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Division of Haematology, Hanson Centre for Cancer Research, IMVS, Adelaide, South Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1-PECAM-1
receptors containing the extracellular and transmembrane portions of
murine FcRIIB1 and the cytoplasmic domain of human PECAM-1. These
chimeric receptors were stably expressed in chicken DT40 B cells either
as wild-type or mutant cells deficient in SHP-1-/-,
SHP-2-/-, SHIP-/-, or
SHP-1/2-/- and then assessed for their ability to inhibit
B cell Ag receptor (BCR) signaling. Coligation of wild-type
FcRIIB1-PECAM-1 with BCR resulted in inhibition of intracellular
calcium release, suggesting that the cytoplasmic domain of PECAM-1 is
capable of delivering an inhibitory signal that blocks BCR-mediated
activation. This PECAM-1-mediated inhibitory signaling correlated with
tyrosine phosphorylation of the FcRIIB1-PECAM-1 chimera, recruitment
of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation
of calcium mobilization responses. Mutational analysis of the two
tyrosine residues, 663 and 686, constituting the immunoreceptor
tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine
residues play a crucial role in the inhibitory signal. Functional
analysis of various PTP-deficient DT40 B cell lines stably expressing
wild-type chimeric FcRIIB1-PECAM-1 receptor indicated that
cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and
SHP-2, were both necessary and sufficient to deliver inhibitory
negative regulation upon coligation of BCR complex with inhibitory
receptor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Ig
subunits. These ITAM motifs
(D/ExxYxxL/Ix6–8YxxL/I) are essential for
initiating a cascade of intracellular biochemical events, including
activation of p60Src and
p72Syk protein-tyrosine kinases, activation of
phosphatidylinositol 3-kinase and phospholipase C-, generation of
phosphoinositide turnover, and calcium mobilization. Balancing the
threshold of cellular activation and termination of BCR-mediated
signals is principally mediated by coligation of inhibitory receptors
such as FcRIIB1, paired Ig-like receptor (PIR-B), Ig-like transcript
ILT2, biliary glycoprotein BGP-1 (CD66), and CD22 (4).
These Ig immunoreceptor tyrosine-based inhibitory motif (ITIM)
receptors belong to the inhibitor receptor superfamily, which are
characterized by the presence of intracytoplasmic ITIM
(I/VxxYxxL/V/Ix>20I/VxxYxxL/V/I) that recruit
and activate protein-tyrosine phosphatases (PTPs) such as SHP-1, SHP-2,
and/or inositol polyphosphate 5-phosphatases, SHIP, and SHIP2
(5). Coligation of BCR with these inhibitory receptors
leads to premature termination of inositol trisphosphate production,
inhibition of extracellular Ca2+ influx, and a
blockage of blastogenesis (6).
Our recent studies and others have defined that platelet endothelial
cell adhesion molecule-1 (PECAM-1/CD31) contains intracytoplasmic ITIM
motifs, which recruit and activate the PTPs, SHP-1 and SHP-2, under
physiologically relevant conditions (7, 8, 9, 10, 11). Our
biochemical analysis suggested that SHP-2 binds to PECAM-1 with high
affinity compared with SHP-1, suggesting that when these PTPs are
located in the same subcellular compartment, SHP-2 may be the preferred
substrate for PECAM-1 (12, 13). However, in vitro
biochemical studies and PTP requirements for inhibitory receptor
signaling in the context of living cells are often quite different. For
example, previous studies have highlighted that while FcRIIB1 ITIM
motif was able to physically associate with SHP-1 and SHIP, its
functional analysis of chimeric receptors expressed in DT40 B cell
lines devoid of SHP-1 or SHIP has demonstrated that SHIP was the
preferred substrate for FcRIIB1 (14, 15). In addition,
functional assessment of PIR-B revealed that SHP-1 and SHP-2 PTPs are
required for PIR-B-inhibitory signaling (16). Based upon
these findings, we wanted to test the functional importance of PECAM-1
cytoplasmic ITIM motifs and the requirement of PTPs for mediating
PECAM-1-inhibitory signaling.
