Molecular mechanisms of T lymphocyte activation: convergence of T cell antigen receptor and IL-1 receptor-induced signaling at the level of IL-2 gene transcription

Georg Varga, Ursula Dreikhausen, Michael Kracht, Annette Appel, Klaus Resch and Marta Szamel

Institute of Molecular Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany

Correspondence to: M. Szamel


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Co-stimulation of murine EL-4 thymoma cells—carrying high numbers of TCR and type I IL-1 receptors (IL-1R)—with anti-CD3 antibodies and IL-1 resulted in synergistic enhancement of IL-2 synthesis. While the extracellular signal-regulated kinase (ERK) cascade was activated by both receptors, IL-1 preferentially stimulated Jun-N-terminal kinases (JNK) and p38 mitogen-activated kinase or microtubule-associated protein kinase (MAPK). Interruption of TCR- or IL-1R-stimulated ERK cascade by PD-98059, a specific inhibitor of MAP/ERK kinase (MEK), resulted in partial suppression of nuclear factor of activated T cells activation and in complete inhibition of IL-1-stimulated NF{kappa}B activation. Suppression of activation of both MEK and p38 MAPK resulted in significant inhibition of IL-2 gene expression. The results show that maximal activation of the IL-2 gene requires activation of at least two different protein kinase cascades, i.e. of the ERK and p38 pathways but presumably also that of JNK which converge at the level of the IL-2 promoter resulting in enhancement of its transcriptional activity.

Keywords: IL-2, protein kinases, signal transduction, T lymphocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activation of T lymphocytes is initiated by the interaction of the TCR–CD3 and its cognate antigen. Stimulation of TCR alone is, however, insufficient to induce proliferation and synthesis of the ultimate mitogen, IL-2, rather causing a state of clonal anergy (reviewed in 1,2). In combination with additional signals—triggered by either accessory molecules or cytokines—T cells become optimally activated (35). Besides the requirement for `accessory signals' via co-receptors, like CD2, CD4/8, CD5 and CD28, the cytokine IL-1 has been shown to synergize with mitogenic lectins or anti-TCR antibodies to induce IL-2 synthesis and cellular proliferation (reviewed in 6). Whether the IL-1 signal plays an enhancing or obligatory role in T cell activation is discussed controversially. More importantly, the molecular mechanisms underlying the co-stimulatory activity of IL-1 still remain unclear.

Both the TCR and IL-1 regulate the expression of the IL-2 gene at the transcriptional level. Several signal transduction cascades have been implied to be involved in the control of the IL-2 enhancer (68). Stimulation of the TCR results in rapid activation of the Ras–Raf– mitogen-activated kinase or microtubule-associated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)–ERK kinase cascade (9). However, activation of the `classical' MAPK cascade appears to be a necessary but not a sufficient signaling pathway for induction of lymphokine, particularly IL-2 synthesis in stimulated T lymphocytes (1013). This is consistent with the finding that T cell activation requires co-stimulatory signals in addition to those activated via the TCR–CD3 complex (14). How these distinct stimuli act synergistically to cause T cell activation has not been fully elucidated.

Besides the best characterized `classical' MAPK subfamily, including ERK-1 and ERK-2, two additional groups of MAPK have been identified recently, i.e. Jun-N-terminal kinases (JNK), also termed stress-activated kinases (SAPK), and p38 MAPK (reviewed in 14).

While it is well established that the `classical' MAPK pathway regulates important TCR induced functions, much less is known about implications of the JNK and p38 MAPK cascades in the course of T cell activation (15). The latter two groups of MAPK (i.e. JNK and p38 MAPK) are effectively activated by IL-1 in several non-lymphoid cells and cell lines (1619). The activation and function of JNK/p38 MAPK is less well characterized in T lymphocytes.

Activation of MAPK pathways results in phosphorylation of transcription factors, thereby transducing signals from cell surface receptors to the nucleus. Some transcription factors are substrates for all three groups of MAPK, whereas others are preferential substrates of distinct groups of protein kinases, like the N-terminal activation domain c-Jun, which is phosphorylated by JNK/SAPK (14). Simultaneous stimulation of distinct MAPK cascades via two different cell surface receptors might therefore amplify a signal by converging on the same effector molecules (e.g. transcription factors) or elicit an enhanced cellular response if distinct effector molecules are activated.

