PDF]
|
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 |
|
 T. T. Chen, E. J. Brown, E. J. Huang, and W. E. Seaman Expression and Activation of Signal Regulatory Protein {alpha} on Astrocytomas Cancer Res., January 1, 2004; 64(1): 117 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bhaduri and R. Sowdhamini A genome-wide survey of human tyrosine phosphatases Protein Eng. Des. Sel., December 1, 2003; 16(12): 881 - 888. [Abstract] [Full Text] [PDF]
|
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L. A. Maile, J. Badley-Clarke, and D. R. Clemmons The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells Mol. Biol. Cell, September 1, 2003; 14(9): 3519 - 3528. [Abstract] [Full Text] [PDF]
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, SHPS-1-Ig 0.1 µg; 
, SHPS-1-Ig 0.5 µg;
, SHPS-1-Ig 1.0 µg; 
, SHPS-1-Ig 4.0 µg). Representative data
are shown in three independent experiments. B, Nalm6
cells (1 x 105) were incubated with SHPS-1-Ig (1
µg/ml) in the presence of an anti-CD47 mAb, followed by
FITC-conjugated goat anti-human Ig. The binding of SHPS-1-Ig was
analyzed by FACSort (solid line). The binding of CD44-Ig (1 µg/ml)
was used as a negative control (dotted line). Representative data are
shown in three independent experiments.
