The p38 Mitogen-Activated Protein Kinase Is Required for
IL-12-Induced IFN-? Expression1
Shangming Zhang and Mark H. Kaplan2
IL-12 is a central immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive
CD4?cells into Th1 cells. We and others have demonstrated that the Stat4 is critical for IFN-? 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. The Journal
of Immunology, 2000, 165: 1374–1380.
inflammatory responses and anti-tumor activity (2). IL-12 stimu-
lates the proliferation of T cells and NK cells and induces the
expression of IFN-?, CD25, IL-18R, and IFN regulating factor-1
(3–6). 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-12R?1 and IL-12R?2, since both chains have
homology to ?-chains of the gp130 family of receptors (8–10).
Both receptor chains associate with members of the Janus kinase
(Jak)3(2) family of tyrosine kinases. The IL-12R ?1-chain, which
contains no tyrosine residues in its cytoplasmic domain, interacts
with Tyk2 (8, 11). The IL-12R ?2-chain contains three tyrosines in
its cytoplasmic domain and interacts with Jak2 (9, 11). The bind-
ing of IL-12 to its receptor leads to activation of Jak kinases and
tyrosine phosphorylation of IL-12R?2, and results in the recruit-
ment and activation of Stat4 (12, 13). Stat4 specifically binds to the
IL-12R?2 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-? pro-
nterleukin-12 is a pleiotropic cytokine composed of disul-
fide-linked p35 and p40 chains. IL-12 is produced mainly by
macrophages and dendritic cells (1). In vivo, IL-12 increases
duction 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 nor-
mal IFN-? expression in activated T cells. Importantly, this path-
way functions by a Stat4-independent mechanism.
Materials and Methods
Wild-type C57BL/6 mice between 6 and 10 wk of age were purchased
from Harlan Bioproducts (Indianapolis, IN). Stat4-deficient mice were gen-
erated as described previously (15), backcrossed to the C57BL/6 back-
ground for eight generations, and intercrossed to generate C57BL/6 Stat4-
deficient mice. Stat4-deficient mice were bred in the animal facility at
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 ?g/ml plate-bound anti-CD3 (145-2C11, purified from hybrid-
oma 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
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 a32P-labeled IFN-?
probe for 16 h at 42°C. The membranes were sequentially washed in 2?
SSC containing 0.1% SDS at 60°C for 20 min and in 0.1? 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 repre-
sented as the fold increase in IFN-? mRNA relative to that in untreated
Department of Microbiology and Immunology, Walther Oncology Center, Indiana
University School of Medicine, Indianapolis, IN 46202; and Walther Cancer Institute,
Indianapolis, IN 46208
Received for publication January 11, 2000. Accepted for publication May 22, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant AI45515. M.H.K.
is a Special Fellow of the Leukemia and Lymphoma Society.
2Address correspondence and reprint requests to Dr. Mark H. Kaplan, Indiana Uni-
versity School of Medicine, Walther Oncology Center, 1044 West Walnut Street,
Room 302, Indianapolis, IN 46202. E-mail address: email@example.com
3Abbreviations 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 tran-
scription 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.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00
cells, using a multi-image light cabinet from Alpha Innotech (San Leandro,
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 ?g/ml) in the presence of 1
ng/ml IL-12 and 10 ?g/ml anti-IL-4 (11B11, purified from hybridoma su-
pernatants 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 ?M 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 in-
struction (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 ?g/ml) as a capture Ab, and IFN-? was detected
using biotinylated anti-IFN-? (PharMingen, San Diego, CA), avidin-alka-
line phosphatase, and p-nitrophenol phosphate (pNPP) as the substrate
(Sigma). Recombinant IFN-? was used as a standard. Similarly, differen-
tiated CD4?Th1 cells were pretreated with 10 ?M SB203580 or DMSO
for 1 h before a restimulation with 2 ?g/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 ?M. 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 ?g/ml pep-
statin, 20 ?g/ml aprotinin, and 20 ?g/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 En-
gland 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 En-
Activated T cells (1 ? 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 ?g/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 ?Ci/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 ?M 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 1? 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.
