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Expression1
Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and Walther Cancer Institute, Indianapolis, IN 46208
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by activated T cells
and Th1 cells. However, several studies have suggested that other
pathways may be involved in IL-12-stimulated IFN- expression. In
this report we demonstrate that IL-12 activates mitogen-activated
protein kinase kinase 3/6 (MKK) and p38 mitogen-activated protein
kinase (MAPK), but not p44/42 (ERK) or stress-activated protein
kinase/c-Jun N-terminal kinase MAPK. The activation of p38 MAPK is
required for normal induction of IFN- mRNA and IFN- secretion by
IL-12 in activated T cells and Th1 cells. Importantly, IL-12-stimulated
p38 MAPK effector functions occur through a Stat4-independent mechanism
and correlate with increased serine phosphorylation of activating
transcription factor-2. The requirement for p38 MAPK in IL-12 function
suggests that this pathway may be an important in vivo target for the
anti-inflammatory actions of p38 MAPK
inhibitors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CD25, IL-18R, and IFN regulating factor-1 (3, 4, 5, 6).
Perhaps most notably, IL-12 promotes the differentiation of naive
CD4+ T cells into the Th1 subset of Th cells
(7).
IL-12 binds specifically to two noncovalently linked receptor chains
expressed on NK cells and activated T and B cells. The chains are
termed IL-12Rß1 and IL-12Rß2, since both chains have homology to
ß-chains of the gp130 family of receptors (8, 9, 10). Both
receptor chains associate with members of the Janus kinase
(Jak)3
(2) family of tyrosine kinases. The IL-12R ß1-chain,
which contains no tyrosine residues in its cytoplasmic domain,
interacts with Tyk2 (8, 11). The IL-12R ß2-chain
contains three tyrosines in its cytoplasmic domain and interacts with
Jak2 (9, 11). The binding of IL-12 to its receptor leads
to activation of Jak kinases and tyrosine phosphorylation of
IL-12Rß2, and results in the recruitment and activation of Stat4
(12, 13). Stat4 specifically binds to the IL-12Rß2
peptide sequence pYLPSNID (where pY represents phosphotyrosine)
(14). In an analysis of mice deficient in Stat4, we and
others have demonstrated that Stat4 is required for IFN- production
by Th1 cells (15, 16). To date, the Jak-STAT pathway is
the only pathway known to be important for IL-12 signaling.
It has recently been reported that IFN- expression by Th1 cells
depends upon the p38 mitogen-activated protein kinase (MAPK) signaling
pathway (17). In the present study we demonstrate that
IL-12 activates p38 MAPK, and this activation is required for normal
IFN- expression in activated T cells. Importantly, this pathway
functions by a Stat4-independent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type C57BL/6 mice between 6 and 10 wk of age were purchased from Harlan Bioproducts (Indianapolis, IN). Stat4-deficient mice were generated as described previously (15), backcrossed to the C57BL/6 background for eight generations, and intercrossed to generate C57BL/6 Stat4-deficient mice. Stat4-deficient mice were bred in the animal facility at Indiana University.
Cell preparation and activation
Total spleen and lymph node cells were treated with RBC lysis solution (Sigma, St. Louis, MO), resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), and activated for 48 h with 2 µg/ml plate-bound anti-CD3 (145-2C11, purified from hybridoma supernatants in our laboratory). Nonadherent cells were washed twice with complete medium and pretreated with DMSO (Sigma) or SB203580, SB202190, and SB202474 (Calbiochem, San Diego, CA) for 1 h and used as indicated.
Northern blot analysis of IFN- mRNA
Cells were activated as described above and pretreated with the
indicated concentrations of SB203580, SB202190, and SB204274 or with
DMSO as a control for 1 h, then stimulated for an additional
4 h with 1 ng/ml of mouse IL-12 (Genzyme, Cambridge, MA). Total
RNA was isolated using TRIzol (Life Technologies, Gaithersburg, MD).