To get insight into the molecular events being responsible for IL-1-induced co-stimulatory activity in T cells, activation of distinct MAPK cascades as well as their involvement in regulation of IL-2 gene transcription have been investigated in the murine thymoma cell line EL4, expressing high numbers of functionally active TCR and IL-1 receptors (IL-1R). Results of this study clearly show that co-stimulation of the TCR and type I IL-1R synergistically enhances IL-2 synthesis. While both receptors activate the `classical' MAPK cascade, IL-1 preferentially activates JNK and p38 MAPK.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hamster anti-mouse CD3{varepsilon} antibody was purified from hybridoma (145-2C11) supernatants by affinity chromatography on protein agarose. Phycoerythrin (PE)-conjugated goat anti-mouse CD3 antibody and rat anti-mouse IL-1R antibody (35F5) were from PharMingen (Hamburg, Germany). Human recombinant (hr)-IL-1ß was from Pharma Biotechnologie (Hannover, Germany). Anti-MEK-1 antibody was purchased from Transduction Laboratories (Lexington, KY). Recombinant ERK-1 was from New England Biolabs (Boston, MA) , hr-ATF-2 and the polyclonal antibody against JNK-1 from Santa Cruz Biotechnology (Santa Cruz, CA), and hr-glutathione-S-transferase (GST)–c-Jun from Alexis (Grünburg, Germany). The anti-p38 MAPK antibody (20) was a kind gift of Dr J. Saklatvala (London, UK) and the mAb raised against the phosphorylated form of p38 MAPK was from New England Biolabs. MAPK activated protein kinase (MAPKAPK)-2 was a kind gift of Dr M. Gaestel (Berlin, Germany). PD-98059 (MEK-1 inhibitor) was from Biomol (Hamburg, Germany). SB-203580 (p38 MAPK inhibitor) was from Alexis. IL-2-specific ELISA was from Genzyme (Cambridge, MA). All other chemicals were from Sigma (St Louis, MO).

Cells and cell culture
The EL-4 subline was a kind gift of Dr R. McDonald (Lausanne, Switzerland). The cells were cultured in DMEM containing 10% FCS, 50 µM ß-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin at a cell density of 105–106 cells/ml. Prior to treatment cells were serum starved for 12–14 h.

Before activation cells were pre-treated with 50 µM PD-98059 (MEK-1 inhibitor) (21), 10 µM SB-203580 (inhibitor of p38 MAPK) (22) or their combination for 90 min. Cells were then stimulated with 2.5 µg/ml anti-CD3 antibody, 2.5 ng/ml IL-1ß or their combination for the times indicated in the experiments.

Isolation of splenic lymphocytes
Spleens from BALB/c mice (6–8 weeks old) were minced and gently smashed in a loose-fitting glass homogenizer for cell release. The cell suspension was filtered through nylon wool and washed 3 times with RPMI medium. Cells were incubated at a density of 5x106 /ml in RPMI 1640 containing 20% FCS, 50 µM ß-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin in plastic flasks overnight to deplete the majority of adherent cells. Before stimulation cells were washed 3 times with RPMI medium then incubated with 2.5 µg/ml anti-CD3 antibody, 2.5 ng/ml IL-1ß or 0.5 M sorbitol for the times indicated in the experiments.

FACS analysis
EL-4 cells (106) were seeded in 96-well microtiter plates, centrifuged and washed 2 times with PBS/1% FCS. Cells were then resuspended in 1% pentaglobin and incubated at room temperature for 5 min. Then 1 µg antibody raised against the IL-1R or 2 µg PE-conjugated anti-CD3 antibody was added for 15 min at room temperature. After repeated washes in PBS/FCS, cells treated with antibodies against IL-1R were incubated with 7.5 µg/ml FITC-conjugated secondary antibody for 15 min, washed twice and all samples resuspended in PBS/FCS. Flow cytometric analysis was carried out with a FACScan (Becton Dickinson, Heidelberg, Germany).

Determination of protein kinase activities
Measurement of ERK phosphorylation by MEK-1.
Samples of 107 cells were washed twice with ice-cold PBS, and resuspended in a buffer containing 20 mM Tris–HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 10 mM NaF, 1 mM DTT, 10 mM ß-glycerophosphate, 1 mM PMSF, 1 mM Na3VO4, 400 nM okadaic acid and 10 µg/ml leupeptin. Cells were sonified at 40 W for 3x10 s. After centrifugation (45 min, 100,000 g, 4°C) the protein concentration of supernatants was determined using the Bradford assay (BioRad, Hercules, CA).

Prior to incubation with a specific antibody preclearing was carried out with Protein A–Sepharose for 1 h. Then 1 mg of cytosolic protein was precipitated with 300 ng of a MEK-1 specific antibody bound to Protein A–Sepharose. Immunoprecipitates were subjected to an in vitro kinase assay using 2 µCi radioactively labeled ATP (300 Ci/mmol; Hartmann, Braunschweig, Germany) and 1 µg of hr-ERK-1 as substrate in `kinase buffer' (10 mM Tris–HCl, pH 7.6, 100 mM NaCl, 10 mM MgCl2 and 1 mM MnCl2) for 15 min at 37°C. Phosphorylated proteins were separated by SDS–PAGE and visualized by autoradiography.

Determination of JNK/SAPK activity.
The assay was performed as described recently (20). Briefly, after stimulation, samples of 107 cells were washed and cytosol prepared as described in (20). Cytoplasmic protein was immunoprecipitated with Protein A–Sepharose-coupled JNK-1-specific antibodies. The activity of the enzyme was determined in an in vitro kinase assay using 2 µCi [{gamma}-32P]ATP and 1 µg GST–c-Jun as substrates. Alternatively, 10 µg cytosolic protein was incubated in the presence of 4 µCi of [{gamma}-32P]ATP and 1 µg GST–c-Jun for 15 min at 30°C, as indicated in the experiments. The reaction was terminated by addition of GSH–Sepharose, equilibrated in lysis buffer containing 1 mM DTT. Beads were recovered at 10,000 g for 5 min and washed twice in lysis buffer. Bound GST–c-Jun was eluted, separated by SDS–PAGE and visualized by autoradiography.

