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Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520
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Introduction |
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phosphorylation, and a sustained calcium flux, while activation with an APL induces transient calcium signaling and differential TCR- chain phosphorylation, concomitant with the absence of ZAP-70 phosphorylation (11, 12, 13, 14, 15, 16). These early biochemical events turn on signaling cascades downstream that ultimately lead to transcription of cytokine genes. However, on the transcriptional level signals induced by APL do not simply result in a lower level of cytokine gene transcription, but rather in transcription of a different set of cytokine genes. Two major signaling pathways that are influenced by TCR signaling and converge at the level of cytokine gene transcription are the calcium-dependent signaling pathway and the protein kinase C/p21ras signaling pathway (17). TCR-induced calcium responses activate the phosphatase calcineurin that subsequently mediates dephosphorylation and nuclear translocation of NFAT (18). NFAT acts synergistically with the transcription factor AP-1 on DNA composite sites. Together, they regulate transcription of many cytokine genes including IL-4 (19, 20, 21, 22, 23). AP-1 is a dimer that consists of proteins from the Fos and Jun family, and its activity is regulated both at the level of gene transcription and by posttranslational modifications of its components (24, 25, 26). AP-1 is composed of either Fos-Jun heterodimers or Jun-Jun homodimers. Its composition may be important for cytokine gene transcription in that differential binding of Fos and Jun proteins has been observed for the IL-2 and IL-4 promoter (22). In addition, homodimers of JunB proteins have been found in effector Th2, but not Th1, T cells (27, 28). Gene transcription of proteins from the Jun family is regulated via the c-Jun N-terminal kinase (JNK) signaling cascade which influences Th differentiation. Mice deficient in JNK1 or JNK2 show preferential differentiation to the Th2 phenotype and/or diminished Th1 differentiation (29, 30, 31, 32).
Transcription of c-fos is regulated via the p21ras/extracellular signal-regulated kinase (Erk) signaling cascade (24). Upon TCR triggering, p21ras becomes activated and recruits raf-1 to the plasma membrane (33, 34, 35, 36). Raf-1 activates the dual specificity mitogen-activated protein (MAP) kinase kinases (MEK)1 and MEK2 (37), which are responsible for the subsequent activation of the MAP kinases, Erk1 and Erk2, by phosphorylation of Tyr and Thr residues (38). Activated Erk translocates to the nucleus to target the ternary complex factor Elk-1, which controls transcription of the c-fos protooncogene (39, 40). More recent studies have also shown a role for Erk in the regulation of expression and DNA binding activity of the Fos proteins, Fra-1 and Fra-2 (41, 42, 43, 44).
Importantly, Erk has been implicated in regulating IL-4 gene expression. A recent study demonstrated that Th2 differentiation was impaired in dominant-negative Ras transgenic T cells (45) and that the Ras/Erk pathway was required for Th2 differentiation. Furthermore, another report showed that IL-4 was decreased, although slightly, when Erk activation was inhibited (46). In contrast, inhibition of the MAP kinases, Erk1 and Erk2, with PD98059 was shown to induce elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 in human peripheral T cells (47). Together, these studies suggest a role of MAP kinases Erk1/2 in the regulation of IL-4 gene expression but the exact mechanism is yet unclear. Furthermore, most of these studies were done with either unfractionated human T cells (47) or murine effector CD8+ T cells (46, 48) and T cell clones (49), so little is known about the contribution of the Erk signaling pathway during Th differentiation in naive CD4+ T cells in response to peptide activation.
In the current study, we investigated the involvement of the Erk signaling pathway in the activation and differentiation of naive CD4+ T cells. Using CD4+ T cells from TCR transgenic mice, we demonstrate that a strong, Th1-inducing TCR signal (agonist peptide) induces sustained Erk activation, whereas a weak, Th2-inducing TCR signal (APL) induces transient Erk activation. To test whether the transient activation of Erk by the APL was important in the regulation of IL-4 gene transcription and Th2 differentiation, Erk activation during agonist peptide stimulation was inhibited. We show that inhibition of the Erk signaling pathway during agonist peptide priming induces Th2-type cytokines in naive CD4+ T cells. The transient activation of Erk correlates with altered AP-1 DNA binding activity and the formation of an altered composition of the AP-1 complex devoid of Fos protein. These events are associated with early IL-4 gene expression and Th2 differentiation.