Ten micrograms of total RNA was fractionated by electrophoresis through
a 1% denaturing agarose gel, transferred to a nylon transfer membrane
(Schleicher & Schuell, Keene, NH), and UV cross-linked. The membranes
were prehybridized for 3 h at 42°C, and hybridization was
performed with a 32P-labeled IFN- probe for
16 h at 42°C. The membranes were sequentially washed in 2x SSC
containing 0.1% SDS at 60°C for 20 min and in 0.1x SSC containing
0.1% SDS at 60°C for 20 min, and then exposed to x-ray film at
-80°C. The membranes were stripped and rehybridized with a TCR
probe to confirm equal RNA loading. Densitometry was determined and is
represented as the fold increase in IFN- mRNA relative to that in
untreated cells, using a multi-image light cabinet from Alpha Innotech
(San Leandro, CA).
Th1 cell differentiation and CD4+ cell isolation
Lymphocytes isolated from wild-type mice prepared as described above were cultured with plate-bound anti-CD3 (2 µg/ml) in the presence of 1 ng/ml IL-12 and 10 µg/ml anti-IL-4 (11B11, purified from hybridoma supernatants in our laboratory) to promote Th1 differentiation (15, 18). In some experiments, cells isolated from wild-type or Stat4-deficient mice were pretreated with 10 µM SB203580 or DMSO for 1 h before Th1 differentiation. Five days following activation, cells were centrifuged over Histopaque-1083 (Sigma) to remove dead cells, followed by isolation of CD4+ cells with MiniMACS beads according to the manufacturer’s instruction (Miltenyi Biotec, Auburn, CA).
IFN- secretion measurement
Activated T cells were pretreated for 1 h with the
indicated drugs at various concentrations and incubated for 36 h
in the presence or the absence of 1 ng/ml IL-12. Supernatants were
harvested to test IFN- production by ELISA (18). ELISAs
were performed using purified monoclonal anti-IFN- Abs (R4/6A2,
2 µg/ml) as a capture Ab, and IFN- was detected using biotinylated
anti-IFN- (PharMingen, San Diego, CA), avidin-alkaline
phosphatase, and p-nitrophenol phosphate (pNPP) as the
substrate (Sigma). Recombinant IFN- was used as a standard.
Similarly, differentiated CD4+ Th1 cells were
pretreated with 10 µM SB203580 or DMSO for 1 h before a
restimulation with 2 µg/ml anti-CD3 for 24 h. Supernatants
were harvested, and IFN- was assayed by ELISA as described
above.
Activation of MKKs, MAPKs, and transcription factors
Activated T cells were exposed to 1 ng/ml of IL-12 for 0, 5, 10, 20, 30, and 60 min with or without 1-h pretreatment with SB202474, SB203580, or SB202190 at 10 µM. Cells were washed twice and lysed in lysis buffer (50 mM Tris (pH 8.0), 0.1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL CA-630 (Sigma), and 10% glycerol) supplemented with 1 mM DTT, 2 µg/ml pepstatin, 20 µg/ml aprotinin, and 20 µg/ml leupeptin at the indicated time points. Cell lysates were used for an analysis of p38 MAPK activity. p38 MAPK activity was determined with a p38 MAP kinase assay kit following the manufacturer’s instructions (New England Biolabs, Beverly, MA). In brief, phosphorylated p38 MAPK was immunoprecipitated by anti-phosphorylated p38 MAPK Ab, and phosphorylated active p38 MAPK was incubated with ATF-2, a substrate of p38 MAPK. ATF-2 phosphorylation was measured by Western blot using anti-phospho-ATF-2 (Thr71) rabbit polyclonal Ab. Densitometry was determined and is presented as the fold increase in p38 MAPK activity relative to that in untreated cells. Phospho-p44/42 MAPK E10 mAb, phospho-SAPK/JNK mAb, and a rabbit affinity-purified polyclonal Ab against SAPK/JNK were purchased from New England Biolabs. A rabbit affinity-purified polyclonal Ab against ERK2 p42 ERK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphorylation of MKK3/6 and MKK4 were detected with Abs (New England Biolabs) according to the manufacturer’s instructions. Blots were stripped and reprobed with total anti-MKK3 as a control. Phosphorylation of endogenous ATF-2, ATF-1, and CREB were determined with Abs as described above or according to the manufacturer’s instructions (New England Biolabs).
Proliferation assay
Activated T cells (1 x 104/well) were pretreated for 1 h with various concentrations of SB203580 or with DMSO as a control, and cultured with 1 ng/ml of IL-12 and 10 µg/ml anti-IL-2 (S4B6) Ab or 30 U/ml of mouse IL-2 (Roche, Indianapolis, IN) in 96-well U-bottom plates. Cells were pulsed for the last 12 h of a 48-h incubation with 0.8 µCi/well of [3H]TdR (New England Nuclear, Boston, MA) and harvested onto glass-fiber filters. [3H]TdR incorporation was analyzed by liquid scintillation counting, and results were expressed as mean counts per minute of triplicate cultures.
