| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI 53233
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
mAbs, anti-PECAM-1 mAbs, or both, and goat
anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium
mobilization induced by CD3 cross-linking was found to be attenuated by
coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated
inhibition of TCR signaling was attributable, at least in part, to
inhibition of release of calcium from intracellular stores. These data
provide evidence that PECAM-1 can dampen signals transduced by
ITAM-containing receptors and support inclusion of PECAM-1 within the
family of ITIM-containing inhibitors of PTK-dependent signal
transduction. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-associated protein of 70 kDa (ZAP-70), which itself
becomes activated by tyrosine phosphorylation and enables transmission
of downstream signals for T cell activation (1). The level
of protein tyrosine phosphorylation is balanced by the activity of
protein tyrosine phosphatases (PTPs), which negatively regulate the
initial phase of T cell activation. The mechanism by which the initial
phase of T cell activation is modulated involves active transmission of
negative signals through membrane-associated molecules, such as CTLA-4
and killer cell inhibitory receptors (KIRs) (2, 3). CTLA-4
contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like
sequence that supports the binding of Src homology 2 domain-bearing PTP
(SHP)-2 (4). Upon docking and activation, SHP-2 associated
with the CTLA-4 cytoplasmic domain dephosphorylates tyrosine residues
in the ITAMs of the TCR -chain (5). Similarly, KIRs
contain an ITIM within their cytoplasmic domain, which, upon tyrosine
phosphorylation, supports the binding of the nontransmembrane PTP,
SHP-1 (6, 7). The substrates for KIR-associated SHP-1 have
not yet been determined.
Platelet-endothelial cell adhesion molecule-1 (PECAM-1), or CD31, is a
130-kDa member of the Ig gene superfamily that is expressed on the
surface of endothelial cells and circulating platelets, monocytes,
neutrophils, and certain T cell subsets. PECAM-1 was originally
assigned to the family of Ig-like cellular adhesion molecules (CAMs)
based on sequence similarity between its six extracellular Ig domains
and those of other Ig domain-containing CAMs (8). An
adhesive function for PECAM-1 is supported by the observations that
amino-terminal domains 1 and 2 mediate homophilic interactions
(9, 10) that facilitate adhesion between
PECAM-1-expressing cells (11). Recent evaluation of
the structure and biochemical properties of its cytoplasmic domain,
however, suggest that PECAM-1 may also belong to the family of Ig-like,
ITIM-containing inhibitory receptors (12). The PECAM-1
cytoplasmic domain contains numerous potential sites for
phosphorylation of serine, threonine, and tyrosine residues
(8). Of the five tyrosine residues in the human PECAM-1
cytoplasmic domain, two are surrounded by sequences that conform to an
ITIM, for which the consensus sequence is (I/V/L)xpYxx(L/V)
(6). The PECAM-1 cytoplasmic domain contains two
ITIMs, one around tyrosine 663 and the other around tyrosine 686
(reviewed in Ref. 12). The long distance between ITIMs in
the PECAM-1 cytoplasmic domain (17 residues) contrasts with the
relatively short distance (6–8 residues) that separates ITAM
sequences, and is consistent with the ability of the PECAM-1
cytoplasmic domain to support interactions with the relatively widely
spaced tandem Src homology 2 (SH2) domains of PTPs, as opposed to the
relatively closely spaced tandem SH2 domains of PTKs (reviewed in Ref.
12). The tyrosine residues at positions 663 and 686 of the
PECAM-1 cytoplasmic domain become phosphorylated in response to
numerous stimuli, including platelet aggregation (13),
cross-linking of PECAM-1-specific mAbs (14, 15),
mechanical stimulation of endothelial cells (16),
engagement of the FcRI on basophils (17), aggregation
of the Ag receptor on T cells (18), and treatment with the
PTP inhibitor pervanadate (19). When phosphorylated, these
tyrosine residues support the binding of SHP-2 (13, 18, 20, 21, 22, 23) and possibly SHP-1 (18, 22, 23). Thus, both
structural and biochemical properties of the PECAM-1 cytoplasmic domain
suggest that it may belong to the Ig-like, ITIM-containing family of
inhibitory receptors.