Previous studies have suggested that PECAM-1 may be expressed on CD19-positive B cells derived from bone marrow (17), reactive plasma cells defined by CD38+++ (18), and as a CD38 ligand found in the lymphoid compartment of follicular mantle B cells and plasma cells (19). Our recent studies using primary human tonsillar tissue have demonstrated that PECAM-1 is expressed on discrete B lymphocyte subpopulations with the phenotype of naive follicular mantle zone cells and not germinal center-specific cells (20). As memory B lymphocytes display several intrinsic differences than naive B lymphocytes, including a lower threshold for cellular activation, an ability to directly present Ag to Th cells, and a longer life span (21), it is likely that the presence of inhibitory receptors such as PECAM-1 in naive B lymphocytes plays a negative regulatory role in their activation and differentiation pathways. At present, there is no evidence to suggest that PECAM-1 may play a regulatory role in B cell activation, maturation, or differentiation.
To test whether PECAM-1 is capable of delivering inhibitory signals in
B cells and the functional requirement of PTPs for this inhibitory
signaling, we generated chimeric FcRIIB1-PECAM-1 receptors
containing the extracellular and transmembrane portions of FcRIIB1
and the cytoplasmic domain of PECAM-1. These chimeric receptors were
stably expressed in DT40 B cells either as wild-type or mutant cells
deficient in SHP-1-/-,
SHP-2-/-, SHIP-/-, or
SHP-1/2-/- and assessed for their ability to
inhibit ITAM-dependent BCR signaling. Our studies demonstrate that the
PTPs, SHP-1 and SHP-2, are required for PECAM-1-mediated inhibitory
signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chicken DT40 B cell lines were maintained in RPMI 1640 medium
supplemented with 10% (v/v) heat-inactivated FCS, 1% (v/v) chicken
serum, 4 mM glutamine, 50 µM 2-ME, and antibiotics. Wild-type mouse
FcRIIB1, pBabe, and pA PURO constructs were kindly provided by
Jeffrey Ravetch (The Rockefeller University, New York, NY) and have
been previously described (14). Human PECAM-1 Y663,686F
construct has been previously described (12). Intact and
F(ab')2 rabbit anti-mouse IgM (Zymed, San
Francisco, CA), mouse anti-chicken IgM mAb M4 (mouse IgM) (Southern
Biotechnology Associates, Birmingham, AL), goat anti-chicken IgM
µ-chain specific (Bethyl Laboratories, Montgomery, TX), 2.4G2
(PharMingen, San Diego, CA), HRP anti-PY Plus Ab (Zymed), and
polyclonal anti-human SHP-2 (Santa Cruz Biotechnology, Santa Cruz,
CA) were purchased. Polyclonal anti-chicken SHP-1 Ab was kindly
provided by Tomohiro Kurosaki (Kansai Medical University, Moriguchi,
Japan). Polyclonal anti-SHIP antiserum was kindly provided by Mark
Coggeshall (Oklahoma Medical Research Foundation, Oklahoma City, OK).
The cDNA of mouse FcRIIB1 and human PECAM-1 receptor were fused at
the carboxyl-terminal residue of the transmembrane domain of each
protein by using overlap PCR with the following junction primers:
FcRIIB1-PECAM-1, 5'-TGGTCTATCTCAGCCAAATGTTATTTTCTG-3', and
PECAM-1-FcRIIB1 primer, 5'-ATAACATTTGGCTGAGATAGACCAAGGATA-3'.
These chimeric cDNAs were then subcloned into pA PURO expression
vector. Sequence integrity was confirmed by automated ABI nucleotide
sequence analysis. A total of 20 µg of expression constructs was
transfected by electroporation at 250 V and 960 µF into the various
wild-type and PTP-deficient chicken DT40 B cell lines. Expression of
chimeric molecules was established by drug selection using 1 µg/ml
puromycin (Sigma, St. Louis, MO) for chicken DT40 B cell lines. Cell
surface expression of each transfected cell line was analyzed by
flow cytometry studies using 1 µg/ml 2.4G2 (anti-FcRIIB1) Ab,
followed by staining with 4 µg/ml FITC-conjugated anti-rat IgG,
and assessed on a FACScan (Becton Dickinson, San Jose, CA).