Determination of p38 MAPK activity.
For immunoprecipitation of p38 MAPK, samples of 107 cells were lysed in a buffer containing 10 mM Tris–HCl, pH 7.05, 30 mM Na pyrophosphate, 50 mM NaCl, 50 mM NaF, 400 nM okadaic acid, 20 mM ß-glycerophosphate, 0.5 mM PMSF, 2 mM Na3VO4, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin and 1% Triton X-100 for 15 min at 4°C. Extracts were cleared by centrifugation (10 min, 100,000 g, 4°C). Protein (1 mg) was immunoprecipitated with p38 MAPK specific antiserum bound to Protein A–Sepharose. Phosphorylation was carried out in kinase buffer containing 2 µCi of [{gamma}-32P]ATP with hr-MAPKAPK-2 or ATF-2 (1 µg of each) as substrates.

Phosphorylated p38 MAPK (pp38 MAPK) was immunoprecipitated and detected by immunoblotting with a pp38 MAPK-specific antibody. Recovery of the enzyme was confirmed by detecting the amount of p38 MAPK by means of a specific antibody.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from samples of 1x107 cells were obtained using a standard protocol (23,24). For the EMSA the following oligonucleotides were used (6): IL-2 distal nuclear factor of activated T cells (NFAT) site: GAT CGC CCA AAG AGG AAA ATT TGT TTC ATA CAG; NF{kappa}B: AAT TCA CAA AGA GGG ACT TTC CCT ACA TCC ATT G. The binding reaction was carried out with radiolabeled double-stranded oligonucleotide (0.1 pmol with 20,000 c.p.m.) and competitor DNA [1 µg/µl poly(dI–dC)] in HP buffer (10 mM Tris, 10 mM EDTA, 10 mM DTT, 500 mM NaCl and 50% glycerol, pH 7.5) for 30 min at 30°C in the presence of 5–7.5 µg protein, as indicated in the experiments. Protein–DNA complexes were separated in a native polyacrylamide gel (4%) and visualized by autoradiography.

Determination of IL-2 synthesis
Cells (1x106) were preincubated with protein kinase inhibitors (50 µM PD-98059 and 10 µM SB-203580) for 90 min. Cells were washed and stimulated with anti-CD3 antibodies, IL-1 or their combination for 18 h. IL-2 concentration of the supernatants was determined by a specific ELISA (Genzyme DuoSet for murine IL-2) according to the manufacturer's instructions (Genzyme, Cambridge, MA).

Isolation of RNA and Northern blot analysis
RNA was prepared using the Quiagen RNeasy kit from Quiagen (Hilden, Germany). First, 15 µg of total RNA was separated by electrophoresis on 1% agarose/formaldehyde gels and then blotted on a nylon membrane (Quiagen) by capillary transfer using standard procedures (24). RNA was cross-linked by UV irradiation, the blot prehybridized in 50% formamide, 5xSSPE, 5xDenhardt's, 10% dextran sulfate and 1 mg/ml salmon sperm DNA for 2 h at 42°C, and then hybridized for 20 h at 42°C with the IL-2-specific cDNA. The membrane was washed twice with 5xSSC/0.1% SDS at 42°C for 10 min followed by incubation for 30 min in 1xSSC/0.1% SDS at 60°C. Hybridization with GAPDH was performed to standardize the signals.

The IL-2-specific cDNA used for Northern hybridization was amplified by reverse transcription from RNA isolated from EL-4 cells. PCR was performed at an annealing temperature of 65°C for 40 cycles with the following primers (Pharmacia, Freiburg, Germany): primer 1: 5'-AAG CTC CAC TTC AAG CTC TAC AGC G-3'; primer 2, 5'-TTG ACA GAA GGC TAT CCA TCT CCT C-3'. The product (412 bp) was separated on 1% agarose gels and eluted using the Quaquick gel extraction kit from Quiagen, following the manufacturer's instructions. The IL-2-specific cDNA was labeled with 30 µCi [{alpha}-32P]dATP (Hartmann, Braunschweig, Germany) using the Randomprime DNA labeling system from Amersham (Braunschweig, Germany).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
EL-4 cells express functionally active TCR–CD3 complexes and type I IL-1R
As shown in Fig. 1Go(A), the EL-4 cells expressed a high number of TCR as measured by staining with a PE-labeled antibody by FACScan. Figure 1Go(B) shows that the EL-4 subline also expressed a high number of IL-1R, as detected with a specific antibody against the type I IL-1R. Practically all cells possessed both receptors (Fig. 1CGo).




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Fig. 1. Expression of TCR–CD3 complex and type I IL-1R on the surface of EL-4 cells. Cells (106) were stained with a PE-conjugated anti-CD3 antibody (A), IL-1R with an antibody raised against the type I receptor and fluorescein (DTAF)-conjugated goat anti-rat IgG (B) or both (C). Flow cytometry was carried out as described under Methods.

 
To ensure that both receptors were functionally active, IL-2 synthesis was measured in activated cells. Upon stimulation with anti-CD3 antibodies the cells synthesized 300 pg/ml IL-2. Similarly, the supernatants of IL-1-stimulated cells contained 250–300 pg/ml of the cytokine. Upon combination of both stimuli IL-2 synthesis was markedly enhanced, i.e. the cells secreted 1000 pg/ml of the lymphokine. These results show that simultaneous activation via TCR and type I IL-1R resulted in a synergistic enhancement of IL-2 synthesis (see also Figs 6D and 7GoGo).