The pattern of PECAM-1 expression on T lymphocytes also suggests a potential inhibitory function for this molecule. Specifically, the majority of CD4+ (24, 25, 26, 27) and half of CD8+ T lymphocytes (24, 25) lose PECAM-1 expression as they make the transition from naive to memory cells. This pattern of expression is consistent with a role for PECAM-1 in dampening the effector function of naive T cells, which are less potent effectors than are memory cells (28). Furthermore, PECAM-1-negative CD4+ T cells provide more effective helper function, in that they respond better to recall Ags, secrete more IL-4, and provide better help for B cell Ig production than do PECAM-1-positive CD4+ T cells (27). Based on these observations, and given the structural and biochemical properties of the PECAM-1 cytoplasmic domain, we hypothesized that PECAM-1 might function as an inhibitory receptor that moderates PTK-mediated signal transduction in T cells. To test this hypothesis, we assessed the impact of PECAM-1 coligation on TCR-mediated calcium mobilization in T cells. We found that the PECAM-1 cytoplasmic domain becomes tyrosine phosphorylated in response to cross-linking either the TCR or PECAM-1 itself, with subsequent recruitment of SHP-2. When PECAM-1/SHP-2 was brought into close proximity with the TCR, it resulted in attenuation of TCR-mediated release of calcium from intracellular stores. These data support inclusion of PECAM-1 within the family of ITIM-containing inhibitory receptors.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The acute T cell leukemia cell line, Jurkat (29), and the mature human T cell lymphoblast line HUT 78 (30, 31), were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in log phase growth at a density of 1–4 x 105 cells/ml in a humidified, 37°C, 5% CO2/95% air atmosphere. Jurkat cells were cultured in RPMI (complete) medium, which is RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 1 mM sodium pyruvate (Life Technologies), 10 IU/ml heparin (Pharmacia/Upjohn, Kalamazoo, MI), 25 mM HEPES (Life Technologies), 50 µg/ml gentamicin (Elkins-Sinn, Cherry Hill, NC), 100 U/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies), containing 10% FBS (Life Technologies). HUT 78 cells were cultured in IMDM (Life Technologies) supplemented with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate (Life Technologies) and 20% FBS.
Antibodies
Peridinin chlorophyl protein (PerCP)-conjugated mouse anti-human CD3 (IgG1 isotype), FITC-conjugated goat anti-mouse IgG (GAM), PerCP-conjugated IgG1, and normal mouse IgG1 (Becton Dickinson, San Jose, CA) were purchased from Becton Dickinson Immunocytometry Systems. The mouse anti-human PECAM-1 Abs, PECAM-1.1 (IgG2a), -1.2 (IgG1), and -1.3 (IgG1), were prepared in our laboratory, and have been previously described (9). The mouse anti-human PECAM-1 Ab, 4G6 (IgG2b), was generously provided by Dr. Steven Albelda (University of Pennsylvania Medical Center, Philadelphia, PA) (32).
The human CD3-specific Ab, UCHT1 (mouse IgG1, 
), containing no
azide and low endotoxin, was purchased from PharMingen (San Diego, CA).
Azide-free GAM F(ab')2 fragments (Accurate
Chemical and Scientific, Westbury, NY) or azide-containing GAM
F(ab')2 fragments (Sigma, St. Louis, MO), from
which azide was removed by dialysis, were used for cross-linking of
cell-surface-associated molecules. HRP-conjugated mouse
anti-phosphotyrosine (clone PY20; Zymed Laboratories, San
Francisco, CA), murine monoclonal anti-SHP-2 (Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-human
PECAM-1 (SEW32-34; prepared in our laboratory), and HRP-conjugated GAM
(Jackson ImmunoResearch, West Grove, PA) or alkaline phosphatase
(AP)-conjugated goat anti-rabbit IgG (Zymed Laboratories) Abs were
used for Western blot analysis.
Induction and assessment of PECAM-1 tyrosine phosphorylation and SHP-2 binding
Jurkat cells in log phase growth were pelleted by centrifugation
at 300 x g for 10 min at room temperature and
resuspended at a final concentration of 107
cells/ml in cold RPMI (complete)/10% FBS. Cell suspensions (1 ml) were
incubated with no Ab, with the human CD3-specific Ab, UCTH1 (1
µg/ml), with a human PECAM-1-specific Ab, or with both
anti-CD3 and anti-PECAM-1 for 30 min on ice. To remove
unbound Ab, cell suspensions were washed twice with 5 ml of cold 0.145
M sodium chloride containing 10 mM HEPES, 2.8 mM KCl, 2 mM
MgCl2, 10 mM D-glucose, and
0.1% BSA (calcium buffer (CB)), and 10 mM CaCl2.
Cells were resuspended in the same buffer at a final concentration of
2 x 107 cells/ml.
To induce PECAM-1 phosphorylation and SHP-2 binding, cell suspensions were first prewarmed at 37°C for 10 min. GAM was added to achieve a final concentration of 20–100 µg/ml, which was empirically determined in preliminary experiments to yield maximal stimulation. Following incubation for an additional 3 min at 37°C, the reaction was stopped by the addition of an equal volume of cold 2x lysis buffer (0.145 M NaCl, 4% Triton X-100, 0.2 mM MgCl2, 30 mM HEPES, 20 mM EGTA, 4 mM sodium orthovanadate, 40 µg/ml leupeptin, 2 mM PMSF (pH 7.4)), and lysates were prepared with gentle rocking for 120 min at 4°C. Lysates were also prepared from unstimulated cells, which were allowed to settle to the bottom of a 15-ml conical tube at 37°C and were not subjected to warming, cooling, or centrifugation. Following cell lysis, the Triton-insoluble fraction was removed by centrifugation at 15,000 x g for 15 min at 4°C.
