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The Journal of Immunology, 2000, 165: 5597-5605.
Copyright © 2000 by The American Association of Immunologists

Cyclic AMP Activates p38 Mitogen-Activated Protein Kinase in Th2 Cells: Phosphorylation of GATA-3 and Stimulation of Th2 Cytokine Gene Expression1

Chang-Hung Chen, Dong-Hong Zhang, Jody M. LaPorte and Anuradha Ray2

Department of Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
cAMP is an important second messenger with immunomodulatory properties. Elevation of intracellular cAMP in T cells, induced by agents such as IL-1{alpha} or PGs, inhibits T cell activation. In effector T cells, an increase in the level of intracellular cAMP inhibits cytokine production in Th1 cells but stimulates cytokine production in Th2 cells. Here we report that cAMP-induced effects in Th2 cells occur independently of the protein kinase A pathway, which is the major mediator of cAMP-induced signaling events in most cell types. Instead, cAMP stimulates activation of p38 mitogen-activated protein kinase in Th2 cells. This appears to be a Th2-selective event because cAMP barely increased p38 phosphorylation in Th1 cells. We show that in Th2 cells, cAMP promotes the production of both IL-5 and IL-13, which play distinct but critical roles in asthma pathogenesis. Our data also show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which we have shown is a critical regulator of Th2 cytokine gene expression and, in turn, of airway inflammation in mice. Thus, Th2-specific GATA-3 expression and p38 mitogen-activated protein kinase activation together provide a molecular basis for the differential effects of cAMP in the two T helper cell subsets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Elevation of intracellular cAMP levels in T cells, induced by treatment with various agents such as IL-1{alpha}, PGE2, cholera toxin, forskolin or by direct stimulation of cAMP pathways with dibutyryl cAMP (bt2cAMP)3, inhibits IL-2 and IL-2 receptor expression thereby blocking cell cycle progression and cell proliferation (1, 2, 3, 4, 5, 6). In effector CD4+ T cells, elevation of intracellular cAMP is associated with inhibition of Th1 cytokine production but augmentation of IL-5 gene expression in Th2 cells (5, 7, 8). In addition, intracellular cAMP-elevating agents have been shown to inhibit IL-12 production (9).

Among the Th2 cytokines, IL-5 has been associated with eosinophilic inflammation in many disease states, including asthma, by promoting the proliferation, differentiation, activation, and survival of eosinophils (10, 11, 12, 13, 14). One unique feature of IL-5 gene expression is that cAMP-elevating agents such as IL-1{alpha}, forskolin, and PGE2 can strongly induce IL-5 gene expression (5, 8, 15, 16), suggesting a positive regulatory role for cAMP in IL-5 production. Interestingly, an analysis of the IL-5 promoter region reveals binding sites for several transcription factors including GATA-3, AP-1, Oct, Ets proteins, and NFAT (17, 18, 19) but no cAMP response element. This raises the possibility that signal transduction pathways other than the well described cAMP-dependent protein kinase A (PKA) pathway are involved in cAMP-mediated IL-5 gene expression in Th2 cells.

In recent years, considerable effort has been mounted to dissect the signaling events in T cells. Full activation of T cells requires at least two signals from TCR and costimulatory receptors (20, 21). Several protein kinases such as the family of mitogen-activated protein kinases (MAPKs) are involved in the transmission of extracellular signals to intracellular targets (22, 23, 24). TCR engagement activates the extracellular signal-related kinase (ERK) pathway, and costimulation through CD28 causes c-Jun N-terminal kinase (JNK) activation, which are required for full activation of T cells (25, 26). T lymphocyte activation by cAMP was shown to be associated with down-regulation of both ERK and JNK pathways (27). Interruption of the mitogen-activated protein/extracellular signal-related kinase kinase (MEK/ERK) pathway with the pharmacological ERK inhibitor PD98059 enhanced Th2 cytokine (IL-4, IL-5, and IL-13) expression induced by PMA/CD3 or by CD3/CD28 (28). The studies using JNK1-deficient mice also showed an enhanced Th2 response (29), suggesting that JNK1 may play a negative role in Th2 cytokine production. Collectively, these studies suggest that the kinases ERK and JNK do not play significant roles in Th2 cytokine gene expression.

In the past few years, several reports have established the importance of p38 MAPK in T cell activation and development. The MKK6-p38 signaling pathway was found to be critical for negative selection of thymocytes during T cell development (30). A specific p38 inhibitor, the pyridinyl imidazole SB203580, has been used to define the cellular functions of p38 in many cell types including Th1 and Th2 effector cells (31, 32, 33, 34, 35, 36). Several studies have investigated the role of p38 kinase using anti-CD3 and anti-CD28 Abs as stimuli of T cells. In murine T cells, while one study showed that blockade of p38 activity partially blocks TCR-stimulated IFN-{gamma} production by mouse Th1 cells without affecting Th2 cytokine (IL-4 and IL-5) production (33), another study observed reductions in IL-2, IL-4, and IFN-{gamma} production by inhibition of p38 activity (36). In human Th2 effector cells, SB203580 inhibited IL-4 production with lesser effects on IFN-{gamma} and IL-2 production in Th1 cells (35). The substrates of p38 include the transcription factors activating transcription factor (ATF)-2 (37, 38), CHOP (38, 39), and MEF2C (40) and the kinases MAPKAP-2 and -3 (41, 42). However, the physiological substrate(s) of p38 MAPK in T cells have not been identified.