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Fig. 6. Inhibition of MEK and p38 MAPK results in suppression of IL-2 synthesis. Cells (106) were stimulated with 2.5 µg/ml anti-CD3 antibody, 2.5 ng/ml IL-1ß or their combination respectively for 12 h (A). Prior to stimulation cells were pre-treated with either 50 µM PD 98059 (specific inhibitor of MEK-1), 10 µM SB 203580 (specific inhibitor of p38 MAPK) or their combination for 90 min. Cells were then washed and stimulated with anti-CD3 antibody (B), IL-1 (C) or their combination (D). IL-2 concentration in cellular supernatants was measured with a commercial ELISA as described under Methods. Results are means of three independent experiments. SD < 5%.

 


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Fig. 7. Inhibition by PD 98059 and SB 203580 of IL-2 mRNA synthesis. EL-4 cells (1.5x107) were pre-treated with specific protein kinase inhibitors as described in Fig. 5Go. Cells were stimulated with anti-CD3 antibody (2.5 µg/ml), IL-1ß (2.5 ng/ml) or their combination for 6 h. RNA was then prepared and Northern blotting performed as described under Methods. Blots were hybridized with a radioactively labeled murine IL-2-specific cDNA for 18 h as described under Methods. Spots were visualized by autoradiography. Results are representative for two independent experiments. Upper part, IL-2; lower part: GAPDH.

 
In order to investigate whether distinct signal transduction pathways were involved in activation of IL-2 synthesis, protein kinase cascades stimulated via the TCR or IL-1R were studied in detail.

Activation of the `classical' MAPK pathway via TCR–CD3 and IL-1 R
As shown in Fig. 2Go(A), stimulation with both IL-1 and anti-CD3 antibody resulted in an increase in the phosphorylation of ERK-1 as measured by an in vitro kinase assay upon immunoprecipitation of MEK-1, one of the specific upstream activators of ERK. Both anti-CD3- and IL-1-activated MEK catalyzed ERK phosphorylation to a comparable extent (Fig. 2AGo). The results imply that at least in the EL-4 subline used both stimuli activated the `classical' MAPK pathway.





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Fig. 2. Activation by TCR and IL-1 of MAPK cascades in EL-4 cells. (A) Activation by TCR and IL-1 of the `classical' MAPK pathway. Cells (107) were incubated with 2.5 µg/ml anti-CD3 antibody or with 2.5 ng/ml IL-1ß for the times indicated. MEK-1 was immunoprecipitated with a specific antibody and its activity measured with hr-ERK-1 as substrate, as described under Methods. After separation by SDS–PAGE phosphorylated proteins were visualized by autoradiography. Results are representative for two independent experiments. (B) Cells (107) were stimulated with IL-1 or anti-CD3 for the times indicated. JNK was immunoprecipitated and subjected to an in vitro kinase assay using hr-GST–Jun fusion protein as substrate according to Methods. Proteins were separated by SDS–PAGE and visualized by autoradiography. Results are representative for four independent experiments. (C) Cells (107) were incubated with 2.5 µg/ml anti-CD3 antibody or with 2.5 ng/ml IL-1ß respectively for the times indicated. p38 MAPK was immunoprecipitated by a specific antibody, then subjected to an in vitro kinase assay with hr-MAPKAPK-2 (left part) or hr-ATF-2 (right part) as substrates. Phosphorylated proteins were processed to SDS–PAGE and autoradiography as described under Methods. Results are representative for two independent experiments.

 
Activation by IL-1 of JNK and p38 MAPK
To investigate the involvement of JNK in IL-1R- and TCR-induced signaling, the respective cytoplasmic proteins from stimulated cells were immunoprecipitated and subjected to an in vitro kinase assay with GST–c-Jun as substrate. Treatment of the cells with IL-1 led to a time-dependent enhancement of c-Jun phosphorylation compared to the control (Fig. 2BGo). In contrast, stimulation with anti-CD3 antibodies did not affect c-Jun phosphorylation (data not shown).

The activity of immunoprecipitated p38 MAPK from anti-CD3- and IL-1-stimulated cells were determined with recombinant MAPKAPK-2 and ATF-2 as substrates. As MAPKAPK-2 is selectively phosphorylated by p38 MAPK, its use allowed to ensure that ATF-2 phosphorylation was not due to contaminating SAP/JNK activities. As shown in Fig. 2Go(C), phosphorylation of both MAPKAPK-2 and ATF-2 by p38 MAPK was markedly enhanced by IL-1 as compared to control. In contrast, stimulation of cells with anti-CD3 antibodies did not affect p38 MAPK activity (Fig. 2CGo, lanes 2–4 and 9–11).

Similar results were obtained in stimulated murine spleen lymphocytes. Upon IL-1 treatment JNK were activated between 10 and 30 min (Fig. 3AGo). Activation by IL-1 also resulted in enhanced phosphorylation and thus activation of p38 MAPK, as detected by a pp38 MAPK-specific mAb. (Fig. 3BGo). Stimulation via TCR did not influence SAPK, i.e. JNK and p38 MAPK in murine lymphocytes (data not shown).




