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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 965-972
IMMUNOBIOLOGY
Switch in the protein tyrosine phosphatase associated with human
CD100 semaphorin at terminal B-cell differentiation stage
Christian Billard,
Stéphanie Delaire,
Emmanuel Raffoux,
Armand Bensussan, and
Laurence Boumsell
From Unit 448, INSERM, Faculté de Médecine Henri Mondor,
Créteil, France.
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Abstract |
Human CD100, the first semaphorin identified in the immune system,
is a transmembrane protein involved in T-cell activation. In the
present study, we showed that activation of peripheral blood or
tonsillar B lymphocytes induced the expression of CD100 in
CD38+CD138 cell populations,
including in CD148+ subpopulations, thus expressing a
memory B-cell-like phenotype. Using an in vitro enzymatic assay, we
found that protein tyrosine phosphatase (PTP) activities
were immunoprecipitated with CD100 in these cell populations, which
were isolated by cell sorting, as well as in most B-cell
lines representing various stages of B-cell differentiation.
Immunodepletion and Western blotting experiments demonstrated that CD45
was the PTP associated with CD100 in cell lines displaying pre-B,
activated B, and pre-plasma cell phenotypes. CD45 also accounted for
PTP activity immunoprecipitated with CD100 in
CD38+CD138 cells sorted after activation
of peripheral blood or tonsillar B lymphocytes. In contrast, no
CD100-CD45 association was observed in plasma cell lines corresponding
to the terminal B-cell differentiation stage. CD148, the other
transmembrane PTP known to be implicated in lymphocyte signaling
pathways, was either only partly involved in the CD100-associated PTP
activity or not expressed in plasma cell lines, indicating the
association of CD100 with another main PTP. Our data show that CD100 is
differentially expressed and can functionally associate with distinct
PTPs in B cells depending on their activation and maturation state.
They also provide evidence for a switch in the CD100-associated PTP at
terminal stage of B-cell differentiation.
(Blood. 2000;95:965-972)
© 2000 by The American Society of Hematology.
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Introduction |
CD100 was defined in our laboratory as a 150 kDa transmembrane homodimeric protein expressed by
various human cells of the immune system, such as thymic T-cell clones,
T and B lymphocytes, natural killer (NK) cells, monocytes, and
neutrophils, but not by eosinophils or immature hematopoietic
precursors.1-3 Evidence for CD100 as a T-cell activation
molecule was provided by our studies showing that its triggering with
monoclonal antibodies (mAbs) delivers a proliferative signal to T cells
in the presence of submitogenic concentrations of anti-CD3 or anti-CD2
antibodies.2,4 Moreover, we demonstrated that CD100 is
associated with CD45, a cell surface protein tyrosine phosphatase (PTP)
considered a key molecule in the T-cell receptor (TCR) activation
process, and that CD100-associated CD45 PTP activity increases upon
T-cell activation.5 In addition, CD100 was found to be
associated with a serine kinase activity in T and NK
cells.6 The molecular characterization of CD100 revealed
that it is a member of the semaphorin family.7 Semaphorins
are transmembrane and secreted proteins involved in axon guidance
during nervous system development.8,9 They are structurally
defined by a conserved 500-amino acid extracellular domain with 16 cysteines (sema domain). The human CD100, like its murine homologue
M-SemaG,10 belongs to the group of transmembrane semaphorins containing an immunoglobulin-like domain in its
extracellular part. The cytoplasmic tail, which has no significant
homology with that of other semaphorins, encodes a site for tyrosine
phosphorylation and multiple sites for serine/threonine
phosphorylation.8 So far, no CD100 ligands have been
described. CD100 can be cleaved from the cell surface to release a
soluble semaphorin.5 From all of these data, CD100 appeared
as the first semaphorin identified in the immune system.7,
11
However, functional or signaling roles of CD100, such as those reported
for T cells, were not investigated in other types of leukocytes. The
only available data were reported on the effects of CD100 transfectants
on B lymphocytes, mimicking a signal delivered by CD100-bearing cells
through interaction with a counter-receptor: CD100 induced B
lymphocytes to aggregate and improved their viability in
vitro.7 In that study, CD100 was found to be expressed in activated B lymphocytes from the germinal center of secondary lymphoid
follicules, where B-cell expansion and differentiation occur.12,13 A more recent study on malignant lymph nodes
from various B-cell non-Hodgkin lymphomas (NHL) indicated that CD100 was generally not expressed in follicular NHL (only 3 positive cases
out of 40), whereas it was detected in 5 out of 5 cases of high-grade,
small, noncleaved B-cell NHL.14 Although suggesting that
CD100 may have a physiological role in the processes of germinal center
formation and B-cell differentiation,7,14 these data also
raise the question of a differential expression of CD100 during B-cell
maturation. Furthermore, the signaling and functional events triggered by CD100 itself on the surface of B cells remain to be established.