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Materials and Methods |
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B10(A)5R mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our facility. AND TCR-
transgenic mice (TCR-specific for the C terminus of moth cytochrome c (MCC)) were derived as described (50) and maintained as heterozygotes on a B10(A)5R or B10.BR background in our animal facility. All mice used were 6- to 8-wk-old.
Preparation of CD4+ T cells and APC
CD4+ T cells were isolated from lymph nodes and spleens from TCR transgenic mice using immunomagnetic negative selection as previously described (51). Briefly, cell suspensions were incubated with Abs against CD8, B220, MHC class II, and FcR, followed by incubation with anti-mouse IgG, anti-mouse IgM, and anti-rat IgG-coated magnetic beads (Polysciences, Warrington, PA). Purity of the V11+ and CD4+ T cells was usually 85–90% as determined by staining with anti-CD4 and anti-V11 mAbs (BD PharMingen, San Diego, CA). In some experiments, the naive CD4+ cell population was further enriched by sorting for Pgp-1low and Mel-14high cells using the FACSVantageSE cell sorter (BD Biosciences, San Jose, CA). Equivalent results were obtained with or without FACS sorting. For the preparation of APCs, spleen cells from B10(A)5R mice were T cell-depleted by Ab-mediated complement lysis of splenocytes as previously described (51), using mAbs against Thy-1, CD4, and CD8. Before use, APC were treated with 50 µg/ml mitomycin C (Boehringer Mannheim, Indianapolis, IN).
Peptides and Abs
Unless otherwise indicated, all mAbs used were purified from supernatants from hybridomas maintained in this laboratory using standard protein A or protein G affinity chromatography. The wild-type peptide, MCC, was a 23-mer peptide coding for residues 81–103 (VFAGLKKANERADLIAYLKQATK) of MCC. Its mutant variant, K99R, had an arginine substitution for lysine at position 99 (VFAGLKKANERADLIAYLRQATK). The sequence of the BSA peptide (residue 141–154) was GKYLYEIARRHPYF. The peptides were synthesized at the W. M. Keck Biotechnology Resource Center (New Haven, CT) and were purified by high performance liquid chromatography before use.
In vitro stimulation of CD4+ T cells
Stimulation of naive CD4+ T cells was performed as described previously (52). Briefly, 5 x 105/ml CD4+ T cells were incubated with 106/ml APC, MCC (5 µg/ml), or K99R (10–15 µg/ml) peptide, 25 U/ml rIL-2 (Boehringer Mannheim), and anti-IFN-
Ab. After 4 days, viable cells were recovered via gradient centrifugation and rested for a 2-day period. Rested cells were restimulated at a final concentration of 5 x 105 cells/ml with 5 µg/ml MCC peptide plus fresh APC (106 cells/ml) for 2 days at which point culture supernatants were harvested and analyzed for the presence of IL-4 and IFN- using ELISA kits from Endogen (Cambridge, MA). For experiments involving PD98059, CD4+ T cells were pretreated with PD98059 (Sigma-Aldrich, St. Louis, MO) at the indicated concentration (5–50 µM) for 1 h at 37°C before activation with APC and peptide. PD98059 was then maintained in the cultures at the appropriate concentration for the duration of priming (i.e., the initial 4 days of culture) as the effect of PD98059 is reversed when the drug is removed from the cell culture. For analysis of cytokine production, cells were then rested and restimulated, in the absence of PD98059, as described above. Effector Th cells were prepared by culturing CD4+ T cells (5 x 105/ml) with MCC (5 µg/ml) and APC (106/ml) in the presence of IL-4 (10 ng/ml), anti-IFN-, and rIL-2 (25 U/ml) or in the presence of IL-12 (5 ng/ml) and rIL-2 (25 U/ml) for 4 days. Cells were rested for 2 days and restimulated for 2 days in the presence of APC (106/ml) and MCC (5 µg/ml).
Protein biochemistry and p21ras precipitation
For biochemical analysis, APC were peptide-pulsed with 100 µg/ml BSA, K99R, or MCC for 4 h at 37°C after which the APC were washed three times. CD4+ T cells (106) were activated with peptide-pulsed APC (106) for the times indicated and subsequently lysed in ice-cold lysis buffer (1% Nonidet P-40, 10% glycerol, 25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, and a mixture of protease inhibitors) for 30 min on ice. Cell nuclei were removed by a 15-min spin at 4°C and cell lysates were subjected to SDS-PAGE. For the extended time course (2 min-24 h), cells were harvested and lysed at the indicated time points. Protein concentrations were determined from lysates using the BCA Protein Assay kit (Pierce, Rockford, IL) and equal amounts of protein were subjected to SDS-PAGE. For experiments involving PD98059, CD4+ T cells were pretreated with 25 µM PD98059 for 1 h at 37°C before activation with peptide-pulsed APC. PD98059 was then maintained with the cells for the duration of activation.
