From the Division of Immunology and Allergy, Department of Pediatrics, Infection, Immunity, Injury, and Repair Program, Research Institute, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario M5G 1X8, Canada
Received for publication, August 9, 2002, and in revised form, December 4, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EphB6 is the most recently identified member of
the Eph receptor tyrosine kinase family. EphB6 is primarily expressed
in thymocytes and a subpopulation of T cells, suggesting that it may be
involved in regulation of T lymphocyte differentiation and functions.
We show here that overexpression of EphB6 in Jurkat T cells and
stimulation with the EphB6 ligand, ephrin-B1, results in the selective
inhibition of TCR-mediated activation of JNK but not the MAPK pathway.
EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in
initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced
secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of
the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for
EphB6-specific signaling. Activation of the JNK pathway and the
establishment of an IL-2/IL-2R autocrine loop have been shown to play a
role in the negative selection of
CD4+CD8+ self-reacting thymocytes. In
agreement, stimulation of murine thymocytes with ephrin-B1 not only
blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but
also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an
important role in regulating thymocyte differentiation and modulating
responses of mature T cells.
Eph receptors represent the largest family of receptor tyrosine
kinases, with at least 14 members (1, 2). Ephs are activated by
a group of ligands, all membrane-anchored either by
glycosylphosphatidylinositol (ephrin-A1-A5) or a trans-membrane
domain (ephrin-B1-B3) (3). Eph receptors are divided into two groups
EphA and EphB according to their ligand binding preference; although
within a group receptor-ligand specificity is degenerate (4). It is a
characteristic of the Eph receptor family that their ligands must be
membrane bound to be active (5-7). This requirement for membrane
anchorage of the ligand makes cell-cell contact an obligatory event for
activation of Eph receptors, and consequently, the activated receptors
are concentrated in the area of cell-cell contact. In accordance with their membrane-anchored nature, ephrins are also involved in the process of reverse signaling. They interact with cytoplasmic signaling molecules and upon stimulation with appropriate receptors transmit signals inside the cell (8). Eph receptors and their ligands are
typically most highly expressed in neural and endothelial cells (4),
and most descriptions of their function concern development of the
nervous system and angiogenesis (9-11). Upon the formation of
cell-cell contact, signaling through the Eph receptors results in
modulation of integrin activity and reorganization of the actin
cytoskeleton. As a result, Ephs generate adhesive or repulsive signals,
and in the neural system guide the movement of axonal growth cones,
cell migration, and synapse formation (12-18).
Uncharacteristically, a recently discovered member of the EphB
subfamily, EphB6 is predominantly expressed in the thymus and a
subpopulation of mature T lymphocytes (19, 20). While structural analysis of EphB6 reveals conservation of the major Eph receptor autophosphorylation sites (Tyr-638 and Tyr-644), there are
several critical alterations in its kinase domain. These include
substitution of a crucial lysine residue in the ATP binding site,
resulting in a receptor without detectable kinase activity (19, 21). We
have previously shown that stimulation with ephrin-B1 induces EphB6
trans-phosphorylation by a catalytically active EphB partner and thus
initiates its signaling (22). The predominant expression of EphB6 in
the thymus (19) and a subset of T lymphocytes (20) indicates that it
may play an important role in regulation of both T cell differentiation
and function. Current evidence suggests that Eph receptors may interact
with the T cell receptor
(TCR)1 signaling pathways as
Eph receptors can regulate the activity of small GTPases (23, 24) and
thus control MAPK pathway and integrin activation, as well as
cytoskeletal rearrangement (23, 25-27), all crucial in TCR-induced
responses (28-35). Moreover, recent observations suggest that EphB6
interacts with a key member of TCR signaling pathways, c-Cbl (22, 36),
and that co-cross-linking of EphB6 and CD3 with antibodies increases
apoptotic cell death in Jurkat T cells (36).
In this paper, we demonstrate that stimulation of thymocytes with the
EphB6 ligand ephrin-B1 (22) prevents major TCR-induced responses such
as IL-2 secretion and up-regulation of IL-2 receptor Antibodies and Recombinant Proteins--
Monoclonal
anti-phosphotyrosine and PAK-1 PBD-agarose beads were obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to EphB6,
Myc, phospho-MAPK, MAPK, JNK, Rac1, and Lck were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-JNK,
anti-phospho-p70 S6 kinase, and anti-p70 S6 kinase were from New
England Biolabs (Beverly, MA). Soluble ephrin-B1 and FGFR-Fc were
purchased from R&D Systems. Anti-human CD3 Immunoprecipitation and Western Blotting--
Cells were lysed
in ice-cold 1% Triton X-100 lysis buffer. Antibodies and protein
G-Sepharose were added to cleared lysates and incubated at 4 °C
overnight. Immunoprecipitates were washed, separated by SDS-PAGE, and
transferred to nitrocellulose (Amersham Biosciences). Membranes
were blocked overnight at 4 °C in 7% non-fat milk (Bio-Rad,
Richmond, CA) in PBS. Immunoblotting antibodies were added at optimal
dilutions in PBS-T (0.1% Tween 20) at 4 °C. Bound antibodies were
detected using horseradish peroxidase-conjugated donkey anti-rabbit or
sheep anti-mouse antibodies (Amersham Biosciences) and LumiGlo
chemiluminescent reagents (Kirkegaard and Perry, Gaithersburg, MD).
