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,
*
Autoimmunity/Diabetes Group, John P. Robarts Research Institute, London, Ontario, Canada; and Departments of
Microbiology and Immunology and
Medicine, University of Western Ontario, London, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induced
by both CD3 and CD3/CD28 ligation and the nuclear expression of the
c-Jun and ATF-2 proteins are each blocked by the p38 MAPK inhibitor
SB203580. Our findings demonstrate that p38 MAPK 1) plays an important
role in signal integration during costimulation of primary mouse T
cells, 2) may be involved in the induction of c-Jun activation and
augmentation of AP-1 transcriptional activity, and 3) regulates whether
T cells enter a state of functional
unresponsiveness. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERKs are activated by agonists for tyrosine-encoded receptors and G protein-coupled receptors that induce mitogenesis or cellular differentiation (2, 3, 4). ERKs mediate the effects of these agonists by phosphorylating and regulating the activity of several proteins, including cytoplasmic enzymes and nuclear factors (1, 2). JNKs phosphorylate the NH2-terminal activation domain of c-Jun and activating transcription factor-2 (ATF-2), increasing their transcriptional activity (4, 10). JNKs are activated preferentially by cellular stress and inflammatory cytokines, but also by G protein-coupled receptor agonists, growth factors, and cytoplasmic oncogenes (4, 10, 11, 12, 13, 14, 15, 16). Similarly, p38 MAPK is activated by cellular stresses, inflammatory cytokines, LPS (14, 17, 18, 19), and G protein-coupled receptors (20), and activated p38 MAPK in turn mediates cytokine production, stress responses, and apoptosis (21, 22, 23, 24, 25, 26). The p38 MAPK substrates include MAPK-activating protein kinase-2 (MAPKAP kinase-2), ATF-2 (18, 19, 27, 28), cAMP response element binding protein (CREB), ATF-1 (29), Elk-1 (25), C/EBP-homologous protein (CHOP) (30), and myocyte-enhancer factor 2C (MEF2C) (31).
TCR engagement activates the ERK cascade in T cells (32, 33, 34). Analyses of Jurkat human T cells and various activated mouse T cell clones have suggested that JNK activation stimulated by TCR engagement requires CD28 coligation (33, 34). p38 MAPK may be fully activated in mouse T cell clones by signaling via either CD3 or CD28, but CD3/CD28 costimulation does not further enhance the amount of p38 MAPK activation (35). In contrast, stimulation of CD28 fails to activate p38 MAPK, but synergizes with CD3 stimulation to fully activate p38 MAPK in preactivated proliferating T cells (36). These results raise the possibility that p38 MAPK may mediate CD28 costimulation in primary naive mouse T cells. The latter possibility is supported by reports that, in T cells from MKK-4-deficient mice, CD28-mediated IL-2 production and proliferation are impaired and CD28 costimulation and PMA/Ca2+ ionophore-induced signaling can stimulate proliferation and IL-2 production independently of JNK activation (37). In addition, in human T cells, the p38 MAPK inhibitor SB203580 blocks CD28-dependent proliferation and IL-2 production (38). Thus, the question of whether p38 MAPK is activated upon either TCR or CD28 stimulation or after TCR/CD28 coligation in primary, unstimulated, naive mouse T cells merits further investigation.
In this study, we determined whether differential regulation of MAPK activation occurs in primary mouse T cells in response to TCR/CD28 or PMA/Ca2+ ionophore costimulation. We show that p38 MAPK activation mediates both TCR- and CD28-induced signaling in primary mouse T cells. Ligation of TCR or CD28 results in only modest p38 MAPK activation, whereas TCR and CD28 synergize upon coligation to elicit enhanced p38 MAPK activation. PMA/Ca2+ ionophore costimulation, which mimics TCR/CD28-mediated signaling, fully activates p38 MAPK in primary mouse T cells. Our results demonstrate that p38 MAPK is involved in both TCR- and CD28-signaling pathways, and that p38 MAPK, but not JNK, is involved in signal integration during costimulation of naive mouse primary T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME), maintained in the Animal Care Facility of the Faculty of Medicine at the University of Western Ontario (London, ON, Canada), and used at 6–10 wk of age.
Abs, proteins, and reagents
The following reagents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): rabbit polyclonal Abs against mouse ERK-1, JNK-1/2, and p38 MAPK; mouse mAbs against c-Jun, c-Fos, and ATF-2; glutathione S-transferase (GST)-c-Jun 1–79(1–79), GST-ATF-2 1–505(1–505), and glutathione-agarose. MBP, IL-2, cycloheximide, and PMA were each obtained from Sigma (St. Louis, MO). Anti-MAPKAP kinase-2 antiserum and recombinant murine heat-shock protein 25 (hsp25) were purchased from Upstate Biotechnology (Lake Placid, NY) and StressGen Biotechnology (Victoria, Canada), respectively. The 145-2C11 anti-CD3 and 37.51 anti-CD28 mAbs were purified by protein G affinity chromatography (Pharmacia Biotech, Uppsala, Sweden) of the supernatants of the B cell hybridomas kindly supplied by Dr. J. Bluestone (University of Chicago, Chicago, IL) and Dr. J. Allison (University of California, Berkeley, CA), respectively. The PV-1 anti-CD28 mAb was kindly supplied by Dr. C. June (Naval Medical Research Institute, Bethesda, MD). The p38 MAPK inhibitor 203580 was generously provided by Dr. P. Young (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). Cyclosporin A (CsA) was supplied by Sandoz Canada (Dorval, Quebec, Canada). The mouse anti-human CD3 (OKT3) and CD28 mAbs were obtained from PharMingen (San Diego, CA). PMA and A23187 was purchased from Calbiochem (San Diego, CA), respectively. IL-2 and cycloheximide were purchased from Sigma.