PECAM-1.1-coated beads were prepared by incubating PECAM-1.1 Ab (25 µg/ml) with protein A-Sepharose beads for 2 h at 4°C with gentle rocking. Ab-coated beads were washed three times with lysis buffer to remove unbound Ab. Cell lysates were incubated with 100 µl of a 50% slurry of PECAM-1.1-coated beads with gentle rocking overnight at 4°C. Immunoprecipitates were washed three times with immunoprecipitation buffer (50 mM Tris (pH 7.4), containing 150 mM NaCl, 2 mM Na3VO4, 1% Triton X-100, and 1 mM PMSF). Pelleted beads were boiled in 60 µl of 2x SDS-sample reducing buffer. Material eluted from the beads was separated on a 7.5% SDS-polyacrylamide gel and transferred to an Immobilon-P membrane, which was subjected to Western blot analysis for the presence of PECAM-1, phosphotyrosine residues, and SHP-2.
Calcium mobilization assays
All procedures for measurement of intracellular calcium concentrations by fluorescence spectrofluorometry were performed in the dark. Jurkat T lymphocytes (107 cells/ml) were incubated for 30 min at 37°C in RPMI (complete) containing 10% FBS and 10 µM INDO-1AM (Molecular Probes, Eugene, OR). Cells were then adjusted to a final concentration of 8 x 106 cells/ml with the addition of cold RPMI (complete)/10%FBS, and cell suspensions were placed on ice. Cell suspensions (0.5 ml) containing 4 x 106 cells/ml were incubated at 4°C for 30 min in the presence of Abs at the indicated final concentrations. To remove unbound Abs, Ab-bound cells were washed three times at 4°C with 3 ml of CB (0.145 m NaCl, 10 mM HEPES, 2.8 mM KCl, ZmM MgCl2, 10 mM D-glucose, 0.1% BSA (pH 7.2)). Cells were resuspended at a final concentration of 2 x 106 cells/ml in cold CB containing either 10 mM CaCl2 or 0.1 mM EGTA and maintained on ice until calcium flux was induced and measured.
Immediately before induction of calcium mobilization, cell suspensions were placed in a 37°C water bath for 2 min. Prewarmed cell suspensions were transferred to a cuvette equipped with a magnetic stir bar and transferred to the sample chamber, maintained at 37°C, of an SLM 8100 spectrofluorometer (SLM-AMINCO, Urbana, IL). To cross-link surface-bound Abs, GAM was added to achieve a final concentration of 20 µg/ml, which was determined in preliminary experiments to induce maximal stimulation. Fluorescence of stirring cell suspensions was excited at 350 nm and emission of fluorescence at wavelengths of 405 and 490 nm was recorded every 3 s over a period ranging from 3 to 10 min in length. Results are presented as plots of the ratio of fluorescence detected at 405, relative to 490, nm as a function of time.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
The HUT78 cell line expressed TCR, but was PECAM-1-negative by both
flow cytometric and immunoblot analyses (data not shown). Therefore,
Jurkat T lymphocytes were chosen for further studies.
|
, PECAM-1, or CD3 and PECAM-1 were bound to the surface of
Jurkat T cells and then cross-linked by addition of
F(ab')2 fragments of a polyclonal goat Ab
specific for murine IgG heavy and light chains (GAM). Cells to which no
primary Abs were bound served as negative controls. As shown in Fig. 2, a slight increase in PECAM-1 tyrosine
phosphorylation was observed in cells stimulated by CD3 cross-linking,
relative to the no Ab control. However, cross-linking of PECAM-1 itself
induced a higher level of PECAM-1 tyrosine phosphorylation, which was
further enhanced upon coligation of PECAM-1 with CD3. Tyrosine
phosphorylation of PECAM-1 resulted in the association of SHP-2, and
the amount of phosphatase coprecipitated correlated with the degree of
PECAM-1 tyrosine phosphorylation (Fig. 2). We also observed a small
amount of the related PTP, SHP-1, coprecipitating with PECAM-1, but
could not rule out that its visualization was due to Ab
cross-reactivity with SHP-2 (data not shown). Therefore, we conclude
that the major PTP associated with PECAM-1 in Jurkat cells is SHP-2.
