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Originally published online as doi:10.1189/jlb.0705418 on February 14, 2006

Published online before print February 14, 2006
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(Journal of Leukocyte Biology. 2006;79:1052-1060.)
© 2006 by Society for Leukocyte Biology

Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity

Olga Kogkopoulou*, Evaggelos Tzakos*, George Mavrothalassitis{dagger}, Cosima T. Baldari{ddagger}, Fotini Paliogianni§, Howard A. Young and George Thyphronitis*,||,1

* Department of Pathophysiology, School of Medicine, University of Athens, Greece;
{dagger} School of Medicine, University of Crete and IMBB-FORTH, Voutes, Heraklion, Greece;
{ddagger} Department of Evolutionary Biology, University of Siena, Italy,
§ Department of Microbiology, School of Medicine, University of Patras, Greece;
Laboratory of Experimental Immunology, Center for Cancer Research, NCI-Frederick, Maryland; and
|| UPR CNRS 9045, Institut André Lwoff, Villejuif, France

1Correspondence: UPR CNRS 9045, Institut André Lwoff, 7 rue Guy Moquet, 94801 Villejuif, France. E-mail: gthyfron{at}vjf.cnrs.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p38 mitogen-activated protein kinase regulates many cellular processes in almost all eukaryotic cell types. In T cells, p38 was shown to regulate thymic development and cytokine production. Here, the role of p38 on interleukin-2 (IL-2) production by human peripheral blood CD4+ T cells was examined. When T cells were stimulated under weak stimulation conditions, pharmaceutical and molecular p38 inhibitors induced a dramatic increase of IL-2 production. In contrast, IL-2 levels were not affected significantly when strong stimulation was provided to T cells. The increase in IL-2 production, following p38 inhibition, was associated with a strong up-regulation of extracellular signal-regulated kinase (Erk)1/2 activity. Furthermore the Erk inhibitor U0126 was able to counteract the effect of p38 inhibition on IL-2 production, supporting the conclusion that p38 mediates its effect through Erk. These results suggest that the p38 kinase, through its ability to control Erk activation levels, acts as a gatekeeper, which prevents inappropriate IL-2 production. Also, the finding that p38 acts in a strength-of-stimulation-dependent way provides an explanation for previously reported, contradictory results regarding the role of this kinase in IL-2 expression.

Key Words: T lymphocytes • protein kinases • transcription factors • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-2 gene expression occurs through a complex cascade of molecular events, which are initiated following T cell receptor (TCR) and CD28 triggering. These molecular events induce the coordinated activation of Ca++, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways, which are involved in the activation of transcription factors important for IL-2 promoter activation such as nuclear factor (NF) of activated T cells, activated protein-1 (AP-1), cyclic adenosine monophosphate response element-binding protein, and NF-{kappa}B [1 ]. IL-2 plays a major role in T cell activation, proliferation, anergy induction, and apoptosis, as well as in the development of CD4+CD25+ regulatory T cells. Therefore, to keep T cell functions in check, IL-2 production should be controlled tightly during the different stages of an immune response. The contribution of TCR/CD28-triggered signaling cascades and transcription factors in the activation and expression of IL-2 has been studied intensely. However, important questions regarding the integration of these signaling cascades into networks that regulate the formation of transcriptional modules controlling IL-2 gene expression remain to be analyzed.

All MAPK [i.e., extracellular signal-regulated kinse (Erk), Jun N-terminal kinase (JNK), and the p38 MAPK] cascades play an important role in regulating IL-2 expression [2 , 3 ]. Increased activation of Erk or of its upstream activators up-regulates IL-2 expression, and the opposite effect was observed when Erk activity was inhibited [4 5 6 7 ]. Recent studies demonstrated that Erk mediates its activity on IL-2 through its main target, Elk-1, which up-regulates transcription of the AP-1 component c-Fos [8 ]. Also, Erk positively regulates the expression and the DNA-binding activity of two other AP-1 complex proteins, Fra-1 and Fra-2 [9 , 10 ], and the nuclear translocation of c-Rel, a member of the NF-{kappa}B family [7 ].

In contrast to Erk, contradictory results regarding the role of the JNK and p38 cascades on IL-2 expression have been reported. JNK phosphorylates c-Jun, a member of the AP-1 transcription factor family, which has been shown to be induced upon T cell stimulation and to be involved in IL-2 gene transcriptional activity. In accordance with these observations, early studies using Jurkat cells showed that JNK is involved in the regulation of IL-2 gene transcription and IL-2 mRNA stability [11 , 12 ]. However, experiments with JNK1–/–/JNK2–/– CD4+ T cells showed that they produce normal levels of IL-2 [13 ].

