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Center for Molecular Medicine and Therapeutics and
Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(or C-X-C) chemokines (IL-8, IFN-
-inducible protein-10, and
stromal cell-derived factor-1
(SDF-1)3) (2, 3, 4, 5, 6, 7) and ß (or
C-C) chemokines (RANTES, macrophage inhibitory protein (MIP-1), and
MIP-1ß) (8, 9). Although stimulation of T cells with anti-CD3 Ab
enhances cell migration toward MIP-1, MIP-1ß (9), and
IFN--inducible protein-10 (3), anti-CD3 Ab treatment of T cells
can also inhibit chemotactic responses. For example, stimulation of
CD45RO+ memory T cells induced a rapid down-regulation of
CCR1 and CCR2 receptor expression, associated with an inhibition of
cell migration in response to RANTES or monocyte chemotactic protein-1
(10, 11). Also, vasoactive intestinal peptide, a T lymphocyte
chemoattractant, induced migration of unstimulated T lymphocytes, and
the response to vasoactive intestinal peptide was decreased by
anti-CD3 activation (12). Pretreatment with MIP-1, was shown to
inhibit the anti-CD3 Ab-induced proliferation of murine splenic T
lymphocytes (13) Therefore, the response to a specific chemokine
appears to be determined by both the differentiation and the activation
state of the responding T cell population. SDF-1 mRNA is expressed constitutively in a wide variety of tissues (14, 15), and this chemokine has been identified as a lymphocyte chemoattractant (16). In keeping with this activity, SDF-1 induces actin polymerization in lymphocytes, a process thought to be a prerequisite for cell motility (16). CXCR4, a seven-transmembrane domain, G protein-coupled receptor predominantly expressed on the unactivated naive subset of T cells (17) is the only known receptor for SDF-1 (16, 18) and has also been shown to serve as an accessory factor for cell entry by T cell-tropic HIV isolates (19, 20, 21, 22). Interestingly, there is evidence that the HIV-1 envelope protein, gp120, makes contact with both the CD4 molecule and the CXCR4 receptor (23), and soluble gp120 has been shown to alter anti-CD3-driven proliferation of CD4+ T cells (24, 25). These results suggest that TCR and CXCR4 signaling may be regulated by T cell contact with gp120, and that this association potentially regulates the responses of these cells to SDF-1.
To gain a greater understanding of the biological responses of T cells to SDF-1, we investigated the effect of SDF-1 pretreatment on T cell activation as well as the effect of TCR activation on SDF-1-induced cellular migration. We demonstrate that anti-CD3 Ab stimulation markedly inhibits the migration of T cells toward SDF-1. This inhibitory effect is much weaker in cell lines lacking essential components of the TCR signaling complex, namely, Lck, CD45, and the TCR ß-chain. TCR activation also led to a down-regulation of CXCR4 surface expression. The preincubation of T cells with SDF-1 reduced the anti-CD3-stimulated phosphorylation of critical downstream effectors of TCR signaling, including ZAP-70, SLP-76, and pp36, providing evidence for cross-talk between CXCR4 and the TCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SDF-1 (residues 1–67), generated using an ABI peptide
synthesizer (Applied Biosystems, Foster City, CA), was provided by I.
Clark-Lewis (16, 18). The following Abs were used: OKT3 (American Type
Culture Collection, Manassas, VA), the ascites containing the C305
monoclonal with specificity against the Jurkat Ti ß-chain (a gift
from A. Weiss), 2C11 purified hamster anti-mouse CD3
mAb
(PharMingen Canada, Mississauga, Canada), anti-phosphotyrosine
monoclonal 4G10 and anti-LAT (linker for activation of T cells;
36/38 kDa) rabbit polyclonal Ab 06-807-MN (Upstate Biotechnology, Lake
Placid, NY), ZAP-70 (LR) rabbit polyclonal Ab (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-ZAP-70 kinase (Z24820) and
pan-ERK (E17120; Transduction Laboratories, Lexington, KY),
anti-human SLP-76 sheep antiserum (26) (provided by G. Koretzky),
phospho-specific MAPK (9101S; New England Biolabs, Beverly, MA), fusin
(CXCR4) R-phycoerythrin (R-PE)-conjugated mouse anti-human mAb
(12G5, PharMingen, San Diego, CA), goat F(ab')2
anti-mouse IgG1 FITC (Cedarlane, Hornby, Canada), goat
anti-mouse and/or anti-rabbit peroxidase-labeled (human
serum-adsorbed) IgG (H+L) (Life Technologies, Grand Island, NY), and
peroxidase-conjugated rabbit anti-sheep IgG (H+L) (Pierce,
Rockford, IL). Bisindolylmaleimide (RO 31-8220) was obtained from Roche
Research Center (Welwyn Garden City, U.K.), and staurosporine was
purchased from Sigma (Oakville, Canada).