Immunoprecipitation and immunoblotting of Stat4
Activated T cells were pretreated with 10 µM SB203580 or DMSO as a control for 1 h, and then stimulated with 1 ng/ml IL-12 at 37°C for 45 min. Cells were immediately washed twice with cold serum-free RPMI 1640 and lysed in cold lysis buffer. Stat4 protein was immunoprecipitated with purified polyclonal rabbit IgG against Stat4 (Santa Cruz Biotechnology). The protein samples were separated on 7.5% SDS-PAGE and transferred to a nitrocellulose transfer membrane (Schleicher & Schuell). The membranes were then blocked with 5% BSA in 1x TBST for 2 h at room temperature, incubated with a mouse monoclonal anti-phosphotyrosine Ab PY99 (Santa Cruz Biotechnology) for 1 h, washed, incubated with secondary Ab for another 1 h, and washed. Specific signals were detected with an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA). The blots were stripped and reprobed with purified polyclonal rabbit IgG against Stat4 to ensure equal protein loading.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression
While Stat4 has been shown to be crucial for IL-12-stimulated
biological activities, the importance of other IL-12-activated pathways
has not been carefully examined. Since p38 MAPK has recently been
implicated in the ability of Th1 cells to express IFN-
(17), we explored the role of p38 MAPK in IL-12 signaling.
We first examined the effects of the p38 MAPK inhibitors SB203580 and
SB202190 (19) on IL-12-induced IFN- mRNA expression.
Total spleen and lymph node cells isolated from wild-type mice were
activated for 48 h with plate-bound anti-CD3. Cells were
pretreated with the indicated inhibitors for 1 h and stimulated
with or without IL-12 for an additional 4 h. As shown by Northern
analysis in Fig. 1
A, cells in
the absence of IL-12 stimulation expressed a low level of IFN-.
IL-12 dramatically stimulated IFN- mRNA expression. This induction
was significantly inhibited by SB203580 and SB202190, but not by
SB202474, an inhibitor analogue that does not affect p38 MAPK activity
(19). It has also been shown that IL-12 stimulates the
activation of ERK MAPK in human cells (20). As a control
we tested whether an inhibitor of the ERK pathway would have a similar
effect. Our results demonstrate that PD98059, a MAPK kinase (MEK)
inhibitor (21), did not affect IL-12 induced IFN- mRNA
expression (Fig. 1B).
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secretion of activated T cells and differentiated
CD4+ Th1 subsets in response to IL-12 stimulation
or 2 µg/ml plate-bound anti-CD3. The data in Fig. 2A show that IL-12-stimulated
activated T cells produced 4-fold higher IFN- compared with
unstimulated cells. IFN- induction by IL-12 was significantly
inhibited by both SB203580 and SB202190 at concentrations ranging from
5–20 µM, but not by SB202474. The inhibition of IFN- production
was not due to nonspecific cytotoxicity, since the presence of SB203580
or SB202190 did not affect the viability of the cells at the time point
when culture supernatants were harvested to determine IFN-
production (data not shown). Th1 cells, pretreated with 10 µM
SB203580 and restimulated with 1 ng/ml of IL-12, had a reduced level of
IFN- production compared with untreated cells (Fig. 2B),
similar to what we and others have observed when anti-CD3-induced
IFN- was examined (Ref. 17 and data not shown). These
results strongly suggest that p38 MAPK is required for IL-12 induced
IFN- expression.
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We next wanted to determine whether IL-12 activates p38 MAPK.
Total spleen and lymph node cells isolated from wild-type mice were
activated for 48 h with plate-bound anti-CD3 (2 µg/ml) and
were stimulated with 1 ng/ml IL-12 for the indicated times with or
without 1-h pretreatment of 10 µM SB202474, SB202190, or SB203580. An
in vitro kinase assay, using ATF-2 as a substrate, demonstrated that
p38 MAPK was activated by 5 min after IL-12 stimulation, peaked at 10
min, and returned to unstimulated levels by 60 min (Fig. 3A). Additionally, there was a
dose-dependent activation of p38 MAPK activation in response to IL-12
(Fig. 3B). As expected, 10 µM SB202190 and SB203580, but
not SB202474, decreased IL-12-induced p38 MAPK activity (Fig. 3C).