Experiments with Jurkat T cells suggested that p38 positively regulates IL-2 gene expression [11 ]. In accordance with these results, SB203580, a specific p38 inhibitor, was shown to inhibit IL-2 production by murine T cells [14 ]. In contrast, in a mouse model of sepsis in which IL-2 expression is suppressed, p38 inhibition restored normal levels of IL-2 production [15 ]. In addition, transgenic mice expressing a dominant-negative p38 (dnp38) transgene were not reported to demonstrate gross defects in IL-2 production [3 ], and studies with primary human T cells indicated that p38 inhibition has no or a minimal positive effect on IL-2 production [16 17 18 ]. In a recent report, we have also shown that p38 regulates IL-2 production in a stimulation-dependent manner [19 ].

Taken together, all these observations suggest that the p38 MAPK has a polyvalent role in IL-2 expression. In light of well-established evidence showing that the strength of the TCR/CD28 signal correlates with the activation of distinct signaling events, we have developed a model explaining the polyvalent, experimental, setting-dependent role of p38 on IL-2 expression. Our model incorporates the property of this kinase to interact with and to regulate the activity of other signaling pathways important for IL-2 production, which are activated quantitatively and/or qualitatively differently, depending on the strength of the triggering signal. To test this hypothesis, we used a combination of pharmacological and molecular approaches to characterize the role and the mechanisms through which p38 regulates IL-2 expression. Our data show that when Erk is weakly activated, which coincides with weak CD3/CD28 ligation, p38 inactivation stimulates strong Erk phosphorylation, which parallels the up-regulation of IL-2 production. Our results strongly suggest that the interaction between the p38 and Erk MAPK is of utmost importance for the regulation of IL-2 production: p38 acts as a gatekeeper, which controls the levels of Erk activation and subsequently IL-2 expression, and Erk activity sets the threshold for IL-2 expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
T cell isolation and culture conditions
The Laiko Hospital Blood Transfusion Center (Athens, Greece) provided buffy coats from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were separated on Ficoll-Paque (Pharmacia Biotech, UK). CD4+ T cells were isolated from mononuclear cells by negative selection using an appropriate mixture of mouse monoclonal antibodies (mAb) against CD21 (Clone 135), CD14 (Clone 63D3), CD11 (Clone OKM1), CD20 (Clone 9645), DR (Clone 145), CD8 (Clone OKT8), and CD56 (Clone 3G8), followed by magnetic separation using ferrous beads, Dynabeads M450, coated with sheep anti-mouse immunoglobulin G antibodies (Dynal, Oslo, Norway). Isolated CD4+ T cells contained <5% contaminating cells, as assessed by flow cytometry. E+ cells were isolated from PBMCs after rosetting with neuraminidase-treated sheep red blood cells [20 ].

Purified CD4+ T, E+, and Hut-78 cells were incubated in RPMI 1640, supplemented with sodium bicarbonate (0.3%), L-glutamine (0.3 mg/ml), gentamycin (0.1 mg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal calf serum (all from Invitrogen, Carlsbad, CA) at 37°C/5% CO2 overnight after isolation. The next day, T cells (2x106 cells/ml) and Hut-78 cells (2x105 cells/ml) were preincubated for 1 h at 37°C/5% CO2 with different concentrations of the p38 inhibitors SB203580, SB202190, or SKF86002, the ERK pathway inhibitor U0126, or the control imidazole compound SB202474 (Calbiochem, La Jolla, CA) at the same concentration as the inhibitors. Dimethyl sulfoxide (DMSO) was used as the diluent control at the same final concentration used for dissolving the inhibitors. The cells were then stimulated with plate-bound anti-CD3, OKT3 at 2 µg/ml, and soluble anti-CD28, 9.3, at 1 µg/ml (kindly provided by Dr. Carl June, University of Pennsylvania, Pittsburgh) or by cross-linking CD3 and CD28 using Dynabeads M450 coated with 0.6/0.3 µg anti-CD3/CD28/106 beads or with calcium ionophore A23187 at 200 ng/ml and phorbol 12-myristate 13-acetate (PMA) at 2 ng/ml (Sigma Chemical Co., St. Louis, MO) in 96-well culture plates (Corning, NY). All cultures were done in triplicates.

Enzyme-linked immunosorbent assay (ELISA) for cytokines
The levels of IL-2, IL-4, IL-10, IL-13, and interferon-{gamma} (IFN-{gamma}) in culture supernatants were determined by ELISA, using pairs of unlabeled and biotin-labeled, specific mAb, according to the manufacturer’s instructions (PharMingen, San Diego, CA).