Subcloning and Mutation of EphB6--
cDNA for EphB6, was
cloned from normal human thymocyte RNA by reverse
transcription-PCR into the expression vector pcDNA3 (Invitrogen) and sequenced. Transfection of Jurkat Cells--
The human T-cell line Jurkat
was transfected with empty pcDNA3, EphB6-M, or Isolation of Murine Thymocytes--
Thymuses were obtained from
female BALB/c mice, 5-7 weeks old. Mononuclear cells were isolated by
Ficoll-Hypaque gradient centrifugation. Adherent cells were removed by
incubation to plastic dishes for 60 min at 37 °C. The resulting
thymocytes are typically >95% CD3. All animal-involved experiments
were conducted according to Canadian federal regulations and Hospital
of Sick Children guidelines.
Stimulation with Ephrin-B1 and Anti-CD3--
Ephrin-B1-Fc and
anti-CD3 were immobilized at the concentration of 20 µg/ml, unless
otherwise indicated, on plastic tissue culture dishes for 1.5 h at
37 °C and remaining soluble protein was removed by three washes with
PBS. Human IgG or irrelevant FGFR-Fc fusion protein were immobilized at
a concentration of 20 µg/ml in all ephrin-B1-negative cases as a
control for the nonspecific effects of the Fc portion of Ephrin-B1-Fc
fusion protein. Concentrations of human IgG were adjusted where
required to keep protein concentration constant. Although murine
ephrin-B1 was utilized, this effectively induced human EphB6
phosphorylation (22).
Analysis of CD25 Expression by Flow Cytometry--
Cells were
incubated in 0.5% serum for 20 h with or without immobilized
ephrin-B1-Fc fusion protein and immobilized anti-CD3 antibody.
Irrelevant antibody or FGFR-Fc fusion protein were used as a control
for immobilized ephrin-B1 where necessary. The expression of CD25 was
then analyzed by staining with phycoerythrin-labeled anti-CD25 and isotype control (Immunotech).
Analysis of IL-2 Secretion--
Cells were stimulated as
indicated for 20 h at 37 °C, and conditioned media collected.
IL-2 concentrations in the collected samples were determined using
enzyme-linked immunosorbent assay kits (R&D Systems), according to the
manufacturer's instructions.
Analysis of Apoptosis Induction--
Cells were stimulated as
indicated for 20 h at 37 °C and stained with annexin V-FITC,
and annexin-positive cells were detected by flow cytometry. Annexin
V-FITC apoptosis detection kits from R&D Systems were used
according to the manufacturer's instructions.
Ephrin-B1 Stimulation Attenuates TCR-mediated Responses in
Thymocytes--
The EphB6 receptor was reported to be predominantly
expressed in thymocytes (19). Complementary expression of its ligands was also detected in the thymus (4), suggesting that EphB6 may play a
role in T cell differentiation. CD4+8+ double
positive thymocytes that express productively rearranged TCR The EphB6 Receptor Inhibits TCR-induced IL-2 Secretion and CD25
Expression--
A high level of EphB6 receptor expression has been
reported not only in thymocytes, but also in a subpopulation of mature T cells and in the T cell line Jurkat (20, 36). The ligands for the
EphB6 receptor, ephrin-B1 and ephrin-B2 (22, 40), are expressed in most
organs and cell types (4), and therefore the potential for T cells to
be activated through EphB6 upon cell-cell contact is high. The
persistent expression of EphB6 across the T cell lineage, combined with
the practically ubiquitous expression of its ligands, suggests that
EphB6 might not be important only during differentiation but also in
mature T cell function.
T lymphocyte homeostasis is precisely regulated with numerous TCR
co-stimulatory events required for finely tuned control of cell fate,
these signals regulating both proliferative and apoptotic
pathways (41). In particular, stimulation of the TCR can lead to the
induction of IL-2 production and CD25 (IL-2R
To determine if the EphB6 receptor could influence responses to TCR
activation, pcDNA3-transfected control and B6-J cells were
stimulated with immobilized anti-CD3 and ephrin-B1 and the secretion of
IL-2 was examined by ELISA. In agreement with recent observations,
immobilized anti-CD3 was sufficient to induce IL-2 expression (38, 42).
However, a significant inhibition of anti-CD3-induced IL-2 production
was observed upon stimulation of control cells with ephrin-B1 (Fig.
3A) in accordance with the high level of endogenous EphB6 expression reported in those cells. Furthermore, the overexpression of EphB6 strongly suppressed induction of IL-2 secretion by anti-CD3 in B6-J cells. This inhibitory effect was
further enhanced upon ephrin-B1 stimulation.
The induction of CD25 expression in control and B6-J cells was also
examined (Fig. 3B). Once again, a significant inhibition of
anti-CD3-induced CD25 up-regulation was observed upon both ephrin-B1
stimulation and overexpression of EphB6. In addition to using human IgG
as a control for the human Fc portion of the recombinant ephrin-B1, an
irrelevant fusion protein control (FGFR-Fc) was included (Fig.
3C). In sum, these findings show that the EphB6 receptor can
inhibit TCR-mediated IL-2 secretion and CD25 expression in mature T
cells, suggesting that EphB6 may be responsible for the similar pattern
of inhibition induced by ephrin-B1 in thymocytes.