Generation of SHP-1-/--, SHP-2-/--, SHP-1/2-/--, and SHIP-/--deficient DT40 cell lines
Knockout PTP-deficient cell lines were created by homologous
recombination of targeting genomic constructs using DT40 chicken B cell
line, as previously described (16). These PTP-deficient
cell lines were purchased from Riken Cell Bank (Tuskuba Science City,
Japan). Evidence of null mutant knockout cell lines was confirmed by
Western blot analysis of cellular lysates using polyclonal
anti-SHP-1, anti-SHP-2, and anti-SHIP Abs that cross-react
with chicken forms of the PTPs, as described above (Fig. 5
A–C).
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A total of 4 x 108 resting chicken DT40 B cells was solubilized in 1 ml of Triton lysis buffer (2% Triton X-100, 10 mM EGTA, 15 mM HEPES, 145 mM NaCl, 0.1 mM MgCl2, 1 mM PMSF, 20 µg/ml leupeptin, and 2 mM sodium orthovanadate, pH 7.4) and constantly mixed on a nutator (Clay Adams) at 4°C for 1 h. Triton-soluble and -insoluble (cytoskeletal) fractions were isolated by centrifugation at 15,000 x g for 15 min at 4°C. Following separation of the 15,000 x g Triton-soluble fraction, the lysate was precleared with 50 µl of 50% slurry of streptavidin-agarose beads for 30 min at 4°C and then centrifuged at 4,000 rpm for 5 min. One milligram of precleared cell lysates was aliquoted into separate Eppendorf tubes and incubated with 10 µg of each respective biotinylated PECAM-1 cytoplasmic domain peptide ± Tyr (PO4) overnight at 4°C. The design and preparation of PECAM-1 phosphopeptides have been previously described (11). Peptido-protein complexes were then isolated by addition of 50 µl of 50% slurry of streptavidin-agarose beads and incubated for 1 h at 4°C, and then beads were washed five times with immunoprecipitation buffer. In some experiments, DT40 cell lysates were precleared with 50 µl of 50% protein G-Sepharose 4B beads and were then sequentially incubated with 4 µg of 2.4G2 IgG and 50 µl of 50% protein G-Sepharose 4 Fast Flow (Pharmacia LKB Biotechnology, Uppsala, Sweden). Bound proteins were eluted from the beads by boiling for 10 min in 30 µl SDS reducing buffer and resolved on a 10% SDS-PAGE gel, then transferred to polyvinylidene difluoride membrane and probed with each respective polyclonal anti-SHP-1, anti-SHP-2, or anti-SHIP Ab. The membrane was additionally incubated with a secondary HRP-conjugated goat anti-rabbit IgG (1:10,000) and visualized by ECL detection.
Calcium mobilization assays and receptor coligation
Cells (5 x 106 cells/ml) were loaded
with 3 µM fura 2-AM (Molecular Probes, Eugene, OR) in medium at room
temperature for 45 min. Cells were washed with PBS twice and
resuspended in the same cell concentration in PBS supplemented with 1
mM CaCl2 and 1 mM MgCl2.
Two milliliters of cell suspension were added to a cuvette with a small
stirrer. Calcium mobilization profiles were recorded at 510 nm emission
wavelength excited by 340 nm and 380 nm using a Perkin-Elmer
Luminescence Spectrophotometer LS-50B (Department of Medical
Biochemistry, Flinders University, Adelaide, Australia). The basis of
coligation of BCR with inhibitory receptor that contains the
extracellular moiety of murine FcRIIB is based upon the fact that
FcRIIB can be effectively cross-linked by Fc portions presented as
multivalent ligands. In this case, the anti-chicken IgM Ab is of
mouse IgM isotype and requires cross-linking with intact rabbit
anti-mouse IgM for coligation purposes, which
F(ab')2 rabbit anti-mouse IgM is not capable
of coligation. Stimulation of chicken DT40 B cells was conducted by
cross-linking BCR with F(ab')2 rabbit
anti-mouse IgM (5 µg) and mouse anti-chicken IgM Ab (2
µg/ml). Coligation of BCR to transfected chimeric receptor was
obtained by preincubation of intact rabbit anti-mouse IgM (5 µg)
with chicken DT40 B cells at room temperature for 10 min, followed by
mouse anti-chicken IgM (2 µg/ml). Maximal
(Rmax) calcium release from intracellular
calcium store was measured in the presence of 20 µM ionomycin, and
minimal calcium release (Rmin) was obtained
by addition of 6.25 mM EGTA. Calibration and calculation of calcium
concentration were performed as described (22). Cumulative
calcium mobilization was evaluated by integration for 4 min of calcium
mobilization over the baseline given before stimulation, as described
by the manufacturer.