Cell isolation and stimulation
B6 and BALB/c thymocytes or splenic T cells were purified
(purity 
98% as determined by FACS analysis of CD3 cell surface
expression) on T cell enrichment columns (R&D Systems, Minneapolis,
MN). Murine T cells were cultured for 5 h at 37°C in complete
RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM
HEPES, 0.1 mg/ml streptomycin, 100 U/ml penicillin, 0.05 µM 2-ME, and
2 mM glutamine (all purchased from Life Technologies, Burlington, ON,
Canada) before stimulation to decrease high basal levels of p38 MAPK
activity. Cells were stimulated for various times with either the
anti-CD3, anti-CD28, or anti-CD3 plus anti-CD28 mAbs,
or with PMA plus the Ca2+ ionophore A23187. Jurkat human T
cells were also grown in complete RPMI 1640 under the same conditions.
After culture, cells (2 x 107/ml) were resuspended at
37°C in complete RPMI 1640, washed twice with serum-free RPMI 1640,
and stimulated as above. Where indicated, SB203580 or CsA was added to
the cells 15 min before stimulation. To obtain proliferating T cells,
splenic T cells were cultured (106/ml) in complete RPMI
1640 in six-well plates precoated with anti-CD3 (1 µg/ml) and
IL-2 (20 U/ml). After 48 h, T cells were removed from the plates
and expanded in IL-2. Proliferating T cells were harvested on day 4 of
culture, and were then stimulated with either anti-CD3,
anti-CD28, or anti-CD3 plus anti-CD28 mAbs.
In vitro kinase assays
After stimulation, cells were lysed in ice-cold lysis buffer
containing 1% Triton X-100, 10 mM Tris (pH 7.5), 150 mM NaCl, 2 mM
EGTA, 50 mM ß-glycerophosphate, 2 mM Na3VO4,
10 mM NaF, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml
apoptinin. Lysates were clarified by centrifugation at 12,000 rpm for
10 min at 4°C, and their protein content was determined by the
Bradford assay using BSA as a standard. Lysates were divided into three
replicate samples; incubated for 1 h at 4°C with either
anti-ERK-1, anti-JNK-1, or anti-p38 MAPK Abs; and further
reacted with protein G agarose (Santa Cruz) or protein A-Sepharose
CL-4B (Pharmacia Biotech, Baie d’Urfe, PQ, Canada) for an additional
1 h at 4°C. Immunoprecipitates were washed three times with
lysis buffer and twice with kinase buffer (20 mM HEPES (pH 7.5), 20 mM
MgCl2, 20 mM MnCl2, 2 mM DTT, 25 mM
ß-glycerophosphate, and 100 nM Na3VO4).
Kinase assays were performed using MBP, GST-c-Jun, and GST-ATF-2 fusion
proteins as substrates for ERK-1, JNK-1, and p38 MAPK, respectively.
Immunoprecipitates were resuspended in 18 µl kinase buffer containing
5 µg MBP, 1 µg GST-c-Jun, and 1 µg GST-ATF-2 fusion proteins in
the presence of 20 µM cold ATP and 20 µCi
[-32P]ATP (Amersham Life Science, Arlington Heights,
IL), and incubated for 30 min at 30°C. Solid-phase JNK assays were
performed essentially as previously described (39). Whole cell extracts
(2 x 107 cell equivalents) were prepared and reacted
for 4 h at 4°C with a GST-c-Jun 1–79(1–79) fusion protein bound to
glutathione-agarose beads to immobilize JNK. The washed beads were then
analyzed for their associated kinase activity by incubation for 30 min
at 30°C in kinase buffer containing 20 µCi
[-32P]ATP. Reactions were terminated by the addition
of SDS sample buffer, samples were boiled, and kinase reaction products
were resolved by SDS-PAGE. The MAPKAP kinase-2 assay was performed as
described (35), using murine recombinant hsp25 as a substrate. Equal
loading of precipitated proteins was confirmed by probing the blots
with specific Abs, and phosphorylation of the substrates was
quantitated using a Molecular Imager System and Molecular Analyst
imaging software (Bio-Rad, Hercules, CA).
T cell proliferation assay
Splenic T cells (106/ml) were resuspended in
complete RPMI 1640 medium in the absence or presence of various
concentrations of SB203580, and then incubated for 15 min at 37°C.
Cells were cultured for 48 h at 37°C in round-bottom 96-well
plates (Nunc) precoated with the 145-2C11 anti-CD3
mAb (1
µg/ml) in the presence or absence of the 37.51 anti-CD28 mAb (1
µg/ml). [3H]thymidine (1 µCi/well; Amersham) was
added 24 h before the end of culture, and cultures were harvested
using a Tomtec Harvester 96 cell harvester (Fisher Scientific, Ottawa,
ON, Canada). The extent of T cell proliferation was proportional to the
amount of [3H]thymidine incorporation, which was
determined using a Wallac 1450 Microbeta Plus beta counter (Fisher
Scientific).