Selective Inhibition of JNK Activation by the EphB6
Receptor--
The JNK pathway is one of the major signaling cascades
initiated upon TCR activation (35, 38, 39, 43). To determine if the
EphB6 receptor could down-regulate TCR-mediated activation of the JNK
pathway, we treated control pcDNA3 cells and B6-J cells with
anti-CD3 alone or in combination with increasing concentrations of
ephrin-B1 and determined the activation status of JNK by Western blotting with anti-phospho-JNK. Stimulation with immobilized anti-CD3 induced JNK activation in agreement with recent findings using pre-complexed anti-CD3 (38). Anti-CD3-induced p54Jun kinase phosphorylation was prominent in control cells but reduced upon stimulation with ephrin-B1 in a concentration-dependent
manner. Induction of JNK phosphorylation was also significantly
inhibited in B6-J cells and further down-regulated upon ephrin-B1
stimulation (Fig. 4, A and
B). TCR-mediated stimulation of the JNK pathway requires
activation of the small GTPase Rac1 by the GEF protein Vav1. This
activation occurs upon direct Vav-Rac1 interaction (38). As Eph
receptors have previously been shown to regulate small GTPases, in
particular through the control of GEF activity (23, 24), we examined
whether the EphB6 receptor could affect Vav interaction with Rac1 in T
cells. In pcDNA3 control cells anti-CD3 stimulation induced
Vav-Rac1 association, which could be detected by co-immunoprecipitation
(Fig. 4C). However, the induction of Vav-Rac1 interaction
was blocked by both ephrin-B1 stimulation and EphB6 overexpression.
Activated Rac1 interacts with the PAK serine/threonine kinase
and induces its autophosphorylation and activation. We could not detect
Rac1 activation in PAK pull-down assays (exploiting PAK1-1 PBD beads)
upon stimulation with anti-CD3, probably due to insufficient
sensitivity of the assay. More potent anti-CD3/anti-CD28 co-stimulation
was found to induce Rac1 activation (Fig. 4D).
Overexpression of the EphB6 receptor and ephrin-B1 stimulation strongly
suppressed anti-CD3/CD28-initiated activation of Rac1. In agreement,
anti-CD3/CD28-induced IL-2 secretion was also inhibited upon EphB6
overexpression (not shown). In sum, these findings suggest that EphB6
inhibits TCR-induced IL-2 secretion and activation of JNK through the
control over Rac1 activity.
In addition to inducing JNK activation, TCR stimulation also results in
the initiation of the MAPK signaling pathway, leading to
phosphorylation of MAPK and its consequent activation. As certain Eph
receptors have been previously reported to down-regulate activation of
the MAPK pathway in non-lymphoid cells (25, 26), we examined the
influence of the EphB6 receptor on anti-CD3-induced MAPK
phosphorylation. Remarkably, attenuation of anti-CD3-initiated
signaling by EphB6 appeared to specifically target the JNK pathway as
TCR-mediated MAPK phosphorylation was not affected by overexpression of
the EphB6 receptor (Fig. 5A).
However, ephrin-B1 stimulation caused a minor inhibition of MAPK
activation, probably mediated by other members of the EphB subfamily
(some of which have been shown to inhibit MAPK activation (26)).
Moreover, anti-CD3-induced phosphorylation of p70 S6 kinase on
Thr-421/Ser-424 residues, crucial for kinase activation and cell cycle
progression, was not affected by the EphB6 receptor either (Fig.
5B).
Thus, EphB6 appears to be a specific negative regulator of TCR-mediated
Rac1 and JNK pathway activation, suggesting that the down-regulation of
TCR-induced IL-2 secretion upon ephrin-B1 stimulation or EphB6
overexpression probably results from the selective inhibition of the
Rac1 GTPase.
EphB6-specific Signaling Is Required for the Suppression of TCR
Responses--
To confirm that the inhibitory effects of EphB6
overexpression were specific to the EphB6 receptor and did not result
from reverse signaling through ephrin-B ligands (potentially expressed in Jurkat cells) or from nonspecific interference with the signaling of
other Eph receptors, we compared the consequences of overexpressing wild type and cytoplasmic domain-deleted ( Unusually for a receptor tyrosine kinase, and particularly for an
Eph receptor, EphB6 is most highly expressed in the T cell lineage (19,
20), and recent findings suggested a potential role for EphB6 in
modulating T-cell responses (36). We have shown here that both the
EphB6 receptor and its ligand ephrin-B1 can regulate TCR-induced IL-2
secretion and IL-2R Activation of the small GTPase Rac1 is the initiating event in the JNK
cascade. Rac1 activation requires interaction with its GEF protein Vav,
which can be induced by TCR stimulation (38). We found that
anti-CD3-induced Rac1-Vav interaction was blocked in B6-J cells or upon
stimulation with ephrin-B1. This strongly suggests that EphB6 inhibits
JNK pathway initiation and induction of IL-2 secretion by preventing
Rac1 activation. This also raises an alternative model of EphB6 action,
whereby suppression of TCR-mediated IL-2 secretion may not be the
consequence of blockage of the JNK pathway, but rather IL-2 secretion
and JNK activation may be down-regulated independently as the result of
the inhibition of the Rac1 GTPase. While we were preparing this
manuscript findings were published, demonstrating multiple effects of
Vav1 deficiency on various anti-CD3-induced responses in Jurkat (44).
Interestingly, activation of JNK and the IL-2 promoter were strongly
inhibited in Vav1-defficient cells, while anti-CD3-initiated MAPK
phosphorylation was unaffected. These observations essentially mimic
the effects of ephrin-B1 stimulation and EphB6 overexpression on
anti-CD3-induced responses observed in our experiments in Jurkat. This
strongly, although indirectly, supports our model suggesting that the
EphB6 receptor may inhibit certain TCR-mediated responses through the
control over Rac1 activity.