NF-AT luciferase assays
A total of 1 x 107 cells in 0.5 ml PBS was electroporated at 250 V and 975 µF in the presence of 20 µg of NF-AT luciferase gene construct, pxpGM55 IL140 x 3 containing NF-AT-p binding sites (23) (supplied by Peter Cockerill, Hanson Center for Cancer Research, Adelaide, Australia) and then recultured in 30 ml of DT40 complete medium. After 24-h incubation at 39°C, cell suspensions were centrifuged and cells were resuspended in 1 ml of medium and aliquoted into six-well plates. For stimulation of DT40 B cells, F(ab')2 (5 µg) anti-mouse IgM with 2 µg/ml anti-chicken IgM (BCR stimulation alone) or intact (5 µg) rabbit anti-mouse IgM with anti-chicken IgM (BCR + inhibitory receptor) was incubated for 6 h at 39°C. Cells were then lysed, normalized for protein content, and measured for luciferase activity using a luciferase reporter assay system (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether the cytoplasmic domain of PECAM-1 is capable of
inhibiting BCR activation, a chimeric molecule expressing the
extracellular and transmembrane portions of murine FcRIIB1 was fused
with the cytoplasmic domain of human PECAM-1 using an overlap PCR
strategy (Fig. 1). As DT40 B cells are of
immature B cell lineage, they are likely to express endogenous chicken
PECAM-1 and FcRIIB1. For this reason, we opted to introduce
a chimeric receptor into the DT40 B cells expressing the cytoplasmic
domain of human PECAM-1 fused to the extracellular and transmembrane
portions of murine FcRIIB1, so that we could control ligand
interactions through species-specific interactions. This chimeric
molecule was transfected into chicken DT40 B cells, and selection
commenced with puromycin to obtain stable transformants. Expression
levels of stable transformants were assessed by flow cytometry analysis
using rat anti-mouse FcRIIB1 mAb, 2.4G2 (Fig. 2A). The expression levels for
the various stable clones of chimeric receptors were similar and
comparable with the levels of PECAM-1 expression on normal naive
primary B cells (19). DT40 cells expressing wild-type
FcRIIB1-PECAM-1 were stimulated by BCR cross-linking alone (Fig. 2B, solid line) or coligation of BCR and wild-type
FcRIIB1-PECAM-1 (Fig. 2B, dashed line), and calcium
mobilization responses were recorded. To exclude the possibility of
spontaneous calcium mobilization in the absence of BCR cross-linking,
we monitored unstimulated DT40 B cells over time and found no evidence
of calcium release (data not shown). In addition, ligation of
FcRIIB1 alone with 2.4G2 mAb did not produce evidence of calcium
release (data not shown). Coligation of wild-type FcRIIB1-PECAM-1
with BCR resulted in inhibition of intracellular calcium release. To
evaluate whether PECAM-1 cytoplasmic domain inhibited BCR-induced
calcium mobilization by release of calcium from intracellular stores
and/or calcium influx, experiments were performed on
DT40-FcRIIB1-PECAM-1 in the absence of extracellular calcium.
Incubation with EGTA before cross-linking of BCR with wild-type
FcRIIB1-PECAM-1 further decreased the calcium mobilization,
indicating that wild-type FcRIIB1-PECAM-1 acts on calcium release
from intracellular stores (Fig. 2C). To test for specificity
of this calcium signal, a downstream effector of B cell
calcium-dependent signaling pathway, NF-AT transcriptional factor
activation was measured, as it is dependent upon the calcium-sensitive
translocation of NF-AT cytosolic component to the nuclear compartment.
In these experiments, transcriptional activation of the NF-AT
luciferase reporter was inhibited by coligation of the BCR to
this wild-type FcRIIB1-PECAM-1 chimeric molecule (Fig. 2D). Our results indicate that the cytoplasmic domain of
PECAM-1 is capable of delivering an inhibitory signal that blocks
BCR-mediated activation in B cells.