After electrophoresis, proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Phosphorylated and total Erk were detected by blotting with anti-phosphoErk Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and total Erk Ab (Upstate Biotechnology, Lake Placid, NY), respectively, followed by rat anti-mouse IgG HRP conjugate and goat anti-rabbit IgG HRP conjugate, respectively (Bio-Rad). All immunoblots were developed with the ECL detection system as described by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). Active p21ras was precipitated using a p21ras Activation Assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. Briefly, CD4+ T cells were activated with peptide-pulsed APC as described above and lysed in ice-cold lysis buffer. Active p21ras was affinity-precipitated using raf-conjugated Sepharose beads that bind active p21ras. After 1 h, precipitates were washed and analyzed by SDS-PAGE and immunoblotting. In addition, 10% of whole cell lysate from each sample was run to assess the total amount of p21ras present.
Preparation of nuclear extracts
After 48 h of primary stimulation of CD4+ T cells, viable cells were recovered by gradient centrifugation. Effector Th cells were restimulated for 4 h with 100 µg/ml MCC-pulsed APC before harvesting for nuclear extract preparation. Cells were washed in ice-cold PBS and resuspended in 200 µl of buffer A (10 mM HEPES, pH 7.9, 3 mM MgCl2, 10 mM NaCl, 0.1 mM EDTA, 300 mM sucrose, 0.5 mM DTT plus a mixture of protease inhibitors). After 10 min on ice, cells were lysed by addition of Nonidet P-40 in a final concentration of 0.1%, and lysates were spun down for 1 min at 800 x g. Cell pellets were washed with 200 µl of buffer A and then resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9, 3 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT plus a mixture of protease inhibitors) for 15 min. The suspension was then briefly vortexed and spun down for 5 min at 14,000 rpm. Supernatants containing the nuclear fractions were stored at -70°C until use. Protein concentrations of the nuclear extracts were determined using the Pierce BCA Protein Assay kit.
EMSA
AP-1 DNA binding was analyzed by EMSA using 2 µg of protein from nuclear extracts that were incubated for 20 min at room temperature with 50,000 cpm 32P-endlabeled AP-1 oligonucleotide (Santa Cruz Biotechnology), 2 µg of poly(dIdC), and a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 1 mg/ml BSA, 3 mM GTP, 1 mM DTT. Following binding, the DNA-protein complexes were separated from free probe by electrophoresis in a nondenaturing 6% polyacrylamide gel (29:1 ratio) at 140 V for 3.5 h in 0.4x Tris-borate EDTA. To supershift AP-1 complexes, nuclear extracts were incubated for 20 min on ice with Abs, before incubation with the AP-1 probe. NFAT EMSAs were performed as described (53). Briefly, 3 µg of protein from nuclear extracts were incubated for 30 min at room temperature with 75,000 cpm 32P-endlabeled probe, 1 µg of poly(dIdC), and a buffer containing 10 mM HEPES, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10% glycerol, 0.1% Nonidet P-40, 0.5 mg/ml BSA, 1 mM benzamidine, 1 mM DTT. DNA-protein complexes were resolved on nondenaturing 5.5% polyacrylamide gels (37.5:1 ratio) in 0.5x Tris-borate EDTA for 2.5 h at 140 V. For detection of NFAT binding, a double-stranded oligonucleotide from the murine IL-4-promoter (-88 to -60) was used: 5'-CTGGTGTAATAAAATTTTCCAATGTAAAC-3'. Oligonucleotides were synthesized at the W. M. Keck Biotechnology Resource Center. For supershifting, nuclear extracts were incubated with NFAT Abs for 20 min on ice before incubation with the probe. The following Abs were used: anti-NFATc (clone 7A6), NFATp (clone 4G6-G5), anti-pan Fos (clone K-25), JunB (clone 11), FosB (clone 102), JunD (clone 329) (all from Santa Cruz Biotechnology). Gels were dried and exposed to autoradiography. Densitometric quantitation was performed using a digital imaging system (model IS-1000; Innotech, San Leandro, CA).