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Fig. 3. Activation by IL-1 of JNK and p38 MAPK in murine spleen lymphocytes. (A) Activation by IL-1 of JNK in murine lymphocytes. Lymphocytes (5x106) were stimulated with 2.5 ng/ml IL-1 for the times indicated and with 0.5 M sorbitol for 10 min. Then 40 µg cytoplasmic protein was subjected to an in vitro kinase assay using hr-GST–Jun fusion protein as substrate according to Methods. The reaction was stopped by the addition of activated GSH–Sepharose. Samples were shaken for 30 min, and beads recovered by centrifugation and washed. Bound GST–c-Jun was eluted by boiling for 5 min in SDS-sample buffer. After centrifugation proteins were separated by SDS–PAGE. Equal recovery of GST–c-Jun was confirmed by Coomassie brilliant blue staining. Phosphorylated GST–Jun protein was visualized by autoradiography. For further details see Methods. Results are representative for four independent experiments. (B) IL-1 activates enhanced phosphorylation of p38 MAPK in murine lymphocytes. Lymphocytes were stimulated as described in (A). p38 MAPK was immunoprecipitated as described above and phosphorylation of p38 MAPK detected by immunoblotting with a pp38 MAPK-specific antibody (upper part). Recovery of the enzyme was confirmed by detecting the amount of p38 MAPK by means of a specific antibody (lower part). Results are representative for four independent experiments.

 
Taken together, the data shown in Figs 2 and 3GoGo demonstrate that specific MAPK cascades were activated by the TCR and IL-1R. While both TCR and IL-1R activated the `classical' MAPK pathway, JNK and p38 MAPK were specifically activated by IL-1 in the murine T cell line EL-4 and, even more importantly, in `physiological' primary T cells.

Activation of transcription factors by TCR and IL-1R
Transcription factors represent important well-characterized targets for all three MAPK pathways activated via TCR–CD3 or IL-1 respectively (14,25). Because IL-2 synthesis was activated synergistically by anti-CD3 and IL-1, and both stimuli activated different MAPK cascades, it was obvious to study the activation of transcription factors involved in the regulation of the IL-2 promoter. One of these, NFAT is an essential transcription factor for several cytokine genes, including IL-2 (6). To test DNA-binding activity of NFAT, oligonucleotides with the specific sequence for co-ordinate binding of NFAT were used. As shown in Fig. 4Go(A), both anti-CD3 antibodies and IL-1 activated DNA binding of NFAT; however, with remarkably different kinetics. In anti-CD3-treated cells elevated NFAT binding was observed after 30 min of stimulation that declined to control levels after 120 min. In IL-1-stimulated cells enhanced NFAT binding was detected later, i.e. between 2 and 3 h of activation. In cells stimulated through both receptors NFAT was activated in a biphasic manner. Early enhancement of NFAT binding was detected between 15 and 60 min, followed by a second phase of enhanced NFAT-binding activity after 3–4 h of stimulation (Fig. 4AGo).




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Fig. 4. TCR–CD3 complex and IL-1R activate NFAT in EL-4 cells. (A) Activation by anti-CD3 and IL-1 of NFAT. Cells (107) were stimulated with 2.5 µg/ml anti-CD3 antibody, 2.5 ng/ml IL-1ß or their combination respectively for the times indicated. Nuclear extracts were prepared and 7.5 µg of protein was incubated with 32P-labeled oligonucleotides (20,000 c.p.m.) coding for the NFAT consensus sequence for 30 min, as described under Methods. Protein–oligonucleotide complexes were separated by PAGE (4% acrylamide) and processed to autoradiography. Results are representative for four independent experiments. (B) Influence of PD-98059 on anti-CD3- and IL-1-induced activation of NFAT. Cells (107) were preincubated with 50 µM PD-98059 for 90 min, and then stimulated with 2.5 µg/ml anti-CD3 antibody or 2.5 ng/ml IL-1ß for the times indicated. Nuclear extracts were prepared and EMSA carried out as described under Methods. Results are representative for five independent experiments.

 
The connection between activation of different protein kinase cascades and transcription factors regulating the IL-2 promoter was investigated by specific protein kinase inhibitors PD-98059, a specific inhibitor of MEK-1, and SB-203580, a specific inhibitor of p38 MAPK. Preincubation with PD-98059 resulted in a partial inhibition of NFAT activation in both anti-CD3- and IL-1-stimulated cells (Fig. 4BGo). In contrast, neither TCR- nor IL-1-stimulated NFAT activation was influenced by SB-203580 (data not shown).

NF{kappa}B is an ubiquitous transcription factor also implied in the regulation of IL-2 gene expression (reviewed in 6,26). As shown in Fig. 5Go(A), stimulation of EL-4 cells by IL-1 led to a rapid activation and DNA binding of NF{kappa}B, i.e. within 15 min, decreasing slowly to control levels between 30 min and 3 h of activation. In contrast, upon treatment with anti-CD3 antibodies NF{kappa}B activation was detectable after 2–3 h of stimulation. In cells stimulated with the combination of anti-CD3 antibody and IL-1, activation of NF{kappa}B was continuously activated between 15 min to 3 h (Fig. 5AGo). Antibodies against the p65 and p50 components competed for NF{kappa}B binding, suggesting that the active factor was composed of the p50/p65 dimer (data not shown).