Cell lines and cell culture
The human acute leukemia T cell line, Jurkat, and derivative clones JE6.1, J.CaM1.6, J45.01, and J.RT3.T3.5 (American Type Culture Collection) were maintained in RPMI 1640 supplemented with 10% FBS, glutamine, 50 µM 2-ME, penicillin, and streptomycin (Life Technologies). Clone JE6-1, selected for lectin-induced IL-2 production, was derived from (Jurkat-FHCRC) (27). J.CaM1.6, a Jurkat clone that does not express Lck (28), was previously selected for a deficiency to mobilize Ca2+ following anti-CD3 Ab stimulation (29). J45.01, which is deficient in CD45 expression, exhibits impaired TCR signaling (30). J.RT3.T3.5, is a CD3- and TCR-negative mutant that lacks the 1.3-kb ß-chain transcript (31). Unfractionated splenocytes were isolated from BALB/c mice, and CD4+ T cells were purified from (CBA/J x C57BL/6)F1 mice using MACS colloidal supermagnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA).
Chemotaxis assay
Jurkat cell lines and murine T cells were washed once in PBS and pelleted by centrifugation at 1500 rpm. Viable cells were resuspended and washed in RPMI 1640 containing 20 mM HEPES, pH 7.2 (Stem Cell Technologies, Vancouver, Canada). Jurkat cells were then resuspended at a cell density of 5.0 x 106/ml in the same medium and were either untreated or treated with 10 µg/ml OKT3/C305 or 100 ng/ml PMA (Life Technologies). Normal splenocytes or T cells isolated from mice were treated with 10 µg/ml 2C11. Cells were delivered (100 µl) to 6.5-mm diameter, 5.0-µm pore size polycarbonate Transwells (Corning-Costar, Cambridge, MA). The lower chamber contained 600 µl of RPMI 1640 medium supplemented with 20 mM HEPES containing SDF-1 (1.5 µg/ml). Transwells were incubated for 3 h in 5% CO2 at 37°C. Cells that migrated to the bottom chamber were transferred to polystyrene tubes. Relative numbers of transmigrated cells were determined by FACScan (Becton Dickinson, Mountain View, CA) on a flow rate setting corresponding to 60 µl/s. Data represent the number of cells collected over 120 s. These values were not extrapolated to the total volume in the bottom chamber (0.6 ml). All experiments were performed in triplicate and were repeated a minimum of three times.
Flow cytometry
Cells (1.0 x 107) were washed once in PBS and pelleted, and viable cells were resuspended in FACS buffer (PBS containing 2% FBS). To assess CXCR4 surface expression, 20 µl of anti-CXCR4-PE was added to 1.0 x 106 cells in a volume of 200 µl. For CD3 levels, OKT3 was added at 1.0 µg/ml for 1 h and following three washes in FACS buffer was incubated with goat F(AB')2 anti-mouse IgG FITC. All incubations were conducted on ice to prevent receptor internalization. After 1 h on ice, cells were washed once with FACS buffer, pelleted by centrifugation, and finally suspended in 500 µl of FACS buffer before analysis on a FACScan (Becton Dickinson) flow cytometer equipped with the LYSIS II software program. For cell surface receptor regulation experiments, cells were washed and counted as described above. However, cells at a density of 1.0 x 107/ml were first preincubated in medium with or without 5 µM RO 31-8220 for 30 min. Cells were then incubated in the presence or the absence of 10 µg/ml OKT3 or 100 ng/ml PMA for the indicated times. Cells were stained and analyzed as described above on a FACScan flow cytometer. For assessment of murine CD4+ lymphocyte purity, 1.0 x 106 cells were incubated for 30 min on ice with R-PE-conjugated rat anti-mouse CD4 (L3T4) mAb and FITC-conjugated rat anti-mouse CD8a (Ly-2) mAb (PharMingen Canada). Cells were washed and analyzed on a FACScan as described above. The CD4+ cells used in the chemotaxis assay were 98% pure.