Cytokine assays
Splenic T cells (106/ml) were pretreated with
different concentrations of SB203580 for 15 min at 37°C, and cultured
in round-bottom 96-well plates coated with anti-CD3 (1 µg/ml) in
the presence or absence of the 37.51 anti-CD28 mAb (1 µg/ml).
Supernatants collected after 48 h were assayed for their cytokine
concentrations by ELISA using a double ligand method. IL-2
concentrations were interpolated from a standard curve using murine
rIL-2 captured by the JES6-1A12 mAb and detected by the biotinylated
JES6-5H4 mAb. IL-4 concentrations were measured using recombinant
murine IL-4, the BVD4-1D11 mAb, and biotinylated BVD6-24G2 mAb, while
IFN- concentrations were detected using recombinant murine IFN-,
R4-6A2 mAb, and biotinylated XMG1.2 mAb (all obtained from PharMingen).
Briefly, flat-bottom 96-well microtiter plates were coated with 50
µl/well of capture mAb (1 µg/ml) in 0.1 M NaHCO3
overnight at 4°C. Nonspecific binding sites were blocked with 3% BSA
for 2 h at 23°C. Standards or samples (50 µl) were added, left
overnight at 4°C, and then incubated with 50 µl/well of
biotinylated detecting mAb (1 µg/ml) for 45 min at 23°C.
Streptavidin-peroxidase conjugates (1 µg/ml; Sigma) in
diethanolamine buffer were successively added to develop the
reaction at 23°C. Plates were read at 405 nm in an automated
microplate reader (Bio-Rad). Cytokine standard curves were
linear in the range of 20–20,000 pg/ml.
Cell viability assay
Splenic T cells were stimulated with an anti-CD3 mAb as above in the presence or absence of SB203580 (100 µM). Cells were harvested after incubation for 48 h, washed in PBS, stained with propidium iodide, and analyzed by flow cytometry.
Effect of SB203580 on nuclear expression of c-Fos, c-Jun, and ATF-2
Thymocytes (4 x 107/ml) were pretreated for 15 min with 10 µM SB203580, and were then stimulated for 5 or 15 min with anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml). Alternatively, thymocytes were stimulated for 4 h at 37°C with either anti-CD3, anti-CD28, anti-CD3 plus anti-CD28, or PMA (50 ng/ml). The cells were then collected, washed with ice-cold PBS, and lysed for 30 min at 4°C in hypotonic lysis buffer (20 mM HEPES (pH 7.5), 5 mM NaCl, 3 mM MgCl2, 1 mM DTT, 5% glycerol, 0.4% Nonidet P-40, 2.5 mM PMSF, 40 µg/ml aprotinin, 40 µg/ml leupeptin, 2 mM EDTA, 1 mM Na3VO4, and 10 mM NaF). Nuclear extraction was performed as previously reported (40). Extracts were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-c-Fos, anti-c-Jun, and anti-ATF-2 mAbs, and the c-Fos, c-Jun, and ATF-2 proteins were detected by chemoluminescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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JNK activation requires costimulation by either CD28 or
Ca2+ ionophore in Jurkat T cells, suggesting that JNK is
involved in signal integration during T cell costimulation (33).
However, it is not known whether activation of JNK and p38 MAPK occurs
in primary naive T cells that are not further stimulated in vitro. To
determine whether CD28 regulates MAPK activation following CD3
ligation, murine thymocytes or purified splenic T cells were stimulated
for 15 min with anti-CD3, anti-CD28, or both mAbs. Cells were
lysed; ERK-1, JNK-1, and p38 MAPK were immunoprecipitated with specific
Abs; and immunoprecipitates were assayed for the activities of
associated MAPKs by their ability to phosphorylate the MBP, c-Jun, and
ATF-2 substrates, respectively. Anti-CD3 stimulation activated ERK-1 in
thymocytes and splenic T cells, and this level of activation of ERK-1
was not increased after CD3/CD28 costimulation (Fig. 1
,
A and B, upper panels), as reported
(33, 34).
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, A and B,
middle panels). Anti-CD3 or anti-CD28 stimulation
induced modest p38 MAPK activity, which was elevated about fivefold
after CD3/CD28 coligation (Fig. 1, A and B,
lower panels). PMA activated ERK-1, JNK-1, and p38 MAPK in
thymocytes and splenic T cells (Fig. 1, A and B).
