Wortmannin inhibits translation of tumor necrosis factor-{alpha} in superantigen-activated T cells

Matilde Ramírez, Neus Fernández-Troy, Maria Buxadé, Ricardo P. Casaroli-Marano1, Daniel Benítez2, Cesar Pérez-Maldonado and Enric Espel

Departament de Fisiologia and
1 Departament de Biologia Cellular, Facultat de Biologia, Universitat de Barcelona, Avenue Diagonal 645, 08028 Barcelona, Spain
2 Servei d'Immunologia, Hospital Clínic Provincial, Villarroel 170, 08036 Barcelona, Spain

Correspondence to: E. Espel


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The superantigen toxic shock syndrome toxin (TSST)-1 can induce tumor necrosis factor (TNF)-{alpha} expression in T cells and monocytes, through different signaling pathways. We have stimulated peripheral blood mononuclear cells with TSST-1 and found that the major cell producers of TNF-{alpha} as detected by cytofluorimetry and immunocytochemistry were CD4+ T lymphocytes. The expression of TNF-{alpha} by CD4+ T cells can be inhibited by either, wortmannin (WN) or LY 294002, two phosphatidylinositol 3-kinase (PI 3-K) inhibitors. The inhibitory effect is not transcriptional as WN does not change the mRNA steady state of TNF-{alpha} at any of the concentrations tested and LY 294002 when preincubated with mononuclear cells at its median inhibitory concentration (IC50 = 1.4 µM) significantly inhibited the expression of TNF-{alpha} but not its mRNA. Immunoprecipitation of pulse-labeled intracellular TNF-{alpha} showed a specific decrease in the synthesis of this cytokine on cells treated with PI 3-K inhibitors. The p38 mitogen-activated protein kinase (MAPK) is involved in control of TNF-{alpha} translation in human macrophages. In T cells, we have found that the p38 MAPK inhibitor SB 203580 significantly decreased the secretion of TNF-{alpha} but not its mRNA. In addition, the combined use of WN and SB 203580 had an additive inhibitory effect on secretion of TNF-{alpha}. Therefore, both PI 3-K and p38 MAPK signaling pathways control TNF-{alpha} production in T cells.

Keywords: LY 294002, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, SB 203580, toxic shock syndrome toxin-1


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha} is a cytokine induced by several stress conditions such as infection. TNF-{alpha} activity is important for mounting the inflammatory response necessary to stop antigen spreading in tissues (reviewed in 1). The pathophysiology of TNF-{alpha} is also associated with serious clinical disorders. The expression of TNF-{alpha} has been described to be regulated at multiple levels, including translation of its mRNA. The 3' untranslated region of murine TNF-{alpha} mRNA contains some elements that confer translatability to a heterologous mRNA expressed in macrophage cell lines stimulated with lipopolysaccharide (LPS) (2). The mechanism of translational control of human TNF-{alpha} mRNA in superantigen or LPS-stimulated monocytes appears to be at the level of ribosome loading (3).

Translation of TNF-{alpha} in a monocytic cell line activated with LPS has been suggested to be under the control of p38 mitogen-activated protein kinase (MAPK) (4). p38 MAPK is the specific target of pyridinyl imidazole compounds such as SB 203580, which inhibit the production of pro-inflammatory cytokines (IL-1ß and TNF-{alpha}) in stimulated monocytes (4). A group of MAPK-regulated protein kinases, MNK, was identified recently (5,6). These kinases are regulated by p38 MAPK and extracellular signal-regulated kinase (Erk), and can phosphorylate eukaryotic initiation factor-4E (eIF-4E) in vitro and in vivo, which indicates an important link between MAPK activation and translational initiation (68). The role of p38 MAPK in TNF-{alpha} expression in T lymphocytes is at present unknown.