Signal transduction cascades driven by tyrosine phosphorylation are
known to regulate important cellular mechanisms, such as proliferation,
differentiation, migration, or cell death. The degree of tyrosine
phosporylation is tightly controlled by the concerted activities of
protein tyrosine kinases and PTPs.15,16 The leukocyte PTP
CD45 positively regulates signaling during not only TCR activation, as
mentioned above, but also during B-cell antigen receptor (BCR)
engagement. CD45 acts by dephosphorylation of specific tyrosine
kinases, allowing their initial activation. Until now, CD45 was
considered to be the only transmembrane PTP involved in lymphocyte
signal transduction.15 Recently, it appeared that the CD148
transmembrane PTP,17,18 corresponding to the previously
described PTP HTP- /DEP-1, is also involved in lymphocyte signal
transduction.19 If the association of CD100 with CD45 on T
cells is also taken into account, this also may be the case on B cells.
To study the signaling and functional roles of CD100 in B cells, we
first quantified the density of CD100 on the surface of peripheral
blood B lymphocytes and B-cell lines, which are blocked at different
B-cell maturation stages, and we then investigated the association of
CD100 with transmembrane PTPs. CD100 was found to be induced by
activation of B lymphocytes and to associate with different PTP
activities during B-cell maturation stages. CD45 was
identified as the CD100-associated PTP in activated B lymphocytes
displaying a memory cell-like phenotype and in cell lines with
phenotypes of pre-B, activated B, and pre-plasma cells, but not in
plasma cell lines corresponding to the terminal differentiation stage.
CD148, which was expressed in some but not all plasma cell lines, did
not appear to be the main CD100-associated PTP, suggesting the
association with a third enzyme at plasma cell stage.
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Materials and methods |
Fourteen human B-cell lines with phenotypes
corresponding to various B-cell differentiation stages were obtained at
the American Type Culture Collection (Rockville, MD), unless otherwise
stated: Nalm-6 and Reh-6, 2 pre-B-cell lines established from common
acute lymphoblastic leukemia; Ramos, Daudi, and Raji, 3 lymphoblastoid cell lines established from Burkitt Lymphoma (BL); DZ, Sanchez (both
isolated in our laboratory), and JY, 3 cell lines of activated B
lymphocytes obtained by Epstein-Barr virus (EBV)-transformation of
normal B lymphocytes; Eskol, a pre-plasma cell line,
established from hairy cell leukemia20 provided by Dr E. F. Srour (University of Indiana, Indianapolis, IN); and 5 plasma cell
lines established from multiple myeloma: LP-1, producing IgA, provided
by Professor J. C. Brouet (Hopital Saint-Louis, Paris, France); U-266,
producing IgE  ; RPMI-8226 ( light chain); and XG-1(IgA  ) and
XG-2 (IgG ), both provided by Dr B. Klein (Unit 291 INSERM,
Montpellier, France). XG-1 was established from peripheral blood after
secondary plasma cell leukemia, and XG-2 from pleural effusion after
relapse.21
Most cell lines were cultured in RPMI-1640 medium containing 10%
heat-inactivated fetal calf serum (FCS), 2 mmol/L
glutamine, 1 mmol/L sodium pyruvate, and antibiotics. This
medium was supplemented with 50 µmol/L
 -mercapto-ethanol and 100 U/mL of interleukin (IL)-6
for XG-1 and XG-2 cell culture. LP-1 cells were grown in Iscove's
modified Dulbecco's medium containing FCS and antibiotics. Cells were
maintained at 37°C in an incubator containing 5% CO2 at densities between 105 and 106
cells/mL by 2 to 3 subcultures a week. All experiments
were performed by 48 hours after subculture at
2 × 105 cells/mL.
After isolating peripheral blood and tonsillar B lymphocytes, we
performed activation and cell sorting. Peripheral blood and tonsil
samples were obtained from healthy donors after informed consent
according to institutional recommendations on biomedical ethics.
Mononuclear cells were isolated by Ficoll-Paque density gradient
centrifugation. Peripheral blood mononuclear cells were depleted of T
cells by E rosette assays with sheep red blood cells treated with amino-ethyl-thiouronium bromide. Peripheral blood E cells
contained E+ cells (fewer than 6% CD2+ cells)
and monocytes, as determined by cell surface staining (see below).
Tonsillar mononuclear cells were purified by 2 successive E rosette
assays and 2 successive depletions of adherent cells. More
than 95% of tonsillar nonadherent E cells were
CD19+. Both cell types were cultured in RPMI-1640 medium
supplemented with 10% FCS, and activation of B lymphocytes was
performed with the use of 300-19 cells transfected with the CD154
complementary DNA in the presence of Staphylococcus aureus
(SAC) (1/5000; Pansorbin, Calbiochem, Meudon, France) and
10 ng/ml IL-4.22 After 7 days of activation, cells were
harvested, and the different populations were analyzed for size,
granulosity, and phenotypic markers with the use of an ELITE cytometer
(Coulter, Miami, FL). Cell sorting was then performed according to 3 parameters: size, granulosity, and CD138 negativity. Recovery rate,
rather than high purity, was privileged. Under these conditions, sorted
cell suspensions contained more than 85% cells of interest. The sorted
cells were recultured for 1 hour before all experiments.