Assessment of cellular phosphotyrosine content
Cells were washed in PBS and pelleted by centrifugation. Cells (1.0 x 107/100 µl) were incubated in serum-free RPMI 1640 medium containing 20 mM HEPES for 30 min at 37°C in 5% CO2 to restore basal levels of protein phosphorylation. Cells were treated with SDF-1 at 50 µg/ml as indicated. In addition, cells were stimulated with Abs at a 1/100 dilution of ascites (C305) or 10 µg/ml OKT3. At the indicated times, stimulated cells were rapidly lysed in 1.0% Nonidet P-40 lysis buffer and flash-frozen in liquid nitrogen.
Immunoprecipitation and immunoblotting
Cells (1.0 x 107/100 µl) were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris (pH 7.5), and 10% glycerol) in the presence of protease inhibitors (100 µg/ml PMSF, 1.0 µg/ml aprotinin, 0.7 µg/ml pepstatin, 0.5 µg/ml leupeptin, 40 µg/ml bestatin, 10 µg/ml soybean trypsin inhibitor (Boehringer Mannheim, Indianapolis, IN), 10 mM sodium fluoride, 1 mM sodium vanadate, and 1 mM sodium molybdate (BDH, Toronto, Canada)). After 30 min on ice, lysates were centrifuged for 30 min at 13,000 rpm, then precleared with 30 µl of protein A-Sepharose 4 Fast Flow, (Pharmacia Canada) for 1 h. Lysates were then centrifuged at 8000 rpm for 1 min. Total protein (300–500 µg) was incubated with 1.0 µg/ml of the indicated immunoprecipitating Ab for 2 h. Immunoprecipitates were collected by centrifugation as described above and were washed three times in 1% Nonidet P-40 buffer containing PMSF and sodium vanadate. Washed immunoprecipitates were resuspended in 1/3 diluted Laemmli buffer (4% SDS, 5.7 M 2-ME, 20% glycerol, and 20% bromophenol blue). Alternatively, lysate volume corresponding to 15 µg of total protein was diluted 3/1 with Laemmli sample buffer. Samples were boiled for 10 min before electrophoresis. Immune complexes or total protein lysates were separated by SDS-PAGE at 150 V and transferred to nitrocellulose paper by electroblotting at 100 V for 1 h (stirred) at 4.0°C in a solution containing 192 mM glycine, 25 mM Tris, and 20% methanol. Filters were incubated overnight in TBST (10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween-20) containing 5% BSA. Filters were subsequently incubated for 60 min at room temperature in TBST containing 1.0% BSA supplemented with primary Ab at a 1/10,000 (4G10), 1/250 (anti-ZAP-70), 1/500 (SLP-76), or 1/1000 (anti-LAT) dilution. After three additional washes in TBST, filters were incubated for 1 h in the appropriate horseradish peroxidase-conjugated secondary Ab and washed again as described, and proteins were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL) and exposure to Eastman Kodak Biomax MR film (Eastman Kodak, Rochester, NY).
Densitometry
Densitometry was performed using a GS 300 densitometer (Hoefer Scientific Instruments, San Francisco, CA), and results were analyzed using the GS370 1-D Data System, version 2.0 for Macintosh.
Statistical methods
We applied Student’s t test (Microsoft Excel) when analyzing the results. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effect of T cell activation on migration in response to SDF-1, chemotaxis assays were performed on Jurkat T cell leukemia cells and a Jurkat clone (JE6.1) that were competent for TCR signaling and on Jurkat mutant clones (J.CaM1.6, J45.01, and J.RT3.T3.5) that were defective in TCR signaling. Conditions for chemotaxis in the transwell system were established by resuspending cells (5 x 105) in serum-free HEPES (20 mM)-buffered RPMI 1640 medium and incubating them in transwells containing SDF-1. The maximum transmigration response to synthetic SDF-1 (residues 1–67) was observed at a concentration of 1.5 µg/ml (187.5 nM) and was essentially complete within 3 h, as established by prior dose-response and time-course experiments (data not shown). As previously shown (16), we also observed a bell-curve chemotactic response with SDF-1 (0.5–3.0 µg/ml). In addition, Jurkat cells demonstrated a chemotactic, as opposed to chemokinetic, response to SDF-1. Maximal transmigration was retained when 0.15 µg/ml SDF-1 was introduced to the upper well with a concentration of 1.5 µg/ml in the bottom well.