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Since the p38 MAPK pathway is only one of three MAPK pathways that
might potentially be activated by IL-12, we next examined the
activation of ERK. In contrast to activation of p38 MAPK, our results
show that IL-12 failed to activate p44/42 (ERK) in mouse activated T
cells (Fig. 4A). This
observation confirms the Northern analysis showing that blocking
activation of ERK using the MEK inhibitor PD98059 did not affect
IL-12-induced IFN- mRNA expression (Fig. 1
B) and suggests
that ERKs are not involved in IL-12 signaling. Since it was reported
that SB203580 could also inhibit JNK2 activity, although with lower
potency than the inhibition of p38 MAPK (22, 23), we also
examined whether IL-12 activates SAPK/JNK. While basal phosphorylation
of SAPK/JNK was detectable, IL-12 did not induce phosphorylation of
SAPK/JNK (Fig. 4B). Thus, of the three MAPK pathways, only
p38 is activated by IL-12.
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To determine whether activation of p38 MAPK by IL-12 occurred via
characterized MAPK kinases, we analyzed the ability of IL-12 to induce
phosphorylation of MAPK kinases known to phosphorylate p38 MAPK,
including MKK3, MKK6, and MKK4 (24, 25, 26, 27). Using an Ab that
detects phosphorylation of MKK3 and MKK6, activation of MKK3/6 was
detected following IL-12 stimulation (Fig. 5). No phosphorylation of MKK4 was
detectable (data not shown), which corresponds to the lack of JNK
activation shown in Fig. 4B following IL-12 stimulation.
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Since Stat4 is required for IFN- production by activated T
cells and Th1 cells (15, 16), we wanted to determine
whether p38 MAPK functions independently of Stat4. Cells activated as
described in Fig. 1 were pretreated with 10 µM SB203580 or DMSO as a
control for 1 h, and then incubated for an additional 45 min in
the presence or the absence of 1 ng/ml IL-12. Stat4 was precipitated
from whole cell extracts for phosphotyrosine analysis. Fig. 6 demonstrates that 10 µM SB203580 did
not affect tyrosine phosphorylation of Stat4 at concentrations that did
inhibit induction of IFN- mRNA and secretion (Figs. 1 and 2). In
addition, 10 µM SB203580 did not affect IL-12-induced tyrosine
phosphorylation of Jak2 (data not shown), consistent with the above
result. These data demonstrate that p38 MAPK inhibitors do not affect
activation of the Jak-STAT pathway.
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expression, we tested
whether p38 MAPK inhibitors would affect IL-12-stimulated proliferation
of activated T cells. Stat4 has been demonstrated to be crucial for
IL-12-stimulated proliferation (15, 16), but as shown in
Fig. 7A, SB203580 did not
affect IL-12-stimulated proliferation. As a control, IL-2-induced
proliferation was significantly inhibited by SB203580, which is
consistent with a previous study (28). This result
provides further evidence that 10 µM SB203580 is not cytotoxic to T
cells and demonstrates the requirement for p38 MAPK signaling in some,
but not all, IL-12-signaled functions.
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(15, 16, 18), which allowed us to assess the role of p38 MAPK in the
generation of Th1-like cells in the absence of Stat4. We differentiated
wild-type and Stat4-deficient cells under Th1-promoting conditions in
the absence or the presence of SB203580. Th1 cultures were then
restimulated with anti-CD3 in the absence of any p38 MAPK
inhibitor. SB203580 decreased IFN- secretion in wild-type
CD4+ cells by about 50%. Similarly, IFN-
production was decreased in
Stat4-/-
CD4+ Th1 cultures (Fig. 7B). These results demonstrate that inhibition of the p38
MAPK pathway does not inhibit all Stat4-dependent functions and that
p38 MAPK inhibitors block IL-12 functions in the absence of Stat4, thus
supporting a role for p38 MAPK, independent of Stat4, in IL-12
signaling.