Quantitative reverse transcriptase-polymerase chain reaction (PCR)
RNA extraction was performed using the RNAwiz solution (Ambion, Austin, TX), according to the manufacturer’s instructions. Total RNA was reverse-transcribed using 0.5 µg/µl oligo-dT (MWG Biotech, Germany), 0.5 mM deoxy-unspecified nucleoside 5'-triphosphates (dNTPs), 10 mM dithiothreitol (DTT), 40 U RNase inhibitor, 200 U Moloney murine leukemia virus, and 5 mM MgCl2 (all from Life Technologies, Gaithersburg, MD) at a total volume of 20 µl. Quantitative real-time PCR was performed in a LightCycler (Roche, Basel, Switzerland). All reactions were carried out in a total volume of 20 µl per capillary. Each reaction mixture contained 0.05 mg/ml bovine serum albumin, 0.5 nmol/ml each primer, 0.2 nmol/ml each dNTP, 0.5 U Taq DNA polymerase (Promega, Madison, WI), and 1x Taq PCR buffer. Syber Green I (Molecular Probes, Eugene, OR) was used at a 1/60,000 dilution. Reactions were performed on cDNA for IL-2 and 18S rRNA. Primers for IL-2 were sense (S): GTC ACA AAC AGT GCA CCT AC, antisense (AS): CCC TGG GTC TTA AGT GAA AG; and for 18S rRNA were S: GTT CCG ACC ATA AAC GAT GC, AS: AAC CAG ACA AAT CGC TCC AC. Standard curves were obtained by amplification of serial dilutions of IL-2 and 18S RNA standards. All results were normalized against 18S rRNA. After the amplification reaction, a melting curve analysis was done by using the LightCycler melting curve analysis software, demonstrating the presence of only one product in the reaction. The presence of a single product was verified further by gel electrophoresis.

RNase protection assay
The Multiprobe RNase protection assay was performed according to the manufacturer’s directions (PharMingen) with the following modifications. For hybridization, probes were synthesized with 33P uridine 5'-triphosphate (70–80 µC/full reaction) using the PharMingen in vitro transcription kit. Following incubation, yeast tRNA and EDTA were added as described by the manufacturer (PharMingen), the reaction was placed on Amersham-Pharmacia G25 Microspin columns, the probe was purified by centrifugation for 2 min at 3000 revolutions per minute (rpm), and 0.5–1.0 x 106 counts per minute were added to each RNA in a final hybridization volume of 10–20 µl (at least 50% PharMingen hybridization buffer). For RNase inactivation, a master cocktail, containing 200 µl Ambion RNase inactivation/precipitation reagent III (Ambion), 50 µl ethanol, 5 µg yeast tRNA, and 1 µl Ambion GycoBlue coprecipitate per RNA sample, was used to precipitate the protected RNA. After adding the individual RNase-treated samples to 250 µl inactivation/precipitation cocktail, the samples were mixed well, placed at –70°C for 15 min, and subjected to centrifugation at 14,000 rpm for 15 min in a room-temperature microcentrifuge. The supernatants were decanted, a sterile cotton swap was used to remove excess liquid, and the pellet was resuspended in 3 µl PharMingen sample buffer prior to gel electrophoresis and autoradiography.

Western blot analysis of MAPK phosphorylation
Cultured T cells or Hut-78 were harvested, placed on ice, and centrifuged at 1500 rpm. Pellets were resuspended in lysis buffer, containing 150 mM NaCl, 1% Igepal CA-630, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 50 mM Tris-(hydroxymethyl)-aminomethan (pH 8), 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (all purchased from Sigma Chemical Co.). Samples were electrophoresed in 10% Tris-glycine polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes by electroblotting. The membranes were reacted with rabbit polyclonal antibodies specific for p38 or Erk or their phosphorylated forms (all from New England Biolabs, Beverly, MA). The reaction was visualized by chemiluminescence and exposure to X-ray film.

In-gel kinase assay
For the in-gel MAPK assay, ERKs were immunoprecipitated with an anti-ERK rabbit polyclonal antibody (New England Biolabs) and analyzed in a 10% polyacrylamide gel containing 0.1 mg/ml glutathione S-transferase-ethylene-responsive transcriptional factor protein, which is a high-affinity target for ERK [21 ]. At the end of the electrophoresis, the gel was washed at room temperature, 2 x 30 min with 20% propanol, 50 mM Tris, pH 8, 2 x 30 min with 50 mM Tris, pH 8, 5 mM 2-mercaptoethanol (2-ME), 2 x 30 min with 6 M guanidine-HCl, and 16 h at 4°C with three changes of 50 mM Tris, pH 8, 5 mM 2-ME, 0.04% Tween 20. The kinase reaction was performed for 1 h at 25°C in 40 mM HEPES, pH 8, 2 mM DTT, 5 mM MgCl2, 0.1 mM EGTA, and 10 µCi/ml [{gamma}-32P]adenosine 5'-triphosphate. The gel was washed 10 x 15 min with 5% trichloroacetic acid and 1% sodium pyrophosphate, dried, and exposed to X-ray film.