Table I
shows the migratory responses of
Jurkat cells (Jurkat, JE6.1) and Jurkat mutant clones (J.CaM1.6,
J45.01, and J.RT3.T3.5) either treated with OKT3 (10 µg/ml) or
untreated. The number of Jurkat and JE6.1 cells that migrated after
treatment with OKT3 was greatly reduced compared with that of untreated
controls. Thus, incubation of Jurkat or JE6.1 cells with OKT3 before
addition of cells to the upper chamber reduced transmigration to 18 and
14% of the control value, respectively. The observed inhibition of
cell migration was statistically significant (p
< 0.0001). In comparison to the chemotaxis response observed in
treated parental cells, OKT3 treatment of Jurkat clones defective for
TCR signaling reduced migration to 63 (J.CaM1.6), 64 (J45.01), and 78%
(J.RT3.T3.5) of that of untreated cells (Table I). There was no
observed change in the number of cells that transmigrated when Jurkat
or J.CaM1.6 cells were incubated in the presence of an isotype control
mAb (data not shown). Furthermore, Jurkat cells incubated in the
presence of OKT3 for 3 h and subsequently examined microscopically
failed to show evidence of aggregation (data not shown). Thus, the
inhibition of cell transmigration appeared to be due to an
OKT3-specific effect and was not a result of Ab-induced cell
aggregation. The results demonstrate that the SDF-1-induced chemotactic
response of Jurkat cells with an intact TCR signaling complex was
inhibited (5- to 7-fold) compared with that of control cells, and that
cells deficient in TCR activation were considerably less sensitive to
OKT3-mediated inhibition (1- to 1.6-fold).
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). Interestingly, OKT3 treatment reduced the level of
spontaneous transmigration occurring in the absence of an SDF-1
gradient, consistent with OKT3 treatment exerting an inhibitory effect
on T cell motility. Background transmigration of clones with limited
capacity for TCR signaling, as expected, was not significantly reduced
by OKT3 treatment. Differences in transmigration might simply be due to
variation in CXCR4 levels, however, as shown in Fig. 1, similar levels of CXCR4 were detected
in all clones. We also examined cell surface expression of CD3 by flow
cytometry. Whereas Jurkat and JE6.1 expressed similar levels of CD3 on
the cell surface, CD3 expression of J.CaM1.6 and J45.01 was three- to
eightfold lower, and the TCR-defective clone (J.RT3.T3.5) showed only
minimal staining (Fig. 1). Although the transmigration response of all
clones was inhibited to varying degrees by OKT3 treatment, the
decreased response to SDF-1 was most evident in clones expressing the
highest levels of CD3. This inhibition was much less apparent in the
Lck-, CD45-, and TCR ß-chain-
clones (Table I). Although the lack of OKT3-mediated inhibition in
these clones may in part have been due to lower CD3 levels, it most
likely reflected the loss of key signaling molecules, such as Lck and
CD45. In summary, the data demonstrate that the inhibition of SDF-1
activity by anti-CD3 Ab treatment requires a functional TCR
signaling complex.
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To establish whether anti-CD3 stimulation would inhibit the
migration of normal cells toward SDF-1, the response of murine
splenocytes and CD4+ cells was evaluated. As shown in Fig. 2, the SDF-1-induced migratory responses
of both unfractionated splenocytes and purified CD4+ T
lymphocytes were inhibited by treatment of the cells with the
anti-CD3 Ab, 2C11. The response of unfractionated splenocytes
pretreated with anti-CD3 was 15% of the migration of cells in
response to SDF-1 alone (p < 0.00005), and the
migration of anti-CD3-treated CD4+ cells was 30% that
of untreated controls (p < 0.00003; Fig. 2).
These results indicate that anti-CD3-mediated TCR activation of
normal murine T cells inhibits transmigration in response to SDF-1.
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Phorbol esters are known to mimic many of the effects of CD3 stimulation of the TCR complex, involving the activation of protein kinase C (PKC) (32, 33). To investigate whether PKC activation would mimic the OKT3-mediated inhibition of chemotaxis to SDF-1, we stimulated JE6.1 cells with PMA (100 ng/ml) and measured cell transmigration as described above. Treatment with PMA inhibited SDF-1-induced transmigration >300-fold compared with that in untreated controls, whereas we observed a 13-fold inhibition of transmigration in OKT3-treated cells. In addition, the level of spontaneous migration of JE6.1 cells following PMA treatment was reduced 30-fold compared with 5.6-fold in OKT3-treated cells (data not shown). These data demonstrated that OKT3-mediated inhibition of T cell migration could be simulated by PMA treatment. However, PMA was a much more effective inhibitor of chemotaxis.