Stimulation of splenic T cells with the Ca2+ ionophore
A23187 did not activate ERK-1 or JNK-1, but rather stimulated p38 MAPK
activity and enhanced the level of p38 MAPK activity induced by PMA
alone (Fig. 1B, lower panel). JNK-2 may bind and
phosphorylate c-Jun more efficiently than JNK-1 (41, 42). To exclude
the possibility that in primary T cells JNK-2 may be solely responsible
for c-Jun phosphorylation, thymocytes were lysed following stimulation
with either anti-CD3, anti-CD28, anti-CD3 plus CD28, or
PMA. JNK-2 activity associated with anti-JNK-2 immunoprecipitates
was examined by c-Jun phosphorylation in an in vitro kinase assay. As
observed for JNK-1, little or no JNK-2 activity was detected in
response to ligation of either CD3, CD28, or both (Fig. 1C,
upper panel). JNK-2 was activated, however, by PMA
stimulation. The failure to activate JNK-1 and JNK-2 by coligation of
CD3 and CD28 was also observed in BALB/c splenic T cells (data not
shown), suggesting that these observations are not mouse strain
dependent. Since several splice variants of JNK-1 and JNK-2 have been
found recently (43), it is possible that the anti-JNK Abs used may
not precipitate all variant forms of JNK. To eliminate this
possibility, GST-c-Jun precoupled to glutathione-agarose beads was used
in a solid-phase JNK assay to precipitate all JNK variants. Similar to
the in vitro kinase assay of JNK-1, c-Jun phosphorylation was not
increased upon CD3, CD28, or CD3/CD28 ligation, but was induced
significantly upon PMA stimulation (Fig. 1C, lower
panel).
To further examine the activation of ERK-1, JNK-1, and p38 MAPK induced
by stimulation of CD3, CD28, or CD3 plus CD28, thymocytes were
pretreated with a constant amount (10 µg/ml) of anti-CD3 together
with variable amounts of anti-CD28. With increasing concentrations
of anti-CD28 mAb (0.1–20 µg/ml), no further augmentation of
ERK-1 activation was observed compared with the amount of ERK-1
activity stimulated by anti-CD3 alone (Fig. 2A, upper panel). A similar result
was obtained for JNK-1 activity. However, p38 MAPK was optimally
activated by anti-CD28 at concentrations of 1–5 µg/ml (Fig. 2A, lower panel).
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B, upper and
lower panels), but JNK-1 was not activated throughout the
entire time course (Fig. 2B, middle panel). This
effect of CD28 costimulation was also analyzed by pretreating
thymocytes with an optimum concentration of anti-CD28 mAb (5
µg/ml) plus variable concentrations of anti-CD3 mAb (0.1–20
µg/ml). ERK-1 activation was gradually enhanced by addition of
increasing concentrations of anti-CD3, with maximal activation
observed at 10 and 20 µg/ml (Fig. 2C,
upper panel). Similarly, p38 MAPK activity was elevated by
increasing concentrations of anti-CD3 and was evident even at very
low concentrations (0.1 µg/ml) of added anti-CD3 (Fig. 2C, lower panel). In contrast, JNK-1 was not
activated significantly at any concentration of added anti-CD3
(Fig. 2C, middle panel). The inability to detect
JNK-1 activation upon CD3/CD28 coligation in primary mouse T cells may
be due to a lack of reactivity of the 37.51 anti-CD28 mAb with the
appropriate CD28 epitope required for JNK-1 activation. However, the
use of another anti-CD28 mAb, PV-1, which recognizes a different
epitope than the 37.51 anti-CD28 mAb as determined by a competitive
binding assay (our unpublished observations), did not activate JNK-1
(Fig. 2D). Note that PMA activated JNK-1 in a positive
control sample. These data indicate that p38 MAPK, but not JNK, is
involved in both TCR- and CD28-mediated signaling pathways, and may
play an important role in signal integration during CD28 costimulation
of primary mouse T cells. Synergistic activation of p38 MAPK requires costimulation by Ca2+ in primary mouse T cells
In Fig. 1B, we showed that the Ca2+
ionophore A23187 (500 ng/ml) activates p38 MAPK, synergizes with PMA to
further enhance p38 MAPK activation, and inhibits JNK-1 activity
induced by PMA. To analyze the relationship of these findings to the
dose of A23187, splenic T cells were stimulated with PMA (50 ng/ml)
plus a range of concentrations (15–1000 ng/ml) of A23187. ERK-1
activity was not enhanced at any of the concentrations of A23187 used
(Fig. 3A, upper panel). JNK-1
activity was inhibited significantly in a dose-dependent manner when
A23187 was added to PMA, and concentrations of 125 ng/ml A23187
abolished virtually all detectable JNK-1 activity (Fig. 3A,
middle panel). Note that this inhibition of JNK by A23187
was not due to different kinetics of JNK activation induced by
PMA/A23187 versus PMA alone. PMA-induced JNK activation was
significantly increased in peripheral T cells after stimulation for 15
min, whereas little or no activation of JNK was induced by PMA/A23187
(Fig. 3B). In contrast, p38 MAPK activity was enhanced upon
exposure of the T cells to increasing doses of A23187 and reached a
maximum at a concentration of 60 ng/ml (Fig. 3A, lower
panel). Interestingly, the latter findings on induced p38 MAPK
activity correlate closely with our observations on the stimulation of
proliferation of splenic T cells by PMA and variable amounts of A23187
(Fig. 3C). The observed inhibition of PMA-induced JNK
activation by A23187 suggests that Ca2+ ionophore may
activate some JNK-specific MAPK phosphatases that inactivate JNK.
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C,
stimulation with anti-CD3 or anti-CD28 alone resulted in only
modest p38 MAPK activation, and p38 MAPK was activated synergistically
by either CD3/CD28 coligation or PMA/A23187 treatment (Fig. 3D, upper panel). These induced p38 MAPK
activities were inhibited appreciably by CsA, with the exception that
PMA- and CD28-induced p38 MAPK activation was CsA resistant.