The p38 MAPK inhibitors decrease IL-12 induced IFN-?
While Stat4 has been shown to be crucial for IL-12-stimulated
biological activities, the importance of other IL-12-activated path-
ways has not been carefully examined. Since p38 MAPK has re-
cently 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. 1A, cells in the absence of IL-12 stimulation ex-
pressed 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 an-
alogue 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
mRNA expression. Total spleen and lymph node cells were activated with
anti-CD3 for 48 h. The activated cells were pretreated with the indicated
doses of SB203580 (A and B), SB202190 (A), SB202474 (A), or PD98059
(B) for 1 h and incubated in the absence or the presence of 1 ng/ml IL-12
for another 4 h. RNA was probed with cDNA for IFN-? or TCR? as a
control. Densitometry was performed and is presented as the fold increase
in IFN-? mRNA relative to that in untreated cells. The result shown here
is representative of three independent experiments.
p38 MAP kinase inhibitors decrease IL-12-induced IFN-?
1375The Journal of Immunology
that PD98059, a MAPK kinase (MEK) inhibitor (21), did not affect
IL-12 induced IFN-? mRNA expression (Fig. 1B).
We next tested whether SB203580 and SB202190 decreased the
IFN-? secretion of activated T cells and differentiated CD4?Th1
subsets in response to IL-12 stimulation or 2 ?g/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
?M, 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 deter-
mine IFN-? production (data not shown). Th1 cells, pretreated
with 10 ?M 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.
IL-12 activates p38 MAPK
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 ?g/ml) and
were stimulated with 1 ng/ml IL-12 for the indicated times with or
without 1-h pretreatment of 10 ?M 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 ?M SB202190 and SB203580, but not SB202474,
decreased IL-12-induced p38 MAPK activity (Fig. 3C).
IL-12 does not affect activation of p44/42 and SAPK/JNK MAP
Since the p38 MAPK pathway is only one of three MAPK path-
ways that might potentially be activated by IL-12, we next exam-
ined 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. 1B) and suggests that ERKs are not in-
volved in IL-12 signaling. Since it was reported that SB203580
cultured with 1 ng/ml IL-12 in the presence or the absence of 10 ?M SB203580 (E), SB202190 (F), or SB202474 (?) for 36 h. Drugs were added for
1 h before the treatment with IL-12. Cell culture supernatants were tested for IFN-? production by ELISA. IL-12 induced IFN-? production (left) and
SB203580 and SB202190, but not SB202474, inhibited IL-12-induced IFN-? production in dose-dependent manner (right). B, Differentiated CD4?Th1
cells were pretreated for 1 h with 10 ?M SB203580 or DMSO and restimulated with 1 ng/ml IL-12 for 24 h. Cell culture supernatants were tested for IFN-?
production by ELISA. The SD for ELISA values was too low to be seen on this scale. The result shown here is representative of three independent
p38 MAP kinase inhibitors specifically inhibit IL-12 induced IFN-? production in activated T cells and Th1 cells. A, Activated T cells were
lated with 1 ng/ml IL-12 for the indicated times. An in vitro kinase assay
measuring the phosphorylation of recombinant ATF-2, following precipitation
of p38 MAPK, was used to analyze p38 MAPK activity, as described in Ma-
terials and Methods. The amount of ATF-2 phosphorylation was quantitated
by densitometry and is presented as the fold increase in p38 MAPK activity
relative to untreated cells. B, Activated T cells were stimulated with the indi-
cated concentrations of IL-12 for 10 min. p38 MAPK activities were analyzed
as described above. C, Activated T cells were treated with DMSO or 10 ?M
an additional 10 min. p38 MAPK activities were analyzed as described above.
Immunoblot analyses of total p38 levels are shown as loading controls for all
panels. The results shown here are representative of three, two, and three
independent experiments for A, B, and C, respectively.
IL-12 activates p38 MAPK. A, Activated T cells were stimu-
1376IL-12 ACTIVATES p38 MAP KINASE
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.
Activation of MAPK kinase 3/6 by IL-12
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 phosphor-
ylate p38 MAPK, including MKK3, MKK6, and MKK4 (24–27).
