Introduction

Signal transduction via mitogen-activated protein (MAP) kinases plays a key role in a variety of cellular responses, including growth factor-induced proliferation, differentiation and cell death. Recently, several parallel MAP kinase signal transduction pathways have been defined in mammalian cells (Whitmarsh and Davis, 1996; Ip and Davis, 1998). These pathways include the extracellular signal related kinase (ERK) (Boulton et al., 1990, 1991), c-Jun N-terminal kinases (JNK, also known as SAPK) (Dérijard et al., 1994; Kyriakis, 1994), and p38 MAP kinase (Han et al., 1994; Lee et al., 1994; Rouse et al., 1994). These MAP kinases are activated by phosphorylation on Thr and Tyr by dual-specificity MAP kinase kinases (Dérijard et al., 1994; Raingeaud et al., 1995). The MAP kinase groups are functionally independent and are implicated in different biological processes (Whitmarsh and Davis, 1996; Ip and Davis, 1998).

The p38 MAP kinase gene was cloned following the purification of a 38 kDa protein that was phosphorylated on tyrosine in response to hyperosmolarity and endotoxic lipopolysaccharide. Independently, human p38 MAP kinase homologs (CSBP1 and CSBP2) were identified as the targets of pyridinyl imidazole compounds, which inhibit the production of pro-inflammatory cytokines [interleukin-1 (IL-1) and tumor necrosis factor (TNF)] in stimulated monocytes (Han et al., 1994; Lee et al., 1994; Rouse et al., 1994). A second gene (p38) that encodes a p38 MAP kinase isoform that is inhibited by pyridinyl imidazole drugs has recently been identified (Jiang et al., 1996; Stein et al., 1996; Enslen et al., 1998).

In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro-inflammatory cytokines (e.g. IL-1 and TNF), lipopolysaccharide and physical–chemical changes in the extracellular milieu caused by environmental stress (heat, osmotic stress, UV irradiation) (Freshney et al., 1994; Han et al., 1994; Lee et al., 1994; Rouse et al., 1994; Raingeaud et al., 1995). More recently, it has been reported that a variety of hemopoietic growth factors, such as colony stimulatory factor-1 (CSF-1), granulocytes/macrophages-colony stimulatory factor (GM-CSF) and IL-3 also activate p38 MAP kinases (Foltz et al., 1997).

The p38 MAP kinase is activated by the MAP kinase kinases MKK3, MKK4 and MKK6 (Dérijard et al., 1995; Han et al., 1996; Moriguchi et al., 1996; Raingeaud et al., 1996). These MKKs phosphorylate p38 MAP kinase on Thr and Tyr within the tripeptide motif TGY in kinase sub-domain VIII, thereby increasing enzymatic activity (Raingeaud et al., 1995). The p38 MAP kinase is implicated in the expression of cytokines and the regulation of cell proliferation and death (Lee et al., 1994; Xia et al., 1995). In vitro studies demonstrate that the transcription factor ATF-2 is phosphorylated and activated by p38 MAP kinase (Dérijard et al., 1995; Raingeaud et al., 1995, 1996). In addition, p38 MAP kinase activates Elk-1, CHOP, MEF2C and SAP-1 transcription factors (Dérijard et al., 1995; Raingeaud et al., 1996; Wang and Ron, 1996; Han et al., 1997; Whitmarsh et al., 1997). p38 MAP kinase also phosphorylates and activates the eIF-4E protein kinases Mnk1 and Mnk2 (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997) and the small heat shock protein hsp27 protein kinase MAPKAP kinase-2 (Freshney et al., 1994; Rouse et al., 1994; Mclaughlin et al., 1996).

The p38 MAP kinase is a selective target for pyridinyl imidazole drugs (Lee et al., 1994). These drugs appear to act by inhibiting p38 MAP kinase activity through competition with ATP at the ATP-binding pocket (Tong et al., 1997; Wilson et al., 1997; Young et al., 1997). These compounds are candidate drugs for the treatment of arthritis, bone resorption and endotoxin shock (Griswold et al., 1988; Lee et al., 1993; Reddy et al., 1994; Badger et al., 1996). The SB203580 compound possesses therapeutic activity in collagen-induced arthritis in DBA/LACJ mice, resulting in significant inhibition of paw inflammation and serum amyloid protein levels (Badger et al., 1996). This anti-arthritic activity is associated with reduced production of pro-inflammatory cytokines, such as IL-1 and TNF, by activated macrophages (Lee et al., 1993; Reddy et al., 1994; Badger et al., 1996; Lee and Young, 1996). However, the role of these inhibitors and the p38 MAP kinase in T-cell mediated responses has not been elucidated.

MAP kinase pathways play a critical role in the activation of T cells during the immune response. Several studies have shown the involvement of the ERK pathway in the positive selection of T cells in the thymus and in T-cell activation (Alberola-Ila et al., 1995; Swan et al., 1995). It has also been shown that JNK is activated during T-cell activation, where this molecule appears to play a role in integrating signals initiated at the T-cell receptor (TcR) complex and the co-stimulatory molecule CD28 (Rincón and Flavell, 1994; Su et al., 1994). In contrast, little is known about the role of p38 MAP kinase. p38 is activated during development in the thymus, suggesting a potentially important role for this MAP kinase in T-lymphocyte development (Sen et al., 1996). More recently, it has been shown that IL-2 and IL-7 activate both JNK and p38 kinases (Crawley et al., 1997).