Interestingly, inhibition of PMA/Ca2+ ionophore-induced p38
MAPK activity by CsA pretreatment occurred in a dose-dependent manner
(Fig. 3D, lower panel). JNK activity was induced
only by PMA, and this PMA-induced JNK activity was inhibited by
Ca2+ ionophore (Fig. 3D, upper
panel). However, CsA did not further inhibit JNK activity induced
by PMA/Ca2+ ionophore (Fig. 3D, lower
panel). Thus, optimal activation of p38 MAPK in primary mouse T
cells requires two signals, one provided by the TCR/CD3 complex or PMA
and another provided by CD28 or Ca2+ ionophore. Different requirements for JNK-1 and p38 MAPK activation in Jurkat T cells, proliferating T cells, and primary T cells
JNK activation requires two signals generated by the TCR and CD28
or PMA and Ca2+ ionophore in Jurkat T cells and mouse T
cell clones (33, 34). To determine whether p38 MAPK activation has the
same requirements in Jurkat T cells, we compared the activation of
ERK-1, JNK-1, and p38 MAPK in response to ligation of CD3,
CD28, CD3 plus CD28, PMA, and PMA plus Ca2+ ionophore in
Jurkat T cells. ERK-1 activation was only seen upon anti-CD3 or PMA
stimulation, and no further augmentation was observed upon CD3/CD28
coligation or exposure to PMA and A23187 (Fig. 4A, upper panel). JNK-1 activation
required CD3/CD28 coligation. PMA induced modest JNK-1
activation, which was significantly enhanced by PMA/A23187 stimulation
(Fig. 4A, middle panel). Similarly, anti-CD3
or PMA weakly activated p38 MAPK, but full activation of p38
MAPK required CD3/CD28 coligation or PMA/Ca2+ ionophore
costimulation (Fig. 4A, lower panel). Next, we
examined the effect of Ca2+/calcineurin on the
activation of ERK-1, JNK-1, and p38 MAPK in Jurkat T cells after a
15-min incubation with CsA (500 ng/ml). CsA did not inhibit
ERK-1 activation in response to CD3/CD28, PMA, or
PMA/Ca2+ ionophore stimulation (Fig. 4B).
However, activation of JNK-1 and p38 MAPK induced by either CD3/CD28
coligation or PMA/A23187 was markedly reduced. Notably, activation of
JNK-1 and p38 MAPK induced by PMA was insensitive to CsA pretreatment.
These data are the first to indicate that p38 MAPK activation in Jurkat
T cells also requires two signals generated by TCR/CD28 or
PMA/Ca2+ ionophore signaling.
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C, upper panel), whereas optimal p38 MAPK
activation required TCR/CD28 costimulation (Fig. 4C,
lower panels). In contrast, JNK-1 was modestly activated by
anti-CD3 stimulation, but no synergistic activation of JNK-1
occurred upon CD3/CD28 coligation. JNK-1 was fully activated, however,
by CD28 ligation (Fig. 4C, middle panel). These
data suggest that Jurkat human T cells, proliferating mouse T cells,
and primary mouse T cells differ in terms of their requirements for
ERK-1, JNK-1, and p38 MAPK activation. p38 MAPK activity is required for CD3 or CD3/CD28 ligation-induced T cell proliferation
The requirement of p38 MAPK activation for T cell proliferation
induced by CD3 or CD3/CD28 ligation was investigated. Splenic T cells
were preincubated with different concentrations of SB203580, a specific
inhibitor of p38 MAPK activity, and were then cultured in complete RPMI
1640 medium with plate-bound anti-CD3 mAb in the presence or
absence of anti-CD28 mAb. SB203580 inhibited T cell proliferation
significantly and blocked T cell proliferation completely at
concentrations of 10 and 100 µM, respectively (Fig. 5A). To exclude the possibility that
inhibition of T cell proliferation results from SB203580-induced cell
toxicity, the capacity of viable T cells to survive after exposure to
SB203580 was measured. As evaluated by propidium iodide staining, we
found that SB203580 (100 µM) completely inhibited T cell death
induced by anti-CD3 stimulation (Fig. 5B). Consistent
with this observation, SB203580 (40–100 µM) also significantly
inhibited TCR-induced cell death in the DO11.10 murine T cell hybridoma
(data not shown). This ability of SB203580 to promote T cell survival
was not attributable to any nonspecific DMSO solvent effects, as
pretreatment of T cells with DMSO alone failed to inhibit p38 MAPK
activity (data not shown). This ability of SB203580 to block T cell
proliferation correlated closely with its capacity to inhibit p38 MAPK
activity, as activities of p38 MAPK and its downstream target MAPKAP
kinase-2 induced by stimulation with either anti-CD3 and
anti-CD3/CD28 were greatly diminished by SB203580 in a
dose-dependent manner (Fig. 5C). Note that since SB203580
reversibly binds to the ATP binding site of p38 MAPK, SB203580 was
present continuously during the kinase assays.