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.
The p38 MAPK functions through a Stat4-independent
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 de-
scribed in Fig. 1 were pretreated with 10 ?M 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 precipi-
tated from whole cell extracts for phosphotyrosine analysis. Fig. 6
demonstrates that 10 ?M SB203580 did not affect tyrosine phos-
phorylation of Stat4 at concentrations that did inhibit induction of
IFN-? mRNA and secretion (Figs. 1 and 2). In addition, 10 ?M
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.
To determine whether p38 MAPK regulates IL-12-stimulated
and Stat4-dependent functions other than IFN-? expression, we
tested whether p38 MAPK inhibitors would affect IL-12-stimu-
lated proliferation of activated T cells. Stat4 has been demon-
strated 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 signif-
icantly inhibited by SB203580, which is consistent with a previous
study (28). This result provides further evidence that 10 ?M
SB203580 is not cytotoxic to T cells and demonstrates the require-
ment for p38 MAPK signaling in some, but not all, IL-12-signaled
To further demonstrate that p38 MAPK functions independently
of Stat4, we tested whether SB203580 would interfere with Th1
differentiation, another IL-12-stimulated function. It has also been
reported that Stat4?/?Th1 cultures make reduced, but detectable,
IFN-? (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-? produc-
tion was decreased in Stat4?/?CD4?Th1 cultures (Fig. 7B).
These results demonstrate that inhibition of the p38 MAPK path-
way 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
phorylation of Stat4. Activated T cells were pretreated with 10 ?M
SB203580 or DMSO as a control for 1 h, and then stimulated with 1 ng/ml
of IL-12 at 37°C for 45 min. Stat4 protein was immunoprecipitated with
purified polyclonal rabbit Abs against Stat4. Stat4 phosphorylation was
analyzed with anti-phosphotyrosine Ab PY99 (top) and reblotted with
Stat4 Ab (bottom). The results shown here are representative of three in-
SB203580 does not affect IL-12-induced tyrosine phos-
JNK MAPK. A, Activated T cells were stimulated with 1 ng/ml IL-12 for
the indicated times. Phosphorylation of p44/42 MAPK (ERK1/ERK2) in
whole cell lysates was analyzed with phospho-p44/42 MAPK E10 mAb
(top), stripped, and reprobed with polyclonal rabbit Abs against ERK2 p42
(bottom). The same amount of protein from lysates of mouse 3T3 fibro-
blasts that were treated with 50 ng/ml of PDGF was used as positive con-
trol. B, Activated T cells were stimulated with 1 ng/ml IL-12 for the in-
dicated times. Phosphorylation of SAPK/JNK in whole cell lysates was
analyzed with phospho-SAPK/JNK mAb (top), stripped, and reprobed with
polyclonal rabbit Abs against SAPK/JNK (bottom). The results are repre-
sentative of two independent experiments.
IL-12 does not affect phosphorylation of p44/42 and SAPK/
with 1 ng/ml IL-12 for the indicated time periods. Phosphorylation of
MKK3/6 in whole cell lysates was analyzed with anti-phospho-MKK3/6
(top), stripped, and reprobed with anti-MKK3 as a loading control. The
results are representative of three experiments.
IL-12 activates MKK3/6. Activated T cells were stimulated
1377 The Journal of Immunology
In vitro studies demonstrated that the transcription factors
ATF-2, Elk-1, CHOP, MEF2C, SAP-1, and CREB are phosphor-
ylated and activated by p38 MAP kinase (23, 24, 27, 29–31). 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 phosphor-
ylation 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 (34–36). 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.
The MAPK signaling pathway plays a key role in a variety of
cellular responses. There are at least three genetically distinct
MAP kinases in mammals, including ERK, JNK (also known as
SAPK), and p38 MAPK. These MAP kinases are activated by
phosphorylation on both threonine and tyrosine residues in a reg-
ulatory TXY loop present in all MAP kinases (37). The physio-
logical function of the ERK kinases is to transmit signals from
mitogens and growth factors to regulate cell proliferation and dif-
ferentiation. JNK and p38 MAPK are both activated by environ-
mental stresses and proinflammatory cytokines, such as TNF-?