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The presence of ITIM-bearing sequences in the PECAM-1 cytoplasmic
domain suggests that upon tyrosine phosphorylation, SH2-containing
PTPs, SHP-1 and SHP-2, or inositol polyphosphate 5'-phosphatase, SHIP,
could be recruited. This hypothesis is supported by recent studies
showing that upon integrin
IIb3-mediated
platelet aggregation, aggregation of the high affinity IgE receptor on
mast cells, or aggregation of the TCR complex on T cells, PECAM-1
becomes tyrosine phosphorylated and recruits SHP-2 and/or SHP-1 in an
ITIM-dependent manner (7, 8, 24). At present, there is no
information on induction of PECAM-1 tyrosine phosphorylation in the
context of B cells. As ITIM motifs and SH2 domain-containing molecules
are highly conserved among various species, we would predict that when
human PECAM-1 ITIM motifs become tyrosine phosphorylated, they would be
capable of recognizing and recruiting the chicken PTPs, SHP-1 and
SHP-2. To test this hypothesis, resting chicken DT40 cell lysates were
incubated with biotinylated human PECAM-1 cytoplasmic domain
peptides ± Tyr(PO4) residues encompassing
the five tyrosine residues known to be present in the human PECAM-1
cytoplasmic domain (Y596, Y636, Y663, Y686, and Y701). These
tyrosine-phosphorylated PECAM-1 peptides mimic activated forms of the
PECAM-1 cytoplasmic domain. Bound SH2 domain-containing protein/peptide
complexes were then recovered with streptavidin-agarose beads, and
proteins resolved on an SDS-PAGE gel, and PTPs identified by respective
Abs in immunoblot analysis. As shown in Fig. 3, only the tyrosine-phosphorylated Y663
and Y686 forms of human PECAM-1 peptides could bind the chicken PTPs,
SHP-1 (A) and SHP-2 (B). However, the recruitment
of chicken SHIP was not phosphotyrosine dependent and was recruited by
residues surrounding the Y686 residue sequence (C). These
results highlight that the SH2 domain-containing PTPs are highly
conserved across species and recognize the human PECAM ITIM motifs.
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RIIB1-wild-type
PECAM-1 chimeric receptor, the recruitment of PTPs was then
confirmed in immunoprecipitation studies under conditions of BCR
stimulation with and without coligation of the inhibitory receptor. As
shown in Fig. 3
D, SHP-1, PTP was recruited upon BCR
stimulation and following coligation of the inhibitory receptor (Fig. 3D, top panel). SHP-2, protein tyrosine
phosphatase, was recruited only upon conditions of coligation of
inhibitory receptor with the BCR complex (Fig. 3D,
middle panel). This apparent difference in kinetics of
assembly between SHP-1 and SHP-2 recruitment by the PECAM-1
cytoplasmic domain has been previously described in other
physiologically relevant conditions such as integrin
IIb3-mediated
platelet aggregation (11). In contrast, SHIP was
constitutively associated with the chimeric receptor consistent with
the peptido-precipitation studies described above (Fig. 3D,
lower panel). Tyrosine residues 663 and 686 in the cytoplasmic domain of PECAM-1 are essential in PECAM-1-mediated inhibitory signaling in B cells
Our previous studies have defined that PECAM-1 contains two ITIM consensus motifs (VQY663TEV and TVY686SEV) involving tyrosine residues, 663 and 686, that upon phosphorylation are capable of recruiting and activating the PTPs, SHP-1 and SHP-2 (7, 11). In vitro surface plasmon resonance studies have suggested differences in affinity between respective ITIM motifs in their association with PTPs such as SHP-2 (12, 13). Mutation of either phosphotyrosine residue leads to loss of association of PTPs, suggesting that both tyrosine residues may be required (12). However, no functional evidence is available to assess the structural importance of each of the phosphotyrosine residues in PECAM-1-mediated inhibitory signaling in B cells.
To test this hypothesis, a chimeric receptor was constructed containing
the extracellular and transmembrane domains of murine FcRIIB1 and
the cytoplasmic domain of human PECAM-1 containing Y663,686F mutant.
This chimeric cDNA receptor was transfected into parental DT40 B cells,
and selection commenced with puromycin to obtain stable transformants.