BD16 (anti-CD100), BJ45, and O509 (anti-CD45), BB27 (anti-CD101), and
O275 (anti-CD2) mAbs were produced in our laboratory as described
previously.2,4 Anti-CD148 (143-41 and A3) and anti-CD22
(5T108) mAbs were obtained through exchanges of the Sixth International
Workshop on White Cell Differentiation Antigens23; 143-41 mAb was also provided by Dr R. Vilella (Hospital Clinic, Barcelona,
Spain). Other mAbs used in this study (CD2, CD3, CD4, CD8, CD14, CD56,
CD19, CD38, and CD138) were purchased from commercial sources.
Polyclonal anti-SHP-1 antibody was purchased from Santa Cruz
Biotechnology (Lake Placid, NY).
The expression of various cell surface molecules was studied either
classically by indirect immunofluorescence with specific mAbs and flow
cytometry analysis according to techniques previously described,24 or by quantitative determination of antigens
with the use of the DAKO QIFIKIT (DAKO A/S, Glostrup, Danemark). The QIFIKIT procedure allows the quantification of antigen density as
follows: cell samples were washed with phosphate-buffered saline (PBS)
and incubated for 30 minutes at 4°C in the dark with the corresponding mAbs and with negative control-isotype-matched mAb at
saturating concentrations. Cells were then washed and incubated for 30 minutes at 4°C in the dark with the
F(ab')2 fragment of fluorescein
isothiocyanate-conjugated goat anti-mouse antibody provided by the
QIFIKIT. After washings, cell samples were immediately analyzed with a
flow cytometer, EPICS (Coulter, Miami, FL). Set-up beads and
calibration beads provided by the QIFIKIT were treated in parallel to
the cell samples and were used to establish the optimal voltage
amplification and draw a calibration curve, respectively. The mean fluorescence intensity was then determined for each population of the calibration beads and for the positive peak of cell samples. From these data, the numbers of primary mAb-binding sites per cell were
calculated. They correspond to the mean numbers of accessible antigenic
sites per cell and are expressed in sites per cell. For the
quantitative determination of antigens expressed by various peripheral
blood lymphocyte subsets, experiments were performed on 100 µL of total blood with primary mAbs (specific for each subset) conjugated with different fluorochromes, and the analysis was
done as previously described after hemolysis of red blood cells.25 In order to evaluate the density of
antigens, cell surface was calculated by measuring cell size with a
Coulter counter.
An in vitro PTP-activity assay, based on a colorimetric enzymatic
reaction using a specific substrate,5 was performed from cell lysates immunoprecipitated with appropriate mAbs in 96-well microtiter plates (Immunoplate Maxisorb, Nunc, Denmark). For
immunoprecipitation, wells were first coated with purified mAbs (10 µg in PBS) overnight, washed extensively, and saturated with 1%
bovine serum albumin in PBS for at least 3 hours. Then, 100 µL of cell lysates (5 × 106 cells
in all experiments unless otherwise stated) were added to each well for
4-hour incubation. Cell lysis was performed with 1% Brij58 in lysis
buffer (20 mmol/L Tris-pH 7.5, 150 mmol/L
NaCl, 1 mmol/L paramethyl sulfonyl fluoride (PMSF), 2 µg/mL aprotinin, 2 µg/mL leupeptin). All
these steps were carried out at 4°C. After immunoprecipitation,
wells were washed 4 times with 20 mmol/L Tris-pH 7.5, 150 mmol/L NaCl, 1 mmol/L PMSF, and 0.1% Brij58. Then, 50 µL of the colorless buffer containing the
substrate para-nitrophenyl phosphate (pNPP) was added to each well (1 mg/mL pNPP, 20 mmol/L Tris-pH 7.5, 150 mmol/L NaCl, 20 mmol/L
ethylenediaminetetraacetic acid [EDTA], 5 mg/mL
di-thiothreitol, and 0.05% Brij58). Microplates were incubated for 40 hours at 37°C with 90% humidity in the dark, and the absorbance
was determined at 405 nm.
We performed immunodepletion experiments, as follows: depletion of
antigens with corresponding mAbs was performed on cell lysates
(5 × 106 cells/100 µL) in all
experiments (unless otherwise stated) in 4 cycles (1 to 2 µg of mAb
for each cycle). Immunodepletion with an irrelevant-isotype-matched
antibody was done simultaneously as a control of depletion.
Immunodepletion of SHP-1 was generally used as a control of depletion
because SHP-1 is a PTP involved in the regulation of BCR activation
through interaction of its SH2 domains with the ITIM
motif of activated substrates, receptor FcR IIB, and B-cell
coreceptor CD2215 and because CD100 cannot bind SHP-1,
owing to the absence of ITIM motif in its intracellular domain.7 In some experiments, CD101 immunodepletion was
also used as a control of depletion because this cell surface
antigen26 was not expressed on the B cells studied. All
steps of the experiments were carried out at 4°C. Cell lysates,
obtained as described above, were incubated with the appropriate
antibodies for 40 minutes. Protein A-coupled Sepharose
CL-4B beads (Pharmacia, Uppsala, Sweden) were then added for 30 minutes
under constant agitation. After centrifugation, supernatants (cell
lysates) were subjected to a second cycle of depletion according to the
same procedure. The third and fourth cycles were performed with the
appropriate antibodies, which were previously adsorbed to protein
A-Sepharose beads. After 40 minutes under agitation, the beads were
eliminated and cell lysates were incubated with protein A-Sepharose
for 30 minutes. Immunodepleted lysates were recovered by centrifugation
and assayed for PTP activity.