We next investigated whether PMA stimulation would down-modulate CXCR4
surface receptor expression in Jurkat cells, perhaps accounting for the
observed inhibition of chemotaxis. Jurkat cells were treated with 100
ng/ml PMA and subsequently stained at various points over a 24-h time
course with anti-CXCR4-PE-conjugated Ab. We observed a marked
down-regulation of CXCR4 surface staining within 4 h of PMA
treatment. Cell surface expression was reduced to 22 and 14% of the
control value after 1 and 2 h of incubation, respectively. After
4 h of PMA treatment, most (94%) CXCR4 cell surface staining was
abolished (Fig. 3 and data not shown).
Our observations agree with those of Signoret et al., who reported
similar results with phorbol ester-treated SupT1 T cells (34).
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One potential mechanism that could account for the decreased
migration of OKT3-activated Jurkat cells would be the down-modulation
of CXCR4. We thus treated JE6.1 cells with OKT3 (10 µg/ml) and
assessed cell surface staining of CXCR4. A representative experiment is
shown in Fig. 4. Total fluorescence
staining of CXCR4 was reduced to 65% of the control value by 1 h
(p < 0.0002), with no further change observed
over a 2-h time course (data not shown). Our data demonstrate an
OKT3-induced down-regulation of CXCR4.
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To establish whether the decreased cell surface expression of
CXCR4 in OKT3-treated cells was dependent on PKC activation, we
pretreated JE6.1 cells with 0.50 µM staurosporine or 1.0 µM RO
31-8220 (a staurosporine analogue) to block PKC activation (35). Both
agents were able to inhibit CXCR4 down-regulation induced by OKT3
(p > 0.10), indicating that the effect of this
Ab was at least in part a consequence of PKC activation (Fig. 4 and
data not shown). Interestingly, an increase in cell surface expression
of CXCR4 was seen in JE6.1 cells treated with RO 31-8220, with peak
fluorescence increasing twofold (Fig. 4), suggesting that PKC may
regulate basal levels of CXCR4 in Jurkat cells. Staurosporine, a
nonspecific kinase inhibitor, failed to modulate basal CXCR4 surface
staining (data not shown), indicating that the two agents probably
inhibit overlapping, but nonidentical, molecules. In summary, our
results strongly suggest that the OKT3-induced down-regulation of CXCR4
is in part mediated via PKC activation.
SDF-1 inhibits the OKT3-stimulated phosphorylation of TCR signaling molecules ZAP-70, SLP-76, and pp36
We demonstrated that OKT3 treatment blocked the chemotactic
response of Jurkat cells. It was therefore of interest to determine
whether SDF-1 was capable of regulating TCR signaling. Thus, we
examined early TCR signaling events triggered by treatment with OKT3 or
C305 in the presence or the absence of SDF-1. Fig. 5 shows the effects of SDF-1 treatment on
Jurkat cells stimulated in combination with C305 Ab over 15–120 s.
Results obtained in separate experiments using OKT3 stimulation were
identical with those using C305 (data not shown). Cells were lysed,
processed, and blotted with anti-phosphotyrosine as described
above. Interestingly, in the presence of SDF-1, tyrosine
phosphorylation of a 70-kDa protein was attenuated at time points up to
30 s. Even at 1–2 min, when phosphorylation due to C305
stimulation was maximal, the level of tyrosine phosphorylation of the
pp70 band was reduced compared to that of control C305-stimulated cells
(Fig. 5a). We did not detect any significant change in
C305-induced p70 phosphorylation when SDF-1 preincubations of 15 and 30
min were compared (compare Fig. 5, b and d).
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, b and
d, compare time course on the left to that on the
right). The kinetics of the 36-kDa protein phosphorylation
were consistent with previous reports (36, 37).
There was a concomitant increase in the tyrosine phosphorylation of the
MAP kinases ERK-1 and ERK-2, which reached a maximum at 120 s, as
demonstrated by reprobing the blot in Fig. 5b with a
phospho-specific MAPK Ab (Fig. 5c). Preincubation of these
cells with SDF-1 resulted in immediate phosphorylation of p42 and p44
MAP kinases at time points as early as 15 s, which remained
constant for up to 1 min and further increased at 2 min. As the 36-kDa
protein remained underphosphorylated at a time when MAPK
phosphorylation was increasing, it suggested that the latter was
primarily the result of SDF-1-stimulated CXCR4 signaling (compare Fig. 5, b to c). Thus, SDF-1 exposure attenuated the
C305- and OKT3-induced tyrosine phosphorylation of proteins of 70 and
36 kDa, which appeared to represent downstream targets of TCR
activation. These results were consistent with the temporal kinetics of
C305-mediated activation of ZAP-70 and pp36 (38).