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10 µM, the SB203580 inhibitor may lose its
selectivity and also block JNK2ß1 and JNK2ß2 (44). To confirm the
specificity of this inhibitor, purified splenic T cells were pretreated
with various concentrations of SB203580 (0–100 µM), then stimulated
with either anti-CD3, anti-CD3 plus anti-CD28, or PMA, and
lysed. Cell lysates were either reacted with GST-c-Jun precoupled to
glutathione-agarose beads or immunoprecipitated with an
anti-anti-ERK-1 Ab in the presence of SB203580. The immobilized
kinases were then incubated with substrates and SB203580 (0–100 µM)
for 30 min at 30°C. ERK-1 and JNK activities remained unaffected in
the presence of increasing concentrations of SB203580 (Fig. 5D). SB203580 inhibits cytokine secretions in primary mouse T cells
We next investigated whether SB203580 inhibits cytokine secretions
induced by CD3 or CD3/CD28 stimulation of primary mouse T cells.
Splenic T cells were pretreated with either different concentrations of
SB203580 in DMSO or equivalent amounts of DMSO as control, and then
stimulated as described above. After 48 h, supernatants were
collected for quantification of IL-2, IL-4, and IFN- by ELISA.
Interestingly, SB203580 inhibited T cell secretion of of IL-2, IL-4,
and IFN- induced by either CD3 or CD3/CD28 stimulation in a
dose-dependent manner (Fig. 6). Since inhibition of T
cell proliferation and cytokine secretion by SB203580 does not result
from cell death, these data suggest that treatment with SB203580
induces T cells to become functionally unresponsive.
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Our data suggest that p38 MAPK, but not JNK, is involved in
signal integration of TCR- and CD28-mediated signaling pathways in
primary mouse T cells. However, JNK is a major kinase responsible for
c-Jun phosphorylation and p38 MAPK cannot phosphorylate c-Jun (28, 31).
Indeed, using in vitro kinase assays, we failed to detect c-Jun
phosphorylation by p38 MAPK (data not shown). Since phosphorylation of
c-Jun and/or ATF-2 is required for c-Jun gene induction, we analyzed
whether blockade of p38 MAPK activity results in the reduced
phosphorylation of c-Jun and ATF-2. Thymocytes were preincubated for 15
min at 37°C with 10 µM SB203580 or an equal amount of
DMSO as control, and were then either stimulated with anti-CD3 plus
anti-CD28 for 5 and 15 min, or left unstimulated. Nuclear extracts
were blotted with anti-c-Jun and anti-ATF-2 mAbs, respectively.
Phosphorylation of ATF-2, as revealed by its reduced mobility, was
evident after 5 and 15 min of stimulation, and these mobility shifts
were significantly inhibited by SB203580 (Fig. 7A, upper panel). In contrast, no
c-Jun phosphorylation was observed (Fig. 7A, lower
panel), consistent with our in vitro data that JNK is not
activated upon CD3 or CD3/CD28 ligation. Note that the failure to
detect c-Jun phosphorylation cannot be attributed to the anti-c-Jun
mAb used because it clearly recognized the phosphorylated form of
c-Jun induced by PMA stimulation, as reported (45). SB203580
can block the induction of the c-Fos and c-Jun genes by diverse stimuli
(45, 46), indicating that p38 MAPK activity may play a role in the
induction of c-Jun gene expression. This may explain our finding of the
nuclear expression of the c-Jun and ATF-2 proteins at later time points
following CD3/CD28 coligation. Interestingly, while SB203580
pretreatment did not alter the nuclear expression of c-Fos
after CD3 or CD3/CD28 stimulation (Fig. 7B, upper
panel), SB203580 pretreatment significantly reduced the
nuclear expression of c-Jun and ATF-2 induced by CD3 or
CD3/CD28 ligation (Fig. 7B, middle and
lower panels). The nuclear expression of c-Jun and ATF-2 was
enhanced more upon CD3/CD28 coligation than ligation of CD3
alone. The nuclear expression of c-Fos, c-Jun, and ATF-2 stimulated by
PMA was not inhibited by SB203580.
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C). The blockade of
c-Jun expression in the nucleus by SB203580 suggests that p38 MAPK
activity plays a role in the induction of expression of the c-Jun
protein. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. These results suggest
that p38 MAPK, but not JNK, is involved in signal integration during
CD28 costimulation of murine primary T cells and may also play an
important role in the induction of T cell anergy. Recently, it has been shown that CD28 and PMA/Ca2+ ionophore-triggered signaling stimulate thymocytes and lymph node T cells to proliferate and produce IL-2 independently of JNK activation in MKK-4-deficient mice (37), suggesting that other MAPK(s) might be involved in primary T cell costimulation. More importantly, impaired CD28-mediated IL-2 production and proliferation by T cells were observed in these mice (37). Since MKK-4 activates both JNK and p38 MAPK (5, 6, 7, 8, 9), this impairment might be attributed to defective p38 MAPK activation in MKK-4-deficient mice. However, p38 MAPK activity was not analyzed in the latter report. Nonetheless, deficient expression of MKK4 does not affect p38 MAPK activation in embryonic stem cells (47), and SB203580 inhibits CD28-dependent proliferation and IL-2 production in human T cells (38). These two sets of evidence suggest a role for p38 MAPK in the costimulation of primary mouse T cells. Although T cell proliferation in response to Con A and PMA/ionomycin stimulation is normal in dominant-negative p38 transgenic mice, TCR/CD28-mediated T cell proliferation and p38 MAPK activity in response to different stimuli were not investigated in this study (48). Additional studies conducted primarily with previously activated T cells have shown that p38 MAPK activation is inducible by CD3, CD28, or CD3/CD28 stimulation (35, 36). Interestingly, however, we observed that MAPK activation differs between primary naive T cells and proliferating preactivated T cells. In proliferating T cells, while ERK-1 and p38 MAPK activity are similar to that in primary naive T cells, JNK-1 is weakly activated by CD3 stimulation and is fully activated only upon CD28 stimulation. It is important to note that a high percentage of proliferating T cells become apoptotic, perhaps due to the continuous exposure to IL-2 (49). Thus, p38 MAPK and JNK-1 activation in proliferating T cells may result in part from activation-induced apoptosis.