The precise mechanism by which EphB6 regulates Vav-Rac1 interaction
remains unclear. Overexpression of the EphB6 receptor did not appear to
affect Vav tyrosine phosphorylation or its association with c-Cbl (not
shown). However, we and others have demonstrated that EphB6 is
constitutively associated with c-Cbl (22, 36), and as Cbl directly
interacts with Vav upon TCR engagement and is required for the
activation of another small GTPase Rap1 (45), EphB6 could potentially
control Vav-Rac1 interaction through regulation of Cbl activity.
In mature T cells blockage of the JNK pathway was reported to enhance
TCR-induced Fas-mediated apoptosis (46). In agreement with the
EphB6-mediated inhibition of the JNK pathway observed in our
experiments, stimulation of Jurkat T cells with an anti-EphB6 antibody
was recently reported to increase anti-CD3-induced Fas-mediated apoptosis (36). Supporting this observation, we found that
overexpression of EphB6 not only prevented TCR-induced IL-2 secretion
but also increased the initiation of Fas-dependent
apoptotic cell death in an ephrin-B1-dependent
manner (not shown). Thus, one of the functions of EphB6 in mature T
cells may be control over the clonal expansion of antigen-activated T
lymphocytes through suppression of Rac1 activation and subsequent
inhibition of the JNK pathway and IL-2 secretion, leading to the
induction of apoptosis without preceding proliferation.
Clearly, whenever an EphB6-positive T lymphocyte encounters and
interacts with a cell expressing ephrins complementary to the EphB6
receptor, signaling through the TCR has the potential to be modified as
a consequence of simultaneous EphB6-ephrin ligation. The EphB6 ligands
ephrin-B1 and ephrin-B2 are widely expressed throughout the body and
therefore only EphB6 expression by T cells should be a limiting factor
in this interaction. This potentially means that the decision to
express EphB6 or not is equivalent to a decision to proliferate or die
upon subsequent TCR stimulation. We propose that in mature T cells,
EphB6 may either help to eliminate potentially harmful clones or simply
control clone expansion.
Thymocytes that reach the double positive
(CD4+8+) stage of maturation and express a
successfully rearranged TCR undergo positive or negative selection in
the cortical area of thymus. Thymocytes that express a TCR that
interacts weakly with self-MHC/antigen are positively selected,
whereas strongly activated thymocytes undergo negative selection. The
formation of an IL-2/IL-2R autocrine loop appears to be one of the
crucial events in initiation of apoptosis in negative selection (37).
By definition, organization of an autocrine signaling loop depends
strictly on both ligand secretion and expression of a complementary
receptor. Stimulation with ephrin-B1 appeared to block TCR-mediated
induction of both IL-2 secretion and IL-2R It is possible that one of the functions of the EphB6 receptor and its
ephrin-B ligands in the thymus is to modulate TCR signaling in a manner
that prevents induction of apoptosis by low affinity TCR interactions
but does not affect TCR survival signals. This possibility is supported
by our observation that in thymocytes (similarly to Jurkat cells)
ephrin-B1 stimulation, although inhibiting IL-2 secretion, does not
significantly affect TCR-mediated MAPK pathway activation (not shown),
a major survival signal in CD4+CD8+ cells (35).
Complementing these observations, expression of the EphB6 receptor was
found by in situ hybridization analysis to be restricted to
the cortex in both murine and human thymus (36)2 where
CD4+CD8+ cells predominate and selection takes
place. In sum, these findings suggest that in thymocytes stimulation
with EphB6 receptor ligands may allow suboptimal TCR stimulation to
switch on survival signals without triggering apoptotic events.
Thus, the ability of the EphB6 receptor to inhibit TCR-mediated JNK
activation and IL-2/IL-2R production without a significant effect on
the MAPK pathway may serve to mark the fine line between the positive
and negative selection of CD4+CD8+ thymocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain (CD25)
expression. In agreement with previously published data (37), the
blockage of IL-2 receptor (IL-2R) signaling in thymocytes significantly
inhibits TCR-mediated apoptotic cell death. This response to
ephrin-B1 is most likely mediated by the EphB6 receptor as
overexpression of EphB6 in Jurkat cells strongly suppresses
TCR-mediated IL-2 cytokine secretion and IL-2R expression, an effect
further enhanced by ephrin-B1 stimulation. We show here that
stimulation of Jurkat T cells with ephrin-B1 or overexpression of the
EphB6 receptor results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing TCR-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade (38). The
requirement for EphB6-specific signaling in modulation of TCR pathways
was confirmed by the inability of the EphB6 receptor with its
cytoplasmic domain deleted (
EphB6) to inhibit TCR-induced JNK
activation and cellular responses. Moreover,
EphB6 demonstrated dominant negative properties and suppressed the inhibitory influence of
the endogenous EphB6 receptor. In sum, these findings strongly suggest
that the EphB6 receptor alters TCR-mediated responses through
inhibition of the Rac1 GTPase.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from
Serotec (Oxford, UK) or BD Biosciences (Mississauga, Ontario, Canada).
EphB6- deletion of the
cytoplasmic tail was created by PCR using cloned cDNA as the
template. The resulting cDNA was cloned and sequenced to confirm
the mutation. Myc-tagged EphB6 was generated by PCR by insertion of a
Myc tag, and constructs were verified by sequencing. Expression of wild type proteins and mutants were examined by transfection in COS-7 cells and Western blotting with appropriate antibodies.