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Fig. 5. Stimulation of NF{kappa}B activation via TCR–CD3 complex and IL-1R in EL-4 cells. (A) Activation by anti-CD3 and IL-1 of NF{kappa}B. Cells (107) were stimulated with 2.5 µg anti-CD3 antibody, 2.5 ng/ml IL-1ß and their combination respectively for the times indicated. Then 5 µg of nuclear protein was incubated with 32P-labeled oligonucleotides (20,000 c.p.m.) containing the NF{kappa}B consensus sequence. Protein–oligonucleotide complexes were separated by PAGE and visualized by autoradiography. Results are representative for four independent experiments. (B) PD 98059 inhibits NF{kappa}B activation in anti-CD3 stimulated cells. Cells (107) were cells were pre-treated with 50 µM PD 98059 (specific inhibitor of MEK-1) for 90 min and then stimulated with 2.5 µg anti-CD3 antibody for the times indicated. Extraction of nuclear proteins and experimental conditions were identical with those described under (A). Results are representative for three independent experiments.

 
The specific inhibitor of MEK-1, PD-98059, proved to be an effective inhibitor of NF{kappa}B activation in anti-CD3-stimulated cells (Fig. 5BGo). In sharp contrast, IL-1-stimulated NF{kappa}B activation was not affected by PD-98059, suggesting that NF{kappa}B activation was regulated by different molecular mechanisms via TCR and IL-1R. Neither anti-CD3- nor IL-1-stimulated NF{kappa}B activation was influenced by the p38 MAPK inhibitor, SB-203580 (data not shown).

Inhibition of different MAPK cascades results in suppression of IL-2 synthesis
Co-stimulation of EL-4 cells with anti-CD3 and IL-1 resulted in a synergistic enhancement of IL-2 secretion (Fig. 6AGo). To establish a link between activation of different protein kinase cascades and regulation of IL-2 synthesis, the influence of specific protein kinase inhibitors on TCR- and IL-1-stimulated IL-2 synthesis has been investigated. The fact that PD-98059, a specific inhibitor of MEK-1-suppressed anti-CD3-stimulated IL-2 synthesis by 50%, while SB-203580, a specific inhibitor of p38 MAPK, had no influence on anti-CD3-stimulated IL-2 production, is consistent with the lack of activation of p38 MAPK by anti-CD3 (Fig. 6BGo). In contrast, IL-1-induced IL-2 secretion was suppressed by both PD-98059 and SB-203580 by ~50% of each. Combination of both inhibitors led to an 80% repression of IL-1-stimulated IL-2 secretion (Fig. 6CGo).

The influence of specific protein kinase inhibitors on IL-2 synthesis induced by co-stimulation with anti-CD3 and IL-1 is shown in Fig. 6Go(D). Both PD-98059 and SB-203580 interfered with IL-2 synthesis, which was reduced by >60% of each. Upon preincubation with both inhibitors IL-2 secretion was suppressed by >80%.

The effects of specific protein kinase inhibitors on IL-2 specific mRNA synthesis are shown in Fig. 7Go. Expression of IL-2 mRNA stimulated by anti-CD3 antibody was inhibited by preincubating the cells with the specific inhibitor of MEK-1. The specific inhibitor of p38 MAPK had no influence on the amount of anti-CD3-induced IL-2 mRNA. Pre-treatment of cells with both protein kinase inhibitors (PD-98059 and SB-203580) did not result in a further reduction of the amount of IL-2 specific mRNA (Fig. 7Go).

IL-1 also induced IL-2 mRNA synthesis; however, to a lower extent as compared to anti-CD3-stimulated cells. Preincubation with the inhibitors of MEK-1, p38 MAPK or their combinations respectively completely abolished IL-1-induced IL-2-specific mRNA production. Co-stimulation via the TCR and the IL-1R led to transcription of very high amounts of IL-2 mRNA. Both the MEK-1 and the p38 MAPK inhibitors reduced the amounts of IL-2 mRNA significantly; however, no complete inhibition of IL-2 gene transcription was observed. Preincubation of the cells with the combination of both inhibitors resulted in a further decrease in IL-2-specific mRNA level (Fig. 7Go).

Correlation of the amount of IL-2 mRNA and IL-2 protein synthesis upon inhibition of distinct MAPK cascades suggests that TCR- and IL-1R-induced signals synergize at the transcriptional level to increase IL-2 gene expression. The data imply that at least two groups of MAPK, i.e. anti-CD3- and IL-1-stimulated ERK, and p38 MAPK(s) activated by IL-1, contribute to activation of the IL-2 gene transcription and synthesis. In summary, the data suggest that convergence of different MAPK cascades activated via TCR and IL-1R is necessary for maximal IL-2 synthesis in stimulated EL-4 cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Results of this study show that signals from the TCR and the IL-1R act in synergy to enhance IL-2 synthesis in EL-4 thymoma cells. While the `classical' MAPK cascade seems to be activated by both receptors, IL-1 preferentially activates JNK and p38 MAPK in thymoma cells, and even more importantly also in primary T cells. The results imply that several protein kinase cascades have to be activated and converge at the level of IL-2 gene transcription for induction of maximal IL-2 synthesis.