Parental DT40 B cells expressing comparable levels of various
FcRIIB1- PECAM-1 wild-type and Y663,686F mutant (Fig. 4A) were stimulated by BCR
alone (solid line) and following coligation of BCR with inhibitory
receptor (dashed line). A significant 95% reversal of inhibition of
the calcium mobilization response was observed by coligation of BCR
with FcRIIB1-PECAM-1 Y663,686F mutant compared with
FcRIIB1-PECAM-1 wild type (Fig. 4B). These results
suggest that both ITIM tyrosine residues, 663 and 686, are capable of
mediating PECAM-1-inhibitory signaling in B cells.
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In vitro peptide-binding experiments have demonstrated that ITIM
consensus sequences within Ig-ITIM-bearing receptors are capable of
binding SHP-1 or SHP-2 PTPs and/or SHIP inositol 5'-phosphatase with
differing affinities. This is in contrast to coimmunoprecipitation
studies from cells stimulated by coaggregation of ITAM-bearing with
ITIM-bearing receptors such as BCR with FcRIIB1, in which the
phosphorylated ITIM-bearing receptor effector interactions may favor a
predominant PTP-phosphotyrosine-dependent interaction or a combination
of PTP-phosphotyrosine-dependent interactions depending upon kinetics
of association and dissociation. The potential role of non-SH2
determinants in PTP modulation of ITIM-bearing receptor inhibitory
signaling still requires clarification. Consistent with these
observations, in vivo functional studies have revealed that ITIM motifs
may deliver inhibitory signals to negatively regulate
ITAM-dependent cell activation by selective PTP-substrate-dependent
pathways. Several examples include FcRIIB1, which uses SHIP to
mediate dephosphorylation of phosphatidylinositol 3'-kinase and
extracellular signal-regulated kinase activation pathways (14, 15), while signal regulatory protein-, killer inhibitory
receptor (KIR), or PIR-B uses SHP-1 and SHP-2 to mediate
dephosphorylation of protein-tyrosine kinases, reduced activation of
mitogen-activated protein kinases, Erk1 and Erk2, and ITAM
dephosphorylation (14, 15, 16, 25). Based upon our biochemical
studies on PECAM-1 ITIM motifs, we would predict that SHP-2 and/or
SHP-1 may be required for delivery of inhibitory signals within the
context of B cells.
To test our hypothesis, chimeric receptors containing the extracellular
and transmembrane domains of murine FcRIIB1 and the cytoplasmic
domain of human PECAM-1 were transfected into wild-type chicken DT40 B
cells and DT40 cells deficient in either
SHP-1-/-, SHP-2-/-,
SHP-1/2-/-, or SHIP-/-
(Fig. 5, A–C). Following
selection with puromycin, stable clones were obtained and refined by
flow cytometry sorting using rat anti-mouse FcRIIB1 Ab, 2.4G2 (2
µg/ml), so that all stable cell lines expressed equivalent levels of
chimeric receptor (Fig. 6A).
In addition, these cell lines were assessed for IgM expression by flow
cytometry using goat anti-chicken IgM (10 µg/ml). Examination of
flow cytometric profiles revealed comparable levels of cell surface IgM
expression and stability of BCR complex before functional analysis
(Fig. 6B). Upon BCR stimulation (solid line) and coligation
of inhibitory receptor with BCR complex (dashed line), the calcium
mobilization responses were reduced in parental and
SHIP-/- DT40 B cells expressing wild-type
FcRIIB1-PECAM-1 (Fig. 6C). This inhibitory effect was
partially reversed in SHP-1-/- and
SHP-2-/- DT40 B cells expressing wild-type
FcRIIB1-PECAM-1, while it was completely abolished in
SHP-1/2-/- DT40 B cells expressing wild-type
FcRIIB1-PECAM-1 (Fig. 6C). Based upon these results, a
quantitative summary of the inhibitory effect of wild-type
FcRIIB1-PECAM-1 chimeric receptor from various genetic background
DT40 B cells was derived from three independent clones of each cell
line, as shown in Fig. 6D. These results suggest that both
SHP-1 and SHP-2 PTPs are required for mediating PECAM-1-inhibitory
signaling in B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB1-inhibitory signaling pathway is dependent on SHIP, but not
SHP-1 and SHP-2, while KIR-, PIR-B-, CD22-, and signal regulatory
protein--inhibitory signaling pathways are dependent upon either
SHP-1 and/or SHP-2, but not SHIP. The fact that PECAM-1 contains two
intracytoplasmic ITIM motifs, and that upon tyrosine phosphorylation
can recruit and activate cytoplasmic SH2-domain-bearing PTPs suggests
that these PTPs may be functionally important for in vivo delivery of
inhibitory signals. To date, little is known of the mechanism of
PECAM-1-inhibitory signaling pathways in the context of various cell
types. As ITIM-bearing molecules have the capacity to negatively
regulate cell activation induced by coaggregation with receptors
bearing ITAM motifs, we would predict that PECAM-1 would be capable of
regulating ITAM-dependent cell activation and receptor tyrosine
kinase-mediated cell proliferation. In this study, we demonstrate that
the PECAM-1 cytoplasmic domain is capable of delivering an inhibitory
negative signal that can down-modulate BCR-mediated cell activation.