EphB6. The Jurkat
cells were electroporated in 400 µl complete RPMI medium with 30 µg
of DNA by pulsing once for 65 ms at 260 V (BTK electrosquare porator,
BTX, Division of Genetronics Inc., San Diego, CA). Cells were incubated
at 37 °C for 24 h before addition of G418 to the medium. After
30 days of selection the resulting oligoclonal cell populations were
screened by immunoprecipitation with anti-Myc and Western blotting with anti-Myc or anti-EphB6 and the EphB6- or
EphB6-expressing cell population was selected.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
are
susceptible to repertoire selection. Double positive thymocytes
reacting with self-MHC/antigen complex with high affinity are
negatively selected and die by apoptosis. A TCR-induced IL-2/IL-2R autocrine loop, among other factors, contributes significantly to the
induction of apoptosis in self-reacting double positive thymocytes
(37). Stimulation of thymocytes with a high concentration of
immobilized anti-CD3 can mimic this process and induce apoptosis in
thymocytes (39). As expected, stimulation of murine thymocytes with
immobilized anti-CD3-induced IL-2 secretion and up-regulation of IL-2R
chain (CD25) expression (Fig. 1,
A and B). To examine the possible involvement of
Eph receptors in the regulation of these responses we simultaneously
stimulated thymocytes with the EphB6 ligand ephrin-B1. Dimeric
ephrin-B1 fused to the Fc domain of human IgG was immobilized to mimic
its membrane-bound nature. Purified human IgG was used in all points
without ephrin-B1 as a specificity control. Co-stimulation with
ephrin-B1 strongly inhibited both TCR-mediated IL-2 secretion and CD25
up-regulation (Fig. 1, A and B). The inhibition
of T cell response was ephrin-B1 concentration-dependant and ligand
immobilization at 2-5 µg/ml was sufficient for significant
suppression of TCR-mediated responses (Fig. 1C). An
additional specificity control with irrelevant FGFR-Fc fusion protein
had no effect even when immobilized at 10 µg/ml (Fig. 1C).
Consistent with its inhibitory effect on TCR-mediated induction of the
IL-2/IL-2R autocrine loop in thymocytes, ephrin-B1 treatment strongly
inhibited TCR-initiated apoptosis (Fig. 1D). Thus, ephrin-B1
and its receptors, potentially including the highly expressed EphB6,
can negatively modulate TCR-induced responses in thymocytes.
View larger version (19K):
[in a new window]
Fig. 1.
Ephrin-B1 co-stimulation inhibits TCR-induced
IL-2 secretion, CD25 up-regulation, and apoptosis in murine
thymocytes. A, purified thymocytes were resuspended in
1% serum RPMI medium and stimulated for 20 h with
plate-immobilized anti-CD3 and ephrin-B1 as indicated. Human IgG was
immobilized in all ephrin-negative points as a specificity control for
the human Fc fragment fused to recombinant ephrin-B1. Conditioned media
were collected and IL-2 secretion was analyzed by ELISA. B,
cells were stimulated as in A, and expression of CD25 was
analyzed by flow cytometry upon staining with PE-labeled anti-CD25
antibody. The percentage of CD25-expressing cells is given after
subtraction of the isotype control. C, thymocytes were
stimulated for 20 h with immobilized anti-CD3 alone or in
combination with various concentrations of ephrin-B1. Corresponding
amounts of human IgG were used to keep protein concentration constant.
Stimulation with immobilized irrelevant FGFR-Fc fusion protein was used
as an extra control for specificity. Expression of CD25 was analyzed as
in B. D, thymocytes were stimulated as in
C, and induction of apoptosis was analyzed by staining with
FITC-labeled annexin V and subsequent flow cytometry.
) expression, resulting
in an autocrine loop and thus the clonal expansion of activated T
cells. To examine whether the EphB6 receptor, specifically, could
down-regulate TCR-mediated induction of IL-2 and IL-2R, we generated
stable overexpression of Myc-tagged EphB6 in the mature T cell line
Jurkat (B6-J cells) (Fig. 2A).
We have previously determined that upon stimulation with ephrin-B1,
EphB6 undergoes tyrosine phosphorylation by catalytically active Eph receptors (22). As a test for functionality of the transfected EphB6
receptor, the oligoclonal B6-J cell line was stimulated with ephrin-B1,
and the overexpressed receptor was indeed observed to undergo tyrosine
phosphorylation (Fig. 2B).
View larger version (28K):
[in a new window]
Fig. 2.
Generation of EphB6 receptor-stable
expression in Jurkat T cells. A, the mature human T-cell
line Jurkat was transfected with empty pcDNA3 or EphB6-M (B6-J).
After 30 days of geneticin selection the resulting oligoclonal cell
populations were examined by immunoprecipitation with anti-Myc and
Western blotting with anti-Myc or anti-EphB6 to confirm EphB6-M
expression. B, transfected EphB6-M is tyrosine
phosphorylated upon stimulation with ephrin-B1. B6-J and pcDNA3
Jurkat cells were stimulated with 1 µg/ml soluble dimerized ephrin-B1
for 15 min at 37 °C, cells lysed, and EphB6-M immunoprecipitated
with anti-Myc. Immunoprecipitates were resolved by SDS-PAGE, and
tyrosine phosphorylation was examined by Western blotting with
anti-phosphotyrosine. Loading of EphB6-M was confirmed by reblotting
with anti-Myc.
View larger version (16K):
[in a new window]
Fig. 3.
The EphB6 receptor is responsible for
modulation of TCR-mediated responses. A, control
pcDNA3-transfected cells and B6-J cells were stimulated in a
serum-free medium as in Fig. 1A, conditioned media were
collected and analyzed for IL-2 by ELISA. B, B6-J and
pcDNA3 cells were resuspended in serum-free medium, stimulated, and
analyzed as in Fig. 1B. C, control pcDNA3
cells were stimulated for 20 h with anti-CD3 alone or
co-stimulated with anti-CD3 and ephrin-B1 or anti-CD3 and recombinant
FGFR-Fc protein. All proteins were immobilized at a concentration of 20 µg/ml. Expression of CD25 was analyzed as in Fig.