SLP-76, which is phosphorylated on tyrosine as a result of TCR
activation, appears to be a substrate of ZAP-70 (37). Equal amounts of
total protein were immunoprecipitated with 4G10 Ab, resolved by
SDS-PAGE, and blotted with either ZAP-70 or SLP-76 Abs. Fig. 6, a and b, shows a
temporally dependent inhibition of ZAP-70 and SLP-76 tyrosine
phosphorylation as a result of SDF-1 treatment. Densitometric analysis
was performed on the autoradiograph represented in Fig. 6, a
and b. The results represent the ratio of the peak band
intensities of SLP-76 and ZAP-70 (normalized to the peak band intensity
of the lower band shown in Fig. 6, a and
b). There was up to a 50% reduction in the SLP-76
phosphotyrosine signal by 60 s. While there was no significant
difference in ZAP-70 at early time points, there was a 30% reduction
in phosphotyrosine signal by 60 s following stimulation with
anti-CD3 (data not shown). These observations suggest that the
4G10-detectable phosphotyrosine levels of a number of different TCR
signaling molecules were reduced by SDF-1 treatment. To further
characterize the 36-kDa protein that we observed to be
underphosphorylated in C305-stimulated Jurkat cells treated with SDF-1,
total protein lysates from the 2 min point in Fig. 5d were
blotted with anti-LAT Ab (Fig. 7b), then stripped and
reprobed with anti-phosphotyrosine Ab (4G10; Fig. 7a).
As shown in Fig. 7 there was a significant reduction (60% of the
control value) in the phosphotyrosine content of the 36-kDa protein in
SDF-1-treated cells. The attenuation of pp36 phosphorylation was much
greater than that of ZAP-70 and SLP-76, suggesting that the effects of
SDF-1 on TCR signaling have a greater impact on events downstream of
ZAP-70 and SLP-76 activation.
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,
lanes 1 and 2) from precleared lysates were
incubated (lanes 3 and 4) with polyclonal
anti-ZAP-70 Ab (Fig. 8). Total lysates or immunoprecipitations were
resolved by SDS-PAGE followed by immunoblotting with 4G10 Ab (Fig. 8, upper panel). The filter was then stripped and reprobed with
rabbit polyclonal ZAP-70 Ab (Fig. 8, lower panel). Our
results suggest that the pp70 protein that is underphosphorylated as a
result of SDF-1 treatment is ZAP-70.
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To identify the 36-kDa protein that was underphosphorylated on
tyrosine in SDF-1-treated cells, total lysates were immunoprecipitated
with anti-LAT Ab (4 µg), resolved by PAGE, and blotted with 4G10
(Fig. 7c). These results strongly suggested that LAT, a
molecular species that is highly phosphorylated on tyrosine in
C305-stimulated Jurkat cells (38), was the 36-kDa protein that was
underphosphorylated in C305-stimulated Jurkat cells following SDF-1
pretreatment.
Given that p70 phosphorylation was reduced in OKT3-treated Jurkat
cells, we investigated whether stimulation with SDF-1 alone would have
an effect on the basal phosphorylation level of p70. SDF-1-treated and
untreated Jurkat cells were thus compared in a time-course experiment
(Fig. 9, a and b).
Interestingly, SDF-1 treatment was associated with a progressive loss
in the phosphotyrosine content of p70 in a time-dependent manner in the
absence of TCR activation (Fig. 9a). In contrast, in the
absence of SDF-1, p70 phosphotyrosine content remained unchanged (Fig. 9b). Furthermore, baseline phosphorylation of the 70- and
36-kDa proteins was markedly reduced when cells were preincubated with
SDF-1 for 6 h (data not shown). Taken together, our results
demonstrate that SDF-1 is capable of inhibiting the tyrosine
phosphorylation of proteins downstream of the TCR/CD3/ZAP-70 complex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is possible that the TCR activation of PKC leads to PLCß3
activation, which, in turn, mediates the inhibition of CXCR4 activity,
an effect that represents an agonist nonspecific heterologous
desensitization pathway (43). Although there is evidence in the
literature that demonstrates that SDF-1 induces CXCR4 coupling to both
Gi (pertussis toxin-sensitive) (5, 39) and Gq
(pertussis toxin-insensitive) (40, 42) family members of G proteins, it
is possible that a given receptor uses more than one G protein subunit
and may initiate several different signaling pathways (44). The
Gq subunit can directly activate PLCß3 (45).