These results extend previous observations (18) by showing
that coligation of PECAM-1 and the TCR results in higher levels of
PECAM-1 tyrosine phosphorylation than is observed upon cross-linking of
the TCR alone. Furthermore, the tyrosine-phosphorylated cytoplasmic
tail of PECAM-1 appears to preferentially bind SHP-2. On the basis
of these results, we conclude that SHP-2, which associates with the
tyrosine-phosphorylated cytoplasmic domain of PECAM-1, can be brought
into proximity with the TCR upon coligation of TCR and PECAM-1.
|
(50 ng/ml)
that induced measurable but suboptimal increases in intracellular
calcium concentrations only when cross-linked with GAM, and an optimal
concentration of GAM (20–100 µg/ml), were identified in preliminary
dose response studies (data not shown). A suboptimal concentration of
anti-CD3 was used to enable detection of either an enhancing or
an inhibitory effect of PECAM-1 on TCR-induced calcium mobilization.
Furthermore, we chose conditions that required GAM cross-linking so
that we could control whether or not PECAM-1 was colocalized with
the TCR from the time of initiation of T cell activation. As shown in
Fig. 3, cross-linking surface-bound
anti-PECAM-1 mAb, PECAM-1.3, failed to induce calcium mobilization,
even though PECAM-1 became tyrosine phosphorylated and bound SHP-2
under these conditions (Fig. 2
). Evaluation of three other
PECAM-1-specific mAbs revealed that none by themselves induced calcium
mobilization in Jurkat T lymphocytes (Table I). In contrast, cross-linking of CD3
induced strong and rapid biphasic calcium mobilization characterized by
a transient, initial rise in intracellular calcium, followed by a lower
but more sustained elevation of intracellular calcium levels (Fig. 3).
Importantly, coligation of PECAM-1/SHP-2 with the TCR attenuated
anti-CD3-mediated calcium mobilization (Fig. 3), an effect that
was proportional to the amount of PECAM-1/SHP-2 brought into the TCR
complex (Fig. 4). The ability of
PECAM-1-specific Abs to inhibit TCR-mediated calcium flux also appeared
to be influenced by the Ig homology domain-specificity of the
anti-PECAM-1 mAb. Thus, two different Ig domain 6-specific mAbs
(PECAM-1.2 and 4G6), and one specific for domain 1 (PECAM-1.3),
effected inhibition of TCR signaling, whereas PECAM-1.1, which is
specific for PECAM-1 Ig domain 5, failed to attenuate the early rise in
intracellular calcium induced by CD3 cross-linking (Table I), most
likely due to the orientation with which PECAM-1.1 interacts with cell
surface PECAM-1 (see Discussion).
|
|
|
).
From these results, we predicted that PECAM-1/SHP-2 acts by inhibiting
calcium release from intracellular stores, but not influx of calcium
from extracellular sources. To test this prediction, we assessed the
inhibitory effect of PECAM-1/TCR coligation in the absence vs the
presence of extracellular calcium. As shown in Fig. 5, the presence of extracellular EGTA did
not affect the inhibitory activity of PECAM-1 on TCR-mediated calcium
mobilization. Rather, the effect of PECAM-1 inhibition was to attenuate
TCR-induced release of calcium from intracellular stores.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Furthermore, we demonstrate that calcium mobilization
induced by TCR cross-linking is attenuated by coligation of the TCR
with PECAM-1, which specifically affects the early rise, but not the
late, sustained elevation of intracellular calcium (Fig. 3).
PECAM-1-mediated attenuation of TCR-induced calcium mobilization is
dose-dependent (Fig. 4), affects release of calcium from intracellular
stores (Fig. 5), and is influenced by the domain-specificity of the
cross-linking Ab (Table I). Together, these findings demonstrate that
PTPs associated with the PECAM-1 cytoplasmic domain can, upon receptor
coligation, be brought into proximity with the TCR and interfere with
PTK-dependent signal transduction events in T cells. The importance of
the PECAM-1 ITIM for this inhibitory function, the components of the
TCR signal transduction pathway that are affected, and the extent to
which PECAM-1 inhibition of TCR signal transduction normally occurs
during the course of Ag stimulation are currently under active
investigation in our laboratory. Nevertheless, these data demonstrate
that PECAM-1 is able to attenuate cellular activation stimulated by an
ITAM-containing stimulatory receptor, and provide the first evidence
that PECAM-1 contains a functional ITIM. The identity of the PTK responsible for phosphorylating tyrosine residues in the PECAM-1 cytoplasmic domain in T cells is not known. We observed a low level of PECAM-1 tyrosine phosphorylation upon cross-linking of the TCR, and higher levels upon cross-linking of PECAM-1 either alone or when coligated with TCR. The PTKs that may be responsible for PECAM-1 tyrosine phosphorylation under these conditions include members of the Src family of PTKs (p59fyn and p56lck) and ZAP-70, all of which can be recruited to the TCR signaling complex and activated following TCR ligation (1). Ample evidence supports a role for members of the Src family of PTKs in PECAM-1 tyrosine phosphorylation. Specifically, Fyn coprecipitates with PECAM-1 (21) and c-Src both coprecipitates with and can phosphorylate the cytoplasmic domain of bovine (16, 21, 43) or murine PECAM-1 (23). In addition, overexpression of Src family members, p56lck, p56lyn, and p53lyn in COS-1 cells resulted in tyrosine phosphorylation of murine PECAM-1 (23). A role for ZAP-70 in PECAM-1 tyrosine phosphorylation is supported by the observation that low levels of PECAM-1 tyrosine phosphorylation in Syk-deficient cells could be reconstituted by the stable transfection of Syk (17), a close homologue of ZAP-70 in non-T cells. However, Cao et al.(23) have recently shown that overexpression of Syk in COS-1 cells is incapable of stimulating PECAM-1 tyrosine phosphorylation. Finally, somewhat surprisingly, members of the Csk family of PTKs are also able to phosphorylate murine PECAM-1 (23). Further studies are required to determine which of the many PTKs capable of phosphorylating PECAM-1 on tyrosine residues actually do so in activated T cells.