In response to pathogens, naïve CD4⁺ T cells differentiate into effector Th1 and Th2 cells. Th1 cells produce mainly IFN and TNF, which are required for cell-mediated inflammatory reactions. Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, which mediate B-cell activation (for review see Paul and Seder, 1994). Development of an inappropriate response can lead to ineffective immunity and may have pathological consequences. One of the most powerful inducers of the differentiation of CD4⁺ T cells into Th1 or Th2 cells appears to be the local cytokine environment. It is clear that the cytokine IL-12 directs differentiation to a Th1 phenotype, while IL-4 can drive differentiation to a Th2 phenotype. We have recently demonstrated that IL-6 is able to initiate the polarization of naïve CD4⁺ T cells to effector Th2 cells by inducing the production of endogenous IL-4 (Rincón et al., 1997a). A large number of studies are currently focused on the identification of intracellular signals and transcription factors specific for either Th1 or Th2 (Rincón and Flavell, 1997a).

Since p38 MAP kinase can be activated by different pro-inflammatory cytokines and has been implicated in the expression of specific cytokines, it is possible that this kinase plays a role in the differentiation or activation of Th1 and Th2 cells. Here, we show that p38 MAP kinase is activated upon stimulation in Th1 cells, but not in Th2 cells. Inhibition of the p38 MAP kinase pathway, either by the specific p38 inhibitor SB203580 or by the expression of dominant-negative (dn) p38 in transgenic mice, results in decreased production of IFN in Th1 cells. Moreover, the expression of MKK6, a constitutive activator of p38 MAP kinase, caused increased IFN expression in transgenic mice. Together, these data demonstrate that the p38 MAP kinase pathway regulates the expression of IFN in Th1 cells.

Results

Inhibition of IFN production by the drug SB203580 in Th1 CD4⁺ T cells

The p38 MAP kinase is implicated in the regulation of cytokine expression and is also regulated by specific cytokines. To determine the potential role of p38 in the regulation of cytokine production by Th1 and Th2 cell subsets, we first analyzed the effect of the p38 MAP kinase inhibitor SB203580, a pyridinyl imidazole drug (Lee et al., 1994; Cuenda et al., 1995), on the production of IL-4 and IFN by Th2 and Th1 CD4⁺ T cells, respectively. To differentiate CD4⁺ T cells in vitro to Th1 and Th2 cells, we followed the protocol that we have used previously in several studies (Rincón et al., 1997a,b). CD4⁺ T cells were activated with Concanavalin A (ConA) and antigen presenting cells (APC) in the presence of either IL-4 to mediate differentiation to Th2 cells, or IL-12 to mediate differentiation to Th1 cells (Le Gros et al., 1990; Swain et al., 1990; Hsieh et al., 1993; Seder et al., 1993). After 4 days, the cells were exhaustively washed and equal numbers of cells were re-stimulated (in the absence of APC or exogenous cytokines) with ConA alone or ConA in the presence of different doses of SB203580. After 24 h the supernatant was harvested and analyzed for cytokine production by ELISA. IL-4 production by Th2 cells was not significantly affected even by the highest concentration of the p38 MAP kinase inhibitor (10 M) (Figure 1A). However, a dose-dependent inhibition of IFN production was obtained in activated Th1 cells in the presence of different concentrations of SB203580 (Figure 1B). The presence of SB203580 did not affect the viability of the cells at the time point (24 h) when culture supernatants were harvested to determine cytokine production (data not shown). No APC were present during the activation of Th1 and Th2 effector cells, since they do not require co-stimulatory signals (Croft et al., 1994). Therefore, inhibition of IFN production by SB203580 was due to a direct effect on the Th1 cells.

p38 MAP kinase activity is selectively induced in Th1 cells

The inhibition of cytokine production (IFN) by SB203580 in Th1 cells suggests that the p38 MAP kinase pathway is differentially regulated in Th1 and Th2 cells. We first analyzed the regulation of endogenous p38 MAP kinase activity during the differentiation of naïve CD4⁺ T cells into Th1 and Th2 cells. No significant differences were observed between the level of p38 MAP kinase activity induced during differentiation of Th2 cells with ConA plus IL-4 and the level of p38 MAP kinase activity during differentiation of Th1 cells with IL-12 (Figure 2A). The activation of p38 MAP kinase was transient and after 4 days of differentiation only low levels of p38 MAP kinase activity were detected. The extent of p38 MAP kinase activation was similar in cells stimulated with IL-12 and IL-4. Nevertheless, the kinetics of p38 MAP kinase activation was consistently advanced when the cells were stimulated with IL-12 compared with IL-4.