On the contrary, PLCß3 phosphorylation by PKC may block activation by
Gq (45). In addition, PLCß3 phosphorylation
was blocked by the PKC inhibitor, bisindolylmaleimide (45). The former
inhibited responses initiated by the G protein-coupled receptor for
platelet-activating factor (45). Given that PKC inhibitors blocked the
OKT3-mediated down-regulation of CXCR4, it is likely that the
down-regulation of receptor expression subsequent to CD3-stimulation
involved the activation of PKC (Fig. 4). PMA treatment ablated
transmigration of Jurkat cells, an inhibition that correlated with the
complete loss of CXCR4 cell surface expression (Fig. 3). It is
unlikely, however, that the relatively modest levels of CXCR4 receptor
down-modulation we observed in OKT3-stimulated Jurkat cells accounted
for the striking inhibition of chemotaxis. It may be hypothesized that
CD3-mediated activation of PKC leads to the phosphorylation PLCß3,
which inhibits the activation of Gq (46) and
thereby produces heterologous desensitization of CXCR4. Our results are
in agreement with such a model and also provide evidence for cross-talk
between the TCR complex and CXCR4. The response of T cells to TCR
activation is probably complex, first involving a migration stop
signal, followed by the down-regulation of CXCR4 activity and cell
surface expression.
Another possibility is that the TCR and CXCR4 signaling pathways share
the same Gq subunit. TCR signaling also
mediates GTP exchange within Gq/11 and leads
to its association with CD3 in Jurkat cells (46). G protein
activation was tyrosine kinase dependent and mediated activation of
PLCß. Stanners et al. demonstrated a G/11-dependent modulation of
CD3 - and 
-chain phosphorylation and that a functionally
deficient G/11 mutant was associated with diminished tyrosine
phosphorylation of CD3 and and of ZAP-70 upon anti-CD3 Ab
stimulation (46). Thus, TCR-CD3 activation could sequester
Gq from CXCR4, thereby preventing
SDF-1-induced effects. SDF-1, on the other hand, might produce a rapid
up-regulation of CXCR4 activity (40), leading to
Gq recruitment. Such a mechanism agrees with a
model proposed by Dustin (47) suggesting that the TCR-induced stop
signal would be dominant in resting T cells when CXCR4 levels are low,
but the signal from SDF-1 may gain dominance once CXCR4 levels on the T
cell blasts are up-regulated. Moreover, a further consequence of a
shift in Gq usage induced by SDF-1 might be
the inhibition of signaling molecules, such as ZAP-70 and pp36, that
lie downstream of the TCR.
Whereas we observed an inhibition of SDF-1-induced chemotaxis and a reduction in CXCR4 cell surface expression during initial activation of lymphocytes, in Jurkat cells others have reported that CXCR4 receptor levels and chemotaxis were up-regulated after either PHA stimulation or IL-2 priming, reaching a peak at 3–6 days. These effects were associated with an increased chemotactic response (17). Moreover, Hesselgesser et al. (39) observed a marginal increase in the chemotactic response of anti-CD3-stimulated Jurkat cells. The discrepancy between these observations and those reported herein probably reflect differences in the TCR stimulation protocols. Thus, treatment of cells for 3–6 h with anti-CD3 Abs before the chemotaxis assay would probably lead to the activation of a number of genes, including those encoding cytokines (32), that might modulate the response to SDF-1. Our experiments, in contrast, examined the effect of initial T cell activation events on the migratory response to SDF-1.
To establish whether the inhibition of chemotaxis seen in
C305-stimulated Jurkat cells would also be a feature of normal T cells,
we assessed the migratory response to SDF-1 of anti-CD3-stimulated,
unfractionated murine splenocytes or CD4+-enriched T cells.
As with Jurkat cells, the SDF-1-induced migratory response of normal T
cells was also inhibited by anti-CD3 treatment. Interestingly,
there was some variation in the response of unfractionated splenocytes
compared with purified CD4+ T cells (Fig. 2), suggesting
either that a non-CD4+ T cell population made up a large
fraction of the migratory population in the unfractionated splenocytes
or perhaps that the CD4 selection procedure altered the chemotactic
response of the purified T cells. In any event, our data clearly
demonstrate that the block of cell migration in response to T cell
activation is probably a mechanism common to both human and murine
species.