Stimulation of EL-4 cells via TCR resulted in activation of the `classical' MAPK pathway, i.e. in elevation of MEK catalysed ERK phosphorylation and in extensive elevation in IL-2 gene transcription and synthesis (Figs 2, 6 and 7GoGoGo). Involvement of the `classical' MAPK cascade in the regulation of IL-2 synthesis was substantiated by the finding that a specific MEK-1 inhibitor, PD-98059, efficiently reduced induction of IL-2-specific mRNA and subsequent IL-2 synthesis in cells stimulated with anti-CD3 (Figs 6 and 7GoGo). These results suggested that interruption of the TCR-induced `classical' MAPK cascade at the level of MEK activation resulted in partial suppression of IL-2 gene transcription (Figs 6 and 7GoGo). In agreement with previous studies these results clearly show that activation of the `classical' MAPK pathway is an important signal for IL-2 gene transcription (913,15). However, stimulation of the MEK/ERK pathway via TCR was not sufficient to induce maximal IL-2 synthesis. Synergistic enhancement in IL-2 mRNA accumulation and synthesis was achieved upon co-stimulation with anti-CD3 and IL-1 (Figs 6 and 7GoGo). As shown in Fig. 6Go(C and D), both IL-1R- and TCR + IL-1R-stimulated IL-2 synthesis were significantly inhibited by PD-98059.

These results imply that in addition to anti-CD3, the IL-1-activated MEK/ERK signaling pathway might be of obligatory importance for IL-2 gene expression and secretion.

IL-1 effectively activates `classical' MAPK in several non-lymphoid cells (2729). Enhanced MEK catalysed ERK phosphorylation was observed upon stimulation with IL-1 in EL-4 cells (Fig. 2AGo). The fact that TCR- and IL-1-induced IL-2 synthesis was inhibited by PD-98059 to a similar extent indicates that IL-1-stimulated ERK activation is of equal functional importance for regulation of IL-2 gene expression.

It should be emphasized that although both TCR and IL-1 activated the `classical' MAPK pathway (Fig. 2AGo), ERK phosphorylation was not further elevated upon co-stimulation with anti-CD3 and IL-1 (data not shown).

As depicted in Figs 2 and 3GoGo, JNK and p38 MAPK were activated to a significant extent by IL-1 both in murine thymoma cells and in splenic lymphocytes. IL-1-induced activation of both protein kinases was less prominent in splenic lymphocytes as compared to thymoma cells. This finding is, however, not surprising considering the significantly lower amounts of JNK and p38 MAPK proteins of splenic lymphocytes compared to thymoma cells (data not shown).

Figure 2Go shows that IL-1 effectively activated p38 MAPK(s) in EL-4 cells as indicated by enhanced phosphorylation of its specific substrates, MAPKAPK-2 and ATF-2 (19,25). The specific p38 MAPK inhibitor, SB-203580, reduced IL-1-stimulated IL-2 synthesis by 50%. Similarly, in cells co-stimulated with anti-CD3 antibody and IL-1, IL-2 synthesis was significantly, but not completely inhibited by SB-203580 (Fig. 6C and DGo). These results argue for a contribution of the p38 MAPK signaling pathway in regulation of the IL-2 gene. It should be emphasized that IL-2 synthesis and secretion induced via the TCR–CD3 complex alone were not influenced by the p38 MAPK inhibitor, ruling out non-specific effects of the substance, and thus also showing that IL-1-stimulated p38 MAPK and IL-2 synthesis were specifically inhibited by the substance. As activation of two important transcription factors, like NFAT and NF{kappa}B, regulating transactivation of the IL-2 promoter was not influenced by inhibition of p38 MAPK, the molecular mechanisms of the regulation of IL-2 gene transcription by p38 MAPK remain to be elucidated. However, it is obvious to speculate that phosphorylation by p38 MAPK of well-defined molecular targets (like ATF-2 or Elk-1) or of some transcription factors not identified so far might participate in transcriptional regulation in T lymphocytes similar to several other cells (30). Furthermore, p38 MAPK might be involved in the post-transcriptional regulation, i.e. in stabilization of IL-2-specific mRNA, thus contributing to elevation of IL-2 synthesis and secretion as shown for IL-8 specific mRNA (manuscript in preparation).

Activation of JNK by IL-1, as measured by enhanced phosphorylation of c-Jun, is shown in Figs 2 and 3GoGo. It is well documented that JNK family protein kinases phosphorylate the N- terminal activation domain of c-Jun resulting in activation of the transcription factor. Besides activating c-Jun, JNK also enhance the transcriptional activity of Jun proteins (14,30,31). Although we have no direct evidence so far for participation of IL-1-activated JNK in the regulation of IL-2 synthesis, because of the lack of specific inhibitors, several reports indicated phosphorylation of c-Jun (and ATF-2), subsequent elevated nuclear binding, and expression of c-Jun protein in different cells and cell lines. Furthermore, the JNK pathway is required for the normal regulation of AP-1 transcriptional activity (14,3234). These results indicate that JNK catalysed phosphorylation of its specific substrates might activate cytokine gene transcription also in T lymphocytes. Thus, activation of JNK might also contribute to elevation of IL-2 synthesis induced by IL-1 and anti-CD3, all the more, as a residual IL-2 secretion was observed after blocking activation of ERK and p38 MAPK (Fig. 6Go).