In this study, we investigated the signaling events after stimulation of murine Th2 effector cells by cAMP. Our results show that cAMP-induced effects in Th2 cells are independent of the PKA pathway but instead are mediated by p38 MAPK. This appears to be a Th2-selective event because cAMP barely increased p38 kinase activity in Th1 cells. We show that cAMP not only promotes IL-5 production but also increases expression of the IL-13 gene in Th2 cells. In addition, we show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which, as we have shown previously, is a critical regulator of Th2 cytokine gene expression and of airway inflammation in vivo (43, 44). We also demonstrate phosphorylation of GATA-3 by active p38 MAPK. Taken together, the differential effect of cAMP on Th1 and Th2 cytokine production can be, at least partly, explained by differential induction of p38 MAPK in Th2 cells which, in turn, triggers phosphorylation of GATA-3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Nuclear run-on transcription analysis

Nuclei were isolated from 5 x 107 unstimulated or stimulated D10 cells, suspended in nuclear storage buffer (40% glycerol; 50 mM Tris-HCl, pH 8.0; 5 mM MgCl2; 0.1 mM EDTA), quick frozen in a dry ice-methanol bath and stored at -80°C until further use. Thawed nuclei were incubated for 30 min at 30°C in a buffer containing 20% glycerol; 20 mM Tris-HCl, pH 8.0; 100 mM KCl; 4.5 mM MgCl2; 2 mM DTT; 0.4 mM each of ATP, GTP, and CTP; and 100 µCi of [{alpha}-32P]UTP (3000 Ci/mmol; Amersham, Arlington Heights, IL). Transcription was stopped by the addition of 50 µl of termination buffer containing 50 mM Tris-HCl, pH 8.0; 0.5 M NaCl; 5 mM EDTA; 2 U DNase I; 40 U RNasin (Promega, Madison, WI); and incubating at 30°C for 15 min. Transcripts were isolated by Trizol LS (Life Technologies, Grand Island, NY) according to the instructions of the manufacturer. Unincorporated radioactivity was removed from the final RNA pellets using spin columns, and the incorporated radioactivity was counted by liquid scintillation. Equal counts of radiolabeled RNA were hybridized to cDNAs linearized with the appropriate restriction enzymes and immobilized as slots on a nylon membrane (Schleicher & Schuell, Keene, NH).

RNA isolation and Northern analysis

Total cellular RNA was prepared using Trizol (Life Technologies) according to the instructions of the manufacturer. Ten micrograms of total RNA from each sample was fractionated on a formaldehyde agarose gel and transferred to a nylon membrane. A ~450-bp SacI-AccI fragment of murine IL-5 cDNA was labeled with [{alpha}-32P]dCTP using a Random Primer DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed using QuikHyb (Stratagene, La Jolla, CA) according to the instructions of the manufacturer.

Analysis of MAPK activation

Rested Th2 cells (D10) were suspended in Bruff’s medium containing 0.5% FBS for 3 h to reduce background phosphorylation. Cells were incubated with or without H89 (Calbiochem, San Diego, CA) for 1 h before stimulating with cAMP or forskolin. Twenty minutes after stimulation, cells were washed once with cold PBS, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4; 250 mM NaCl; 2 mM EDTA; 50 mM NaF; 0.1 mM Na3VO4; 0.5% Nonidet P-40; and protease inhibitors), and incubated for 20 min on ice. The insoluble cell debris was pelleted by centrifugation at 4°C at 14,000 x g for 10 min. The protein concentration in cell lysates was determined (Bio-Rad, Hercules, CA). Equal amounts of total protein were suspended in SDS-PAGE sample buffer, fractionated on 4–20% gradient SDS polyacrylamide gels (Bio-Rad), and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). To analyze the activation of the individual MAPKs, p38, ERK, and JNK, the blots were blocked with 5% nonfat dry milk in TBST (25 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20) for 1 h at room temperature and subsequently incubated with rabbit anti-phospho-p38, anti-phospho-ERK, and anti-phospho-JNK Abs (New England Biolabs, Beverly, MA) followed by anti-rabbit IgG-HRP conjugate. Blots were then visualized by chemiluminescence (Pierce, Rockford, IL). To ensure similar protein loading in each lane, the phospho blots were stripped and the membranes were reprobed with Abs against p38, ERK, and JNK.