In vitro studies demonstrated that the transcription factors ATF-2,
Elk-1, CHOP, MEF2C, SAP-1, and CREB are phosphorylated and activated by
p38 MAP kinase (23, 24, 27, 29, 30, 31). Since both ATF-2 and
CREB have been implicated in IFN- regulation (32, 33)
we examined whether IL-12 increased the endogenous levels of serine
phosphorylation of either of these factors. Fig. 8 demonstrates that there was a 4-fold
increase in ATF-2 phosphorylation following IL-12 stimulation, but no
corresponding increase in ATF-1 or CREB phosphorylation. The timing of
endogenous ATF-2 phosphorylation by IL-12 corresponded to the timing of
phosphorylation of other endogenous factors following p38 MAPK
activation (34, 35, 36). Thus, ATF-2 offers a potential target
of p38 MAPK following IL-12 stimulation and is a potential mediator of
Stat4-independent, IL-12-signaled functions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1 (37). Several hemopoietic growth
factors, including IL-2, IL-3, IL-7, GM-CSF, and steel locus factor,
but not IL-4, have been reported to activate p38 MAPK (28, 38). Regulation of p38 MAPK function has been shown to be
important for anti-CD3-induced IFN- in Th1 cells, T cell
homeostasis, and thymic development (17, 39, 40). In this
paper we demonstrate that IL-12 activates p38 MAPK activity, and that
this activation is required for normal IL-12-induced IFN-
expression.
The effector of IL-12-stimulated p38 MAPK activity is still unclear,
although Stat4 would be the most obvious candidate. Stat1 and Stat3
have been shown to be phosphorylated on serine residues by p38 MAPK
(35, 36, 41, 42). A recent study has also demonstrated
that a p38 MAPK inhibitor reduced serine phosphorylation of Stat1
induced by IL-2 in combination with IL-12 (41). However,
our study differs from that study in that they examined a restricted
population of CD8+
CD18bright T cells and found no effect of IL-12
alone, in contrast to our study, where IL-12 effects were seen even in
the absence of IL-2 (Fig. 7A). It is possible that p38 MAPK
is involved in serine/threonine phosphorylation of Stat4.
Serine/threonine phosphorylation of human Stat4 was previously shown to
affect migration of Stat4 in an SDS-PAGE gel (43).
However, we did not see any altered migration of murine Stat4 in a
similar system following activation with IL-12 or IL-12 plus p38 MAPK
inhibitors or by treating extracts in vitro with a serine/threonine
phosphatase. Whether this indicates that mouse Stat4 is not
serine/threonine phosphorylated, that Stat4 serine/threonine kinases
are not activated by our protocols, or that we simply cannot detect
serine/threonine-phosphorylated murine Stat4 by this method is unclear.
We also demonstrate that SB203580 does not affect tyrosine
phosphorylation of Stat4, suggesting that Jak kinase activity and
receptor recruitment are normal in the presence of p38 MAPK inhibitors.
Importantly, p38 MAPK inhibitors do not interfere with all
Stat4-dependent functions of IL-12 (Fig. 7A) and did reduce
IFN- secretion in Stat4-deficient Th1 cultures, strongly supporting
a Stat4-independent role for p38 MAPK in IL-12 signaling. This also
provides a pathway for Stat4-independent development of Th1-like cells
(18).
Many transcription factors may regulate IFN- expression and are
potential targets of p38 MAPK. In vitro studies demonstrated that the
transcription factors ATF-2, Elk-1, CHOP, MEF2C, SAP-1, and CREB are
phosphorylated and activated by p38 MAP kinase (23, 24, 27, 29, 30, 31). Two regulatory elements have been defined in the
IFN- promoter, termed proximal and distal elements, that contain
ATF/CREB DNA binding sequences and bind ATF-2 and related bZIP
transcription factors (32, 33). Indeed, we observed
increased serine phosphorylation of endogenous ATF-2 following IL-12
stimulation (Fig. 8). Thus, it is possible that p38 MAPK-mediated
phosphorylation of ATF-2 or related family members is responsible for
p38 MAPK-stimulated IFN- gene transcription. Other transcription
factors have been implicated in IFN- gene regulation, including
NF-AT, NF-
B, YY-1, CREB, AP-1, GATA-3, and Stat4
(44, 45, 46, 47, 48, 49). However, none of these is expressed in a
strictly Th1-dependent manner. Most recently, T-bet has been described
as a regulator of IFN- expression (50), although
whether it becomes phosphorylated and might be a target of p38 MAPK has
not been determined.