For Western blot analysis, cells were lysed with 1% Brij58, 50 mmol/L Tris-pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 2 µg/mL aprotinin, and 2 µg/mL leupeptin
for 30 minutes at 4°C. Cell lysates were precleared with protein A Sepharose for 1 hour, then immunoprecipitated for 2 hours with 10 µg of the appropriate mAbs and protein A-Sepharose beads under
agitation. After washings, immunoprecipitates were eluted from beads by
boiling in Laemmli buffer for 3 minutes. Samples were electrophoresed by sodium dodecyl sulfate 7% polyacrylamide gel electrophoresis (SDS-PAGE), and separated proteins were electrotransferred overnight at
4°C on PVDF membranes (Bio-Rad Laboratories,
Hercules, CA). Membranes were saturated with 3% nonfat dried milk in
PBS containing 0.2% Tween 20, then incubated for 1 hour with
antibodies recognizing the molecule of interest at adequate dilution in
blocking conditions. After washings, membranes were incubated with goat
anti-mouse horseradish peroxidase complex to reveal the immunoblotted
proteins. Membranes were washed again and developed with the use of an
enhanced chemoluminescence system (Amersham Pharmacia Biotech, Rainham, UK).
| |
Results |
CD100 was previously shown to be expressed on the
surface of most hematopoietic cells, including
lymphocytes.1-3 The levels of CD100 expression were first
quantified in resting peripheral blood B lymphocytes and compared with
those of different subsets of T and NK lymphocytes (Table
1). While 4000 to 9000 CD100 antigenic sites per cell were found in CD56+, CD8+, or
CD4+ subsets, approximately 1000 CD100 sites per cell were
detected in CD19+ cells. Therefore, the number of CD100
sites per cell was weak on resting peripheral blood B lymphocytes and
much lower than on resting T and NK lymphocytes.
Further, peripheral blood B lymphocytes were activated in vitro with
CD154 presented by transfected cells in the presence of SAC and IL-4
for 7 days. Cells were then analyzed for their size, granulosity, and
expression of surface markers (Figure 1). Populations of granular cells were observed (Figure 1A). These populations were negative for CD100 and CD38, and in addition, they
included dead cells, as shown by propidium iodine staining (data not
shown). More interestingly, 2 main populations of nongranular cells
were clearly defined: a population of large B lymphocytes (LBL) and a
population of small B lymphocytes (SBL). Both LBL and SBL populations
were positive for CD100, CD45, and CD38 (Figure 1B), but negative for
CD138 (data not shown). In each of these 2 populations, a subpopulation
also expressed the CD148 PTP (Figure 1B): a large subpopulation of SBL
cells but only a small subpopulation of LBL cells were
CD148+. The possibility of a contamination of the LBL
population with SBL cells was excluded since CD148 positivity remained
unchanged by the use of different gates for analysis of the cytometric
profiles shown in Figure 1A.
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| Fig 1.
Induction of CD38+CD138
cell populations by activation of peripheral blood B lymphocytes.
E cells were activated for 7 days with CD154-transfected 300-19 cells
in the presence of SAC (1/5000) and 10 ng/mL IL-4. (A)
Analysis of cell size and granulosity with the use of a cytometer
according to forward scatter/side scatter (FS/SS). We could clearly
define 2 populations of nongranular small B lymphocytes (SBL) and large
B lymphocytes (LBL); cell populations with high granulosity included
cells positive for propidium iodine staining. (B) Expression of surface
markers in LBL and SBL populations identified with the use of indirect
immunofluorescence with specific mAbs and flow cytometry analysis. Dark
histograms represent the fluorescence obtained with negative control
isotype-matched mAbs. Top panels: SBL population. Bottom panels: LBL
population. All experimental procedures are described in "Materials
and Methods." Both LBL and SBL populations were negative for CD138
(not shown).
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Quantification experiments showed that CD100 was expressed at a high
level on LBL cells (more than 10 000 sites/cell) and that several
thousand CD148 sites per cell were found in a subpopulation. The number
of CD100 (or CD148) sites per cell was lower in SBL cells (Table
2). This was due to a difference in CD100
density but not to the different cell size, as evaluated by measuring the cell surfaces (data not shown). These results demonstrated that
activation of peripheral blood B lymphocytes induced CD100 expression
in cell populations with a CD38+CD138
phenotype, in which subsets of cells coexpressed CD148. Note that no
CD100 induction was observed when IL-4 was replaced by IL-10, or in the
absence of SAC.
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Table 2.