To examine the regulation of p38 MAP kinase activity during activation of Th1 and Th2 effector cells, CD4⁺ T cells were cultured in the presence of ConA plus IL-4 or IL-12. After 4 days, the cells were washed, counted and restimulated with ConA for different periods of time. No significant increase in p38 MAP kinase activity was detected during the activation of Th2 effector cells (Figure 2B). In contrast, p38 MAP kinase was rapidly activated in Th1 effector cells upon stimulation. These data show that in effector cells the p38 MAP kinase pathway is specific to Th1-cell activation. To determine whether the deficiency in p38 activation in Th2 cells was a TcR proximal or distal event, we examined p38 activity in response to a TcR-independent stimulus, a combination of phorbol myristate acetate (PMA) plus ionomycin. Like ConA, PMA plus ionomycin strongly induced p38 activity in Th1 cells, while only a minor increase was observed in Th2 cells (Figure 2C). Together these results indicated that the p38 MAP kinase pathway was differentially regulated in Th1 and Th2 cells and suggested that its activation in Th1 cells could be critical for the production of IFN in these cells.

p38 MAP kinase is required for IFN gene transcription

To determine whether inhibition of IFN production in Th1 cells by SB203580 was due to a specific effect on IFN gene expression, we examined the level of mRNA by competitive reverse transcriptase (RT)–PCR (Reiner et al., 1993). Specific primers for HPRT (positive control) and IFN were used to amplify the cDNA obtained from stimulated Th1 cells. The level of IFN mRNA detected in Th1 cells upon ConA-activation were decreased (45–60%) when the activation was performed in the presence of different doses of SB203580 (Figure 3A). These results indicated that the blockade of p38 MAP kinase by SB203580 in Th1 cells inhibited IFN gene expression. We cannot, however, exclude the possibility that IFN translation may also be affected by SB203580, as reported for certain cytokine mRNAs (Prichett et al., 1995; Pietersman et al., 1997).

ATF/CREB motifs have been identified in two critical regulatory elements of the IFN promoter, termed 'proximal' and 'distal' elements (Penix et al., 1993, 1996; Cippitelli et al., 1995; Aune et al., 1996). Since the ATF family of transcription factors are known to be substrates of p38 MAP kinase (Dérijard et al., 1995; Raingeaud et al., 1995, 1996), we considered it probable that at least one of the effects of p38 MAP kinase was directly on the IFN promoter. To test the direct effect of p38 MAP kinase on IFN gene transcription, we first analyzed the effect of SB203580 on transcription mediated by the IFN promoter. Jurkat T cells were transiently transfected with a reporter construct containing the luciferase gene driven by the IFN promoter (-538 to +64 from transcription start site). Luciferase activity was measured in cells activated with PMA and ionomycin in the absence or presence of different doses of SB203580. A dose-dependent inhibition of the transcription mediated by the IFN promoter was observed in the presence of the p38 MAP kinase inhibitor SB203580 (Figure 3B), suggesting that activation of p38 MAP kinase is required for IFN gene transcription.

To examine further the involvement of the p38 MAP kinase pathway in transcription of the IFN gene, we investigated the effect of expression of dn p38 MAP kinase. This construct was generated by replacing the activating phosphorylation sites Thr¹⁸⁰ and Tyr¹⁸² with Ala and Phe, respectively (Raingeaud et al., 1995). This inactive form of p38 MAP kinase binds endogenous substrates, thereby inhibiting signaling by the endogenous p38 MAP kinase pathway. Expression of this dn p38 MAP kinase by transient transfection in Jurkat T cells did not affect cell viability (data not shown) and did not significantly modify transcription mediated by a constitutive promoter, such as the Rous sarcoma virus (RSV) long terminal repeat (data not shown). However, overexpression of dn p38 MAP kinase inhibited transcription driven by the IFN promoter in activated Jurkat cells in a dose-dependent manner (Figure 3C). In contrast, a dominant-negative form of JNK (Dérijard et al., 1994) did not affect IFN promoter transcriptional activity (Figure 3C), indicating the specific involvement of p38 MAP kinase in the regulation of IFN gene transcription.

Normal T-cell proliferation in dn p38 transgenic mice

To examine the role of p38 MAP kinase in Th1 responses, we generated transgenic mice expressing dn p38 MAP kinase. The cDNA encoding dn p38 was linked to the distal lck promoter (Figure 4A) which, though weaker than the proximal lck promoter used extensively by Perlmutter and colleagues (Alberola-Ila et al., 1995; Swan et al., 1995), expresses in both peripheral T cells and thymocytes (Wildin et al., 1991). Three different founder mice (lines #18, #25 and #27) were obtained and back-crossed to generate stable lines. Expression of the transgene was examined in both negative and positive littermates by RT–PCR (Figure 4B). Positive littermates from all the lines expressed the dn p38 transgene. High levels of dn p38 expression were observed in T cells and in the thymus and lower levels in the spleen; no expression was observed in B cells (Figure 4C).