T lymphocytes are major producers of TNF-{alpha}. Several T cell populations are able to secrete large amounts of TNF-{alpha} upon activation and some of them may have a role in pathological situations like rheumatoid arthritis (910). Following T cell stimulation the levels of several translation factors (11,12) and their activities (11,13) are increased. Up to 13% of mRNA species seem to be translationally regulated on antigen-stimulated T cells (14). Human T cell activation with ionomycin and phorbol myristate acetate leads to inactivation of glycogen synthase kinase-3 and stimulation of translation initiation factor eIF2B (13). Stimulation of murine CTLL-20 T cells or a human leukemic T cell line with IL-2 leads to the activation of p70S6 kinase in a mechanism sensitive to phosphatidylinositol 3-kinase (PI 3-K) inhibitors (15,16). Similarly, ligation of the T cell co-stimulatory receptor CD28, which is necessary for a higher secretion of IL-2 and other cytokines, activates PI 3-K and protein kinase B (PKB) activities (17). Moreover, transfection of constitutively active forms of PI 3-K and PKB is sufficient for activation of p70S6 kinase in T cells (16). Inhibition of p70S6K with the immunosuppressant rapamycin inhibits specific protein synthesis (18). In other cell types, PI 3-K and PKB are required for the inhibition of the translational repressor 4EBP1 (19). Whereas this data suggests a link between PI 3-K activation and translational control, there is no presently known evidence relating PI 3-K activity to control of TNF-{alpha} translation.

Effective inhibitors of PI 3-K may help to define the role of PI 3-K in cells. The best characterized subfamily of PI 3-K comprises heterodimers containing an 85 kDa regulatory subunit and a catalytic 110 kDa subunit. p110 is covalently modified and potently inhibited by the fungal metabolite wortmannin (WN) (20,21). The compound LY 294002, unrelated structurally to WN is also a potent competitive inhibitor of p110 (22). WN inhibits CD28-mediated co- stimulation of IL-2 production in resting and activated human T cells (2325). In an attempt to study the regulation of the expression of TNF-{alpha} in superantigen-induced T cells, we describe that translation of TNF-{alpha} mRNA can be specifically inhibited, whereas its mRNA levels are maintained constant. We have found that both PI 3-K inhibitors WN and LY 294002, inhibit the expression of TNF-{alpha} at the level of protein synthesis, implicating the PI 3-K signaling pathway in the translational control of this cytokine. Additionally, we have found that the p38 MAPK inhibitor SB 203580, inhibits TNF-{alpha} secretion but not its mRNA steady state.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chemicals and antibodies
WN (Calbiochem, La Jolla, CA) at 1 mM, LY294002 (Calbiochem) at 100 mM, SB 203580 (a generous gift of Dr Lee, SmithKline Beecham, King of Prussia, PA) at 20 mM and rapamycin (ICN, Aurora, OH) at 100 µg/ml were all dissolved in DMSO, aliquoted and stored at –20°C. Brefeldin A (Epicentre, Madison, WI) at 10 mg/ml in ethanol and toxic shock syndrome toxin (TSST)-1 (T5662, Sigma-Aldrich Química, Madrid, Spain) at 500 µg/ml dissolved in DMEM were aliquoted and stored at –20°C. Mowiol mounting medium was from Calbiochem (La Jolla, CA). The secondary antibodies were: TRITC-conjugated swine anti-rabbit Ig was from Dakopats (Grostrup, Denmark), Cy2-conjugated goat anti-mouse Ig was purchased from Amersham (Arlington Heights, IL) and FITC-conjugated goat anti-mouse (55520; Cappel). Paraformaldehyde fixative was from Merck (Darmstadt, Germany). For lymphocyte detection we used mAb directed against CD4 (IgG1, clone 193-19, Workshop VI), CD8 (IgG1, clone 143-44, Workshop IV) and for monocytes a mAb against CD14 (IgG1, clone 47-3D6, Workshop II), all a generous gift of Dr Ramon Vilella (Servei d'Immunologia, Hospital Clínic, Barcelona). The anti-TNF-{alpha}–phycoerythrin (PE) antibody for intracellular detection was from Caltag (Burlingame, CA). The mAb to HLA class I was kindly provided by Dr J. A. García Sanz (CNB-UAM, Madrid). Recombinant human TNF-{alpha} was a generous gift of Dr Geert Plaetinck (Roche, Gent, Belgium).

Cell isolation and culture.
Human peripheral blood mononuclear cells (PBMC) were obtained from buffy coats from normal individual donors from the Hospital Clinic of the Barcelona Blood Bank service (Barcelona, Spain). PBMC were isolated by density gradient centrifugation through Ficoll (Ficoll Histopaque-1077; Sigma-Aldrich Química). Cells were resuspended in DMEM (Sigma-Aldrich Química), supplemented with 10% heat-inactivated FCS (F-7524, Sigma-Aldrich Quimica), seeded into 24-well plates at 4x106 cells/well, and cultured overnight at 37°C and 5% CO2. Cells were then subjected to different experimental treatments as indicated.