Tyrosine phosphorylation of PECAM-1 results in the creation of sites with the potential for binding the tandem SH2 domain-containing PTPs, SHP-1 and SHP-2. Our data show that, in T cells, SHP-2 preferentially associates with the tyrosine-phosphorylated cytoplasmic domain of PECAM-1. These results are consistent with those of numerous studies demonstrating coprecipitation of SHP-2 with tyrosine-phosphorylated PECAM-1 (13, 18, 20, 22, 23). Although we occasionally observed, as have others (18, 23), coprecipitation of small amounts of SHP-1 with PECAM-1, SHP-2 appears to bind preferentially, most likely due to the presence of specific amino acid residues surrounding the phosphorylated tyrosines in the PECAM-1 ITIM, with which the SH2 domains of SHP-1 and SHP-2 must interact. The tandem SH2 domains of SHP-1 and SHP-2 are structurally similar, enabling both to recognize the consensus ITIM sequence (44). The PECAM-1 ITIM sequence of VxpY663xxVx17TxpY686xxV (12) is like that of another member of the Ig-like, ITIM-containing family of inhibitory receptors, Ly49, in that it possesses a valine instead of a leucine residue at the +3 position relative to the tyrosine residues in its ITIM (45). The ability of the Ly49 ITIM to activate SHP-1 is reduced relative to that of another inhibitory receptor, p58 KIR, and this difference has been attributed to the substitution of valine for leucine at ITIM position +3 (45). In contrast, the binding of SHP-2 to phosphorylated ITIMs appears not to be affected by the substitution of valine for leucine at position +3, as peptides containing either amino acid at this position were selected equally well from a degenerate peptide library by the SH2 domains of SHP-2 (44). Thus, the preferential binding of SHP-2, relative to SHP-1, to the PECAM-1 ITIM may similarly be attributable to the existence of valine, as opposed to leucine, residues at the +3 positions of the PECAM-1 ITIM.
We found that PECAM-1 oligomerization resulted in PECAM-1 tyrosine phosphorylation and SHP-2 binding, but not calcium mobilization in Jurkat T cells. Our observations are consistent with the previous observation of PECAM-1 tyrosine phosphorylation in response to binding of the PECAM-1-specific Ab, PECAM-1.2 (15). They are, however, inconsistent with the previous observation that the PECAM-1-specific mAb, 4G6, induces calcium mobilization in human endothelial cells and PECAM-1-transfected REN cells (46). This difference could be due to different signal transduction properties for PECAM-1 in T cells relative to endothelial cells, or to a difference in the assays used for assessment of calcium mobilization. We conclude that PECAM-1 tyrosine phosphorylation and SHP-2 binding, at least in T cells, are not coupled to signal transduction pathways that elevate intracellular calcium. Thus, though a positive signaling function for SHP-2 has been proposed based upon its ability to bind Grb2/SOS and activate the Ras/MAPK signal transduction pathway (47, 48, 49, 50, 51), our results suggest that positive signaling effected by SHP-2 is either not active in T cells or, if active, is not coupled to signaling cascades that induce calcium mobilization. In either case, our results support the hypothesis that induction of protein tyrosine phosphorylation and activation of signal transduction pathways that result in calcium mobilization can be independently regulated (52).
Whereas PECAM-1 oligomerization, tyrosine phosphorylation and SHP-2
binding were not sufficient to induce calcium mobilization in T
lymphocytes, they did interfere with calcium mobilization induced by
TCR cross-linking. Only one of four PECAM-1-specific Abs tested,
PECAM-1.1, failed to produce this effect. It is possible that the
orientation of PECAM-1.1, when bound to PECAM-1, imposes geometrical
constraints on the receptor that interfere with the ability of the GAM
cross-linker to either dimerize PECAM-1 or to bring it productively
into proximity with Ab-bound CD3. All of the other PECAM-1-specific
Abs tested, however, supported PECAM-1-mediated inhibition of
TCR-induced calcium mobilization.