The developing thymocytes of dn p38 transgenic mice appeared normal. The ratio of double negative CD4^-CD8^-, double positive CD4⁺CD8⁺, and single positive CD4⁺CD8^- and CD8⁺CD4^- thymocytes was similar in both transgene positive and negative littermates from line #18 (Figure 5A) and the other lines (data not shown). Development and homeostasis of lymphocytes in the periphery also appeared to occur normally, since similar ratios of CD4⁺ and CD8⁺ T cells were observed in lymph nodes of dn p38 and wild-type mice (Figure 5A). To test the effect of dn p38 on T-cell activation, splenocytes from positive and negative littermates from all the lines were activated with either ConA or PMA plus ionomycin. No significant differences were observed in the proliferative response (Figure 5B). Moreover, proliferation of purified CD4⁺ T cells in response to ConA or anti-CD3 monoclonal antibody (mAb) in the presence of APC, was similar for cells obtained from dn p38 and wild-type mice (Figure 5C). Thus, the ability of T cells to produce IL-2 (data not shown) and enter the cell cycle appears to be normal in the dn p38 animals.

Activation of Th1 cells is impaired in the dn p38 transgenic mice

To analyze the activation of Th1 cells in the dn p38 transgenic mice, CD4⁺ T cells from negative control and dn p38 transgenic littermates were activated with ConA and APC in the presence of either IL-4 (Th2) or IL-12 (Th1) for 4 days. The cells were then exhaustively washed and equal numbers were re-stimulated with ConA alone for 24 h before harvesting the supernatants to analyze cytokine production. In correlation with the results obtained with the p38 MAP kinase inhibitor drug SB203580, the production of IFN was significantly inhibited by Th1 cells from dn p38 transgenic mice from line #18 (Figure 6A). In contrast, no significant differences were observed in the production of IL-4 and IL-5 in Th2 cells (Figure 6A). Similarly, the production of IL-10, another Th2 type cytokine, was unaffected (data not shown). A slight increase in IL-4 production was also observed in Th1 cell populations from the dn p38 mice, probably as a consequence of the decrease of IFN production, since IFN can suppress IL4 production. The reduction of IFN production in Th1 cells from dn p38 transgenic mice was consistent in several independent experiments performed using two different transgenic mouse lines (Figure 6B). This effect was caused by the specific overexpression of the dn p38 transgene, since no modifications were observed in Th1 cells from transgenic mice overexpressing dn JNK1 (our unpublished data).

Increased p38 MAP kinase activity was observed during activation of Th1 cells, but not Th2 cells. We examined the effect of expression of dn p38 on endogenous p38 MAP kinase activity. Rapid and transient activation of p38 MAP kinase was observed in wild-type Th1 cells (Figure 6C). Significant reduction of endogenous p38 MAP kinase activity was observed in Th1 cells from the dn p38 transgenic mice. The grade of inhibition of the endogenous p38 MAP kinase activity (40–50%) correlated with the inhibition of IFN production (50–60%) observed in these mice. As expected, an inhibition of p38 MAP kinase was also detected in Th2 cells from the dn p38 transgenic mice, since the distal lck promoter drives equal expression in both Th1 and Th2 cells. However, in correlation with the results obtained in the presence of SB203580, the inhibition of p38 MAP kinase did not affect IL-4 production in Th2 cells (Figure 6B). We also analyzed the regulation of p38 activity in response to a TcR-independent stimulus. The p38 MAP kinase activity induced by PMA plus ionomycin was also inhibited in Th1 cells from dn p38 transgenic mice (Figure 6D).

To determine whether p38 MAP kinase activation was required to trigger Th1 responses by specific antigens, we crossed the dn p38 transgenic mice with cytochrome c (Cyt c) TcR transgenic mice (Kaye et al., 1989). These mice express the and chain of the TcR from a CD4⁺ T-cell clone that recognizes a peptide of Cyt c in the context of MHC class II I-E^k. CD4⁺ T cells were isolated from both Cyt c TcR transgenic mice and dn p38Cyt c TcR double transgenic mice, and differentiated with specific Cyt c peptide alone or Cyt c peptide in the presence of IL-12. After 4 days, cells were washed and restimulated with peptide alone. Supernatants were harvested after 24 h. Although low levels of cytokines were produced when the cells were differentiated with the peptide in the absence of the driving cytokine, a preferential Th1 response was observed in Cyt c TcR transgenic mice. This Th1 response was significantly reduced in dn p38Cyt c TcR double transgenic mice (Figure 6E). Th1 cells differentiated with Cyt c peptide in the presence of IL-12 produce higher levels of IFN (Figure 6E). Despite the high production of IFN, 50% inhibition was observed in dn p38Cyt c TcR double transgenic mice (Figure 6E).

Together, these data indicate that the inhibition of the p38 MAP kinase pathway in CD4⁺ T cells causes an impairment of Th1 responses.