T cell lines
Allogeneic donors were tested for optimal in vitro allogeneic production of TNF-{alpha}. An alloreactive MHC class II+ T cell line was derived from PBMC of a normal donor stimulated once every 3 weeks with irradiated allogeneic Epstein–Barr virus (EBV)-transformed B cells and maintained in RPMI 1640 (Gibco BRL, Paisley, UK) medium supplemented with 10% heat-inactivated FCS (ICN), 2 mM L-glutamine and 20 U/ml recombinant IL-2 (Boehringer Mannheim, Mannheim, Germany).

Superantigen stimulation
Where indicated, cells were treated with WN or rapamycin for 4 h and with SB 203580 or LY 294002 for 1 h prior to superantigen stimulation (TSST-1). Cells that did not receive inhibitors received control solvent (DMSO). When kinetic studies were performed, WN was added every 5 h. After 4 h of stimulation with superantigen, supernatants were harvested and assayed for levels of cytokine by ELISA assay. From the cell pellet, total RNA was isolated and levels of TNF-{alpha} mRNA quantified by RNase protection assay.

For T cell lines, the stimulation with superantigen was performed at the 14th day after the last incubation with irradiated EBV B cells and recombinant IL-2.

Cytokine assay
TNF-{alpha} levels in supernatants were determined by specific sandwich ELISA using mAb raised against human TNF-{alpha} (Immunokontact, Frankfurt, Germany). Microtiter plates were coated with 50 µl (5 µg/ml in TBS ) of anti-cytokine capture mAb (2TNF-H34A; Immunokontact) overnight at 4°C. After washing 3 times with wash buffer (PBS/0.05% Tween 20), blocking reagent (Blocking reagent for ELISA; Boehringer Mannheim) was added and incubated at 37°C for 1 h. After washing 3 times with wash buffer, 50 µl of supernatants or a serial dilution (31.0–1000 pg/ml) of recombinant human TNF-{alpha} to generate a standard curve (Roche) was added and incubated at 37°C for 1 h. After washing 4 times, 50 µl (2 µg/ml in TBS) of biotinylated anti-cytokine detecting mAb (2TNF-H33B; Immunokontact) was added and incubated at 37°C for 1 h. After several washings to remove the unbound antibody, the bound antibodies were then incubated for 1 h at 37°C with 50 µl (2:10,000 dilution in TBS) of streptavidin–alkaline phosphatase conjugate (Boehringer Mannheim). After washing 4 times with wash buffer and twice with diethanolamine buffer (1 M diethanolamine, 5 mM MgCl2, pH 9.8), the amount of specifically bound streptavidin–alkaline phosphatase conjugate was determined by assaying for alkaline phosphatase activity with freshly prepared p-nitrophenylphosphate solution (10 mM in diethanolamine buffer). Then 50 µl of substrate solution was added into each well and incubated for 45 min at 37°C. The absorption at 405 nm was determined with an automatic EIA analyzer.

Preparation and analysis of RNA
Total RNA was prepared using the guanidium thiocyanate–acid method (26). Analysis of specific mRNA was performed using a T7 antisense transcript corresponding to the human TNF-{alpha} cDNA [HindIII (1003)–PstI(1158)] together with a T7 antisense transcript corresponding to the human GAPDH cDNA (USB, Cleveland, Ohio) as probes. Hybridization and digestion were performed as described (3). Protected fragments were separated on a 6% sequencing gel and the relative amounts of TNF-{alpha} and GAPDH were quantified using a BioRad molecular imager.