RNase protection assay (RPA)
CD4+ T cells were activated as described above and viable cells were harvested after 48 h of primary culture by gradient centrifugation. Total cellular RNA was isolated using TRIzol reagent (Life Technologies, Frederick, MD). Cytokine RNA levels were analyzed by RPA using the RiboQuant multiprobe kit with the mCK-1 template (BD PharMingen). Five micrograms of total RNA were used in each reaction. To ensure equal loading, cytokine transcript levels were normalized against the housekeeping gene, L32, by phosphoimaging analysis with a GS-525 Molecular Imager system and Molecular Analyst software (Bio-Rad).
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Results |
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We have previously shown that naive CD4+ T cells can be induced to differentiate into either Th1 or Th2 cells depending on the affinity of the peptide for the TCR (6, 7, 10). As seen in Fig. 1A, the high affinity peptide, MCC, induced a strong CD4+ T cell response leading to Th1 differentiation with IFN- and little IL-4 production, while the low affinity peptide, K99R, induced a weak response resulting in predominantly IL-4 production and Th2 differentiation. To investigate whether Erk is differentially activated upon stimulation, we analyzed the kinetics of Erk phosphorylation with MCC, K99R or an irrelevant peptide, BSA. As seen in Fig. 1B, both MCC and K99R peptides induce Erk activation, although the pattern is quite different. Whereas phospho-Erk in MCC-primed T cells is still present 30 min after activation, active Erk, induced by K99R, is transient and strongly diminished by 10 min. As a control, BSA clearly fails to elicit any Erk activity. Because TCR-induced Erk activation follows signaling via p21ras (34), analysis of p21ras exhibits a similar activation pattern as that observed for phospho-Erk (Fig. 1C). Given that Erk has been shown to regulate several transcription factors (24, 41, 42, 43, 44, 54), we next examined whether this pattern of Erk activation extended over a prolonged time course which might allow Erk to influence those factors important for IL-4 expression and Th2 differentiation. As shown in Fig. 1D, Erk activation with MCC peptide is sustained over at least 8 h. In contrast, the Erk activation pattern during K99R stimulation remains weak and transient. Together, these data show that a strong TCR response elicited by the high affinity peptide, MCC, correlates with sustained p21ras and Erk activation, whereas a weak TCR response by the APL, K99R, generates transient p21ras and Erk activation.
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Because transient Erk activity correlates with differentiation of Th2 cells, we questioned whether sustained Erk activity inhibits Th2 differentiation. PD98059 has been shown to specifically inhibit Erk activation by blocking activation of MEK, while leaving activation of other members of the MAP kinase pathway, JNK and p38, intact (55, 56). Our approach was to treat CD4+ T cells with a dose of PD98059 before stimulation with MCC peptide that would reduce Erk function. To ensure that this treatment resulted in reduced Erk activity, cell lysates of treated and untreated CD4+ T cells were analyzed by SDS-PAGE and immunoblotted for active Erk. In lysates of MCC-primed T cells, Erk activity was reduced significantly at a dose of 25 µM PD98059 and this inhibition was maintained over an extended period of time (Fig. 2A).
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production (data not shown). To determine when Erk influences IL-4 production, cells were treated with PD98059 at day 0, 1, or 2 of priming and the drug was kept in the culture until the rest period on day 4. Strikingly, as shown in Fig. 2D, IL-4 production is induced only when PD98059 is present from the initiation of priming and not when the drug is added later, suggesting that Erk inhibition early during priming is critical for the induction of IL-4. Moreover, the addition of PD98059 on different days had no significant effect on IFN- production (Fig. 2D). Interestingly, PD98059 treatment of cells before activation with the low affinity peptide, K99R, also increased IL-4, suggesting that Erk activity may not be necessary, and is possibly inhibitory, for IL-4 production (data not shown). Both MCC- and K99R-stimulated cells showed a 10- to 20-fold increase in IL-4 production when Erk was inhibited with PD98059. Thus, the reduced Erk activation in CD4+ T cells upon treatment with PD98059 induced IL-4 secretion in response to a high affinity peptide. Reduced Erk activation induces early IL-4 expression in naive CD4+ T cells
To exclude the possibility that contaminating memory or effector CD4+ T cells accounted for the increase in IL-4 during PD98059 treatment, CD4+ T cell populations were sorted for the naive T cell markers, Pgp-1low and Mel-14high. Naive, sorted CD4+ T cells are still capable of differentiation into IL-4-producing cells in the presence of PD98059, showing that contaminating effector cells are not responsible for the increase in IL-4 production (data not shown). Furthermore, we examined whether the increase in IL-4 production in the secondary culture by PD98059-treated CD4+ T cells was due to an increase in early IL-4 gene expression during Th2 differentiation. To examine this, total cellular mRNA was harvested from sorted, naive CD4+ T cells stimulated with peptide for 48 h and analyzed by RPA. As seen previously (57), MCC-primed CD4+ T cells express no IL-4 mRNA at 48 h. However, IL-4 gene expression is induced when Erk activity is inhibited by PD98059 (Fig. 3A). Interestingly, this level of IL-4 mRNA is very similar to the level of IL-4 message in K99R-primed CD4+ T cells (Fig. 3). Thus, decreased activation of the Erk signaling pathway correlates with early IL-4 gene expression in naive CD4+ T cells.