Cell transfections, cell sorting, and luciferase (luc) assay
Expression vectors for wild-type and dnp38, containing mutations at threonine 180 and tyrosine 182 to alanine and phenylalanine, respectively, were provided by Dr. Helmut Holtmann (Medical School Hanover, Germany) [22 ]. The IL-2 promoter (–326 to +45) luciferase construct IL-2/luc was a kind gift of Dr. Michael Lenardo (National Institutes of Health, Bethesda, MD) via Dr. Dimetrios Boumpas (University of Crete, Greece) [23 ]. The enhanced green fluorescent protein (EGFP) expression vector was purchased from Clontech (Palo Alto, CA). Hut-78 T cells were transfected by electroporation using a gene pulser (Bio-Rad, Hercules, CA) at 220V/950 µF. For transfections with IL-2/luc, 10 µg plasmid was used. For contransfections with dnp38 and EGFP, 10 µg and 3 µg of the two constructs were used, respectively. After transfections, cells were rested for 24 h and then stimulated as described above or sorted using the FACSDiva cell sorter (BD Biosciences, San Jose, CA) to ~90% purity. Sorted cells were rested overnight and then stimulated. For luciferase assays, cells were harvested and washed twice with cold phosphate-buffered saline, and luciferase activity was determined with a LabSystem luminometer using the Bright-Glo luciferase assay system (Promega) following the manufacturer’s directions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased IL-2 production and IL-2 gene promoter activation are induced following p38 inhibition in CD4+ cells
Purified CD4+ cells were stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml) in the presence or absence of the specific p38 inhibitor SB203580. This treatment dramatically increased IL-2 production, and in accordance with previous studies, IL-4, IL-5, IL-10, and IL-13 productions were decreased (Fig. 1A ). The effect of SB203580 on these cytokines was consistent in four independent experiments, and IFN-{gamma} production was variably affected (data not shown). Similar results were obtained with two more p38 inhibitors, SB202190 and SKF86002 (data not shown). RNase protection analysis of RNA extracted 6 or 24 h after initiation of cultures also demonstrated an increase in steady-state IL-2 mRNA levels in the presence of SB203580, in contrast to IL-5 and IL-13 mRNA levels, which diminished (Fig. 1B and 1C) . To examine whether costimulation was necessary for up-regulation of IL-2 production following p38 inactivation, CD4+ T cells were treated with different doses of SB203580 and then stimulated with plate-bound anti-CD3 (2 µg/ml) in the presence or absence of soluble anti-CD28 (1 µg/ml). Similar to costimulation (Fig. 2A ), all doses of SB203580 from 0.5 to 5 µM up-regulated IL-2 production (Fig. 2B) . Also, SB203580 up-regulated IL-2 mRNA expression in anti-CD3-stimulated cells at 6 and 24 h time-points and had the opposite effect on IL-5 and IL-13 mRNA (Fig. 1B and 1C) . Increased IL-2 production, following p38 inhibition, was observed in all of more than 10 independent experiments and ranged from two- to tenfold in the anti-CD3/CD28 and from eight- to 60-fold in the anti-CD3-stimulated cultures. IL-2 production, in the absence of p38 inhibitors, ranged from 0.25 to 8.0 ng/ml and from 25 to 123 pg/ml, respectively. It is important to note that in the anti-CD3-stimulated cultures, increase in IL-2 production was far more dramatic in comparison with T cell cultures costimulated with anti-CD28.