There are four mammalian isoforms of p38 MAP kinase: p38, p38 ß,
p38, and p38
(51, 52, 53, 54, 55, 56, 57). p38 and p38 are not
inhibited by SB203580 (51, 53). Thus, it is likely that
IL-12 activates p38, since p38ß is only expressed at low levels in
CD4+ T cells (58). Notably, it has
most recently been shown that p38 MAP kinase is strongly activated by
CD3/CD28 coligation (59). It may also be interesting to
examine whether TCR or IL-12 activates distinct isoforms of p38 MAPK or
whether Th1 and Th2 cells differentially express isoforms of p38
MAPK.
There is specificity in the activation of MAPK by IL-12. It is
noteworthy that we did not see IL-12-stimulated tyrosine
phosphorylation of ERK, which differs from observations in human cells
(20). Whether this is a species difference and whether ERK
plays a functional role in human cells is still unclear. We have also
shown that IL-12 does not activate SAPK/JNK. This is in apparent
contrast to evidence from JNK1- and JNK2-deficient mice (60, 61), which demonstrate that JNKs are required for Th1
differentiation. Importantly, both JNK and ERK are activated following
T cell stimulation and are probably involved in TCR signaling
(62, 63). While IL-12 signaling was affected in
JNK2-deficient cells, this was found to be due to a lack of IFN-
induced expression of IL-12Rß2 (61). When IFN- was
replaced in the cultures, IL-12 signaling was recovered, supporting our
finding that SAPK/JNK is not involved in IL-12 responses. IL-12 has
also been shown to activate p56lck
(lck) in human cells (64). However, we
have observed normal IL-12-induced IFN- production and Th1
differentiation in lck-deficient T cells (unpublished
observation). This confirms the lack of a role of lck in Th1
differentiation seen in transgenic mice expressing a dominant-negative
lck (65). Thus, the IL-12-activated signaling
molecules important for biological functions appear to be limited to
the Jak-STAT and MKK3/6-p38 MAPK pathways.
SB203580 is a well-characterized anti-inflammatory drug. This
anti-inflammatory activity is associated with reduced production of
proinflammatory cytokines, such as IL-1ß and TNF-, by activated
macrophages (19, 66, 67). More recent data using p38 MAPK
inhibitors as well as dominant negative p38 MAPK transgenic mice and
MKK3-deficient mice demonstrate that the p38 MAPK pathway may be
involved in IL-12 production by macrophages and IFN- production by
anti-CD3-stimulated T cells (17, 68). Since IFN- is
an important mediator of delayed-type hypersensitivity
(69), the data in this report suggest that p38 MAPK
inhibitors may also function in vivo by inhibiting Th1 differentiation
and IL-12-induced IFN- expression in activated T and Th1 cells and
subsequently decrease delayed-type hypersensitivity. The demonstration
that MKK3/6 and p38 MAPK are activated by IL-12 and are involved in
some IL-12-activated functions in primary T cells may also explain the
T cell defect seen in MKK3-deficient mice (68).
Our data demonstrate, for the first time, that IL-12 activates MKK3/6
and p38 MAPK, but not p44/42 and SAPK/JNK MAPK. IL-12-activated p38
MAPK is required for IL-12-induced IFN- expression and Th1
development in a Stat4-independent pathway. Importantly, these data
demonstrate that the anti-inflammatory activity of p38 MAPK
inhibitors may occur through the inhibition of production and signaling
of multiple proinflammatory cytokines, including IL-12.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Indiana University School of Medicine, Walther Oncology Center, 1044 West Walnut Street, Room 302, Indianapolis, IN 46202.
3 Abbreviations used in this paper: Jak, Janus kinase; ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; SAPK, stress-activated protein kinase; ATF, activating transcription factor; CREB, cAMP response element binding protein; CHOP, C/EBP homologous protein; MEF2C, monocyte enhancer factor 2C; SAP-1, serum response factor accessory protein-1.