To examine kinase activation in Th1 and Th2 cells derived from primary CD4+ T cells, CD4+ T cells from DO11.10 TCR transgenic mice were prepared by positive selection using mAbs to CD4 coupled to magnetic and a magnetic-activated cell sorting (MACS) separation column- beads (Miltenyi Biotec, Auburn, CA). Syngeneic T cell-depleted splenocytes were used as APCs, which were prepared by depletion of CD4 and CD8 T cells using Ab-coupled magnetic beads, and the cells were subsequently treated with mitomycin C. Cultures were set up in flasks containing CD4+ T cells and T cell-depleted APCs together with pOVA323–339 (5 µg/ml), IL-2 (50 U/ml), anti-IL-4 and IL-12 (5 ng/ml) for Th1 polarization, and anti-IFN-{gamma} and IL-4 (200 U/ml) for Th2 polarization for 7 days. At the end of this period, cells were recovered and stimulated with bt2cAMP for 20 min. The phosphorylation of p38 MAPK was analyzed as described above.

Analysis of cytokine production

D10 cells (2 x 106) were cultured at 37°C for 1 h in the presence or absence of kinase inhibitor (SB203580). Cells were incubated with bt2cAMP and brefeldin A (1 µg/ml) for 4 h, and IL-5 production was evaluated by intracellular staining procedures. After washing twice in staining buffer, cells were fixed and permeabilized with Cytofix/Cytoperm solution for 20 min at 4°C. PE-anti-IL-5 was used to detect cytokine expression. Samples were then analyzed by FACS (Becton Dickinson, Mountain View, CA). The forward scatter and side scatter properties of the cells were used to exclude dead cells from the analysis.

Spleens were harvested from DO11.10 TCR transgenic mice, and CD4+ T cells were prepared by positive selection using mAbs to CD4 coupled to magnetic beads (MACS; Miltenyi Biotec) and a MACS separation column. Syngeneic T cell-depleted splenocytes were used as APCs, which were prepared by depletion of CD4 and CD8 T cells using Ab-coupled magnetic beads (MACS; Miltenyi Biotec), and the cells were subsequently treated with mitomycin C. Cultures were set up in flasks containing CD4+ T cells and T cell-depleted APCs together with pOVA323–339 (5 µg/ml), IL-2 (50 U/ml), anti-IFN-{gamma}, and IL-4 (200 U/ml) for 7 days. At the end of this period, cells were stimulated with PMA+bt2cAMP or PMA+ ionomycin for 48 h in the presence or absence of SB203580. Culture supernatants were assayed for the presence of IL-4, IL-5, or IL-13. The lower limits of detection for the cytokines were IL-4, 5 pg/ml; IL-5, 5 pg/ml (Endogen, Cambridge, MA); and IL-13, 1.5 pg/ml (R&D Systems, Minneapolis, MN).

Transfection assays

IL-5 and IL-4 reporter constructs have been described previously (43). For the (GATA)2-Luc construct, the GATA binding doublet present in the IL-5 -> promoter (CTATCTGATTG) was dimerized and linked to the minimal <- IL-5 promoter (5' border at -40) using BglII and BamHI introduced into PCR primers as cloning sites. Rested D10 cells were washed once and resuspended in serum-free RPMI 1640 medium, and transfections were conducted as described previously (43, 44). The cells were left on ice for 15 min and then diluted in 10 ml of Bruff’s medium at 37°C overnight. Cells were harvested 20 h poststimulation with PMA+bt2cAMP or PMA+ionomycin for reporter gene assays.

Cell labeling and immunoprecipitation

D10 cells were starved in phosphorus-free RPMI 1640 medium containing 5% dialysed FCS (Life Technologies) for 1 h. 32P-labeled orthophosphate (10 mCi/ml, ICN Pharmaceuticals, Costa Mesa, CA) was added to cells (1 mCi/10 x 106 cells) in the presence or absence of bt2cAMP, and the cells were incubated for 3 h. Cells were washed and lysed in lysis buffer on ice for 20 min. Cell extracts were harvested by centrifugation at 14,000 x g for 10 min to remove cell debris. GATA-3 protein was immunoprecipitated using agarose-conjugated monoclonal anti-GATA-3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) by incubation at 4°C overnight. The immunoprecipitates were washed three times in washing buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, 0.5% Nonidet P-40, and protease inhibitor). The samples were then boiled in 2x SDS sample buffer and fractionated by SDS-PAGE. The gel was stained by Coomassie blue before autoradiography.

Production of recombinant GATA-3 and in vitro kinase assay

An EcoRI/XbaI (tagged by PCR) fragment of murine GATA-3 cDNA was cloned into the insect cell expression vector pIZT/V5.His (Invitrogen, Carlsbad, CA). To express recombinant GATA-3 protein, Sf9 cells were transfected with the construct pIZT/G3.V5.His using the insectinPlus transfection kit (Invitrogen) according to the instructions of the manufacturer. Protein expression was monitored by GFP expression, and, at 60 h posttransfection, cells were lysed and GATA-3 fusion protein was purified using a nickel-chelated column. To perform in vitro kinase assay, 2 µg of GATA-3 fusion protein or 10 µg of rATF-2 (New England Biolabs) was incubated at 30°C for 30 min with active p38 MAPK (Stratagene) in the presence of 1 µCi of [{gamma}32P]ATP (>3000 Ci/mmol; NEN, Boston, MA). Following incubation, the phosphorylated GATA-3 fusion protein was immunoprecipitated with anti-GATA-3 Ab, the immunoprecipitates were fractionated by SDS-PAGE, and the gels were analyzed by a Phosphorimager (Cyclone; Packard, Meriden, CT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of IL-5 mRNA expression by cAMP is PKA independent in Th2 effector cells