Figure 1
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  		Figure 1. p38 inhibition selectively up-regulates IL-2 production. Purified CD4+ T cells were preincubated for 1 h with SB203580 at 5 µM or DMSO or SB202474 as controls. Cells were then stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml). All cultures were done in triplicates. (A) Cytokine production was assayed by ELISA on Day 2 of culture. One representative experiment out of four from different donors is shown. Differences in cytokine production among the triplicates were less than 10%. Results are presented as percent of cytokine production in SB203580-treated in comparison with SB202474-treated cultures. In this experiment, cytokine production in SB202474-treated cultures was IL-2, 280 pg/ml; IL-4, 38 pg/ml; IL-5, 420 pg/ml; IL-10, 1350 pg/ml; and IL-13, 2250 pg/ml, and was considered as 100% (dotted line). (B) Cells were treated as indicated and then left unstimulated (Lane 1) or stimulated with plate-bound anti-CD3 (Lanes 2–4) or as in A (Lanes 5–7). RNA was extracted 6 h (left panel) or 24 h (right panel) after initiation of cultures and assessed by the RNase protection assay for the expression of cytokine mRNA. The L32 ribosomal gene product was used as control for equal loading and for normalization of the results. NS, Nontreated, nonstimulated cell cultures. (C) Densitometry analysis of 24 h IL-5, IL-13, and IL-2 mRNA gel blot shown in B. Results are presented as percent of cytokine mRNA in SB203580-treated in comparison with control SB202474-treated cultures considered as 100% (dotted line). (A and C) Tables below express cytokines or mRNA increase or decrease shown in the respective histograms as percent of control.

 

Figure 2
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  		Figure 2. Dose-response effect of SB203580 on IL-2 production. Purified CD4+ T cells were preincubated for 1 h with different concentrations of SB203580 or with equivalent amounts of SB202474 as a control and then stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml; A) or plate-bound anti-CD3 only (B). IL-2 production was assayed after 48 h of culture by ELISA. The data are representative of one out of more than 10 independent experiments from different donors. All cultures were done in triplicates. Differences in IL-2 production among the triplicates were 5–10%. IL-2 production by SB202474-treated cells was comparable with that of nontreated cells. The numbers on the top of the bars show the IL-2 concentration in culture supernatant.

 
We have previously shown that the p38 inhibitors up-regulated IL-2 production in the Hut-78 T cell line [19 ]. Therefore, to further confirm that the inhibitors mediated their effect through p38, Hut-78 cells were cotransfected with GFP and a dnp38, and then transfected cells were sorted and stimulated with plate-bound anti-CD3 (2 µg/ml) plus soluble anti-CD28 (1 µg/ml). Green fluorescent cells produced higher amounts of IL-2 in comparison with sorted, nonfluorescent cells or nonsorted cells (Fig. 3A ). In addition, when GFP/dnp38-positive cell cultures were treated with SB203580, no increase in IL-2 production was observed, further confirming that SB203580 acts specifically by inactivating the endogenous p38 (Fig. 3B) . Taken together, these experiments show that p38 inactivation with pharmacological or molecular inhibitors has a profound, stimulating effect on IL-2 production.


Figure 3
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  		Figure 3. Increased IL-2 production by dnp38-transfected Hut-78 cells, which were transiently transfected with expression vectors encoding EGFP (3 µg) together with dnp38 (10 µg). Twenty-four hours after transfection, green fluorescent cells were separated from nonfluorescent cells by fluorescein-activated cell sorter and were then stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml; A) in the presence or absence of 5 µM SB203580 (B). One representative experiment out of three is displayed. One hundred percent is calculated as the IL-2 production by GFP/dnp38-negative cells stimulated in the absence of the p38 inhibitor.

 
In light of previous reports demonstrating that IL-2 expression is regulated at the transcriptional and at the mRNA stability level, experiments were designed to determine whether p38 inactivation up-regulates IL-2 expression through transcriptional or post-transcriptional mechanisms. Hut-78 cells were transfected with a reporter plasmid containing the minimal IL-2 promoter driving the expression of the luciferase gene or the corresponding control vector. As shown in Figure 4A , IL-2 promoter activity was up-regulated significantly in the presence of SB203580. In contrast, p38 inhibition did not affect IL-2 mRNA stability (Fig. 4B) . Therefore, we concluded that p38 mediates its effects on IL-2 expression by acting at the transcriptional level.


Figure 4
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  		Figure 4. p38 inactivation stimulates IL-2 gene promoter activity but does not affect IL-2 mRNA stability. (A) Hut-78 cells were transfected with 10 µg IL-2/luc. Twenty-four hours later, cells were left untreated or treated for 1 h with 5 µM SB203580 or SB202474 as a control and then stimulated or not with plate-bound anti-CD3 (2 µg/ml) plus soluble anti-CD28 (1 µg/ml). Luciferase activity was determined after 16 h of culture (mean±SD, n=3). (B) CD4+ T lymphocytes were left untreated or treated with SB203580 for 1 h and then stimulated as in A. Six hours after initiation of the cultures, actinomycin D (5 µg/ml) was added. Cells were collected at Time 0 and every 30 min thereafter. RNA was extracted and analyzed by real-time PCR in triplicates for IL-2 mRNA. Results were normalized against 18 s rRNA. One representative experiment out of two is shown.