Quantified expression of CD100, CD148, and CD45 in
CD38+CD138 SBL and LBL populations of
activated peripheral blood B lymphocytes
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Finally, activation of purified tonsillar B lymphocytes (97% of
CD19+ and 86% CD23+ cells after activation),
under the same experimental conditions as for peripheral blood cells,
also induced CD100 expression in a population of
CD38+CD138 large cells: in 2 independant
experiments with different donors that gave the same results, 87% of
cells were CD100+, with similar fluorescence intensity as
in peripheral blood cells (data not shown).
To confirm that CD100 was differentially expressed during
B-cell activation and maturation, CD100 expression was quantified in 14 B-cell lines which are characteristic of various B-cell differentiation
stages (Table 3). The number of CD100
antigenic sites per cell was low (2000) in pre-B cell lines and was
higher in 2 of 3 BL lymphoblastoid cell lines. The highest levels (more than 10 000 sites/cell) were found in cell lines derived from EBV-transformed normal B lymphocytes, corresponding to the phenotype of
activated B lymphocytes, and in a pre-plasma cell line and some plasma
cell lines that express more mature phenotypes. Note also
that CD45 expression was high in most cell lines, but low or hardly
detectable levels in 4 out of 5 plasma cell lines (Table 3). We
concluded that CD100 was expressed at different levels on B-cell lines,
depending on their activation and differentiation phenotypes.
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Table 3.
Quantified expression of CD100, CD45, and CD148 and
their associated PTP activities in B-cell lines at various stages of
differentiation
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CD100 was previously reported to be associated with PTP activity in
human T cells.5 To test the possibility of such an association in B cells, PTP activity was assayed in CD100
immunoprecipitates obtained from 12 B-cell lines. The enzymatic
activity of the transmembrane PTP CD45 was measured simultaneously as a
positive control. CD101 immunoprecipitates were also assayed in
parallel as negative controls, since the cell surface molecule CD101
was not expressed in these cell lines.26 As
shown in Table 3, PTP activity was detected in CD100 immunoprecipitates
over CD101 negative control values in most cell lines, with 3 exceptions: 1 BL cell line (Sanchez), and 2 plasma cell lines
(RPMI-8226 and XG-2). The levels of PTP activity associated with CD100
depended on each cell line regardless of its differentiation phenotype
and did not seem to be related to the number of CD100 sites per cell.
To identify the PTP associated with CD100, we tested the hypothesis of
an association with CD45, because most cell lines with phenotypes
ranging from pre-B to pre-plasma cell stages expressed high numbers of
CD45 sites per cell and displayed high levels of CD45 PTP activity
(Table 3). To this end, CD45 immunodepletion experiments were performed
on cell lysates before immunoprecipitations and PTP assays. As
exemplified with the Eskol cell line in Figure 2, CD45 depletions induced nearly a
complete loss of PTP activity in CD100 immunoprecipitates and CD45
immunoprecipitates, as compared with the values obtained with negative
control depletions (SHP-1; see "Materials and Methods"). As
expected, these depletion control values were reduced compared with the
basal levels of PTP activity found in both CD100 and CD45
immunoprecipitates from undepleted lysates, probably owing to protein
loss and dilution during the 4 cycles of depletion. Because CD22 is a
cell surface molecule known to associate with CD45,15 CD22
immunodepletions were also performed in parallel (Figure 2). PTP
activity was detected in CD22 immunoprecipitates. As expected, this
activity was completely abolished upon CD22 or CD45 depletions, whereas
PTP activity measured in CD100 immunoprecipitates was not greatly
modified by CD22 depletion. Thus, the data obtained with pre-plasma
Eskol cell line clearly showed that (1) PTP activity immunoprecipitated
with CD100 resulted from CD100-CD45 association and (2) this
association did not seem to involve the CD22 molecule. Similar data
(not shown) were obtained with a number of other cell lines, such as
Reh-6 (pre-B), DZ (EBV-activated normal lymphocytes), or Ramos
(lymphoblastoid). Therefore, CD100 was associated with CD45 PTP
activity in most B-cell lines with phenotypes ranging from pre-B to
pre-plasma cell stages, including the phenotype of activated B
lymphocytes.
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| Fig 2.
CD100-bound PTP activity is due to association of CD100
with CD45 PTP in Eskol cells.
Cell lysates were CD45-immunodepleted through the use of 4 cycles of
depletion with O509 mAb. Negative control depletions consisted of SHP-1
immunodepletions (see "Materials and Methods"). Experiments of
CD22 immunodepletion were done in parallel. Then, depleted lysates were
immunoprecipitated with BJ45 (anti-CD45), BD16 (anti-CD100), or an
anti-CD22 mAb, and the immunoprecipitates were assayed for PTP
activity. Immunoprecipitates from undepleted lysates were also assayed
to determine the basal levels of PTP activity. Negative control values
of PTP activity were obtained by using depleted and undepleted lysates
that were immunoprecipitated with an anti-CD101 mAb (BB27); this
procedure was based on the observation that this cell surface molecule
was not expressed on Eskol cells. All procedures are
detailed in "Materials and Methods."