Using the chicken DT40 B cell model, stable expression of chimeric
FcRIIB1-PECAM-1 receptors enabled us to test whether the PECAM-1
cytoplasmic domain was capable of producing an inhibitory negative
signal upon coligation of BCR complex with inhibitory receptor. Our
studies with the chimeric B cell IgG coreceptor, FcRIIB1, containing
the cytoplasmic domain of human PECAM-1 have established a critical
role for peptide sequences within the PECAM-1 cytoplasmic domain in
mediating an inhibitory negative signal to suppress release of calcium
from intracellular stores (Fig. 2). Our studies have also revealed that
this inhibitory signaling was dependent upon the integrity of the
PECAM-1 intracytoplasmic ITIM motifs, as mutation of
Tyr663 and Tyr686 to Phe
residues abrogated the inhibitory negative signal (Fig. 4). In
addition, analysis of various PTP-deficient DT40 B cell lines either
singly or in combination indicated that cytoplasmic SH2
domain-containing phosphatases, SHP-1 and SHP-2, were both necessary
and sufficient to mediate inhibitory signaling by PECAM-1 ITIM motifs
(Fig. 6). Based upon these findings, we can propose a model for
PECAM-1-inhibitory signaling in the context of B cells. Upon BCR
stimulation and coligation of inhibitory receptor with BCR complex,
protein-tyrosine kinases are activated and induce progressive
phosphorylation of tyrosine residues within PECAM-1 ITIM motifs, which
act as a molecular "scaffold" to coordinate the docking and
activation of cytoplasmic SH2-tandem phosphatases, SHP-1 and SHP-2. The
functional consequence of these PTP interactions involves
dephosphorylation of target substrates to prevent release of calcium
from intracellular stores to negatively regulate BCR-mediated cell
activation. Our studies demonstrate that like the KIR and PIR-B class
of molecules, the PECAM-1 cytoplasmic domain appears to inhibit
ITAM-mediated B cell signaling by preventing release of calcium from
intracellular stores only without affecting calcium influx from the
extracellular compartment, as observed for FcRIIB1 (14, 16, 27).
Our in vitro studies have revealed that PECAM-1 has differing kinetics
of association and dissociation for SHP-1 and SHP-2 PTPs. Typically,
PECAM-1 precipitates higher amounts of SHP-2 and forms a more stable
complex than with SHP-1. Under physiological conditions, it would
appear that the interaction of PECAM-1 with SHP-1 is of a transient
nature. This observation is further supported by the fact that a
catalytically inactive C453S SHP-1 could be coimmunoprecipitated with
PECAM-1 reconstituted in COS-7 cells (10). In this study,
we have demonstrated that deficiency of SHP-2 is associated with a more
significant reversal in PECAM-1-inhibitory signaling than SHP-1,
suggesting that it may be the preferred PTP functionally used by
PECAM-1. However, our studies also demonstrate that SHP-1 contributes
to the attenuation of the calcium mobilization response (Fig. 6). In
contrast, our functional data demonstrate that SHIP is dispensable for
the PECAM-1-mediated inhibitory response. Therefore, under conditions
of ITIM-dependent phosphorylation of PECAM-1, SHP-1 and SHP-2 appear to
act in concert to augment the inhibitory negative signaling. Our
finding that SHIP is constitutively associated with PECAM-1 via a
nonphosphotyrosine-dependent interaction with 681–691 aa residues may
implicate the possibility of a second regulatory mechanism under basal
conditions (Fig. 3, C and D). Interestingly,
previous studies have demonstrated that loss of
Tyr686 from exon 14 of PECAM-1 by mutation of
Tyr686
Phe or phosphorylation of the tyrosine
residue results in a change in ligand specificity from heterophilic to
homophilic binding (28).