Received for publication January 11, 2000. Accepted for publication May 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducing factor in enhanced production of IFN-. J. Immunol. 159:2125. expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17:2817.[Medline]
promoter confer activation specific expression in T cells. J. Exp. Med. 178:1483. promoter mediates selective expression in T cells. J. Biol. Chem. 271:31964. uses a different signaling pathway. Proc. Natl. Acad. Sci. USA 96:13956.: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781.[Abstract]
B and NFAT with the interferon- promoter. J. Biol. Chem. 272:30412. promoter in primary CD4+ T lymphocytes. J. Immunol. 160:3642. promoter in T cells. J. Biol. Chem. 273:34775., ß, , and in inflammatory cell lineages. J. Immunol. 162:4246. in delayed-type hypersensitivity mediated by Th1 clones. J. Immunol. 143:2887.[Abstract]
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J. E. Comer, A. K. Chopra, J. W. Peterson, and R. Konig Direct Inhibition of T-Lymphocyte Activation by Anthrax Toxins In Vivo Infect. Immun., December 1, 2005; 73(12): 8275 - 8281. [Abstract] [Full Text] [PDF]
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 T. Higashi, J. Tsukada, Y. Yoshida, T. Mizobe, F. Mouri, Y. Minami, H. Morimoto, and Y. Tanaka Constitutive tyrosine and serine phosphorylation of STAT4 in T-cells transformed with HTLV-I Genes Cells, December 1, 2005; 10(12): 1153 - 1162. [Abstract] [Full Text] [PDF]
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G. J. Renukaradhya, T. J. R. Webb, M. A. Khan, Y. L. Lin, W. Du, J. Gervay-Hague, and R. R. Brutkiewicz Virus-Induced Inhibition of CD1d1-Mediated Antigen Presentation: Reciprocal Regulation by p38 and ERK J. Immunol., October 1, 2005; 175(7): 4301 - 4308. [Abstract] [Full Text] [PDF]
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B. Schmeck, W. Beermann, V. van Laak, J. Zahlten, B. Opitz, M. Witzenrath, A. C. Hocke, T. Chakraborty, M. Kracht, S. Rosseau, et al. Intracellular Bacteria Differentially Regulated Endothelial Cytokine Release by MAPK-Dependent Histone Modification J. Immunol., September 1, 2005; 175(5): 2843 - 2850. [Abstract] [Full Text] [PDF]
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W.-M. Yu, S. Wang, A. D. Keegan, M. S. Williams, and C.-K. Qu Abnormal Th1 Cell Differentiation and IFN-{gamma} Production in T Lymphocytes from Motheaten Viable Mice Mutant for Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-1 J. Immunol., January 15, 2005; 174(2): 1013 - 1019. [Abstract] [Full Text] [PDF]
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A. Mavropoulos, G. Sully, A. P. Cope, and A. R. Clark Stabilization of IFN-{gamma} mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells Blood, January 1, 2005; 105(1): 282 - 288. [Abstract] [Full Text] [PDF]
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J. C. Deng, X. Zeng, M. Newstead, T. A. Moore, W. C. Tsai, V. J. Thannickal, and T. J. Standiford STAT4 Is a Critical Mediator of Early Innate Immune Responses against Pulmonary Klebsiella Infection J. Immunol., September 15, 2004; 173(6): 4075 - 4083. [Abstract] [Full Text] [PDF]
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K. A. Papadakis, J. L. Prehn, C. Landers, Q. Han, X. Luo, S. C. Cha, P. Wei, and S. R. Targan TL1A Synergizes with IL-12 and IL-18 to Enhance IFN-{gamma} Production in Human T Cells and NK Cells J. Immunol., June 1, 2004; 172(11): 7002 - 7007. [Abstract] [Full Text] [PDF]
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 A. O'Sullivan, H.-C. Chang, Q. Yu, and M. H. Kaplan STAT4 Is Required for Interleukin-12-induced Chromatin Remodeling of the CD25 Locus J. Biol. Chem., February 20, 2004; 279(8): 7339 - 7345. [Abstract] [Full Text] [PDF]
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J. J. Yu, C. S. Tripp, and J. H. Russell Regulation and Phenotype of an Innate Th1 Cell: Role of Cytokines and the p38 Kinase Pathway J. Immunol., December 1, 2003; 171(11): 6112 - 6118. [Abstract] [Full Text] [PDF]