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A recent study had shown that Erk can inhibit NFATc nuclear translocation by phosphorylation of Ser residues that regulate subcellular localization (54). In addition, it was shown that PD98059 treatment was sufficient to trigger NFATc nuclear translocation. Furthermore, we showed recently that the level of nuclear NFATc and NFATp and the ratio of NFATc/NFATp DNA binding activity are critical parameters for IL-4 transcription in naive CD4+ T cells in that high levels of NFATc and low levels of NFATp promote transcription of IL-4 (57). Therefore, it was possible that reduced Erk activation would induce more nuclear accumulation of dephosphorylated NFATc forms resulting in an increase in the ratio of NFATc/NFATp DNA binding activity. Nuclear extracts of CD4+ T cells activated for 48 h with MCC in the presence or absence of PD98059 were analyzed by a gel-shift mobility assay. As shown in Fig. 4A, various doses of PD98059 do not alter the level of NFATc or NFATp DNA binding activity. Although at 25 µM PD98059, both NFATc and NFATp DNA binding are slightly decreased (Fig. 4B), the ratio NFATc/NFATp does not change (Fig. 4C). Thus, reducing Erk activation does not influence NFATc nuclear localization nor the NFATc/NFATp DNA binding ratio.
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Because reduced Erk activity did not change NFAT nuclear localization and because Erk is known to regulate c-fos transcription (24), a component of the transcription factor AP-1, we postulated that Erk activity influences AP-1 DNA binding activity. To investigate this, mobility shift assays were performed with nuclear extracts from CD4+ T cells that were stimulated for 48 h with MCC in the presence or absence of PD98059. During normal activation, a distinct band is observed representing the AP-1 DNA binding complex (Fig. 5A, lane 1). However, in the presence of PD98059 the AP-1 DNA binding activity is altered and a lower migrating AP-1 complex is induced (Fig. 5A, lane 6). According to Rincon et al. (27), this lower complex is composed of JunB homodimers and is present in effector Th2 cells. Analysis of the AP-1 complex using Abs to supershift the complex indicated that only the upper migrating band could be supershifted with pan Fos Abs (Fig. 5A, lanes 2 and 7). Furthermore, we were able to supershift the lower migrating band with JunB- and JunD-specific Abs. This suggests that the lower migrating complex is deficient of Fos proteins and consists of JunB and JunD dimers. Strikingly, this lower Jun-Jun complex is more highly expressed in nuclear extracts of K99R-primed CD4+ T cells (Fig. 5B, left panel) and, as expected, effector Th2 cells (Fig. 5B, right panel). Quantitation of the Fos-Jun and Jun-Jun dimers showed that there is both a decrease in Fos-Jun dimers and an increase of Jun-Jun dimers (Fig. 5C) resulting in a decrease of the ratio of Fos-Jun/Jun-Jun. More importantly, a low ratio of Fos-Jun/Jun-Jun correlated with the production of IL-4 (Fig. 5D). These data indicate that Erk activation alters AP-1 DNA binding activity and the formation of distinct AP-1 complexes, thereby influencing IL-4 gene expression and Th2 differentiation.