Increased IFN production in Th1 cells from constitutively-activated MKK6 transgenic mice

To confirm the results obtained with the dn p38 transgenic mice, we generated transgenic mice expressing constitutively-activated MKK6, an upstream activator of p38 MAP kinase (Han et al., 1996; Moriguchi et al., 1996; Raingeaud et al., 1996). MAP kinase kinases are activated by dual phosphorylation within subdomain VIII. These phosphorylation sites are conserved in MKK6 (Ser²⁰⁷ and Thr²¹¹). The mechanism of MKK6 activation may therefore be mediated by increased negative charge on these phosphorylation sites. Thus, the replacement of these two residues with Glu, which provides a constitutive negative charge, generates an activated MKK6 [MKK6(Glu)] that is able to phosphorylate p38 MAP kinase in the absence of prior stimulation (Raingeaud et al., 1996). To generate transgenic mice in which p38 MAP kinase is constitutively activated, the cDNA encoding MKK6(Glu) was linked to the distal lck promoter (Figure 7A) and microinjected into fertilized eggs. Three transgenic mouse lines were obtained and characterized. To determine the expression of the transgene, we directly examined p38 MAP kinase activity, since expression of MKK6(Glu) should result in an increased level of p38 MAP kinase activity in the absence of stimulation. We first examined p38 MAP kinase activity in total thymocytes isolated from negative and positive littermates from the different transgenic mouse lines. In the three transgenic lines, the level of p38 MAP kinase activity in the transgenic thymocytes was 2- to 4-fold higher than negative control thymocytes (Figure 7B). MKK6 is a specific activator of p38 MAP kinase that activates neither JNK nor ERK (Han et al., 1996; Moriguchi et al., 1996; Raingeaud et al., 1996). In correlation, no significant changes were observed in the level of JNK activity in total thymocytes from MKK6(Glu) transgenic mice compared with wild-type mice (Figure 7B).

The analysis of cell-surface staining showed no major differences in the distribution of thymic subpopulations in MKK6(Glu) transgenic mice. The number and the phenotype of CD4⁺ T cells from lymph nodes and spleen from these mice were also normal (data not shown). To examine the expression of MKK6(Glu) in peripheral T cells, we first isolated the CD4⁺ population from spleen and lymph nodes from negative littermate control and MKK6(Glu) transgenic mice. p38 MAP kinase and JNK activities were examined in these cells prior to stimulation. The level of p38 MAP kinase activity was higher in CD4⁺ T cells from MKK6(Glu) transgenic mice (Figure 7C). No significant increase in JNK activity was observed.

CD4⁺ T cells from control and MKK6(Glu) transgenic mice were differentiated into Th1 and Th2 cells with ConA in the presence of IL-12 or IL-4, respectively. After 4 days, the cells were washed and equal numbers of cells were restimulated with ConA. Cytokine production was then analyzed. In contrast to the dn p38 transgenic mice, an increase in IFN production was observed in Th1 cells from the MKK6(Glu) transgenic mice compared with the negative littermates (Figure 7D). Reduced IL-4 production was also observed in Th2 cells from the MKK6(Glu) transgenic mice (Figure 7D). These data indicate that the MKK6(Glu) transgenic mice exhibit polarization of the effector T cells to the Th1 phenotype with reduced IL-4 secretion and increased IFN production. Since the expression of MKK6(Glu) caused constitutive activation of p38 MAP kinase in unstimulated CD4⁺ T cells, we also examined the production of IFN by these cells during their differentiation into Th1 cells. CD4⁺ T cells were stimulated with ConA and IL-12 and culture supernatants were harvested at several time points during the differentiation. Increased IFN production in CD4⁺ T cells from MKK6(Glu) mice was detected by day 3 (Figure 7E).

Together, the data obtained from experiments using the p38 MAP kinase inhibitor SB203580 and the two transgenic mouse models indicate that that p38 MAP kinase pathway plays an important role in Th1 responses.

Discussion

In response to pathogens, naïve CD4⁺ T cells differentiate into effector Th1 and Th2 cells. The development of an inappropriate response can lead to ineffective immunity and may lead to pathological consequences. The balance of Th1 and Th2 responses is critical for the generation of physiologically appropriate immune responses and alterations of this balance are associated with several immune disorders, such as diabetes, leishmaniasis, arthritis and experimental encephalomyelitis. A large number of studies are currently focused on the identification of intracellular signals and transcription factors specific for either Th1 or Th2 cells (Rincón and Flavell, 1997a). While several transcription factors have been described to be specifically active in one of these subsets (principally in Th2 cells), little is known about the regulation of the intracellular signals that lead to the activation of these factors in Th1 and Th2 cells. p38 MAP kinase can be activated by pro-inflammatory cytokines and is involved in the expression of specific cytokine genes. In this study, we have used three different experimental approaches (p38 MAP kinase specific inhibitors, dn p38 transgenic mice and constitutively-activated MKK6 transgenic mice) to show that the p38 MAP kinase pathway is critical for the development of Th1 responses.

Pyridinyl imidazole compounds were initially identified as powerful anti-arthritic drugs (Griswold et al., 1988; Lee et al., 1993; Reddy et al., 1994; Badger et al., 1996). These drugs also constitute the most specific inhibitors of p38 MAP kinases that have been identified to date (Lee et al., 1994). However, the anti-arthritic activity of these drugs has been exclusively associated with blocking the production of pro-inflammatory cytokines such as IL-1, IL-6 and TNF by macrophages (Lee et al., 1993; Reddy et al., 1994; Badger et al., 1996; Beyaert et al., 1996; Lee and Young, 1996). We have shown here, for the first time, that inhibition of IFN production by CD4⁺ Th1 cells is another mechanism by which the pyridinyl imidazole compounds could modify the course of arthritis. The blockade of Th1-cell activation was not due to an indirect effect of SB203580 on the APC, since no APC were present during the restimulation of Th1 cells. Furthermore, these results were corroborated by the inhibition of IFN production observed in the dn p38 transgenic mice where the transgene is expressed only in T cells (distal lck promoter) and wild-type APC were used. The description of the role of these drugs in the Th1/Th2 balance is essential due to their potential therapeutic use in the treatment of arthritis. Although the contribution of Th1/Th2 responses in arthritis is not fully established, a predominant Th1 response has been found in several animal models of arthritis, such as collagen-induced and Lyme arthritis, but the implication of a Th1 response is less clear in human rheumatoid arthritis.