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 de-
velopment (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 un-
clear, 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 demon-
strated that a p38 MAPK inhibitor reduced serine phosphorylation
of Stat1 induced by IL-2 in combination with IL-12 (41). How-
ever, our study differs from that study in that they examined a
restricted population of CD8?CD18brightT 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 pre-
viously 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/thre-
onine 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 in-
terfere 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-indepen-
dent 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,
Stat4. A, Activated cells were pretreated for 1 h with various concentrations
of SB203580, then cultured with 1 ng/ml of IL-12 along with 10 ?g/ml
anti-IL-2 S4B6 Ab (F) or 30 U/ml of IL-2 alone (E) in a 96-well U-bottom
plate. Cells were pulsed for the last 12 h of a 48-h incubation with [3H]thy-
midine. Data (mean ? SD) based on triplicate culture are shown. The
background counts per minute in the absence of IL-12 and IL-2 was
2010 ? 269. The result shown here is representative of two independent
experiments. B, Total spleen and lymph node cells isolated from wild-type
and Stat4-deficient mice were pretreated with SB203580 or DMSO for 1 h
and cultured for 5 days under conditions promoting Th1 development.
CD4?cells were isolated by MiniMACS beads and restimulated with
plate-bound anti-CD3 for 24 h. Supernatants were tested for IFN-? pro-
duction by ELISA. The SD for ELISA values was too low to be seen on
this scale. The results shown here are representative of three independent
IL-12-activated p38 MAPK functions independently of
vated T cells were stimulated with 1 ng/ml IL-12, and total cell extracts
were isolated at the indicated time points. Extracts were immunoblotted
with Abs for anti-phospho-ATF-2 (top) and anti-phospho-CREB/ATF-1
(bottom). Blots were then stripped and reprobed with Abs reactive with
total ATF-2 or total CREB, respectively, as a loading control. Densitom-
etry analysis was performed, and the fold increase in the level of phospho-
proteins (ATF-2 and CREB) relative to that in unstimulated cells was
IL-12 stimulates serine phosphorylation of ATF-2. Acti-
1378 IL-12 ACTIVATES p38 MAP KINASE
SAP-1, and CREB are phosphorylated and activated by p38 MAP
kinase (23, 24, 27, 29–31). Two regulatory elements have been
defined in the IFN-? promoter, termed proximal and distal ele-
ments, 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 (44–49). 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 ?, p38?, and p38? (51–57). p38? and p38? are not inhibited
by SB203580 (51, 53). Thus, it is likely that IL-12 activates p38?,
since p38? 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 phos-
phorylation 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 differen-
tiation. 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-12R?2 (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 ob-
served normal IL-12-induced IFN-? production and Th1 differen-
tiation in lck-deficient T cells (unpublished observation). This con-
firms 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 func-
tions appear to be limited to the Jak-STAT and MKK3/6-p38
SB203580 is a well-characterized anti-inflammatory drug. This
anti-inflammatory activity is associated with reduced production of
proinflammatory cytokines, such as IL-1? and TNF-?, by acti-
vated macrophages (19, 66, 67). More recent data using p38
MAPK inhibitors as well as dominant negative p38 MAPK trans-
genic mice and MKK3-deficient mice demonstrate that the p38
MAPK pathway may be involved in IL-12 production by macro-
phages and IFN-? production by anti-CD3-stimulated T cells (17,
68). Since IFN-? is an important mediator of delayed-type hyper-
sensitivity (69), the data in this report suggest that p38 MAPK
inhibitors may also function in vivo by inhibiting Th1 differenti-
ation 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 pri-
mary 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 ac-
tivity of p38 MAPK inhibitors may occur through the inhibition of
production and signaling of multiple proinflammatory cytokines,
We thank Dr. Michael J. Grusby for kindly providing Stat4-deficient breed-
ing pairs. We also thank Drs. Randy Brutkiewicz and Lindsey Mayo for
helpful comments and suggestions on the manuscript.
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