The requirements for p38 MAPK and JNK-1 activation in murine primary T cells were found to differ from those in Jurkat cells, a human T cell line frequently used to identify pathways of T cell signaling. In Jurkat cells, optimal activation of both p38 MAPK and JNK-1 requires TCR/CD28 coligation or PMA/Ca2+ ionophore costimulation. In primary mouse T cells, however, stimulation by either CD28 or Ca2+ ionophore activates p38 MAPK, and p38 MAPK can be activated synergistically by CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation. Whereas high concentrations of Ca2+ ionophore inhibit JNK-1 activity in primary T cells, these same concentrations of Ca2+ ionophore augment p38 MAPK activity. This enhancement of p38 MAPK activity correlates closely with T cell proliferation induced by optimal concentrations of PMA and Ca2+ ionophore. Furthermore, CsA inhibited p38 MAPK activation by anti-CD3, anti-CD3 plus CD28, or PMA plus Ca2+ ionophore. Although the levels of activation of ERK-1 and p38 MAPK in CD3- or CD3/CD28-stimulated proliferating and naive primary mouse T cells are similar, JNK-1 is fully activated by CD28 ligation alone in proliferating T cells. These data demonstrate that the requirements for full activation of JNK-1 and p38 MAPK differ among primary T cells, Jurkat T cells, and proliferating T cells. Such differences may arise from the stage of activation of a T cell. Jurkat T cells are transformed activated cells in continuous growth in culture, and these cells appear to differ somewhat in their signaling pathways from fresh isolated primary T cells that presumably consist mainly of T cells in a resting state.
As JNK-1 activation was not detected after CD3/CD28 costimulation in primary T cells, we further analyzed the reactivity and epitope specificity of the 37.51 anti-CD28 mAb used for T cell stimulation. Costimulation by a wide concentration range of this mAb plus a single dose of anti-CD3 did not induce JNK-1 activation. In contrast, optimum conditions of CD3/CD28 costimulation elicited the full activation of p38 MAPK. While recent studies indicate that JNK-1 may be activated by CD3/CD28 coligation in mouse lymph node T cells, this was achievable only after preactivation of the T cells in the presence of plate-bound anti-CD3 and anti-CD28 mAbs for 40 h (50). Replacement of the 37.51 anti-CD28 mAb with the PV-1 anti-CD28 mAb, which differs in its CD28 epitope specificity from the 37.51 mAb, also did not stimulate JNK-1 activation. Furthermore, the 37.51 anti-CD28 mAb elicited full JNK-1 activation in preactivated proliferating mouse T cells. Importantly, the results obtained from a solid-phase JNK assay were similar to those observed using in vitro kinase assays of JNK-1 and JNK-2. This suggests that the failure to detect JNK activation in response to CD3 and CD28 ligation is not due to the inability of the anti-JNK Abs used to immunoprecipitate all isoforms of JNK. Note that the gel loading of equivalent amounts of JNK proteins was confirmed by anti-JNK immunoblotting, which excludes the possibility that these observations result from unequal amounts of kinase proteins loaded. It is evident therefore that the failure to detect JNK activation in response to CD3/CD28 coligation in primary mouse T cells is not due to the use of an inappropriate anti-CD28 mAb. Thus, JNK does not appear to mediate CD28 costimulatory signaling in primary mouse T cells.
Additional supportive evidence for a role for p38 MAPK in T cell
costimulation was derived from the use of SB203580, a highly specific
pyrinidyl imidazole inhibitor of p38 MAPK (17). SB203580 effectively
blocked T cell proliferation, cytokine (IL-2, IL-4, and IFN-)
production, and p38 MAPK activation following CD3 or CD3/CD28
stimulation. These results lend credence to the idea that p38 MAPK
plays an important role in TCR/CD28 costimulation of proliferation and
cytokine production in primary T cells. Since inhibition of T cell
proliferation and cytokine secretion by SB203580 does not result from T
cell death or T cell toxicity, SB203580 treatment appears to induce the
functional unresponsiveness of T cells. The latter observation supports
the notion that p38 MAPK may regulate the entry of T cells into a state
of anergy. Consistent with this observation, SB203580 has been shown to
inhibit anti-CD3-induced deletion of
CD4+CD8+ thymocytes in fetal thymic organ
culture (51).