Little is known about the role of p38 MAP kinase in T cells. p38 MAP kinase is constitutively activated in immature thymocytes (Sen et al., 1996), suggesting a role for this pathway in thymic development. We could not find significant alterations in the distribution of thymic subpopulations in dn p38 or MKK6(Glu) transgenic mice at the age that experiments were performed (6–12 weeks). Further experiments are required to determined whether p38 MAP kinase is involved in positive or negative selection of immature thymocytes.

Activation of p38 MAP kinase has been associated with the induction of apoptosis in neuronal cells (Xia et al., 1995; Kawasaki et al., 1997; Kummer et al., 1997). Expression of activated MKK3 in PC12 cells (Xia et al., 1995) or activated MKK6 in Jurkat T cells (Huang et al., 1997) causes increased apoptosis. Conversely, dn MKK3 (Xia et al., 1995) or dn MKK6 (Huang et al., 1997) can inhibit apoptosis. These data strongly support the hypothesis that the p38 MAP kinase pathway contributes to apoptotic responses. Nevertheless, p38 MAP kinase does not appear to be universally required for apoptosis. Thus, it has been shown that p38 MAP kinase activation by Fas in Jurkat T cells is dependent on the activation of caspase proteases (Juo et al., 1997), suggesting a role of p38 MAP kinase downstream of apoptotic events. Similarly, p38 MAP kinase is not required for TNF-induced apoptosis of murine L cells, although this pathway is required for TNF-induced IL-6 gene expression (Beyaert et al., 1996). Furthermore, it is possible that some of the apoptotic effects of p38 MAP kinase activators (e.g. MKK6) are not mediated by p38 MAP kinase (Huang et al., 1997). In our analysis of Th1 cells, we observed increased apoptosis during the differentiation of Th1 cells from MKK6(Glu) transgenic mice compared with wild-type mice, but no significant cell viability differences were detected between Th1 cells from wild-type and dn p38 transgenic mice (data not shown). These data do not support a major role for the p38 MAP kinase pathway in the apoptosis of Th1 cells under the conditions of our experiments. However, our study does implicate the p38 MAP kinase pathway in the regulation of IFN production by Th1 cells.

In addition to the activation of p38 MAP kinase by pro-inflammatory cytokines (IL-1 and TNF) and physical–chemical stresses, several growth factors can also stimulate p38 MAP kinase pathway. Hematopoietic growth factors including IL-2, IL-3, IL-7, GM-CSF and Steel locus factor (SLF) induce p38 MAP kinase activity (Crawley et al., 1997; Foltz et al., 1997). Furthermore, IL-2 -stimulated proliferation is inhibited by SB203580 (Crawley et al., 1997). In contrast, IL-4 does not affect p38 MAP kinase activity (Foltz et al., 1997). The potential role of p38 MAP kinase in the IL-12 pathway has not yet been addressed. Our data show no significant effect of the dn p38 transgene on T-cell proliferation. The drug SB203580 did not affect cell recovery during the activation of Th1 and Th2 cells; however, SB203580 did cause partial inhibition of CD4⁺ T-cell proliferation during differentiation to Th1 and Th2 cells, but it is unclear whether this was a direct effect on the T cells or whether it was indirectly mediated by an effect of SB203580 on the APC.

Pyridinyl imidazole drugs can affect the secretion of several cytokines by interfering with the translation of the specific mRNAs (Prichett et al., 1995; Pietersman et al., 1997). These drugs can also decrease the level of cytokine mRNA, for example IL-6 (Beyaert et al., 1996). Here we demonstrate that the inhibition of IFN production by Th1 cells caused by SB203580 is associated with reduced IFN mRNA expression. Furthermore, we demonstrate that transcription mediated by the IFN promoter is inhibited by treatment of cells with SB203580. The expression of dn p38 in CD4⁺ Th1 cells caused inhibition of IFN expression. Moreover, the constitutive activation of p38 MAP kinase caused by expression of MKK6(Glu) in CD4⁺ Th1 cells resulted in increased production of IFN. Together, these results demonstrate the importance of the p38 MAP kinase pathway for the expression of the IFN gene in Th1 cells. Several substrates for p38 MAP kinase have already been identified, including ATF-2, Elk-1, CHOP, MEF2C and SAP-1 transcription factors (Dérijard et al., 1995; Raingeaud et al., 1995, 1996; Wang and Ron, 1996; Han et al., 1997; Whitmarsh et al., 1997). p38 MAP kinase also phosphorylates and activates the eIF-4E protein kinases Mnk1 and Mnk2 (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997) and the small heat shock protein hsp27 protein kinase MAPKAP kinase-2 (Freshney et al., 1994; Rouse et al., 1994; Mclaughlin et al., 1996). Two regulatory elements have been defined in the IFN promoter, termed proximal and distal elements (Penix et al., 1993, 1996; Cippitelli et al., 1995; Aune et al., 1996). These regulatory elements bind several nuclear factors. These elements include ATF/CREB motifs present in both the proximal and distal regulatory regions of the IFN promoter which bind ATF-2 and related bZIP transcription factors. Thus, it is possible that p38 MAP kinase-stimulated IFN gene transcription is mediated, in part, by regulatory phosphorylation of ATF2 or related proteins by p38 MAP kinase.