To determine whether cAMP induces nascent transcription of the IL-5 gene, nuclear run-on assays were conducted. The mitogen Con A, which is a potent inducer of IL-5 gene expression, was used as a positive control. As shown in Fig. 1GoA, both Con A and bt2cAMP induced new transcription of the IL-5 gene in Th2 (D10) cells. Because PKA is the major mediator of cAMP-induced effects in many cell types, we first investigated its role in cAMP-induced IL-5 mRNA expression. As shown in Fig. 1GoB, cAMP-induced IL-5 gene expression (lane 2) was not inhibited in the presence of the PKA inhibitor H89 (lanes 3 and 4) or HA1004 (data not shown), suggesting that the PKA pathway is not critical for cAMP-mediated IL-5 production in Th2 effector cells. However, the PKA signaling pathway was previously implicated in IL-5 promoter activation in the murine thymoma line EL-4 (45). Expression of the catalytic subunit of PKA in EL-4 cells was shown to efficiently activate the IL-5 promoter in the presence of phorbol ester (45). As shown in Fig. 1GoB, the involvement of the PKA pathway in EL-4 cells was also evident in our experiments where PMA- and bt2cAMP-stimulated IL-5 mRNA expression was potently inhibited by H89. Thus, unlike in EL-4 cells, cAMP alone is sufficient to efficiently activate IL-5 gene expression in Th2 cells without dependence on the PKA signaling pathway.



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  		FIGURE 1. cAMP induces IL-5 gene expression in Th2 cells through a PKA-independent signaling pathway. A, Nuclear run-on analysis of IL-5 gene transcription induced by bt2cAMP. Nuclei were isolated from D10 cells that were either left untreated or incubated for 6 h with Con A (5 µg/ml) or bt2cAMP (1 mM). Nascent RNA molecules initiated in vivo were 32P-labeled by elongation in vitro in isolated nuclei. Equal counts of radiolabeled RNA were hybridized to cDNAs linearized with the appropriate restriction enzymes and immobilized as slots on nylon membrane (Schleicher & Schuell). Binding to the Bluescript plasmid (BSK; Stratagene) is illustrative of nonspecific binding, whereas the GAPDH cDNA was used as a control for equal RNA counts applied to each slot. The membrane was washed, and specific binding was evaluated by autoradiography. B, D10 cells were stimulated with bt2cAMP alone, whereas EL4 cells were stimulated with PMA and bt2cAMP for 6 h after preincubation with H89 (0.5 and 1 µM). Total RNA was isolated from cells and analyzed by Northern blotting techniques.

 
cAMP activates p38 MAPK in Th2 but not Th1 cells

The MAPKs, p38, JNK, and ERK, have been implicated in T cell activation. cAMP has been shown to inhibit JNK and ERK activation in T cell lines (Jurkat and EL4 cells) (27, 46), but no study has yet addressed the effect of cAMP on p38 MAPK activation. We investigated the effect of cAMP on the activation of MAPKs in D10 cells. As shown in Fig. 2GoA, a low level of basal phospho-p38 was detected in D10 cells, and stimulation with bt2cAMP enhanced p38 phosphorylation. However, bt2cAMP did not induce phosphorylation of either ERK or JNK. In fact, the phosphorylation of JNK decreased slightly after cAMP stimulation. Our initial examination of the kinetics of phosphorylation showed optimal p38 phosphorylation at 20 min poststimulation; therefore, this time point was chosen in all subsequent experiments. We did not detect ERK or JNK phosphorylation at any of the other time points examined (from 1 to 60 min of stimulation). These results indicated that in Th2 cells, cAMP preferentially activates p38 kinase but not ERK or JNK. Forskolin, which elevates intracellular cAMP by stimulating adenylate cyclase, also enhanced p38 phosphorylation in D10 cells (Fig. 2GoB). However, when cells were pretreated with H89, there was no effect on cAMP- or forskolin-induced p38 phosphorylation. If anything, H89 enhanced p38 phosphorylation in unstimulated cells. This suggests that the PKA pathway and the p38 MAPK signaling pathway are mutually antagonistic in Th2 cells such that inhibition of the PKA pathway raises the basal phosphorylation of p38. Taken together, these data suggested that cAMP induces p38 MAPK in Th2 cells in a PKA-independent fashion.