 
The strength-of-stimulation conditions determines the effect of p38 inhibition on IL-2 production
Next, we examined the effects of SB203580 treatment when CD4+ T cells were cultured under different stimulation conditions by cross-linking CD3 and CD28 using bead-bound antibodies or with PMA plus ionomycin. SB203580 treatment had a limited effect on IL-2 production in these cultures (Fig. 5A ). This observation was confirmed in four independent experiments, in which SB203580 treatment had a minimal (±20%) effect on IL-2 production. In the same experiments, a three- to sevenfold increase of IL-2 was observed in plate-bound anti-CD3 plus soluble anti-CD28-stimulated cultures, which were treated with SB203580 in comparison with nontreated cells. It is important to note that CD3/CD28 cross-linking or PMA plus ionomycin stimulation induced IL-2 production, which was two orders of magnitude higher in comparison with that observed in plate-bound anti-CD3 plus soluble anti-CD28-stimulated cultures (Fig. 5A) . This observation indicated that p38 inhibition exerts its stimulating effect on IL-2 production, only when suboptimal stimulation is provided to T cells. This premise was supported further by experiments in which CD4+ cells were stimulated with plate-bound anti-CD3 and increasing amounts of soluble anti-CD28. In these experiments, the stimulating effect of SB203580 on IL-2 production was inversely proportional to the amount of the anti-CD28 antibody (Fig. 5B) . Thus, although with 0.03 µg/ml anti-CD28, IL-2 production increased by 59-fold in the presence of the SB203580 inhibitor, at a concentration of 3 µg/ml, no increase was observed. Together, these results suggest that the effect of p38 inhibition on IL-2 production is strength-of-stimulation-dependent.


Figure 5
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  		Figure 5. Stimulation-dependent effect of p38 inhibition on IL-2 production. (A) Purified CD4+ T cells were stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml), PMA plus calcium ionophore (P+I), or by cross-linking anti-CD3 and anti-CD28 (crs CD3/CD28) with antibody-coated beads. (B) Alternatively, cells were pretreated with 5 µM SB203580 or SB202474 as a control and then stimulated with plate-bound anti-CD3 (2 µg/ml) plus different doses of soluble anti-CD28 as indicated. IL-2 production was assayed on Day 2 of culture by ELISA. The numbers on the top of the bars in B show fold increase of IL-2 in SB203580-treated cultures as compared with SB202474-treated cells. IL-2 production by SB202474-treated cells was comparable with that of nontreated cells. All cultures were done in triplicates. Differences in IL-2 production among the triplicates were 5–10%. Representative results from one out of four (A) or three (B) experiments are displayed.

 
Increased IL-2 production upon p38 inactivation correlates with increased Erk1/2 activity
To understand the mechanisms implicated in the distinct outcomes of p38 inactivation on IL-2, p38 phosphorylation was compared in CD4+ cell cultures stimulated with plate-bound anti-CD3 (2 µg/ml) plus soluble anti-CD28 (1 µg/ml) bead-bound antibodies or PMA plus ionomycin. Phospho-p38 levels in these three stimulation protocols revealed no obvious differences, which would explain the distinct effects of p38 inhibition on IL-2 production (data not shown and ref. [19 ]). In addition, p38 was equally sensitive to SB203580 inhibition in all three protocols (data not shown). As p38 phosphorylation and the effect of the inhibitor were independent of the stimulation protocol, a likely possibility was that the inhibitors interfere, directly or indirectly, with the activity of some other signaling molecule(s), which contribute to IL-2 expression and whose activity is stimulus-dependent. Erk was a likely candidate, as increased Erk activity, following p38 inactivation in nonimmune cells, was reported in several studies [24 25 26 ] and as activated Erk regulates IL-2 gene expression in a positive way [4 , 6 , 27 ]. Analysis of the Erk phosphorylation status by immunoblotting revealed strong Erk phosphorylation after PMA plus ionomycin or bead-bound anti-CD3/CD28 stimulation (Fig. 6A , middle and bottom panels). In contrast, after stimulation with plate-bound anti-CD3 (2 µg/ml) plus soluble anti-CD28 (1 µg/ml), a small, transient increase in Erk phosphorylation was detected in primary T cells (Fig. 6A , top panel and data not shown).