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Discussion |
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Several studies examining the role of Erk in cytokine gene expression and cytokine production have reported outcomes that seem contradictory to each other. Although some reported no effect on IL-4 production while inhibiting Erk activity (49), others reported a decrease (45, 46) or increase (47) in IL-4 production. One explanation of the different outcomes in these studies is the types of cells used in each of these experimental systems. However, in the present study we used naive CD4+ T cells expressing a transgenic TCR. This enabled us to examine TCR-induced Erk activation using peptides with differing affinities for the TCR, rather than activators that bypass TCR signaling such as PMA and ionomycin. We intervened in the downstream biochemical events during strong TCR signaling by inducing a transient Erk activation pattern using the MEK inhibitor PD98059. This was sufficient to induce IL-4 gene expression as early as 48 h after primary stimulation. A similar result has been reported with human T cells where PD98059 treatment correlated with an increase of IL-4 mRNA after activation with anti-CD3 and anti-CD28 Abs (47). These and our results seem to be in contrast with a study by Badou et al. (49) in which PD98059 had no effect on IL-4 production in long-term Th2 clones. It is likely, though, that once T cells have committed to a Th phenotype, some signaling pathways become dispensable or unresponsive, in this case the Erk signaling cascade. In fact, we also found that PD98059 treatment of effector Th2 cells had little or no effect on IL-4 production (data not shown). Moreover, we show that PD98059 only promotes IL-4 production when added within the first 24 h of priming naive CD4+ T cells. The significance of Erk signaling in the generation of Th2 cells is further explained by our observation that a weak and transient Erk activation pattern early during priming, by either PD98059 treatment or a low affinity TCR signal, is critical for the induction of IL-4. This time frame may be necessary to induce expression and/or to regulate activity of transcription factors which will ultimately lead to detectable levels of IL-4 mRNA by 48 h. Interestingly, PD98059 treatment of cells before activation with the low affinity peptide, K99R, also increased IL-4 (data not shown), suggesting that Erk activity may not be necessary, and is possibly inhibitory, for IL-4 production.
Unlike most other studies investigating the role of Erk activity in the context of cytokine production, we have taken an additional step by investigating which downstream transcription factors targeted by the Erk pathway account for the induction of IL-4 expression. Our previous study demonstrated that STAT6 signaling and GATA-3 up-regulation are not necessary for early IL-4 transcription during weak TCR signaling (57). This led us to ask whether changes in NFAT or AP-1 could explain the increased early IL-4 expression that we observed in the presence of PD98059. It had been shown that Erk is able to phosphorylate NFATc on critical Ser residues, thereby preventing its nuclear translocation and IL-4 transcription (54). We showed previously that elevated levels of nuclear NFATc relative to NFATp are necessary for early IL-4 transcription (57). Our gel shift assays show that NFATc DNA binding has not increased in the presence of PD98059 even though IL-4 expression has increased, suggesting that Erk activity has not affected NFATc translocation. This can potentially be explained by studies showing that all three kinases, Erk, p38, and JNK play redundant roles in negatively regulating NFATc nuclear localization (54, 58). Still, IL-4 expression is induced, an indication that other IL-4 transcription factors are sensitive to Erk activation. Because Erk is known to regulate a component of the AP-1 complex (24) and because NFAT proteins are known to cooperate with the AP-1 complex on DNA sites in the IL-4 promoter (22), the AP-1 complex was selected for further investigation.
AP-1 consists of either Fos-Jun heterodimers or Jun-Jun homodimers which can be regulated at the level of transcriptional or posttranslational modifications of the individual components. Analysis of AP-1 DNA binding activity in primary CD4+ T cells indicates that decreased Erk activation, either by a low affinity TCR signal or by PD98059 treatment with a strong TCR signal, induces a faster migrating AP-1 band in gel shift assays that is devoid of Fos proteins. Interestingly, this faster migrating band has been shown by Rincon et al. (27) to preferentially accumulate in Th2, and not Th1, cells. Moreover, this faster migrating AP-1 DNA binding complex was shown to consist primarily of JunB dimers which are thought to enhance AP-1 transcriptional activity in Th2 cells (28). Rooney et al. (22) also showed that the complex on the AP-1 binding site on the IL-4 promoter contains proteins from the Jun family including JunB, but not c-Fos.