Several transcription factors have been described to function selectively in Th2 cells, and to be required for Th2 responses. STAT-6 is induced by IL-4 and the disruption of this gene results in an impairment of Th2 responses (Kaplan et al., 1996; Shimoda et al., 1996; Takeda et al., 1996). GATA-3 expression is maintained in Th2 cells and is eliminated in Th1 cells (Zheng and Flavell, 1997). c-Maf appears to be induced late in the differentiation of Th2 cells (Ho et al., 1996). Although NFAT and AP-1 complexes are detected in both Th1 and Th2 cells, these transcription factors mediate stimulus-dependent transcription only in Th2 cells (Rincón and Flavell, 1997b; Rincón et al., 1997b). An accumulation of JunB in the AP-1 complexes has been found in Th2 cells (Rincón et al., 1997b). Defects in NFATp cause decreased expression of Th2-related cytokines (Schuh et al., 1997). No intracellular signaling pathways triggered in Th2 cells have yet been identified.

In contrast to the progress that has been made towards understanding Th2-specific responses, little is known about the transcription factors and intracellular signals that control Th1 responses. STAT-4 is required for the initiation of Th1 responses (Kaplan et al., 1996; Thierfelder et al., 1996); however, it has not yet been established whether the IFN gene is regulated by STAT-4. More recent studies have demonstrated an important role of IRF-1 in Th1 responses through induction of IL-12 expression in macrophages (Lohoff et al., 1997; Taki et al., 1997). Here, we have shown that p38 MAP kinase is selectively activated in Th1 cells in response to both TcR-dependent and -independent stimuli, while significant induction was not observed in Th2 cells. These results indicate that not only transcription factors, but also upstream intracellular signaling pathways, are differentially regulated in Th1 and Th2 subsets. It is not clear at present whether p38 MAP kinase is selectively activated in Th1 cells or selectively repressed in Th2 cells. Th1-specific expression of specific p38 MAP kinase family members or upstream activators represents one potential mechanism. Although the mechanism of differential regulation in Th1 and Th2 cells is unclear, our analysis strongly implicates p38 MAP kinase signaling as an important check-point in the control of Th1 responses.

Our study shows, for the first time, that the p38 MAP kinase pathway in CD4⁺ T cells is not required for Th2 responses; however, p38 MAP kinase plays an important role in the regulation of Th1 responses. Since inhibition of p38 MAP kinase can diminish the progression of several models of arthritis, our results provide an additional mechanism that may contribute to the inflammatory effects of p38 MAP kinase signaling. Furthermore, our observations indicate a role for the p38 MAP kinase pathway in the maintenance of the balance between Th1 and Th2 responses in physiological and pathophysiological immune responses.

Materials and methods

Generation of the dn p38 and MKK6(Glu) transgenic mice

The dn p38 mutant was generated by replacing Thr¹⁸⁰ and Tyr¹⁸² by Ala and Phe, respectively (Raingeaud et al., 1995). The constitutively-activated MKK6 cDNA was generated by replacing Ser²⁰⁷ and Thr²¹¹ by Glu [MKK6(Glu); Raingeaud et al., 1996]. These cDNA were subcloned downstream of the distal lck promoter (Wildin et al., 1991). The DNA fragments containing the distal lck promoter, the dn p38 or MKK6(Glu) genes and the human growth hormone (hGH) intron and polyadenylation signals was injected into fertilized (C57BL6/C3H) F2 eggs and transgenic mice were generated as previously described (Hogan et al., 1986). Expression of the transgene was analyzed by slot blot using a BamHI–SacI 0.5 kb fragment from the hGH gene. Three founders were back-crossed into B10.BR (Jackson Laboratory, Bar Harbor, Maine) background to establish stable transgenic mouse lines.

Cell preparation and surface staining

Total CD4⁺ T cells were isolated from spleen and lymph nodes from wild-type or transgenic littermates by negative selection as previously described (Kamogawa et al., 1993; Rincón and Flavell, 1997b; Rincón et al., 1997a,b). Mitomycin C-treated (50 g/ml) syngeneic splenocytes were used as the source of APC.