1B.
View larger version (38K):
[in a new window]
Fig. 4.
EphB6 inhibits TCR-mediated activation of the
JNK pathway. A, EphB6 inhibits phosphorylation of JNK.
Cells were stimulated with immobilized anti-CD3 and ephrin-B1 as
indicated, and the phosphorylation of JNK was examined by Western
blotting of cell lysates with anti-phospho-JNK. JNK expression was
confirmed by Western blotting with anti-p54JNK. B,
densitometry analysis of the JNK phosphorylation blot presented in
A. C, B6-J and pcDNA3 cells were stimulated
with plate-immobilized anti-CD3 or co-stimulated with anti-CD3 and
ephrin-B1 as indicated. Each protein was immobilized at 20 µg/ml.
Vav-Rac1 association was detected by Western blotting of Rac1
immunoprecipitates with anti-Vav antibody. Equal sample loading was
confirmed by Western blotting with anti-Rac1. Vav expression in B6-J
and pcDNA3 cells was controlled by Western blotting of total
lysates with anti-Vav. Human IgG was immobilized at 20 µg/ml, as a
specificity control in all ephrin-negative points. D, cells
were co-stimulated with anti-CD3 and anti-CD28 in the presence or
absence of ephrin-B1. Anti-CD3 and ephrin-B1 were immobilized as in
Fig. 3, and anti-CD28 was immobilized at 10 µg/ml. Activated Rac1 was
pulled down from cell lysates using PAK-1 PBD-agarose beads. Samples
were resolved by SDS-PAGE, transferred, and blotted with anti-Rac1.
Rac1 expression was confirmed by Western blotting of cell lysates with
anti-Rac1.
View larger version (23K):
[in a new window]
Fig. 5.
Overexpression of EphB6 does not affect MAPK
phosphorylation. A, B6-J and pcDNA3-transfected
control cells were stimulated with anti-CD3, ephrin-B1 were immobilized
at 20 µg/ml as indicated, and phosphorylation of MAPK was detected by
Western blotting with anti-phospho-MAPK. The presence of MAPK was
confirmed by Western blotting with anti-MAPK. Densitometry analysis of
the MAPK phosphorylation blots are presented in arbitrary units.
B, phosphorylation of p70 S6 kinase on Thr-421/Ser-424
residues was examined by Western blotting with anti-phospho-p70S6K.
Expression of p70S6K was confirmed by reblotting with anti-p70S6K. In
all experiments human IgG was immobilized at the appropriate
concentrations as a specificity control.
EphB6) forms of EphB6 (Fig. 6A). While oligoclonal
B6-J populations were utilized in previous experiments, this should
also eliminate the possibility that the effects observed with B6-J
cells were due to clonal variation. The functional status and
expression level of
EphB6 was verified by the ability of
EphB6-transfected Jurkat cells (
B6-J) to bind recombinant
ephrin-B1-Fc ligand, which was greater than control cells and
equivalent to B6-J cells (not shown). Equal CD3
expression on B6-J,
B6-J, and pcDNA3 cells was confirmed by flow cytometry (not
shown). In contrast to the overexpression of wild-type EphB6, expression of
EphB6 did not inhibit TCR-mediated responses.
Moreover, although we did not detect any increase in Vav-Rac1
association in
B6-J (not shown), probably due to low sensitivity of
the assays,
B6-J cells demonstrated significantly increased
responses to anti-CD3 stimulation relative to control cells (Fig. 6,
B, C, and D). This suggests that
EphB6 may act as dominant negative toward endogenous EphB6
receptors, releasing the cell from their basal inhibitory effects. In
agreement,
EphB6 also reduced to varying degrees the inhibitory
effects of ephrin-B1 stimulation. In sum, these findings strongly
suggest that an EphB6-specific signaling cascade is responsible for the
attenuation of TCR-mediated responses.
View larger version (18K):
[in a new window]
Fig. 6.
Cytoplasmic domain-deficient EphB6 does not
inhibit TCR-mediated responses. A, stable expression of
truncated EphB6. Jurkat T cells were transfected with Myc-tagged
EphB6 (EphB6 with the cytoplasmic domain deleted) in the pcDNA3
expression vector. Selection was performed as in Fig. 2A.
Expression of the
EphB6 receptor in the resulting cell population
(
B6-J) was confirmed by immunoprecipitation with anti-Myc and
Western blotting with anti-EphB6. B,
EphB6 does not
prevent induction of CD25 expression. B6-J,
B6-J, and pcDNA3
cells were stimulated and analyzed as Fig. 3B. C,
IL-2 secretion is not inhibited by
EphB6. Cells were stimulated as
in Fig. 3A, and conditioned media were analyzed for IL-2 by
ELISA. D, expression of
EphB6 does not inhibit the JNK
pathway. Cells were stimulated with immobilized anti-CD3 and ephrin-B1
as indicated, and phosphorylation of JNK was examined by Western
blotting of cell lysates with anti-phospho-JNK. Equal protein loading
was confirmed by Western blotting with the protein-specific antibodies
(not shown). Blots were analyzed by densitometry, and the results for
each cell line were presented as a percentage relative to its
unstimulated control. Human IgG was immobilized at 20 µg/ml as a
specificity control in all ephrin-negative points.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain expression in T lymphocytes. Ephrin-B1
treatment and EphB6 overexpression also inhibited stimulation of the
JNK pathway in anti-CD3-activated cells. The inhibitory properties of
EphB6 were confirmed by the ability of a truncated EphB6 receptor,
missing its cytoplasmic portion, to suppress the inhibitory activity of
the endogenous EphB6 receptor. The inhibition of TCR responses by EphB6
specifically targeted the JNK pathway as essentially no effect on
another major signaling cascade, the MAPK pathway, was observed upon
either increased EphB6 expression or ephrin-B1 stimulation. Similarly there was no inhibition of anti-CD3-induced p70 S6 kinase
phosphorylation. While there is evidence to suggest that production of
IL-2 upon TCR engagement requires activation of the JNK signaling
cascade (38, 39, 43), contradictory reports also exist and the precise role of JNK in T cells is currently controversial (35).