Expression and DNA binding activity of Fos proteins are to a large extent regulated by the Erk signaling pathway (24, 25, 26). Our data imply that Erk plays an active negative role in regulating IL-4 expression by mediating the expression or the activation of Fos proteins. One target of activated Erk is the ternary complex factor Elk-1, whose transcriptional activity increases upon phosphorylation by Erk, leading to binding and activation of the c-fos promoter. Newly synthesized c-Fos combines with proteins from the Jun family to form the AP-1 complex (24). Erk has also been shown to induce expression of Fra-1 and Fra-2 (42, 44). Furthermore, Erk can directly phosphorylate both Fra-1 and Fra-2, thereby enhancing their DNA binding activity (41). In addition, a recent study showed that AP-1 transactivation required Erk-dependent activation of Fra-1 (43). Together, this raises the possibility that the transient levels of Erk activity are critical to maintain low levels of Fos proteins or low levels of Fra-1 and/or Fra-2 DNA binding activity. In both scenarios, an environment would be created that favors the formation of AP-1 complexes consisting of Jun-Jun dimers which is the preferred complex to cooperate with NFAT during IL-4 gene transcription. These possibilities are the subject of further investigation.
In summary, our results indicate that naive CD4+ T cells activated with high potency ligand can convert to IL-4-producing T cells by inhibiting TCR-induced Erk activation. This correlates with the induction of an AP-1 complex with altered composition which is devoid of Fos proteins and consists of Jun-Jun dimers. Our findings suggest that reducing Erk activity either by a weak TCR signal or by using the pharmacological inhibitor PD98059 during strong TCR signaling, alters the expression or activity of the Fos proteins in such a manner that an environment is created that favors the formation of Jun-Jun dimers. We propose that this complex is favorable to cooperate with NFAT during IL-4 transcription. In a physiological system, it is ultimately the potency of the TCR signal that directs the extent of Erk activation and subsequent IL-4 expression and Th2 differentiation.
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Acknowledgments |
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Footnotes |
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2 P.J.J. and J.L.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Kim Bottomly, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street LH408, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address: Kim.Bottomly{at}yale.edu
4 Abbreviations used in this paper: APL, altered peptide ligand; JNK, c-Jun N-terminal kinase; Erk, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP kinase kinase; MCC, moth cytochrome c peptide; RPA, RNase protection assay.
Received for publication June 17, 2002. Accepted for publication December 19, 2002.
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References |
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by T cells. Proc. Natl. Acad. Sci. USA 92:9510. and lack of ZAP-70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
Phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. production. Eur. J. Immunol. 31:2487.[Medline]
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X. Wang, L. Simeoni, J. A. Lindquist, J. Saez-Rodriguez, A. Ambach, E. D. Gilles, S. Kliche, and B. Schraven Dynamics of Proximal Signaling Events after TCR/CD8-Mediated Induction of Proliferation or Apoptosis in Mature CD8+ T Cells J. Immunol., May 15, 2008; 180(10): 6703 - 6712. [Abstract] [Full Text] [PDF]
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 B. Cassani, M. Mirolo, F. Cattaneo, U. Benninghoff, M. Hershfield, F. Carlucci, A. Tabucchi, C. Bordignon, M. G. Roncarolo, and A. Aiuti Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients Blood, April 15, 2008; 111(8): 4209 - 4219. [Abstract] [Full Text] [PDF]
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 S. E. Dunn, S. S. Ousman, R. A. Sobel, L. Zuniga, S. E. Baranzini, S. Youssef, A. Crowell, J. Loh, J. Oksenberg, and L. Steinman Peroxisome proliferator-activated receptor (PPAR){alpha} expression in T cells mediates gender differences in development of T cell-mediated autoimmunity J. Exp. Med., February 19, 2007; 204(2): 321 - 330. [Abstract] [Full Text] [PDF]
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F. D'Acquisto, A. Merghani, E. Lecona, G. Rosignoli, K. Raza, C. D. Buckley, R. J. Flower, and M. Perretti Annexin-1 modulates T-cell activation and differentiation Blood, February 1, 2007; 109(3): 1095 - 1102. [Abstract] [Full Text] [PDF]