Total or naïve CD4⁺ T cells were cultured at 10⁶ cells/ml in the presence of syngeneic APC (510⁵ cells/ml) with ConA (Boehringer Mannheim, Germany) or synthetic moth Cyt c peptide (Yale University) plus IL-12 (kindly provided by Genetics Institute) or IL-4 (kindly provided by DNAX). After 4 days, effector Th cells were exhaustively washed, counted and restimulated at 10⁶ cells/ml with ConA (in the absence of APC) or Cyt c peptide plus APC (510⁵ cells/ml).

The distribution of populations in the thymus and in lymph nodes was examined by cell surface staining and flow-cytometry (EPICS, Coulter). A red⁶¹³-conjugated anti-CD4 mAb and a fluorescein isothiocyanate (FITC)-conjugated anti-CD8 mAb (Pharmingen, San Diego, CA) were used.

The proliferative response was examined after 2 days by measurement of [³H]thymidine (Amersham Corp.) incorporation for 18 h.

RT–PCR

To determine the expression of transgenes, isolated cytoplasmic RNA (Tugores et al., 1992) was treated with RNase-free DNase I (Promega, Madison, WI) and used (5 g) to generate single strand cDNA by using Superscript RT (Gibco-BRL, Gaithersburg, MD). One-fifth of the reaction was used for PCR amplification of the dn p38 transgene cDNA and 1/25 was used to amplify -actin as a control in a total volume of 50 l. A primer located in dn p38 MAP kinase (5' primer, 5'-GCCCTTGCACATGCCTACTTTGC-3') and a second primer corresponding to the human growth hormone polyadenylation sequences (Brinster et al., 1988) of the transgene (3' primer, 5'-CAGAACCCCCAGACCTCCCTC-3') were used to analyze the expression of the dn p38 MAP kinase transgene. A -actin 5' primer (5'-CACCCTGTGCTCACCCGAGGCC-3') and 3' primer (5'-CCACACAGATGACTTGCGCTCAGG-3') were used to examine -actin expression by RT–PCR. Fifteen l/sample were examined by gel electrophoresis and detected by ethidium-bromide staining.

To examine cytokine gene expression, total RNA was extracted as described (Chomczynski and Sacchi, 1987) from 210⁶ cells. Briefly, after dilution (1:5) of reverse transcription reaction mixture, 1 l was assayed for levels of IFN or HPRT transcripts using the competitor (5 pg) in the presence of specific primers by competitive RT–PCR as described (Reiner et al., 1993). The percentage of inhibition by SB203580 was stimulated by the ratio of IFN:competitor transcript levels, as determined by densitometry.

Cytokine production

ELISAs were performed using purified anti-IL-4, anti-IL-5 and anti-IFN mAbs (3 g/ml) as primary antibodies and the corresponding biotinylated anti-IL-4, anti-IL-5 and anti-IFN mAbs (1 g/ml) (Pharmingen), horseradish peroxidase-conjugated avidin D (2.5 g/ml; Vector Laboratories, Burlingame, CA), the TMB microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD), following the recommended protocol (Pharmingen). Recombinant IL-4 (DNAX) and IL-5 and IFN (Gibco-BRL) were used as standards.

p38 and JNK kinase assays

T cells were lysed with buffer A (20 mM Tris pH 7.5, 10% glycerol, 1% Triton X-100, 0.137 M NaCl, 25 mM -glycerophosphate, 2 mM EDTA, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM Na pyrophosphate, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), as described (Dérijard et al., 1994; Rincón et al., 1997b). Endogenous p38 and JNK were immunoprecipitated using anti-p38 polyclonal antibody (Raingeaud et al., 1995) or anti-JNK mAb (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitates were washed twice with buffer A and twice with kinase buffer (25 mM HEPES pH 7.4, 25 mM -glycerophosphate, 25 mM MgCl₂, 0.5 mM dithiothreitol, 0.1 mM sodium orthovanadate). The protein kinase reactions were initiated by addition of 1 g of recombinant substrate proteins (GST–ATF2 for p38 and GST–Jun for JNK) and 50 M [-³²P]ATP (10 Ci/mmol). The reactions were terminated after 30 min at 30°C by addition of Laemmli sample buffer. Phosphorylation of the substrate proteins was examined after SDS–PAGE by autoradiography and PhosphorImager analysis (Molecular Dynamics Inc.).

IFN promoter activity

Jurkat T cells were transiently transfected with IFN (-538 bp) promoter luciferase reporter plasmid by electroporation as described (Penix et al., 1993, 1996). After transfection, the cells were stimulated with PMA and ionomycin for 18 h, harvested and lysed to examine luciferase activity as recommended by the manufacturer (Promega).

Acknowledgements

We thank C.Hughes and D.Buktus for generating the transgenic mice; Genetics Institute and DNAX for kindly providing the IL-12 and IL-4, respectively; S.Hedrick for providing the Cyt c transgenic mice; R.Perlmutter for the distal lck promoter construct; T.Barrett for technical help; C.Charland for flow-cytometry analysis; and F.Manzo for secretarial assistance. This work was supported in part by National Institute grant P01 AI36529 (R.A.F.), P30 HD27757 (L.P.), and R01 CA65861 (R.J.D.). R.A.F. and R.J.D. are Investigators of the Howard Hughes Medical Institute.

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