There was no significant difference in the cell surface expression of
CXCR4 among the various Jurkat clones used in our study (Fig. 1). There
was, however, some variability in SDF-1-induced transmigration among
these clones (Table I). While the reason(s) for this is probably
complex, the finding of reduced chemotaxis in the lines deficient in
Lck and CD45 raises the possibility that these molecules play some
facilitatory role in either SDF-1 signaling or cell motility. Taken
together with the inhibitory effect of T cell activation on
SDF-1-induced chemotaxis, these observations raise the possibility that
reciprocal regulation of TCR activation by SDF-1 might also occur. We
therefore examined the effect of SDF-1 treatment on the signaling
events subsequent to TCR activation. These events have been well
characterized (for review, see Refs. 48 and 49). Thus, engagement of
the TCR results in the activation of Lck/Fyn, members of the Src family
of protein tyrosine kinases. Phosphorylation of TCR- leads to the
recruitment, phosphorylation, and activation of ZAP-70 protein tyrosine
kinase (50, 51, 52) and subsequent phosphorylation of other key molecules,
such as SLP-76 (26, 37) and LAT (38). Events downstream and parallel
with the activation of SLP-76 and LAT eventually culminate in T cell
activation, proliferation, and cytokine production (49, 53, 54). The
uncoupling of this cascade at initial phosphorylation events would be
predicted to have a great impact on the magnitude of the downstream
responses. For example, a reduction in ZAP-70 activity could greatly
alter the character of the activation response. In keeping with this
hypothesis, we demonstrate a reduction in the phosphotyrosine content
of ZAP-70 and SLP-76 following SDF-1 treatment. This effect is, in
turn, associated with a pronounced inhibition of pp36 phosphorylation.
The 36- to 38-kDa protein LAT was shown to be a substrate for ZAP-70
(38). Moreover, pp36–38 has been shown to associate with Grb2 and
PLC1 in C305-stimulated Jurkat cells (38). Our results further
demonstrate that the 36-kDa protein that is underphosphorylated in
Jurkat cells treated with SDF-1 represents LAT, a molecule that appears
to be critical to T cell activation (38). Our results suggest that
SDF-1 is capable of inhibiting critical components of the TCR signaling
cascade. The precise level at which SDF-1 exerts its inhibitory effect
on TCR signaling remains to be determined. Interestingly, the naive,
CD26low, CD45RA+, CD45RO- T cells
express high levels of CXCR4 protein and mRNA (17, 55). Given the
ability of SDF-1 to suppress phosphorylation of ZAP-70, pp36, and
SLP-76, there is a possibility that this chemokine may regulate the
activation of naive cells in vivo.
In addition to having a role in lymphocyte chemotaxis, CXCR4 is a
coreceptor for T cell tropic HIV-1 strains (56). Although CXCR4
signaling does not appear to be required for viral entry into the cell
(57), it is not clear whether gp120 binding induces CXCR4 signaling.
HIV-1 gp120 is known to contact both CD4 and CXCR4, probably forming a
complex (23), which, in turn, may modulate both T cell activation (58)
and migration (59). Several studies have indicated that gp120 is able
to inhibit TCR signaling, an effect that is accompanied by a reduction
in downstream and subsequent phosphorylation events (25, 58, 60, 61).
Moreover, T cells obtained from early asymptomatic and symptomatic/AIDS
patients demonstrated a reduction in the tyrosine kinase activity of
Lck, Fyn, and ZAP-70 associated with a decrease in -chain
phosphorylation (62). In view of our results suggesting that CXCR4
signaling may regulate TCR signaling, it would be important to
establish whether the effects of gp120 on T cells might be due at least
in part to binding of this molecule to CXCR4.
In conclusion, we have demonstrated in Jurkat and normal murine T cells that anti-CD3 stimulation of the TCR complex inhibits SDF-1 function. Jurkat clones deficient in TCR signaling were much less sensitive to the effects of anti-CD3 treatment, confirming the role of TCR components in this process. Anti-CD3 Ab stimulation of Jurkat cells also reduced CXCR4 cell surface expression, a process that was inhibited by staurosporine and RO 31-8220. These results suggest that TCR complex-mediated activation of PKC is involved in CXCR4 receptor down-modulation. We have also shown that SDF-1 treatment of C305-stimulated cells is associated with reduced phosphorylation of critical TCR downstream effectors, ZAP-70, SLP-76, and pp36. Taken together, these observations are suggestive of reciprocal regulation between TCR and CXCR4. The latter finding suggests that the SDF-1 chemokine is potentially capable of regulating the threshold of T cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frank R. Jirik, Center for Molecular Medicine and Therapeutics, 950 West 28th Ave., Vancouver, British Columbia, Canada V5Z 4H4. E-mail address:
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; MIP-1, macrophage inhibitory protein-1; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; R-PE, R-phycoerythrin; PKC, protein kinase C; PLCß3, phospholipase Cß3.
Received for publication June 8, 1998. Accepted for publication September 21, 1998.
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