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  		FIGURE 2. p38 MAPK, but not ERK or JNK, is activated by cAMP in Th2 cells. A, D10 cells were incubated with bt2cAMP for 20 min at 37°C. Cell lysates were resolved by SDS-PAGE, and the phosphorylations of p38, ERK, and JNK were detected by probing with the specific anti-phospho-kinase Ab. The same membranes were reprobed with anti-kinase Ab. B, D10 cells were incubated with bt2cAMP (1 mM) or forskolin (10 µM) for 20 min at 37°C in the presence or absence of H89 (1 µM). Phospho-p38 was detected as in A. C, AE7 cells (Th1 cells) were stimulated with bt2cAMP in the presence or absence of H89 for 20 min at 37°C. Total cell lysates were analyzed as in A. D, Th1 and Th2 cells were generated by in vitro differentiation of CD4+ T cells isolated from DO11.10 mice. After 6 days, the primed CD4+ T cells were recovered and restimulated with bt2cAMP for 20 min. Total cell lysates were harvested and used for the analysis of phosphorylation of p38 MAPK as in A. Results shown are representative of three independent experiments.

 
To test whether this cAMP-induced p38 phosphorylation is Th2 specific, we tested the effect of cAMP on p38 phosphorylation in Th1 cells using the Th1 clone, A.E7. Addition of bt2cAMP only marginally augmented p38 phosphorylation in Th1 cells and, unlike in Th2 cells, H89 reduced phospho-p38 detected in the presence of cAMP alone (Fig. 2GoC). The results were essentially the same at other time points post stimulation. The Th2-specific activation of p38 MAPK was also evident in Th1 and Th2 cells generated by differentiation of primary CD4+ T cells (Fig. 2GoD). These results indicate that cAMP-induced p38 phosphorylation is a Th2-specific event and strongly suggest that cAMP-induced signaling pathways are distinct in Th1 and Th2 cells. Thus, the p38 MAPK pathway is the dominant cAMP-induced signaling pathway stimulated in Th2 but not in Th1 cells.

Inhibition of p38 MAPK inhibits cAMP-induced IL-5 and IL-13 production in Th2 cells

Having observed the Th2-specific activation of p38 kinase by cAMP, we next examined the role of this kinase in cAMP-induced IL-5 gene expression. D10 cells were stimulated with PMA and cAMP for 4 h, and IL-5 protein production in unstimulated and stimulated cells was estimated by intracellular staining. As shown in Fig. 3GoA, the majority of the unstimulated cells displayed a mean fluorescence intensity (MFI) of <35, whereas 64% of the cells had a MFI of >100 in cAMP-stimulated cells. However, in the presence of SB203580 IL-5 production was greatly reduced, with 21% of the cells displaying an MFI of 54 (Fig. 3GoA). Instead of bt2cAMP alone, PMA+ bt2cAMP was used in this experiment as well as in the following experiment (Fig. 4Go) because the combination is a more potent stimulus for IL-5 production. However, the overall effect of SB203580 was the same whether bt2cAMP or PMA+ bt2cAMP was used to induce IL-5 gene expression.



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  		FIGURE 3. Blockade of p38 activation by SB203580 inhibits IL-5 and IL-13 expression in Th2 cells. A, Analysis of IL-5 expression in D10 cells with or without SB203580 by intracellular cytokine staining. D10 cells were stimulated with PMA+bt2cAMP (for IL-5 expression) in the presence or absence of SB203580 (10 µM) for 4 h in the presence of brefeldin A to block cytokine secretion. PE-anti-IL-5 Ab was used to detect cytokine expression, and samples were analyzed by FACS. Comparable results were obtained in three independent experiments. B, Analysis of cytokine expression in bt2cAMP-stimulated Th2 cells generated by in vitro differentiation of CD4+ T cells isolated from DO11.10 mice in the presence or absence of SB203580. Splenic CD4+ T cells were isolated from DO11.10 mice and stimulated with the antigenic peptide pOVA323–339 and syngeneic T cell-depleted APCs under Th2-differentiating conditions. After 7 days, the primed CD4+ cells were restimulated with PMA+bt2cAMP with or without SB203580 (1 and 10 µM) or PMA+ionomycin with or without SB203580 (10 µM) for 48 h. Culture supernatants were harvested and assayed for IL-4, IL-5, and IL-13 expression by ELISA. Shown is a representative experiment of two.

 


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  		FIGURE 4. Inhibition of GATA-3-dependent promoter activities in D10 cells by SB203580. D10 cells were transfected with 30 µg of DNA (10 µg of reporter plasmid, 2 µg of RSV-{beta}-galactosidase (CMV-{beta}-gal) plasmid, and carrier plasmid). Cells were left unstimulated or stimulated with PMA+bt2cAMP (P+C) or PMA+ionomycin (P+I) in the presence or absence of SB203580 (1 and 10 µM) or PD98059 (10 and 50 µM). Cells were harvested after 20 h and assayed for luciferase and {beta}-galactosidase activity. The average absolute 100% luciferase activities when the IL-5 and the IL-4 promoters were stimulated with P+C or P+I were 13,820 and 22,200 relative light units, respectively. Results were normalized for {beta}-galactosidase activity. Shown is a representative experiment of two with <5% deviation between experiments.