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H.-C. Chang, S. Zhang, I. Oldham, L. Naeger, T. Hoey, and M. H. Kaplan STAT4 Requires the N-terminal Domain for Efficient Phosphorylation J. Biol. Chem., August 22, 2003; 278(34): 32471 - 32477. [Abstract] [Full Text] [PDF]
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J. H. Bream, R. E. Curiel, C.-R. Yu, C. E. Egwuagu, M. J. Grusby, T. M. Aune, and H. A. Young IL-4 synergistically enhances both IL-2- and IL-12-induced IFN-{gamma} expression in murine NK cells Blood, July 1, 2003; 102(1): 207 - 214. [Abstract] [Full Text] [PDF]
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X. Guo, R. E. Gerl, and J. W. Schrader Defining the Involvement of p38{alpha} MAPK in the Production of Anti- and Proinflammatory Cytokines Using an SB 203580-resistant Form of the Kinase J. Biol. Chem., June 13, 2003; 278(25): 22237 - 22242. [Abstract] [Full Text] [PDF]
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K. B. Nguyen, T. P. Salazar-Mather, M. Y. Dalod, J. B. Van Deusen, X.-q. Wei, F. Y. Liew, M. A. Caligiuri, J. E. Durbin, and C. A. Biron Coordinated and Distinct Roles for IFN-{alpha}{beta}, IL-12, and IL-15 Regulation of NK Cell Responses to Viral Infection J. Immunol., October 15, 2002; 169(8): 4279 - 4287. [Abstract] [Full Text] [PDF]
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J. Kwang Yoo, J. Ho Cho, S. Woo Lee, and Y. Chul Sung IL-12 Provides Proliferation and Survival Signals to Murine CD4+ T Cells Through Phosphatidylinositol 3-Kinase/Akt Signaling Pathway J. Immunol., October 1, 2002; 169(7): 3637 - 3643. [Abstract] [Full Text] [PDF]
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 A. Morinobu, M. Gadina, W. Strober, R. Visconti, A. Fornace, C. Montagna, G. M. Feldman, R. Nishikomori, and J. J. O'Shea STAT4 serine phosphorylation is critical for IL-12-induced IFN-gamma production but not for cell proliferation PNAS, September 17, 2002; 99(19): 12281 - 12286. [Abstract] [Full Text] [PDF]
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 A. Ariel, D. Novick, M. Rubinstein, C. A. Dinarello, O. Lider, and R. Hershkoviz IL-12 and IL-18 induce MAP kinase-dependent adhesion of T cells to extracellular matrix components J. Leukoc. Biol., July 1, 2002; 72(1): 192 - 198. [Abstract] [Full Text] [PDF]
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 C. Du, Q. Guan, M. W. Khalil, and S. Sriram Stimulation of Th2 Response by High Doses of Dehydroepiandrosterone in KLH-Primed Splenocytes Experimental Biology and Medicine, December 1, 2001; 226(11): 1051 - 1060. [Abstract] [Full Text] [PDF]
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R. E. Curiel, C. S. Garcia, L. Farooq, M. F. Aguero, and I. Espinoza-Delgado Bryostatin-1 and IL-2 Synergize to Induce IFN-{gamma} Expression in Human Peripheral Blood T Cells: Implications for Cancer Immunotherapy J. Immunol., November 1, 2001; 167(9): 4828 - 4837. [Abstract] [Full Text] [PDF]
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S. J. White, G. H. Underhill, M. H. Kaplan, and G. S. Kansas Cutting Edge: Differential Requirements for Stat4 in Expression of Glycosyltransferases Responsible for Selectin Ligand Formation in Th1 Cells J. Immunol., July 15, 2001; 167(2): 628 - 631. [Abstract] [Full Text] [PDF]
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G. S. Yap, R. Ortmann, E. Shevach, and A. Sher A Heritable Defect in IL-12 Signaling in B10.Q/J Mice. II. Effect on Acute Resistance to Toxoplasma gondii and Rescue by IL-18 Treatment J. Immunol., May 1, 2001; 166(9): 5720 - 5725. [Abstract] [Full Text] [PDF]
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V. A. Lawless, S. Zhang, O. N. Ozes, H. A. Bruns, I. Oldham, T. Hoey, M. J. Grusby, and M. H. Kaplan Stat4 Regulates Multiple Components of IFN-{gamma}-Inducing Signaling Pathways J. Immunol., December 15, 2000; 165(12): 6803 - 6808. [Abstract] [Full Text] [PDF]
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), SB202190 (•), or SB202474
(
) for 36 h. Drugs were added for 1 h before the treatment
with IL-12. Cell culture supernatants were tested for IFN-