Figure 6
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  		Figure 6. Erk activity is up-regulated in SB203580-treated T lymphocytes. E+ T cells were stimulated as indicated and then lysed on ice. The lysates were electrophoresed, transferred to nitrocellulose membranes, and immunoblotted with antiphospho-Erk antibody or with an anti-Erk antibody to determine total Erk, as indicated. (A) Kinetics of phospo-Erk expression after stimulation with plate bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml; top panel) or by cross-linking anti-CD3 and anti-CD28 with antibody-coated beads (middle panel) or with PMA plus calcium ionophore (bottom panel). E+ T cells were from the same donor in all stimulation protocols. (B) Phospho-ERK levels in E+ T cells stimulated for 15 min by cross-linking CD3 and CD28 (left panel) or with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml) in the presence or absence of SB203580 and/or U0126 or SB202474 as control. (C) Kinase assay for Erk in Hut-78 cells stimulated for 15 min by cross-linking CD3 and CD28 (left panel) or with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of SB203580 and/or U0126. An aliquot of each lysate was analyzed by immunoblotting (Wb) for total Erk (lower panel).

 
Next, the effect of p38 inhibition on Erk phosphorylation was examined. Treatment of plate-bound anti-CD3 plus soluble anti-CD28-stimulated T cells with SB203580 induced Erk phosphorylation at levels similar to those observed in the cross-linking protocol (Fig. 6B) . Further increase in Erk phosphorylation also occurred in PMA plus ionomycin or bead-bound anti-CD3/CD28-stimulated cultures after p38 inactivation (Fig. 6B and data not shown). Addition of U0126, which blocks Erk activation by inhibiting its upstream activators MAPK kinase 1 (MEK1) and MEK2 [28 ], strongly reduced the intensity of the phospho-Erk-specific bands (Fig. 6B) , demonstrating the specificity of the reaction. Kinase assay analyzed in Hut-78 cells further confirmed that SB203580 treatment of T cells increased Erk activity (Fig. 6C) . Therefore, we conclude that in peripheral blood T cells and similar to nonimmune cells [29 , 30 ], p38 inactivation exerts a stimulatory effect on Erk activation. To directly assess the role of Erk in regulating IL-2 production following SB203580 treatment, CD4+ T cells were treated with or without SB203580 or with SB203580 plus U0126 and stimulated with plate-bound anti-CD3 (2 µg/ml) plus soluble anti-CD28 (1 µg/ml). In fact, U0126 was able to reverse the effect of SB203580 on IL-2 in a dose-dependent manner (Fig. 7A ). In addition, the degree of IL-2 inhibition paralleled the levels of the remaining phosphorylated Erk (Fig. 7B) , as determined by Western blot analysis of cellular extracts. Together, these results demonstrate that the up-regulation of IL-2 in the SB203580-treated cultures was a result, at least in part, of the induction of Erk activity.


Figure 7
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  		Figure 7. The Erk inhibitor U0126 antagonizes the effect of SB203580 on IL-2 production. (A) Purified CD4+ T cells were treated for 1 h with 5 µM SB202474 as a control (–) or with 5 µM SB203580 alone or in the presence of U0126 at different concentrations as indicated. Cells were then stimulated with plate-bound anti-CD3 (2 µg/ml) and soluble anti-CD28 (1 µg/ml), and IL-2 production was assayed on Day 2 of culture. One representative experiment out of two from different donors is shown. (B) Total cell lysates were extracted from parallel cultures and analyzed by immunoblotting for phospho-Erk (upper panel). Membranes were stripped and probed against total Erk as a loading control (lower panel). Densitometry units are shown as percent of nontreated anti-CD3 plus anti-CD28-stimulated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that the potency of the TCR/CD28 ligation results in quantitative and qualitative differences in signal transduction and in subsequent cytokine gene expression [31 ]. Here, we present data consistent with a polyvalent role of p38 on IL-2 gene expression and production; p38 plays a negative role in IL-2 production, mainly when low-strength activation signals are provided to T cells through the TCR/CD3 and the CD28 molecules, and it has a limited role under strong stimulation conditions. Several concordant findings in our study strongly support the conclusion that the effects of p38 on IL-2 are mediated through its ability to regulate the activity of the Erk MAPK. First, as we have previously shown, the distinct outcomes of p38 inhibition on IL-2 production were not associated with differences in the level of its activation or of its sensitivity to SB203580 inhibition, as under weak or strong CD3/CD28 ligation, p38 was activated equally and inhibited consistently by SB203580. In contrast to p38, Erk activation was sensitive to the potency of the stimulating signal, and the level of its activation correlated well with the amount of IL-2 that was produced. In addition, PMA plus ionomycin stimulation, which bypasses the membrane signals, triggered strong Erk activation (mostly the p42 isoform) and also induced high IL-2 production. It is most important that when weak CD3/CD28 stimulation was provided, Erk activity was increased significantly in SB203580-treated CD4+ T cells, and this increase paralleled SB203580-induced IL-2 up-regulation. Finally, the Erk activation inhibitor, U0126, antagonized SB203580 by reducing IL-2 production, demonstrating that the effect of the p38 inhibitor was mediated through its ability to up-regulate Erk activity.