 
It is now well established that the three cytokines, IL-4, IL-5, and IL-13, play critical and distinct roles in asthma pathogenesis. Therefore, we investigated the effect of cAMP on the production of all of these cytokines in Th2 cells obtained by differentiation of primary CD4+ T cells. In these experiments, CD4+ T cells were isolated from DO11.10 mice, which are transgenic for the TCR recognizing the OVA peptide 323-339 (pOVA323–339). Seven days after priming with Ag and APCs, the CD4+ T cells were stimulated with PMA and bt2cAMP in the presence or absence of SB203580, and culture supernatants were harvested 48 h later for analysis of cytokine production. As shown in Fig. 3GoB, PMA+bt2cAMP increased IL-5 and IL-13, but not IL-4, production in effector Th2 cells (DO11.10), and the increase in cAMP-induced IL-5 and IL-13 production was suppressed by SB203580 in a dose-dependent fashion. The inhibitor SB203580 did not affect basal expression of any of the cytokines in the cells. To show the specific effect of cAMP on IL-5 and IL-13 expression, the effector Th2 cells (DO11.10) were stimulated with PMA and ionomycin, an efficient inducer of IL-4 gene expression. As shown in Fig. 4GoB, IL-4 production was inhibited in the presence of SB203580, suggesting the involvement of p38 MAPK in calcium-induced IL-4 production. These data confirm that cAMP is a poor inducer of IL-4 but a strong inducer of IL-5 and suggest that full induction of IL-4 gene expression requires more than p38 MAPK activation. These results also show for the first time that cAMP can induce IL-13 production and provide evidence that p38 kinase is involved in cAMP-induced IL-5 and IL-13 production in effector Th2 cells.

Inhibition of GATA-3-dependent promoter activation in Th2 cells

To further examine the role of p38 in IL-4 and IL-5 gene expression, we tested the ability of SB203580 to inhibit IL-4 and IL-5 promoter activation in Th2 cells. As shown in Fig. 4GoA, the p38 MAPK inhibitor SB203580, but not the ERK inhibitor PD98059, resulted in a dose-dependent decrease in IL-5 promoter activity with a 50% decrease at the highest dose of the inhibitor (10 µM) (Fig. 4GoA). However, there was no inhibition of PMA+ionomycin-induced IL-4 promoter activity in the presence of either SB203580 or PD98059 (Fig. 4GoA). These results indicate that cAMP-induced p38 kinase is involved in IL-5 promoter activation probably through phosphorylation of specific transcription factor(s) activated by this kinase. It is to be noted that multiple isoforms of p38 kinase, {alpha}, {beta}, {gamma}, and {delta} have been described and the p38 kinase inhibitor SB203580 can only inhibit the {alpha} and {beta} forms of p38. Recently, CD4+ T cells were shown to preferentially express p38{alpha} and p38{delta} (47). Thus, the inability of SB203580 to completely inhibit IL-5 promoter activity may be due to residual cAMP-stimulated phospho-p38{delta} activity in D10 cells.

The selective inhibition of IL-5 promoter activity by SB203580 together with our previous studies demonstrating a direct activation of the IL-5 but not the IL-4 promoter by GATA-3 (48) raised the possibility that GATA-3 was the downstream substrate of the p38 signaling pathway activated by cAMP. To test this hypothesis, a (GATA)2-luciferase gene construct was transfected into D10 cells. Interestingly, (GATA)2-directed luciferase activity could be induced by stimulation with PMA+bt2cAMP (or bt2cAMP alone) but not with PMA+ionomycin (Fig. 4GoB), suggesting that the activation was mediated by a specific selective pathway induced by cAMP. Similar to its effects on the IL-5 promoter, SB203580 caused a dose-dependent decrease in (GATA)2-luciferase activity (Fig. 4GoB). As discussed earlier, the inability of the p38 inhibitor to fully repress reporter gene activation is probably because of residual activities of other p38 isoforms such as p38{delta} in the cells that may also be activated by cAMP. In contrast, the ERK inhibitor PD98059 did not affect (GATA)2-luciferase activity.

Phosphorylation of GATA-3 by activated p38

Inhibition of GATA-3-dependent promoter activity by the p38 kinase inhibitor indicated that p38 kinase may contribute to GATA-3 activation either directly or indirectly. Because there was no report on posttranslational modifications of GATA-3, we first investigated whether GATA-3 could be phosphorylated after cAMP stimulation of Th2 cells. D10 cells were metabolically labeled with inorganic 32P in the presence or absence of cAMP, and GATA-3 protein was recovered by immunoprecipitation. As shown in Fig. 5GoA, a phosphorylated protein of expected molecular size (55 kDa) was obtained using anti-GATA-3 but not control Ab. Although a basal level of GATA-3 phosphorylation was observed in Th2 cells, cAMP augmented the level of phosphorylation. These results suggested that GATA-3 exists as a phosphoprotein in Th2 cells, and cAMP signaling is able to increase GATA-3 phosphorylation.