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To confirm the physical association between CD100 and CD45 molecules,
Western blotting experiments using an anti-CD45 mAb were performed on
CD100 immunoprecipitates from cell lines with pre-B to pre-plasma cell
phenotype. Results obtained with pre-plasma Eskol cells showed clearly
that CD45 coprecipitated with CD100 (Figure
3). The intensities of the bands were
similar to those detected with CD22 immunoprecipitates, although weaker
than the intensities of the bands observed with CD45 immunoprecipitates (positive controls). The different negative controls used (CD2 immunoprecipitates or blotting of CD100 immunoprecipitates with an
anti-SHP-1 antibody) remained repeatedly negative, even after prolonged exposure of the blots (Figure 3 and not shown). Similar results were observed with other cell lines, such as Reh-6 (pre-B), Raji (lymphoblastoid), or JY (EBV-activated normal lymphocytes). These
results demonstrated the molecular interaction between CD100 and CD45
in cell lines with pre-B to pre-plasma cell phenotypes.
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| Fig 3.
Co-immunoprecipitation of CD45 with CD100 in Eskol cells.
Cells lysates were immunoprecipitated with different mAbs: BD16
(anti-CD100), an anti-CD2 (O275) as a negative control, and an
anti-CD22 (a molecule known to associate with CD45) and an anti-CD45
(O509) as positive controls. Immunoprecipitates (IP) and
cell lysate were subjected to SDS-PAGE, and proteins were transferred
on PDVF membrane and blotted with a mixture of anti-CD45 mAbs (O509 and
BJ45). Immunoblotted proteins were revealed with goat anti-mouse
horseradish peroxidase complex and a system of enhanced
chemoluminescence. For detailed experimental procedures, see
"Materials and Methods." We show 2 distinct Western blots: (A)
Western blot exposed for 1 minute. (B) Western blot in which cell
lysate and CD45 IP were exposed for 5 seconds while CD100 and
CD22 IP were exposed for 30 seconds.
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The association of CD100 with CD45 PTP activity was then investigated
in the XG-1 plasma cell line that expressed a high number of CD45 sites
per cell (Table 3). Results showed clearly that the levels of PTP
activity immunoprecipitated with CD100 remained unchanged after CD45
depletion compared with depletion control values (Figure
4) and that no CD100-CD45 coprecipitation
could be visualized in Western blotting experiments (data not shown). In addition, 2 plasma cell lines (U-266 and LP-1) also displayed CD100-associated PTP activity, despite no or hardly detectable CD45
expression (Table 3). Consequently, CD100 was not associated with the
CD45 PTP in plasma cell lines corresponding to the terminal stage of
differentiation.
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| Fig 4.
CD100-bound PTP activity is not due to association of
CD100 with CD45 PTP in XG-1 cells.
Cell lysates were CD45-immunodepleted before CD100 or CD45
immunoprecipitations and PTP assays, as described in Figure 2 and
detailed in "Materials and Methods." Immunodepletions of CD101
(not expressed on XG-1 cells) with the BB27 mAb were used as negative
control depletions. Basal levels of PTP activity were measured in
immunoprecipitates from undepleted lysates. CD101 immunoprecipitates
from depleted and undepleted lysates were also assayed for PTP activity
to determine the negative control values.
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This indicated the association of CD100 with another PTP. The possible
association between CD100 and CD148, a recently identified cell surface
molecule with PTP activity,17-19 was therefore examined. We
first investigated CD148 expression and PTP activity in various B-cell
lines. We found that CD148 was expressed in some but not all of the
B-cell lines tested and that there was a good relationship between the
number of CD148 antigenic sites per cell and the levels of PTP activity
immunoprecipitated with CD148 (Table 3). No CD148 expression was
detected on LP-1 cells, indicating that CD148 was not the
CD100-associated PTP in this cell line. In contrast, elevated numbers
of CD148 sites per cell were found on XG-1 and U-266 cells (12 500 and
4800, respectively). We further investigated the effects of CD148
immunodepletion on the PTP activity immunoprecipitated with CD100 in
these 2 cell lines. In both cases, CD148 depletion reduced by 20% to
25% the PTP activity measured in CD100 immunoprecipitates, while this
activity was completely abolished in CD148 immunoprecipitates, as
exemplified for XG-1 cells in Figure 5.
Although the effect of CD148 depletion was rather weak, it was
reproducibly observed in repeated experiments. Thus, CD148 could
participate only partly in the CD100-associated PTP activity in XG-1
and U-266 cells. These data provided evidence for the association of
CD100 with at least another major PTP, yet unidentified, in plasma
cells.
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| Fig 5.
Effect of CD148 immunodepletion on CD100-associated PTP
activity in XG-1 cells.
CD148 immunodepletions with a mixture (1:1) of 2 anti-CD148 mAbs
(143-41 and A3) and CD101 immunodepletions (negative control
depletions) were performed on cell lysates. Depleted and undepleted
lysates were immunoprecipitated with either BD16 (anti-CD100), BB27
(anti-CD101), or a mixture (1:1) of the 2 anti-CD148 mAbs, and the
immunoprecipitates were then assayed for PTP activity. All experimental
procedures are described in "Materials and Methods."