The characteristics of PECAM-1-mediated inhibition of TCR-induced
calcium mobilization differed somewhat from the effects of other
Ig-ITIM inhibitory receptors. Specifically, PECAM-1/TCR coligation
inhibited release of calcium from intracellular stores only
incompletely and failed to block or reduce the late, sustained
elevation in intracellular calcium concentration that is normally
observed after TCR ligation. This pattern of inhibition differs from
that of Fc
RIIb, which inhibits B cell receptor (BCR)-induced influx
of calcium from extracellular sources, a property that is consistent
with the ability of FcRIIb, upon tyrosine phosphorylation, to
recruit the 5'-inositol phosphatase, SHIP (reviewed in Ref.
53). The inhibitory effect of PECAM-1 differed less
dramatically from those of inhibitory receptors that, like PECAM-1,
recruit protein tyrosine phosphatases to the membrane upon tyrosine
phosphorylation. Thus, p58 KIR (34) and PIR-B (35, 36) functioned similarly to PECAM-1 in that they inhibited,
respectively, FcR- or BCR-mediated release of calcium from
intracellular stores. Unlike PECAM-1, however, the levels of
intracellular calcium sustained later in these responses were also
reduced upon coligation of p58 KIR with FcR (34) or
upon coligation of the BCR with either PIR-B (35, 36) or
CD22 (37).
The absence of an effect of PECAM-1 on the late, sustained elevation of intracellular calcium concentrations may determine the manner in which T cell effector function is inhibited by PECAM-1. The late, sustained calcium signal is important for nuclear localization of NF-AT (54), induction of IL-2 transcription (55), and irreversible commitment of T cells to full activation (56). Thus, the inability of PECAM-1 coligation to reduce the magnitude of the late, sustained calcium signal may influence its ability to affect the magnitude of a T cell effector response. However, stimuli that delay the onset of a rise in intracellular calcium following initial T cell activation, as does PECAM-1, also delay the onset of a T cell proliferative response (57). Thus, we predict that PECAM-1 coligation with the TCR may delay the onset of T cell effector responses, but not affect their ultimate magnitude. That the overall level of inhibition caused by PECAM-1/TCR coligation is incomplete is consistent with the observation that PECAM-1-deficient mice do not display any overt defects in T cell maturation or homing (58). It is possible that an effect of PECAM-1 on T cell responses other than maturation and migration may have been missed in preliminary characterizations of the PECAM-1-deficient mice. However, it is also important to consider that T cell activation is controlled by other inhibitory receptors, including KIRs and CTLA-4. If the function of PECAM-1 in T cells is subtle, so as to delay the onset of, or raise the threshold required for activation, it is possible that the effect of PECAM-1 deficiency may be observable only in the absence of these other inhibitory receptors. Studies of T cell function in PECAM-1-deficient mice that are also heterozygous for CTLA-4 deficiency are currently ongoing in our laboratory to explore this possibility.
Upon interaction with appropriate APC, T cells receive signals through both activating and inhibitory receptors. The relative strength of the signal delivered by each of these two classes of receptors determines whether the degree of activation of the signal transduction pathway reaches a critical threshold required for commitment to activation. In general, the function of inhibitory receptors is to recruit phosphatases that counteract the activity of kinases recruited to activating receptors, thus making it more difficult for a given T cell to reach the critical activation threshold. We show here that PECAM-1, a molecule that is expressed on the surfaces of both endothelial cells and certain T cell subsets, and that mediates homophilic interactions, is capable of interfering with TCR-mediated signal transduction. On the basis of this understanding, we propose that one context in which PECAM-1 exerts its inhibitory function may involve T cell transmigration across endothelial cell barriers, during which process PECAM-1 may serve to prevent T cells from becoming aberrantly activated. Studies of in vitro T cell effector activity, as well as in vivo studies involving comparisons of the immune reactivity of wild-type and PECAM-/- mice, are currently ongoing in our laboratory to test the validity of this newly demonstrated function for PECAM-1.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Debra K. Newton-Nash, Blood Research Institute, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address:
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; AP, alkaline phosphatase; BCR, B cell receptor for Ag; CB, calcium buffer; GAM, goat anti-mouse IgG; KIR, killer inhibitory receptor; PECAM-1, platelet-endothelial cell adhesion molecule-1; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SHP, Src homology 2 domain-bearing PTP; SH2, Src homology 2; PerCP, peridinin chlorophyl protein.