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  		FIGURE 5. cAMP stimulates GATA-3 phosphorylation in Th2 cells. A, Phosphorylation of GATA-3 in intact cells. D10 cells were labeled with [32P]orthophosphate for 3 h in the presence or absence of bt2cAMP. Total cell lysates were prepared, and GATA-3 was immunoprecipitated using agarose-conjugated anti-GATA-3 mAb or control Ig. The immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. B, GATA-3 is phosphorylated by p38 in vitro. Ten micrograms of recombinant GATA-3 protein produced in insect cells (Sf9) or recombinant ATF-2 was incubated with an active form of p38 kinase (Stratagene) for 30 min at 30°C. GATA-3 was immunoprecipitated with anti-GATA-3 Ab. The phosphorylated proteins were resolved by SDS-PAGE and visualized using a phosphorimager. Shown is a representative of three independent experiments.

 
To address whether p38 MAPK can directly phosphorylate GATA-3, recombinant His-tagged GATA-3 was used as a substrate in an in vitro kinase assay using an active form of recombinant p38 kinase. Recombinant ATF-2 was used as a positive control in these assays. Because insect cells have high levels of His-rich proteins, the recombinant GATA-3 protein preparation had low levels of contaminant proteins. Therefore, GATA-3 was immunoprecipitated with anti-GATA-3 Ab after termination of the kinase reaction. As shown in Fig. 5GoB, phosphorylation of GATA-3 and ATF-2 occurred in a p38 kinase-dependent fashion; no phosphorylation of either protein was detected in the presence of only [{gamma}-32P]ATP. Also, no phosphorylated band was detected in the absence of any added substrate.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies published to date have shown that an increase in intracellular cAMP induces Th2 cytokine production but decreases Th1 cytokine production (5, 7, 8, 49). In these studies, we investigated the molecular basis for the increased production of Th2 cytokines in response to cAMP. Our studies summarized schematically in Fig. 6Go highlight the following points: 1) cAMP activates p38 MAPK in a dominant fashion in Th2 but not Th1 cells; and 2) GATA-3 is a substrate of p38 kinase, which explains, at least partly, the distinct effect of cAMP in Th2 cells.



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  		FIGURE 6. Schematic representation of the differential effects of cAMP in Th1 and Th2 cells. Elevation of intracellular cAMP elicited by different agents inhibits cytokine production in Th1 cells but stimulates IL-5 and IL-13 production in Th2 cells. cAMP-induced cytokine production in Th2 cells is independent of the PKA pathway but is dependent on the p38 MAPK pathway. X denotes a presently uncharacterized signaling intermediate. In contrast to Th2 cells, p38 kinase phosphorylation is barely augmented by cAMP in Th1 cells. Our study shows that p38 kinase can phosphorylate the Th2-specific factor GATA-3, which may explain the differential effects of cAMP in Th1 and Th2 cells.

 
The PKA pathway plays a major role in cAMP-induced responses in many cell types. Several isozymes of PKA have been described, and it has been shown that the type I isozyme is the major form present in T cells (50). Interestingly, the regulatory subunit of PKA, which is the intracellular receptor for cAMP, is deficient in T cells isolated from patients with systemic lupus erythematosus (51). This raises the possibility that the absence of PKA involvement in Th2 cells, as observed in our studies, is due to a down-modulation of PKA-I activity in these cells. Alternatively, the PKA and the p38 MAPK pathways may be antagonistic in T cells, and the balance of these pathways may be different in Th1 and Th2 cells. It is important to note that although the PKA pathway is not involved in cAMP-induced IL-5 gene expression in Th2 cells, it appears to regulate cAMP-induced IL-5 promoter activation in the thymoma line EL-4 (45). Expression of the catalytic subunit of PKA was shown to efficiently activate the IL-5 promoter in EL-4 cells in a PKA-dependent fashion (45). Thus, in EL-4 cells, the PKA pathway and, most likely, the phorbol ester-induced PKC pathway synergistically activate the IL-5 promoter. In contrast, in untransformed Th2 cells, cAMP alone can induce IL-5 gene expression in a PKA-independent fashion via the p38 MAPK pathway. In Th1 cells, the low level of cAMP-induced p38 phosphorylation was inhibited by the PKA inhibitor H89. It will be interesting to determine whether 1) the weak p38 phosphorylation in these cells is directly mediated by PKA or due to cross-talk with the p38 MAPK pathway; and 2) the PKA pathway is antagonistic to the p38 MAPK pathway.