-chain expression in
thymocytes. As expected, anti-CD3-induced apoptosis was similarly
disrupted. Since each ephrin can activate multiple members of the Eph
family, we cannot be absolutely certain that the EphB6 receptor was
predominantly responsible for the transduction of ephrin-B1 inhibitory
signals in thymocytes. However, EphB6 is activated by stimulation with ephrin-B1 (12), and the highest level of EphB6 expression is found in
thymocytes (19). Furthermore, overexpression of EphB6 enhances
ephrin-B1-induced inhibition of TCR-mediated responses in Jurkat cells,
strongly suggesting that EphB6 is likely to be the primary ephrin-B1
effector in thymocytes.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Eldad Zacksenhaus for technical help.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the National Cancer Institute of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Donald and Audrey Campbell Chair of Immunology. To whom correspondence should be addressed: Division of Immunology and Allergy, Department of Pediatrics, Infection, Immunity, Injury and Repair Program, The Research Institute of Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-8623; Fax: 416-813-8624; E-mail: chaim.roifman@sickkids.on.ca.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M208179200
2 C. Rashotte, T. Grunberger, and C. M. Roifman, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TCR, T cell receptor; MAPK, mitogen-activated protein kinase; FGFR, fibroblast growth factor receptor; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; IL, interleukin; GEF, guanine exchange factor; JNK, c-Jun NH2-terminal kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Ann. Rev. Cell Biol. 10, 251-337[CrossRef] |
2. | Pasquale, E. B. (1997) Curr. Opin. Cell Biol. 9, 608-615[CrossRef][Medline] [Order article via Infotrieve] |
3. | Drescher, U. (1997) Curr Biol. 7(12) |
4. | Zhou, R. (1998) Pharmacol. Ther. 77, 151-181[CrossRef][Medline] [Order article via Infotrieve] |
5. | Davis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak, V., Pawson, T., Goldfarb, M., and Yancopoulos, G. D. (1994) Science 266, 816-819[Medline] [Order article via Infotrieve] |
6. | Sakano, S., Serizawa, R., Inada, T., Iwama, A., Itoh, A., Kato, C., Shimizu, Y., Shinkai, F., Shimizu, R., Kondo, S., Ohno, M., and Suda, T. (1996) Oncogene 13, 813-822[Medline] [Order article via Infotrieve] |
7. | Winslow, J. W., Moran, P., Valverde, J., Shih, A., Yuan, J. Q., Wong, S. C., Tsai, S. P., Goddard, A., Henzel, W. J., Hefti, F., et al.. (1995) Neuron 14, 973-981[Medline] [Order article via Infotrieve] |
8. | Boyd, A. W., and Lackmann, M. (2001) Science's STKE, http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/112/re20 |
9. |
Frisen, J.,
Holmberg, J.,
and Barbacid, M.
(1999)
EMBO J.
18,
5159-5165 |
10. | Wilkinson, D. G. (2001) Nat. Rev. Neurosci. 2, 155-164[CrossRef][Medline] [Order article via Infotrieve] |
11. | Cheng, N., Brantley, D. M., and Chen, J. (2002) Cytokine Growth Factor Rev. 13, 75-85[CrossRef][Medline] [Order article via Infotrieve] |
12. | Drescher, U., Kremoser, C., Handwerker, C., Loschinger, J., Noda, M., and Bonhoeffer, F. (1995) Cell 82, 359-370[Medline] [Order article via Infotrieve] |
13. | Hsueh, Y. P., and Sheng, M. (1998) Neuron 21, 1227-1229[Medline] [Order article via Infotrieve] |
14. | Krull, C. E., Lansford, R., Gale, N. W., Collazo, A., Marcelle, C., Yancopoulos, G. D., Fraser, S. E., and Bronner, F. M. (1997) Curr. Biol. 7, 571-580[Medline] [Order article via Infotrieve] |
15. | Nakamoto, M., Cheng, H. J., Friedman, G. C., McLaughlin, T., Hansen, M. J., Yoon, C. H., O'Leary, D. D., and Flanagan, J. G. (1996) Cell 86, 755-766[Medline] [Order article via Infotrieve] |
16. | Mellitzer, G., Xu, Q., and Wilkinson, D. G. (1999) Nature 400, 77-81[CrossRef][Medline] [Order article via Infotrieve] |
17. | Xu, Q., Mellitzer, G., Robinson, V., and Wilkinson, D. G. (1999) Nature 399, 267-271[CrossRef][Medline] [Order article via Infotrieve] |
18. | Mellitzer, G., Xu, Q., and Wilkinson, D. G. (2000) Curr. Opin. Neurobiol. 10, 400-408[CrossRef][Medline] [Order article via Infotrieve] |
19. | Gurniak, C. B., and Berg, L. J. (1996) Oncogene 13, 777-786[Medline] [Order article via Infotrieve] |
20. | Shimoyama, M., Matsuoka, H., Tamekane, A., Ito, M., Iwata, N., Inoue, R., Chihara, K., Furuya, A., Hanai, N., and Matsui, T. (2000) Growth Factors 18, 63-78[Medline] [Order article via Infotrieve] |
21. | Matsuoka, H., Iwata, N., Ito, M., Shimoyama, M., Nagata, A., Chihara, K., Takai, S., and Matsui, T. (1997) Biochem. Biophys. Res. Comm. 235, 487-492[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Freywald, A.,
Sharfe, N.,
and Roifman, C. M.