Received for publication March 4, 1999. Accepted for publication April 29, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
signaling complex in human natural killer (NK) cells. J. Exp. Med. 184:2243.RIIb. Curr. Opin. Immunol. 10:306.[Medline]
This article has been cited by other articles:
|
|
 |
|
  B. Dasgupta, E. Dufour, Z. Mamdouh, and W. A. Muller A Novel and Critical Role for Tyrosine 663 in Platelet Endothelial Cell Adhesion Molecule-1 Trafficking and Transendothelial Migration J. Immunol., April 15, 2009; 182(8): 5041 - 5051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. I. Azevedo, M. V. D. Soares, J. T. Barata, R. Tendeiro, A. Serra-Caetano, R. M. M. Victorino, and A. E. Sousa IL-7 sustains CD31 expression in human naive CD4+ T cells and preferentially expands the CD31+ subset in a PI3K-dependent manner Blood, March 26, 2009; 113(13): 2999 - 3007. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J.A. Korporaal, C. A. Koekman, S. Verhoef, D. E. van der Wal, M. Bezemer, M. Van Eck, and J.-W. N. Akkerman Downregulation of Platelet Responsiveness Upon Contact With LDL by the Protein-Tyrosine Phosphatases SHP-1 and SHP-2 Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 372 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kohler and A. Thiel Life after the thymus: CD31+ and CD31- human naive CD4+ T-cell subsets Blood, January 22, 2009; 113(4): 769 - 774. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Goel, B. R. Schrank, S. Arora, B. Boylan, B. Fleming, H. Miura, P. J. Newman, R. C. Molthen, and D. K. Newman Site-Specific Effects of PECAM-1 on Atherosclerosis in LDL Receptor-Deficient Mice Arterioscler Thromb Vasc Biol, November 1, 2008; 28(11): 1996 - 2002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bergom, C. Paddock, C. Gao, T. Holyst, D. K. Newman, and P. J. Newman An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling J. Cell Sci., April 15, 2008; 121(8): 1235 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Garnacho, V. Shuvaev, A. Thomas, L. McKenna, J. Sun, M. Koval, S. Albelda, V. Muzykantov, and S. Muro RhoA activation and actin reorganization involved in endothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role for tyrosine 686 in the cytoplasmic tail of PECAM-1 Blood, March 15, 2008; 111(6): 3024 - 3033. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Rui, X. Liu, N. Li, Y. Jiang, G. Chen, X. Cao, and J. Wang PECAM-1 Ligation Negatively Regulates TLR4 Signaling in Macrophages J. Immunol., December 1, 2007; 179(11): 7344 - 7351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Goel, B. Boylan, L. Gruman, P. J. Newman, P. E. North, and D. K. Newman The proinflammatory phenotype of PECAM-1-deficient mice results in atherogenic diet-induced steatohepatitis Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1205 - G1214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bosco, K. L. McKenna, C. J. Devitt, M. J. Firth, P. D. Sly, and P. G. Holt Identification of novel th2-associated genes in T memory responses to allergens. J. Immunol., April 15, 2006; 176(8): 4766 - 4777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Falati, S. Patil, P. L. Gross, M. Stapleton, G. Merrill-Skoloff, N. E. Barrett, K. L. Pixton, H. Weiler, B. Cooley, D. K. Newman, et al. Platelet PECAM-1 inhibits thrombus formation in vivo Blood, January 15, 2006; 107(2): 535 - 541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. O'Brien, G. Cao, A. Makrigiannakis, and H. M. DeLisser Role of immunoreceptor tyrosine-based inhibitory motifs of PECAM-1 in PECAM-1-dependent cell migration Am J Physiol Cell Physiol, October 1, 2004; 287(4): C1103 - C1113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Albelda, K. C. Lau, P. Chien, Z.-Y. Huang, E. Arguiris, A. Bohen, J. Sun, J. A. Billet, M. Christofidou-Solomidou, Z. K. Indik, et al. Role for Platelet-Endothelial Cell Adhesion Molecule-1 in Macrophage Fc{gamma} Receptor Function Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 246 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Feng, J. A. Nagy, K. Pyne, H. F. Dvorak, and A. M. Dvorak Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium J. Histochem. Cytochem., January 1, 2004; 52(1): 87 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Rathore, M. A. Stapleton, C. A. Hillery, R. R. Montgomery, T. C. Nichols, E. P. Merricks, D. K. Newman, and P. J. Newman PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets Blood, November 15, 2003; 102(10): 3658 - 3664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kogata, M. Masuda, Y. Kamioka, A. Yamagishi, A. Endo, M. Okada, and N. Mochizuki Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells Mol. Biol. Cell, September 1, 2003; 14(9): 3553 - 3564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. M. Relou, G. Gorter, I. A. Ferreira, H. J. M. van Rijn, and J.