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In order to know whether CD100 was also associated with PTP activity in
peripheral blood B lymphocytes, resting cells were activated as
described above, and the CD100+ positive cells were
isolated through cell sorting. Activated B lymphocytes were negatively
selected for CD138 and granulosity to avoid triggering of CD100 or
CD38. The sorted cells corresponded to
CD100+CD38+CD138 cells from
LBL and SBL populations. PTP activity was then assayed in CD100, CD148,
and CD45 immunoprecipitates. Results of the experiments performed with
9 different donors are shown in Table 4.
PTP activity was consistently found at various levels in CD148 and CD45
immunoprecipitates. PTP activity immunoprecipitated with CD100 was
detected in 6 out of 9 donors (donors 3 to 8) although at rather low
levels, but not in donors 1, 2, and 9.
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Table 4.
Phosphatase activities in sorted
CD100+CD38+CD138 cells
induced by activation of peripheral blood B lymphocytes
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CD45 and CD148 immunodepletion experiments were carried out to
characterize the PTP activity immunoprecipitated with CD100 in the
sorted CD38+CD138 cells. CD45 depletion
was found to reduce PTP activity in CD100 immunoprecipitates to levels
corresponding to negative control values (CD101 immunoprecipitates), as
shown by the example of donor 8 in Table 5.
In contrast, CD148 depletion had no effect, with the exception of 1 donor for which a decrease of 30% was detected (data not shown). In
addition, CD45 depletion reduced CD100-immunoprecipitated PTP activity
to negative control levels in
CD100+CD38+CD138 cells
sorted after activation of purified tonsillar B lymphocytes, under the
same experimental conditions of activation and cell sorting as for
peripheral blood cells. Results of 1 of 2 similar experiments are shown
in Table 5. We concluded that CD100-associated PTP activity resulted
mainly from the association between CD100 and CD45 in
CD38+CD138 cells induced by activation
of peripheral blood as well as tonsillar B lymphocytes.
View this table:
[in this window]
[in a new window]
|
Table 5.
Effect of CD45 immunodepletion on PTP activity
immunoprecipitated with CD100 in
CD38+CD138 cells sorted from activated
peripheral blood and tonsillar B lymphocytes
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Discussion |
In the present study, we show that the human leukocyte semaphorin
CD100 is differentially expressed and can functionally associate with
distinct PTPs in B cells, depending on their activation and differentiation stage.
The induction of CD100 during T-cell activation has been previously
reported.2-5 Our quantification experiments demonstrate for
the first time that CD100, which was weakly expressed on resting peripheral blood B lymphocytes (1000 sites/cell) and weaker than on
resting T lymphocytes and NK cells (several thousand sites per cell),
was induced at a high density (more than 10 000 sites per cell) upon
activation in a population of large B lymphocytes positive for CD38 but
negative for CD138. CD100 was also induced but at a lesser extent in a
population of SBLs displaying the same
CD38+CD138 phenotype. Furthermore, in
each of these cell populations, a subpopulation coexpressed CD148,
which was recently shown to clearly identify the memory B-cell
phenotype.27 Thus, these results indicated that CD100 was
induced during activation and maturation of circulating B lymphocytes
toward a memory B-cell phenotype. Interestingly, CD100 induction was
also observed in a population of activated tonsillar B lymphocytes
exhibiting the CD38+CD138 phenotype.
That CD100 was differentially expressed during B-cell maturation was strengthened by our results on B-cell lines: the number
of CD100 sites per cell was low (2000) in pre-B cell
lines in agreement with the previous observation that
CD100 was weakly represented on immature hematopoietic
precursors,2,3 but was high (more than 10 000 sites/cell)
in cell lines with a phenotype of activated lymphocytes and with more
mature phenotypes (pre-plasma and some plasma cell lines).
Our data suggested a role for CD100 in cell surface signaling events
involved in B-cell activation and differentiation. Therefore, we
investigated whether CD100 was associated with transmembrane PTPs known
to be implicated in these processes. CD100 was found to be associated
with PTP activity in activated peripheral blood B lymphocytes from most
donors as well as in most B-cell lines. We clearly identified CD45 as
the CD100-associated PTP in cell lines, with phenotypes ranging from
pre-B to pre-plasma cell stages. We also found that CD100 was
associated with CD45 PTP activity in
CD38+CD138 cell populations of activated
peripheral blood and tonsillar B lymphocytes, as previously reported
for activated T cells.5 In contrast, CD100 was not
associated with CD45 in plasma cell lines representing the terminal
stage of differentiation, including 1 expressing CD45 at a high level
(XG-1). Consequently, at least another PTP was involved in the high
CD100-associated PTP activity observed in several plasma cell lines. We
further examined the possible association of CD100 with CD148, a
transmembrane PTP previously reported to be expressed by some but not
all B-cell lines.19 While poorly or not expressed on BL and
EBV-transformed cell lines, CD148 was present at a rather elevated
level on a plasma cell line and 2 of 5 plasma cell lines. In these
latter, however, CD148 did not seem to play a crucial role in
CD100-associated PTP activity. This indicated the involvement of
another PTP in plasma cells, as evidenced by the observation that a
plasma cell line (LP-1) showing a high level of
CD100-immunoprecipitated PTP activity did not express either CD148 or CD45.