(2002)
J. Biol. Chem.
277,
3823-3828 |
23. | Shamah, S. M., Lin, M. Z., Goldberg, J. L., Estrach, S., Sahin, M., Hu, L., Bazalakova, M., Neve, R. L., Corfas, G., Debant, A., and Greenberg, M. E. (2001) Cell 105, 233-244[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Dodelet, V. C.,
Pazzagli, C.,
Zisch, A. H.,
Hauser, C. A.,
and Pasquale, E. B.
(1999)
J. Biol. Chem.
274,
31941-31946 |
25. | Miao, H., Wei, B. R., Peehl, D. M., Li, Q., Alexandrou, T., Schelling, J. R., Rhim, J. S., Sedor, J. R., Burnett, E., and Wang, B. (2001) Nat. Cell Biol. 3, 527-530[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Elowe, S.,
Holland, S. J.,
Kulkarni, S.,
and Pawson, T.
(2001)
Mol. Cell. Biol.
21,
7429-7441 |
27. |
Zou, J. X.,
Wang, B.,
Kalo, M. S.,
Zisch, A. H.,
Pasquale, E. B.,
and Ruoslahti, E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13813-13818 |
28. | Holsinger, L. J., Graef, I. A., Swat, W., Chi, T., Bautista, D. M., Davidson, L., Lewis, R. S., Alt, F. W., and Crabtree, G. R. (1998) Curr. Biol. 8, 563-572[Medline] [Order article via Infotrieve] |
29. |
Abraham, C.,
Griffith, J.,
and Miller, J.
(1999)
J. Immunol.
162,
4399-4405 |
30. | Bleijs, D. A., de Waal-Malefyt, R., Figdor, C. G., and van Kooyk, Y. (1999) Eur. J. Immunol. 29, 2248-2258[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Wulfing, C.,
and Davis, M. M.
(1998)
Science
282,
2266-2269 |
32. |
Wulfing, C.,
Sjaastad, M. D.,
and Davis, M. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6302-6307 |
33. |
Vivinus-Nebot, M.,
Ticchioni, M.,
Mary, F.,
Hofman, P.,
Quaranta, V.,
Rousselle, P.,
and Bernard, A.
(1999)
J. Cell Biol.
144,
563-574 |
34. |
Zhang, J.,
Shehabeldin, A.,
da Cruz, L. A.,
Butler, J.,
Somani, A. K.,
McGavin, M.,
Kozieradzki, I.,
dos Santos, A. O.,
Nagy, A.,
Grinstein, S.,
Penninger, J. M.,
and Siminovitch, K. A.
(1999)
J. Exp. Med.
190,
1329-1342 |
35. | Rincon, M., Flavell, R. A., and Davis, R. J. (2001) Oncogene 20, 2490-2497[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Luo, H.,
Wan, X.,
Wu, Y.,
and Wu, J.
(2001)
J. Immunol.
167,
1362-1370 |
37. |
Bassiri, H.,
and Carding, S. R.
(2001)
J. Immunol.
166,
5945-5954 |
38. |
Kaminuma, O.,
Deckert, M.,
Elly, C.,
Liu, Y. C.,
and Altman, A.
(2001)
Mol. Cell. Biol.
21,
3126-3136 |
39. |
Sabapathy, K.,
Kallunki, T.,
David, J. P.,
Graef, I.,
Karin, M.,
and Wagner, E. F.
(2001)
J. Exp. Med.
193,
317-328 |
40. | Munthe, E., Rian, E., Holien, T., Rasmussen, A., Levy, F. O., and Aasheim, H. (2000) FEBS Lett. 466, 169-174[CrossRef][Medline] [Order article via Infotrieve] |
41. | Chambers, C. A., and Allison, J. P. (1997) Curr. Opin. Immunol. 9, 396-404[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Nemorin, J. G.,
Laporte, P.,
Berube, G.,
and Duplay, P.
(2001)
J. Immunol.
166,
4408-4415 |
43. |
Nishina, H.,
Bachmann, M.,
Oliveira-dos-Santos, A. J.,
Kozieradzki, I.,
Fischer, K. D.,
Odermatt, B.,
Wakeham, A.,
Shahinian, A.,
Takimoto, H.,
Bernstein, A.,
Mak, T. W.,
Woodgett, J. R.,
Ohashi, P. S.,
and Penninger, J. M.
(1997)
J. Exp. Med.
186,
941-953 |
44. |
Cao, Y.,
Janssen, E. M.,
Duncan, A. W.,
Altman, A.,
Billadeau, D. D.,
and Abraham, R. T.
(2002)
EMBO J.
21,
4809-4819 |
45. | Schmitt, J. M., and Stork, P. J. (2002) Mol. Cell 9, 85-94[Medline] [Order article via Infotrieve] |
46. |
Nishina, H.,
Radvanyi, L.,
Raju, K.,
Sasaki, T.,
Kozieradzki, I.,
and Penninger, J. M.
(1998)
J. Immunol.
161,
3416-3420 |