B cell coreceptors have been demonstrated to suppress membrane Ig (mIg)
signaling via FcRIIB1 coligation with mIg by IgG-Ag complexes or
through cross-linking of mIg complex to induce tyrosine phosphorylation
of a constitutively associated B cell coreceptor such as CD22 or CD72
(29, 30). In this study, suppression of mIg signaling was
initiated by coligation of a chimeric FcRIIB1-PECAM-1 receptor with
mIg by soluble immune complexes. This approach enabled us to gain an
insight into the mechanism of inhibition by human PECAM-1 cytoplasmic
domain. However, the mechanism of mIg signaling via endogenous PECAM-1
coligation in primary B cells is not clear. Whether PECAM-1 is
constitutively associated with the BCR remains to be determined and
could not be addressed in this investigation.
Examination of human tonsillar B cells has demonstrated a developmental
pattern of PECAM-1 expression found in naive resting B cells, which is
lost upon differentiation from an immature B cell into a germinal
center memory B cell (20). These observations taken
together with results of this study would support the concept that
PECAM-1 acts as a B cell coreceptor in increasing the threshold of
sensitivity to Ag that accompanies the differentiation of immature B
cells. As B cell coreceptors have been shown to modulate the signaling
threshold for B cell tolerance as well as B cell activation, it is
possible that PECAM-1 serves to control maintenance of self-tolerance.
The important regulatory role of B cell coreceptors has been
demonstrated by targeted disruption of coinhibitory receptor genes in
mice, in which the absence of an inhibitory receptor leads to
uncoupling of signaling pathways responsible for control of B cell
tolerance and activation. Immunological defects observed include
enhanced humoral Ab and anaphylactic responses in
FcRIIB1-/- mice, constitutively activated T
cells and lymphoproliferation in CTLA-4-/-
mice, and hyperresponsive B cells and autoantibody production in
CD22-/- mice (31, 32, 33, 34, 35, 36). The
phenotype of PECAM-1-/- B cells is currently
under investigation.
In conclusion, as PECAM-1 belongs to the newly defined Ig-ITIM superfamily of receptors that possess intracytoplasmic (I/V)xYxx(L/V) ITIM motif(s) and its cytoplasmic domain has the capacity to induce an ITIM- and PTP-dependent inhibitory negative signal, we propose that it serves as a B cell coreceptor to negatively regulate the amplitude and duration of ITAM-mediated BCR signaling responses upon coligation of BCR complex with inhibitory receptor. Our studies indicate that PECAM-1 may operate as a negative regulator in hemopoietic cells that coexpress PECAM-1 and ITAM-bearing receptors providing they are positioned in close proximity. This concept is supported by recent observations of negative regulation of TCR-mediated calcium mobilization responses by PECAM-1 in Jurkat T cells (25). Additional studies will be required to elucidate the physiological significance of PECAM-1 interactions in primary B cell biology.
|
Acknowledgments |
|---|
RIIB1-pA
PURO cDNA constructs; and Dr. Mark Coggeshall (Oklahoma Medical
Research Foundation) for supply of polyclonal anti-SHIP Ab. We
thank Andrew Bert for technical advice on NF-AT luciferase assays, and
Denise Barbulesca for technical assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Denise Jackson, FAIMS, Division of Haematology, Hanson Centre for Cancer Research, IMVS, Frome Road, Adelaide, South Australia. 5000.
3 Abbreviations used in this paper: BCR, B cell receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer inhibitory receptor; mIg, membrane Ig; PECAM-1, platelet endothelial cell adhesion molecule-1; PIR-B, paired Ig-like receptor B; PTP, protein-tyrosine phosphatase; SHIP, Src homology 2 domain containing inositol polyphosphate 5’-phosphatase; SHP, Src homology 2 domain containing protein-tyrosine phosphatase.
Received for publication September 18, 2000. Accepted for publication December 22, 2000.
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References |
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RIIb1 require distinct phosphatases. J. Exp. Med. 186:473.RII-deficient mice. Nature 379:346.[Medline]
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