-W. N. Akkerman Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Inhibits Low Density Lipoprotein-induced Signaling in Platelets J. Biol. Chem., August 29, 2003; 278(35): 32638 - 32644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Newman and D. K. Newman Signal Transduction Pathways Mediated by PECAM-1: New Roles for an Old Molecule in Platelet and Vascular Cell Biology Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 953 - 964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Righi, S. Deaglio, C. Pecchioni, A. Gregorini, A. L. Horenstein, G. Bussolati, A. Sapino, and F. Malavasi Role of CD31/Platelet Endothelial Cell Adhesion Molecule-1 Expression in in Vitro and in Vivo Growth and Differentiation of Human Breast Cancer Cells Am. J. Pathol., April 1, 2003; 162(4): 1163 - 1174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kabat, F. Borrego, A. Brooks, and J. E. Coligan Role That Each NKG2A Immunoreceptor Tyrosine-Based Inhibitory Motif Plays in Mediating the Human CD94/NKG2A Inhibitory Signal J. Immunol., August 15, 2002; 169(4): 1948 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wilkinson, A. B. Lyons, D. Roberts, M.-X. Wong, P. A. Bartley, and D. E. Jackson Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) acts as a regulator of B-cell development, B-cell antigen receptor (BCR)-mediated activation, and autoimmune disease Blood, June 17, 2002; 100(1): 184 - 193. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kimmig, G. K. Przybylski, C. A. Schmidt, K. Laurisch, B. Mowes, A. Radbruch, and A. Thiel Two Subsets of Naive T Helper Cells with Distinct T Cell Receptor Excision Circle Content in Human Adult Peripheral Blood J. Exp. Med., March 18, 2002; 195(6): 789 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Miyakawa, K. Obaru, K. Maeda, S. Harada, and H. Mitsuya Identification of Amino Acid Residues Critical for LD78beta , a Variant of Human Macrophage Inflammatory Protein-1alpha , Binding to CCR5 and Inhibition of R5 Human Immunodeficiency Virus Type 1 Replication J. Biol. Chem., February 8, 2002; 277(7): 4649 - 4655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cicmil, J. M. Thomas, M. Leduc, C. Bon, and J. M. Gibbins Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets Blood, January 1, 2002; 99(1): 137 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. RayChaudhury, M. Elkins, D. Kozien, and M. T. Nakada Regulation of PECAM-1 in Endothelial Cells during Cell Growth and Migration Experimental Biology and Medicine, July 1, 2001; 226(7): 686 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. K. Newman, C. Hamilton, and P. J. Newman Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56lck Blood, April 15, 2001; 97(8): 2351 - 2357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Patil, D. K. Newman, and P. J. Newman Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen Blood, March 15, 2001; 97(6): 1727 - 1732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Henshall, K. L. Jones, R. Wilkinson, and D. E. Jackson Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatases, SHP-1 and SHP-2, Are Required for Platelet Endothelial Cell Adhesion Molecule-1/CD31-Mediated Inhibitory Signaling J. Immunol., March 1, 2001; 166(5): 3098 - 3106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Zhao and P. J. Newman Integrin Activation by Regulated Dimerization and Oligomerization of Platelet Endothelial Cell Adhesion Molecule (Pecam)-1 from within the Cell J. Cell Biol., January 8, 2001; 152(1): 65 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Fournier, L. Chalus, I. Durand, E. Garcia, J.-J. Pin, T. Churakova, S. Patel, C. Zlot, D. Gorman, S. Zurawski, et al. FDF03, a Novel Inhibitory Receptor of the Immunoglobulin Superfamily, Is Expressed by Human Dendritic and Myeloid Cells J. Immunol., August 1, 2000; 165(3): 1197 - 1209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Botella, A. Puig-Kroger, N. Almendro, T. Sanchez-Elsner, E. Munoz, A. Corbi, and C. Bernabeu Identification of a Functional NF-{kappa}B Site in the Platelet Endothelial Cell Adhesion Molecule-1 Promoter J. Immunol., February 1, 2000; 164(3): 1372 - 1378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-j. Xu, R. Zhao, and Z. J. Zhao Identification and Characterization of Leukocyte-associated Ig-like Receptor-1 as a Major Anchor Protein of Tyrosine Phosphatase SHP-1 in Hematopoietic Cells J. Biol. Chem., June 2, 2000; 275(23): 17440 - 17446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cicmil, J. M. Thomas, T. Sage, F. A. Barry, M. Leduc, C. Bon, and J. M. Gibbins Collagen, Convulxin, and Thrombin Stimulate Aggregation-independent Tyrosine Phosphorylation of CD31 in Platelets. EVIDENCE FOR THE INVOLVEMENT OF Src FAMILY KINASES J. Biol. Chem., August 25, 2000; 275(35): 27339 - 27347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Cao, C. D. O'Brien, Z. Zhou, S. M. Sanders, J. N. Greenbaum, A. Makrigiannakis, and H. M. DeLisser Involvement of human PECAM-1 in angiogenesis and in vitro endothelial cell migration Am J Physiol Cell Physiol, May 1, 2002; 282(5): C1181 - C1190. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