Although we cannot exclude the possibility that some results on B-cell
lines may be due to intrinsic properties that were not directly related
to the differentiation state, our data suggest a switch in the PTP
associated with CD100 at the terminal stage of B-cell differentiation.
The following model can be proposed: CD100 is weakly expressed in pre-B
cells and resting B lymphocytes and is induced at high levels upon
B-cell activation. Until this stage, CD100 is associated with the CD45
PTP. This is still the case when B lymphocytes are committed to
differentiate toward either memory cells or pre-plasma cells. However,
at terminal differentiation into plasma cells, a switch would occur in
the CD100-associated PTP from CD45 to another, yet unidentified PTP. In
certain differentiating B-cell subsets that express CD148 at elevated
levels, the CD100-associated PTP might also switch from CD45 to CD148.
Finally, in some terminally differentiated cells, CD100 could be no
longer associated with a PTP (as illustrated by RPMI-8226 or XG-2 cells).
According to this model, the selective interactions of CD100 with
several PTPs might be critical for B-cell activation and differentiation signaling. The association of CD100 with CD45 might
have a permissive effect on the positive signals delivered by CD45
during B-cell activation and proliferation. In support of this
hypothesis, serine phosphorylation of CD45 is known to enhance its PTP
activity,28 and it has been recently reported that CD100
can associate with serine kinase activity in T cells.6 However, it cannot be excluded that CD100 might negatively regulate the
CD45 PTP activity. In this view, CD100, which was shown to exist as a
dimeric form,2 might induce CD45 dimerization, known to
inhibit its PTP activity.29 Therefore, it would be of
interest to determine whether CD100 triggering results in stimulation
or inhibition of B-cell proliferation. Additional questions remain to
be elucidated regarding the association of CD100 with CD45. For
instance, we do not know whether CD100 is actually a receptor (for a
ligand presented by other cells) that would be involved in the positive
or negative regulation of CD45 activity or is instead a substrate for
CD45 since CD100 has a tyrosine residue in its intracellular domain.
Neither do we know whether CD100 interacts with CD45 directly or
through other molecules.
After switching, the association of CD100 with the unidentified PTP
activity might be involved in such processes as termination of
proliferation signals or initiation of terminal differentiation signals
leading to the formation of plasma cells. The characterization of this
CD100-associated PTP activity will determine whether it corresponds to
a novel transmembrane PTP or to a previously described enzyme. At
present, the possibility that this PTP activity corresponds to SHP-1,
known to deliver a negative signal during B-cell activation, can be
excluded since CD100 does not contain an ITIM motif and thus cannot
bind SHP-1.7,15 Furthermore, the molecular basis for
CD100-associated PTP switching also remains to be investigated. Among
several hypotheses, it can be assumed that the unidentified PTP, either
induced or activated from a preexisting inactive form, could have a
better affinity for CD100 than CD45. Alternatively, structural changes
of CD100 might be involved.
In other respects, our observations that CD148 was expressed and
showed a functional enzymatic activity in particular
subpopulationsof differentiating B cells (activated lymphocytes
with a memory cell-like phenotype) or in B-cell lines
representing terminal differentiation stages (pre-plasma and plasma
cell lines) seem of interest. Indeed, although the exact role of CD148
in B-cell activation and differentiation has not been described so far,
it was recently reported that CD148 negatively regulates T-cell
activation.30
Finally, by showing the selective and functional interactions of CD100
with several PTPs in B cells, the present work enlarges the field of
CD100 partners: CD100 associates with not only protein kinases31 but also phosphatases in B cells, as in T
cells.5,6 Thus, CD100 appears to be the only transmembrane
semaphorin clearly behaving as a cell surface receptor. No similar data
are available regarding transmembrane semaphorins of the nervous system
although Sema VIb, whose function is unknown, could bind
c-src.32 Concerning soluble semaphorins whose function in
the nervous system is known, they all act as ligands for specific
receptors on target cells.33-35 CD100 may also have effects
through binding to a counter-receptor at B-cell surface, as reported
elsewhere using CD100-transfected cells.7 In this view, a
soluble form of CD100 can be cleaved from T-cell surface,5
and interestingly, the first evidence for a semaphorin receptor in the
immune system has been recently provided.36 Therefore,
CD100 turns out to be a discrete semaphorin.
| |
Acknowledgments |
We are indebted to Dr Ramon Vilella for his generous gift of monoclonal
antibody, Dr Bernard Klein and Professor Jean-Claude Brouet for kindly providing cell lines, and Professor
Stuart F. Schlossman for CD154-transfected cells and helpful
discussions. We gratefully acknowledge Dr Jean-Pierre Roy for preparing
monoclonal antibodies and Dominique Charue for flow cytometry analysis.
| |
Footnotes |
Supported by INSERM, Sidaction, the Association pour la
Recherche sur le Cancer and the University of Paris 12.
C.B. and S.D. contributed equally to this work.
Submitted April 26, 1999; accepted September 22, 1999.
Reprints: Christian Billard, INSERM Unit 448, Faculté de
Médecine, 8 rue du général Sarrail, F-94010
Créteil (cedex), France.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction