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The Journal of Immunology, 2000, 165: 1374-1380.
Copyright © 2000 by The American Association of Immunologists

The p38 Mitogen-Activated Protein Kinase Is Required for IL-12-Induced IFN-{gamma} Expression1

Shangming Zhang and Mark H. Kaplan2

Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and Walther Cancer Institute, Indianapolis, IN 46208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{gamma} production by activated T cells and Th1 cells. However, several studies have suggested that other pathways may be involved in IL-12-stimulated IFN-{gamma} 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-{gamma} mRNA and IFN-{gamma} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a pleiotropic cytokine composed of disulfide-linked p35 and p40 chains. IL-12 is produced mainly by macrophages and dendritic cells (1). In vivo, IL-12 increases inflammatory responses and anti-tumor activity (2). IL-12 stimulates the proliferation of T cells and NK cells and induces the expression of IFN-{gamma}, 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-{gamma} 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-{gamma} 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-{gamma} expression in activated T cells. Importantly, this pathway functions by a Stat4-independent mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

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-{gamma} 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-{gamma} 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{alpha} probe to confirm equal RNA loading. Densitometry was determined and is represented as the fold increase in IFN-{gamma} 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-{gamma} 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-{gamma} production by ELISA (18). ELISAs were performed using purified monoclonal anti-IFN-{gamma} Abs (R4/6A2, 2 µg/ml) as a capture Ab, and IFN-{gamma} was detected using biotinylated anti-IFN-{gamma} (PharMingen, San Diego, CA), avidin-alkaline phosphatase, and p-nitrophenol phosphate (pNPP) as the substrate (Sigma). Recombinant IFN-{gamma} 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-{gamma} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The p38 MAPK inhibitors decrease IL-12 induced IFN-{gamma} 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-{gamma} (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-{gamma} 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. 1GoA, cells in the absence of IL-12 stimulation expressed a low level of IFN-{gamma}. IL-12 dramatically stimulated IFN-{gamma} 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-{gamma} mRNA expression (Fig. 1GoB).



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  		FIGURE 1. p38 MAP kinase inhibitors decrease IL-12-induced IFN-{gamma} 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-{gamma} or TCR{alpha} as a control. Densitometry was performed and is presented as the fold increase in IFN-{gamma} mRNA relative to that in untreated cells. The result shown here is representative of three independent experiments.

 
We next tested whether SB203580 and SB202190 decreased the IFN-{gamma} 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. 2GoA show that IL-12-stimulated activated T cells produced 4-fold higher IFN-{gamma} compared with unstimulated cells. IFN-{gamma} 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-{gamma} 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-{gamma} 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-{gamma} production compared with untreated cells (Fig. 2GoB), similar to what we and others have observed when anti-CD3-induced IFN-{gamma} was examined (Ref. 17 and data not shown). These results strongly suggest that p38 MAPK is required for IL-12 induced IFN-{gamma} expression.



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  		FIGURE 2. p38 MAP kinase inhibitors specifically inhibit IL-12 induced IFN-{gamma} production in activated T cells and Th1 cells. A, Activated T cells were cultured with 1 ng/ml IL-12 in the presence or the absence of 10 µM SB203580 ({circ}), SB202190 (•), or SB202474 ({square}) for 36 h. Drugs were added for 1 h before the treatment with IL-12. Cell culture supernatants were tested for IFN-{gamma} production by ELISA. IL-12 induced IFN-{gamma} production (left) and SB203580 and SB202190, but not SB202474, inhibited IL-12-induced IFN-{gamma} production in dose-dependent manner (right). B, Differentiated CD4+ Th1 cells were pretreated for 1 h with 10 µM SB203580 or DMSO and restimulated with 1 ng/ml IL-12 for 24 h. Cell culture supernatants were tested for IFN-{gamma} 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 experiments.

 
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 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. 3GoA). Additionally, there was a dose-dependent activation of p38 MAPK activation in response to IL-12 (Fig. 3GoB). As expected, 10 µM SB202190 and SB203580, but not SB202474, decreased IL-12-induced p38 MAPK activity (Fig. 3GoC).



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  		FIGURE 3. IL-12 activates p38 MAPK. A, Activated T cells were stimulated 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 Materials 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 indicated 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 µM SB202474, SB202190, or SB203580 for 1 h, followed by IL-12 stimulation for 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 does not affect activation of p44/42 and SAPK/JNK MAP kinases

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. 4GoA). This observation confirms the Northern analysis showing that blocking activation of ERK using the MEK inhibitor PD98059 did not affect IL-12-induced IFN-{gamma} mRNA expression (Fig. 1GoB) 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. 4GoB). Thus, of the three MAPK pathways, only p38 is activated by IL-12.



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  		FIGURE 4. IL-12 does not affect phosphorylation of p44/42 and SAPK/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 fibroblasts that were treated with 50 ng/ml of PDGF was used as positive control. B, Activated T cells were stimulated with 1 ng/ml IL-12 for the indicated 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 representative of two independent experiments.

 
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 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. 5Go). No phosphorylation of MKK4 was detectable (data not shown), which corresponds to the lack of JNK activation shown in Fig. 4GoB following IL-12 stimulation.



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  		FIGURE 5. IL-12 activates MKK3/6. Activated T cells were stimulated 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.

 
The p38 MAPK functions through a Stat4-independent mechanism

Since Stat4 is required for IFN-{gamma} 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. 1Go 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. 6Go demonstrates that 10 µM SB203580 did not affect tyrosine phosphorylation of Stat4 at concentrations that did inhibit induction of IFN-{gamma} mRNA and secretion (Figs. 1Go and 2Go). 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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  		FIGURE 6. SB203580 does not affect IL-12-induced tyrosine phosphorylation 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 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 independent experiments.

 
To determine whether p38 MAPK regulates IL-12-stimulated and Stat4-dependent functions other than IFN-{gamma} 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. 7GoA, 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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  		FIGURE 7. IL-12-activated p38 MAPK functions independently of 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 µg/ml anti-IL-2 S4B6 Ab (•) or 30 U/ml of IL-2 alone ({circ}) in a 96-well U-bottom plate. Cells were pulsed for the last 12 h of a 48-h incubation with [3H]thymidine. 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-{gamma} production 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 experiments.

 
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-{gamma} (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-{gamma} secretion in wild-type CD4+ cells by about 50%. Similarly, IFN-{gamma} production was decreased in Stat4-/- CD4+ Th1 cultures (Fig. 7GoB). 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-{gamma} regulation (32, 33) we examined whether IL-12 increased the endogenous levels of serine phosphorylation of either of these factors. Fig. 8Go 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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  		FIGURE 8. IL-12 stimulates serine phosphorylation of ATF-2. Activated 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. Densitometry analysis was performed, and the fold increase in the level of phospho-proteins (ATF-2 and CREB) relative to that in unstimulated cells was determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 regulatory TXY loop present in all MAP kinases (37). The physiological function of the ERK kinases is to transmit signals from mitogens and growth factors to regulate cell proliferation and differentiation. JNK and p38 MAPK are both activated by environmental stresses and proinflammatory cytokines, such as TNF-{alpha} 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-{gamma} 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-{gamma} 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. 7GoA). 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. 7GoA) and did reduce IFN-{gamma} 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-{gamma} 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-{gamma} 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. 8Go). Thus, it is possible that p38 MAPK-mediated phosphorylation of ATF-2 or related family members is responsible for p38 MAPK-stimulated IFN-{gamma} gene transcription. Other transcription factors have been implicated in IFN-{gamma} gene regulation, including NF-AT, NF-{kappa}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-{gamma} 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{alpha}, p38 ß, p38{gamma}, and p38{delta} (51, 52, 53, 54, 55, 56, 57). p38{gamma} and p38{delta} are not inhibited by SB203580 (51, 53). Thus, it is likely that IL-12 activates p38{alpha}, 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-{gamma} induced expression of IL-12Rß2 (61). When IFN-{gamma} 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-{gamma} 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-{alpha}, 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-{gamma} production by anti-CD3-stimulated T cells (17, 68). Since IFN-{gamma} 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-{gamma} 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-{gamma} 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.


   Acknowledgments
 
We thank Dr. Michael J. Grusby for kindly providing Stat4-deficient breeding pairs. We also thank Drs. Randy Brutkiewicz and Lindsey Mayo for helpful comments and suggestions on the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI45515. M.H.K. is a Special Fellow of the Leukemia and Lymphoma Society. Back

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. Back

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. Back

Received for publication January 11, 2000. Accepted for publication May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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