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A. Agrawal, S. Dillon, T. L. Denning, and B. Pulendran ERK1-/- Mice Exhibit Th1 Cell Polarization and Increased Susceptibility to Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 2006; 176(10): 5788 - 5796. [Abstract] [Full Text] [PDF]
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 O. Kogkopoulou, E. Tzakos, G. Mavrothalassitis, C. T. Baldari, F. Paliogianni, H. A. Young, and G. Thyphronitis Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity J. Leukoc. Biol., May 1, 2006; 79(5): 1052 - 1060. [Abstract] [Full Text] [PDF]
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S. E. Dunn, S. Youssef, M. J. Goldstein, T. Prod'homme, M. S. Weber, S. S. Zamvil, and L. Steinman Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin J. Exp. Med., February 21, 2006; 203(2): 401 - 412. [Abstract] [Full Text] [PDF]
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L. Cannarile, F. Fallarino, M. Agostini, S. Cuzzocrea, E. Mazzon, C. Vacca, T. Genovese, G. Migliorati, E. Ayroldi, and C. Riccardi Increased GILZ expression in transgenic mice up-regulates Th-2 lymphokines Blood, February 1, 2006; 107(3): 1039 - 1047. [Abstract] [Full Text] [PDF]
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 J. Rubenfeld, J. Guo, N. Sookrung, R. Chen, W. Chaicumpa, V. Casolaro, Y. Zhao, V. Natarajan, and S. Georas Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L66 - L74. [Abstract] [Full Text] [PDF]
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 S. N. Georas, J. Guo, U. De Fanis, and V. Casolaro T-helper cell type-2 regulation in allergic disease Eur. Respir. J., December 1, 2005; 26(6): 1119 - 1137. [Abstract] [Full Text] [PDF]
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R. J. Salmond, G. Huyer, A. Kotsoni, L. Clements, and D. R. Alexander The src Homology 2 Domain-Containing Tyrosine Phosphatase 2 Regulates Primary T-Dependent Immune Responses and Th Cell Differentiation J. Immunol., November 15, 2005; 175(10): 6498 - 6508. [Abstract] [Full Text] [PDF]
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H. Yamane, J. Zhu, and W. E. Paul Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment J. Exp. Med., September 19, 2005; 202(6): 793 - 804. [Abstract] [Full Text] [PDF]
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T. Nekrasova, C. Shive, Y. Gao, K. Kawamura, R. Guardia, G. Landreth, and T. G. Forsthuber ERK1-Deficient Mice Show Normal T Cell Effector Function and Are Highly Susceptible to Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2005; 175(4): 2374 - 2380. [Abstract] [Full Text] [PDF]
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S. Sehra, D. Patel, S. Kusam, Z.-Y. Wang, C.-H. Chang, and A. L. Dent A Role for Caspases in Controlling IL-4 Expression in T Cells J. Immunol., March 15, 2005; 174(6): 3440 - 3446. [Abstract] [Full Text] [PDF]
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H. Inoue, R. Kato, S. Fukuyama, A. Nonami, K. Taniguchi, K. Matsumoto, T. Nakano, M. Tsuda, M. Matsumura, M. Kubo, et al. Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness J. Exp. Med., January 3, 2005; 201(1): 73 - 82. [Abstract] [Full Text] [PDF]
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 L. Sebkova, A. Pellicano, G. Monteleone, B. Grazioli, G. Guarnieri, M. Imeneo, F. Pallone, and F. Luzza Extracellular Signal-Regulated Protein Kinase Mediates Interleukin 17 (IL-17)-Induced IL-8 Secretion in Helicobacter pylori-Infected Human Gastric Epithelial Cells Infect. Immun., September 1, 2004; 72(9): 5019 - 5026. [Abstract] [Full Text] [PDF]
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D. Jankovic, M. C. Kullberg, P. Caspar, and A. Sher Parasite-Induced Th2 Polarization Is Associated with Down-Regulated Dendritic Cell Responsiveness to Th1 Stimuli and a Transient Delay in T Lymphocyte Cycling J. Immunol., August 15, 2004; 173(4): 2419 - 2427. [Abstract] [Full Text] [PDF]
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T. Luft, E. Maraskovsky, M. Schnurr, K. Knebel, M. Kirsch, M. Gorner, R. Skoda, A. D. Ho, P. Nawroth, and A. Bierhaus Tuning the volume of the immune response: strength and persistence of stimulation determine migration and cytokine secretion of dendritic cells Blood, August 15, 2004; 104(4): 1066 - 1074. [Abstract] [Full Text] [PDF]
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W. Duan, J. H. P. Chan, C. H. Wong, B. P. Leung, and W. S. F. Wong Anti-Inflammatory Effects of Mitogen-Activated Protein Kinase Kinase Inhibitor U0126 in an Asthma Mouse Model J. Immunol., June 1, 2004; 172(11): 7053 - 7059. [Abstract] [Full Text] [PDF]
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