A characteristic feature of Jun (and Fos) family proteins is that they bind co-operatively with NFAT family proteins resulting in more stabile NFAT–AP-1 complexes at the NFAT sites of the IL-2 promoter (6,35,36). As shown in Fig. 4Go, both TCR and IL-1 activated DNA binding of NFAT with remarkably differential kinetics. It remains to be elucidated whether TCR and IL-1 activated different members of the NFAT family or, alternatively, nuclear export and import, or rephosphorylation of NFAT were responsible for activation of the transcription factor after 3–4 h in co-stimulated cells (37,38). In spite of this, NFAT proteins seem to be obvious targets of both TCR- and IL-1-stimulated signaling cascades resulting in their activation, nuclear translocation and finally up-regulation of IL-2 gene transcription.

The fact that interruption of the `classical' MAPK cascade at the level of MEK-1 resulted only in partial inhibition of both TCR- and IL-1-stimulated NFAT activation is consistent with the finding that besides the MEK/ERK cascade additional signal transduction pathways participate in activation of NFAT and in subsequent induction of IL-2 gene expression (39).

Binding of NF{kappa}B to the IL-2 promoter is required for IL-2 gene transcription in stimulated T cells (2,6,26). As shown in Fig. 5Go, both TCR and IL-1 activated NF{kappa}B. However, activation via IL-1R resulted in fast and transient activation of NF{kappa}B, upon co-stimulation with IL-1 + anti-CD3 a continuous NF{kappa}B activation was observed (Fig. 5Go). Thus, stimulation via TCR and IL-1R might prolong NF{kappa}B binding to the IL-2 promoter thereby resulting in enhanced IL-2 synthesis. Although proinflammatory cytokines, e.g. IL-1, are most efficient NF{kappa}B activators (40,41), the data in Fig. 5Go show that TCR-induced signaling significantly prolonged activation and nuclear binding of NF{kappa}B.

The fact that IL-1-stimulated NF{kappa}B activation was not influenced by the specific MEK inhibitor PD-98059 or the p38 MAPK inhibitor SB-203580 suggest that NF{kappa}B activation via IL-1R represents a distinct signaling pathway independent of ERK and p38 MAPK activation. This finding is consistent with recent data demonstrating that IL-1-induced activation of NF{kappa}B involves activation of a kinase complex that phosphorylates the I{kappa}B and is distinct of the known MAPK pathways (42,43).

In sharp contrast, anti-CD3-stimulated NF{kappa}B activation was completely inhibited upon interruption of the MEK/ERK pathway by PD-98059 (Fig. 5Go) suggesting that TCR-induced activation of NF{kappa}B obviously depends on activation and functioning of the `classical' MAPK pathway. Putative involvement of MAP (ERK) kinases in activation of I{kappa}B phosphorylation and degradation was discussed recently (44). More recent data indicated that the Cot/Tlp-2 serine–threonine kinase was an upstream activator of the NF{kappa}B-inducing kinase in TCR–CD28-stimulated T lymphocytes (45). In respect to the finding that the MAPK kinase kinase, Cot/Tlp2 participates in activation of the ERK pathway (46) and effectively enhances IL-2 synthesis in anti-CD3-stimulated cells (47), raising the possibility that Cot/Tlp2 and its downstream targets might be involved in TCR-stimulated NF{kappa}B activation.

Although the molecular mechanisms of TCR-induced NF{kappa}B activation as well as participation of the `classical' MAPK pathway in activation of NF{kappa}B remain to be elucidated, the data presented in this paper imply that different and independent signaling pathways activate NF{kappa}B via the TCR and IL-1R. However, it should be mentioned that although all NF{kappa}B family members are present in EL-4 cells, both TCR and IL-1 stimulation resulted in activation of the p50/p65 heterodimer (data not shown).

In summary, the data clearly show that signaling pathways activated via TCR and IL-1R converge at the level of the IL-2 gene transcription, resulting in synergistically enhanced IL-2 synthesis and secretion. Maximal activation of the IL-2 gene requires activation of at least two different protein kinase cascades, e.g. ERK, p38 and presumably JNK kinases activated via the TCR and IL-1R respectively. The co-stimulatory effect of IL-1 in T cell activation might be due to convergence of different signaling pathways at the level of the IL-2 promoter resulting in enhancement of its transcriptional activity. The results also suggest that convergence of TCR and IL-1 induced signaling cascades is obligatory for maximal IL-2 synthesis in co-stimulated cells.


    Acknowledgments
 
The competent technical assistance of Mrs M. Golombek is gratefully acknowledged. The authors thank Mrs K. Erdogan for her excellent typing assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 265 /A9)


    Abbreviations
 
EMSA electrophoretic mobility shift assay
ERK extracellular signal-regulated kinase
GST glutathione-S-transferase
IL-1R IL-1 receptor (type I)
JNK Jun-N-terminal kinase
MAPK mitogen-activated kinase or microtubule-associated protein kinase
MAPKAPK MAPK-activated protein kinase
MEK MAP/ERK kinase
NFAT nuclear factor of activated T cells
PE phycoerythrin
SAPK stress-activated protein kinase

    Notes
 
Transmitting editor: M. Reth

Received 11 December 1998, accepted 5 August 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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