The protein complex that binds to the AP-1 transcriptional activation site of the IL-2 gene promoter region may comprise either a c-Jun-c-Jun homodimer or c-Jun-Fos heterodimer, and formation of the AP-1 complex is critical for the regulation of IL-2 gene expression (33). Decreased binding of the AP-1 protein complex to the IL-2 promoter has been implicated in the molecular basis of T cell anergy (52). Phosphorylation of c-Jun at Ser63 and Ser73 activates c-Jun gene transcription (10, 53), and this phosphorylation is mediated by JNK, but not p38 MAPK (28). We found that CD3/CD28 or PMA/Ca2+ ionophore costimulation did not result in appreciable JNK-1 or JNK-2 activation in primary mouse T cells; rather, PMA/Ca2+ ionophore costimulation inhibited JNK-1 activity in these T cells. Although we did not detect any c-Jun phosphorylation by p38 MAPK in CD3/CD28-costimulated mouse thymocytes, c-Jun protein synthesis induced by CD3 or CD3/CD28 ligation was completely inhibited by SB203580. Consistent with our findings, SB203580 is known to block the induction of the c-Fos and c-Jun genes by various stimuli despite the lack of an effect of SB203580 on c-Jun and ATF-2 phosphorylation (45, 46). This raises the possibility that the phosphorylation of other transcription factors by p38 MAPK may be essential for c-Jun gene activation. We have shown that ATF-2 can be phosphorylated by p38 MAPK in vitro, and phosphorylation of ATF-2 in the nucleus is inhibited by SB203580. ATF-2 may therefore mediate the induction of c-Jun gene activation. Furthermore, in monocytes that migrate to a site of inflammation, phosphorylation of MEF2C by p38 MAPK increases c-Jun transcription and inhibition of p38 MAPK activity diminishes LPS-induced c-Jun transcription (31). Taken together, our data suggest that p38 MAPK may be the major kinase responsible for the induction of c-Jun activation in primary mouse T cells. Although JNK-3 has been identified (54), JNK-3 is selectively expressed in the nervous system, and it is unlikely that JNK-3 is involved in the phosphorylation and induction of c-Jun gene expression in T cells. The lack of an effect of SB203580 on nuclear c-Fos expression may be explained by the fact that CD3 ligation also activates ERK-1, which is not inhibited by SB203580.
In summary, our results suggest that p38 MAPK is involved in both TCR-
and CD28-signaling pathways, and that p38 MAPK, but not JNK, plays an
important role in signal integration during costimulation of primary
mouse T cells. p38 MAPK activity may be involved in the induction of
c-Jun activation, augmentation of AP-1 transcriptional activity, and
stimulation of IL-2, IL-4, and IFN- production. Inhibition of p38
MAPK activity may lead to the induction of T cell anergy.
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Acknowledgments |
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Footnotes |
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2 J.Z. and K.V.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Autoimmunity/Diabetes Group, John P. Robarts Research Institute, 1400 Western Road, London, Ontario N6G 2V4, Canada. E-mail address:
4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ATF-2, activating transcription factor-2; CsA, cyclosporin A; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; hsp, heat-shock protein; JNK, c-Jun NH2-terminal kinase; MAPKAP, MAPK-activating protein; MBP, myelin basic protein; MEK, MAP kinase/ERK kinase; MKK, MAP kinase kinase.
Received for publication August 10, 1998. Accepted for publication December 21, 1998.
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K. V. Salojin, J. Zhang, C. Meagher, and T. L. Delovitch ZAP-70 Is Essential for the T Cell Antigen Receptor-induced Plasma Membrane Targeting of SOS and Vav in T Cells J. Biol. Chem., February 25, 2000; 275(8): 5966 - 5975. [Abstract] [Full Text] [PDF]
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E. Fernandez Thymocyte development past the CD4+CD8+ stage requires an active p38 mitogen-activated protein kinase Blood, February 15, 2000; 95(4): 1356 - 1361. [Abstract] [Full Text] [PDF]
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S.-C. Hsu, M. A. Gavrilin, M.-H. Tsai, J. Han, and M.-Z. Lai p38 Mitogen-activated Protein Kinase Is Involved in Fas Ligand Expression J. Biol. Chem., September 3, 1999; 274(36): 25769 - 25776. [Abstract] [Full Text] [PDF]
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 L.A. SMYTH, L. REYNOLDS, R. TYRRELL, O. WILLIAMS, T. NORTON, T. SCHUMACHER, V. TYBULEWICZ, S.C. LEY, and D. KIOUSSIS Signaling in Primary Thymocytes after Presentation of Altered Peptide Ligands by Antigen-presenting Cells or MHC-peptide Tetramers Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 275 - 282. [Abstract] [PDF]
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T. A. Fischer, A. Palmetshofer, S. Gambaryan, E. Butt, C. Jassoy, U. Walter, S. Sopper, and S. M. Lohmann Activation of cGMP-dependent Protein Kinase Ibeta Inhibits Interleukin 2 Release and Proliferation of T Cell Receptor-stimulated Human Peripheral T Cells J. Biol. Chem., February 16, 2001; 276(8): 5967 - 5974. [Abstract] [Full Text] [PDF]
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Y. Abe, S. Matsumoto, K. Kito, and N. Ueda Cloning and Expression of a Novel MAPKK-like Protein Kinase, Lymphokine-activated Killer T-cell-originated Protein Kinase, Specifically Expressed in the Testis and Activated Lymphoid Cells J. Biol. Chem., July 7, 2000; 275(28): 21525 - 21531. [Abstract] [Full Text] [PDF]
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 T. Zell, A. Khoruts, E. Ingulli, J. L. Bonnevier, D. L. Mueller, and M. K. Jenkins Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo PNAS, September 11, 2001; 98(19): 10805 - 10810. [Abstract] [Full Text] [PDF]
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