At least two nonexclusive possibilities may explain the strength-of-stimulation-conditioned effect of p38 on IL-2. First, under weak stimulation conditions, p38 represses Erk activation below a threshold necessary for full IL-2 gene activation. Release of the inhibitory effect of p38, using its specific inhibitor, induces increased Erk activation, ultimately leading to increased IL-2 gene activation. In contrast, under stimulation conditions in which initial levels of Erk activity are above that threshold, p38 inactivation and the subsequent increase in Erk activity would not affect IL-2 gene expression. Second, under low TCR/CD28 occupancy, Erk is activated by signaling pathways sensitive to p38 inhibition, whereas under high receptor occupancy, Erk may be activated by additional signaling pathways insensitive to p38 inhibition. In fact, the degree of receptor occupancy seems to play an important role in Erk activation and in the ability of upstream effector molecules to activate Erk in nonimmune cells. For example, in Swiss 3T3 cells, at low platelet-derived growth factor receptor occupancy, an active phosphatidylinositol-3 kinase (PI-3K) pathway is required for Erk activation, and under high receptor occupancy, Erk activation dissociates from the PI-3K pathway, as additional signaling pathways (e.g., the PKC pathway) are activated [32 ].

In accordance with our findings, recent studies demonstrated that the level, duration, and kinetics of Erk activation are dependent on the strength of TCR ligation and the T cell activation state. According to these studies, Erk seems to act as an integrator of the TCR-emanating signal to control important T cell functions, including c-fos expression and T helper cell type 1 (Th1)/Th2 differentiation [7 , 33 , 34 ]. In nonimmune cells, such as fibroblasts and the PC12 cell line, the duration of Erk activation also seems to be a key factor, which controls important biological functions including proliferation and differentiation [35 , 36 ]. In our culture conditions, we observed that SB203580 treatment induced sustained Erk activation, detectable at least 4 h after stimulation (O. Kogkopoulou, G. Thyphronitis, unpublished observations). Whether this is important for the observed up-regulation of IL-2 production is presently under evaluation. It should be noted that although this is the first report showing that interaction between p38 and Erk regulates important functions in T cells, in several previous studies, this interaction has been implicated in directing important biological outcomes in diverse cell types, including myoblast differentiation, low-density lipoprotein expression in hepatic cells, and apoptosis [29 , 30 , 37 , 38 ]. Yet, it is not clear whether p38 interacts directly with Erk or acts indirectly through activation of protein phosphatase 1 and 2A to inhibit the upstream activators of Erk, MEK1, and MEK2 [38 , 39 ].

Overall, the multiple effects, which p38 exerts on IL-2, may be explained by the possibility that this kinase has the potential to participate in different signaling networks and to regulate the activation of different transcription modules involved in IL-2 gene activation. Thus, quantitative differences in the level of activation of the transcriptional modules and in their composition, depending on the strength of the activation signals, may determine the importance of p38 and its role in IL-2 gene activation.

Overall, our results imply that Erk is a rate-limiting factor for IL-2 gene expression, and p38 acts as a gatekeeper, which restricts IL-2 production under conditions of weak TCR/CD28 ligation as a result of its ability to inhibit Erk activation. The demonstration that the effect of p38 inactivation on IL-2 production is stimulus-dependent provides an explanation and a molecular basis for the previously reported, apparently contradictory results. The p38-Erk interaction in T cells may be of paramount importance for preventing inopportune IL-2 production and subsequent T cell activation. Also, the observation that in T cells treated with SB203580 and stimulated with anti-CD3, IL-2 production increased at levels close to those obtained after anti-CD3 plus anti-CD28 stimulation indicates that the p38 activity may be important for anergy induction. Further studies in vivo and in an antigen-specific setting are needed to fully evaluate the significance of these findings.


   ACKNOWLEDGEMENTS
 
This work was supported by the Hellenic Secretariat for Research and Technology, programs EPET II and PENED 01ED486. O. K. and E. T. contributed equally to the work. We thank Drs. H. Holtmann, M. Lenardo, D. Boumpas, and C. June for providing us with plasmids and antibodies, Drs. H. Moutsopoulos and O. Georgopoulou for helpful discussions, and Z. Mishal for expert cell-sorting.

Received July 28, 2005; revised December 19, 2005; accepted December 23, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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