A novel finding in our studies is the differential activation of p38 MAPK in Th2 and Th1 cells in response to cAMP (Fig. 6Go). p38 MAPK was first identified as a major tyrosine-phosphorylated protein of 38 kDa when murine macrophage cell lines were induced by LPS (52). Thus far, four mammalian isoforms have been described: p38{alpha}, p38{beta}, p38{gamma}, and p38{delta} (26, 31, 41, 52, 53, 54, 55, 56, 57, 58, 59, 60). Although p38 MAPK was first implicated in the production of proinflammation cytokines (e.g., TNF-{alpha} and IL-1{beta}) by monocytes/macrophages (31), the activation of p38 kinase has also been described in activated T cells in both human and murine systems. An interesting finding in these studies is that p38 is an important integrator of TCR/CD28 stimulation of cell proliferation and cytokine production in primary naive mouse CD4+ T cells (36). Thus, stimulation of naive primary CD4+ T cells through the TCR in the presence of costimulation was shown to activate this signaling pathway, and its blockade by SB203580 reduced the production of IL-2, IL-4, and IFN-{gamma} (36). In effector cells, although activation of p38 kinase was shown to be a critical requirement for Th1 (IFN-{gamma}) but not Th2 cytokine production in response to TCR/CD28 stimulation in murine cells (33), studies of human Th2 effector cells show a requirement for this kinase in IL-4 production in response to TCR/CD28 costimulation (35). This difference has been attributed to the dependence of IL-4 on CD28 costimulation in human but not murine effector Th2 cells (36). Our data demonstrate the importance of p38 kinase in cAMP-induced effects in Th2 cells.

The p38 inhibitor SB203580 blocks the activity of p38 by binding to the ATP-binding site on p38 MAPK (31). However, this inhibitor is specific for p38{alpha} and p38{beta} and does not inhibit p38{gamma} and p38{delta} (55, 56, 60, 61, 62). A recent study showed that both p38{alpha} and p38{delta} isoforms are highly expressed in T cells (47). Thus, the inability of SB203580 to completely inhibit reporter gene activity (Fig. 4Go) may be due to the fact that D10 cells express both isoforms, and both may contribute toward activation of GATA-3. However, the complete inhibition of IL-5 and IL-13 production by SB203580 in in vitro differentiated Th2 cells suggests that the major form of p38 in Th2 cells is p38{alpha}.

In previous studies, we and others demonstrated Th2-specific expression of the transcription factor GATA-3 (43, 63, 64) and showed a critical role for this transcription factor in IL-5 gene expression (19, 43, 64). Furthermore, we and others have shown that GATA-3 directly activates the IL-5 but not the IL-4 promoter (48, 65). It is important to note that GATA-3 is also important for IL-4 and IL-13 gene expression as we have recently demonstrated using transgenic mice expressing a dominant-negative mutant of GATA-3 (44). However, the mechanisms involved in GATA-3-induced IL-4 and IL-13 gene expression are not well understood. The transcription factor c-Maf has been shown to be critical for IL-4 gene expression and directly activates the IL-4 promoter (66, 67). However, c-Maf does not appear to directly regulate IL-5 or IL-13 gene expression (67). Thus, the differential responsiveness of the IL-4 and the IL-5/IL-13 genes to cAMP in effector Th2 cells may be due to the inability of cAMP to activate c-Maf. Also, although cAMP-induced p38 MAPK activation alone is not sufficient to cause IL-4 gene expression, calcium-induced IL-4 production involves p38 MAPK.

Six members of GATA family have been cloned, and GATA-1 and GATA-2 have been shown to exist as phosphoproteins (68, 69). By phosphorylation mapping, GATA-1 was shown to be phosphorylated on six serine residues in uninduced cells and, after stimulation, a seventh serine residue became phosphorylated (68). Similarly, the phosphorylation of GATA-2 was increased after IL-3 stimulation (69). Here we show that GATA-3 also exists as a phosphoprotein in unstimulated cells. Most importantly, we demonstrate that cAMP can induce GATA-3 phosphorylation via p38 MAPK and stimulate GATA-3-dependent promoter activity. It is unlikely that phosphorylation will affect the DNA-binding ability of GATA-3 because the GATA-3 protein recovered from unstimulated Th2 cells or in vitro translated GATA-3 are able to bind the IL-5 GATA doublet with high affinity. The p38 kinase-induced phosphorylation of GATA-3 may be important for its ability to interact with other regulatory factors to promote IL-5 gene expression or to make the IL-4/IL-13 locus more accessible to other factors. The optimum consensus sequence for MAPK has been identified as PPTP, and one such motif exists in the GATA-3 coding region. The retention of wild-type activity by the GATA-3 mutant T155A suggests that this site may not be the correct site for the response to cAMP (J. M. LaPorte and C.-H. Chen, unpublished observations). There are at least 12 potential phosphorylation sites ((S/T)P) for MAPK on GATA-3, and which of these sequences is important for phosphorylation by p38 kinase is currently under investigation. It is also possible that p38 MAPK phosphorylates an unconventional site in GATA-3. The signaling intermediate that couples intracellular cAMP to the p38 MAPK pathway in Th2 cells remains to be identified.


   Acknowledgments
 
We thank P. Ray for helpful discussions.


   Footnotes
 
1 This work was supported by grants HL60995, HL 56843, and P50-HL56389 (to A.R.) from the National Heart, Lung, and Blood Institute of the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Anuradha Ray, Department of Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, 333 Cedar Street, LCI 105, New Haven, CT 06520. Back

3 Abbreviations used in this paper: bt2cAMP, dibutyryl cAMP; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; ATF, activating transcription factor; MACS, magnetic-activated cell sorting; MFI, mean fluorescence intensity. Back

Received for publication March 10, 2000. Accepted for publication August 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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