Analysis of TNF-{alpha} biosynthesis
Biosynthesis rates of TNF-{alpha} were quantified by immunoprecipitation of cytoplasmic extracts from human PBMC after a 20 min pulse label with [35S]methionine. Human PBMC (4x106) were stimulated as described. After 3 h of TSST-1 stimulation, cells were washed 3 times with methionine- and cysteine-free DMEM, and incubated in the same medium for another 30 min. Cells were incubated with 2.4 mCi of [35S]methionine (Redivue; Amersham) for 20 min at 37°C, then washed with cold PBS and lysed in 1% Triton-X lysis buffer containing protease inhibitors, for 15 min at 4°C. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C and an aliquot of cytoplasmic extracts was used to determine protein concentration. Supernatants were sequentially incubated with rabbit pre-immune serum, with rabbit anti-human TNF-{alpha} antibody (Labgen, Frankfurt, Germany), with anti-human MHCI (W6/32) mAb or anti-human MHCII (3B12) and 33 µl of Protein G–Sepharose (Sigma, Poole, UK) for 3 h at 4°C. Sepharose beads were washed several times and bound proteins were eluted with 30 µl of 1xSDS-loading buffer containing 5% mercaptoethanol. Samples were boiled for 5 min and run on a 12% SDS–PAGE. Gels were incubated with Enhance (DuPont, Brussels, Belgium) and exposed to a film (X-Omat AR; Eastman Kodak, Rochester, NY). Specific bands were quantified using a BioRad molecular imager. An aliquot of cell lysate was also loaded on the same gel to quantify the overall increase in protein synthesis in all the conditions of cell stimulation.

Immunofluorescence and flow cytometry experiments
Cells were detached from dishes by gentle scrapping. For flow cytometry procedures and immunofluorescence detection for confocal analysis, samples with 1x106 cells were rinsed in PBS 0.1 M (100 mM phosphate, 150 mM NaCl, pH 7.4) containing 5% FCS (PBS/FCS), pelleted at 300 g for 5 min and incubated with mAb directed against cell membrane antigens (CD), on ice for 20 min. Cells were then washed twice in PBS/FCS solution and incubated with a FITC- or Cy2- conjugated goat anti mouse. Cells were washed again and fixative reagent (Gas003; Caltag, Burlingame, CA) was applied for 15 min at room temperature. After washing, cells were incubated with antibodies directed against intracellular TNF-{alpha}, conjugated with PE (for flow cytometry) or not (for immunofluorescence), diluted in permeabilization medium (Caltag) for 15 min at room temperature in darkness. Secondary TRITC-conjugated swine anti-rabbit Ig was applied for TNF-{alpha} detection in immunofluorescence confocal experiments. After successive washes, cells were pelleted and maintained in 1% paraformaldehyde in 0.1 M phosphate buffer solution, at 4°C in darkness before flow cytometry studies.

Flow cytometric experiments were carried out using an Epics XL flow cytometer (Coulter, Miami, FL). Excitation of the sample was done using a standard 488 nm air-cooled argon-ion laser at 15 mW power. The instrument was set up with the standard configuration. Forward scatter, side scatter, green (525 nm) fluorescence for FITC- or Cy2-conjugated antibody and orange (575 nm) fluorescence for PE conjugated antibodies were acquired. Green fluorescence was collected with a 550 dichroic long filter and a 525 band pass filter. Orange fluorescence was collected with a 600 dichroic long filter and a 575 band pass filter. Optical alignment was based on the optimized signal from 10 nm fluorescent beads (Immunocheck, Epics Division). Gates were set on a dot plot of forward versus side scatter. Fluorescence was projected on a 1024 channel histogram. Between 1x104 and 4x104 cells from each sample were analyzed. Time was used as a control of the stability of the instrument. Lymphocytes and monocytes were gated on forward and side scatter.

Confocal scanning laser microscopy
For confocal analysis cell pellets were resuspended in 10 µl of Mowiol and mounted in glass coverslips. Control coverslips were carried out by omitting primary antibodies and incubating with fluorocrom-conjugated antibody solution to observe secondary antibody specificity. Cells were analyzed on a Leica TCS 4D (Leica Lasertechnik, Heidelberg, Germany) confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope and a x63 (NA 1.4, oil) Leitz Plan Apo objective. Green (cell membrane antigens) and red (intracellular TNF) fluorescence were simultaneously excited at 488 and 568 nm lines with an argon–krypton laser (75 mW). We made six serial optical sections with voxel dimensions of 0.31 µm lateral and 0.96 µm axial. For co-localization analysis (MultiColor Software; Leica Lasertechnik) a combined image of each single section was created using original fluorescent images. For the co-localization images we made a projection of two serial intermediate cell sections. Images were photographed on a high-resolution color video printer Mitsubishi CP2000E.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TSST-1 stimulation of PBMC induces TNF-{alpha} secretion in T cells
PBMC can be activated to secrete cytokines and proliferate by incubation with bacterial superantigens. PBMC were stimulated with the superantigen TSST-1 and the secretion of TNF-{alpha} was rapidly induced in cell supernatant (see Fig. 3AGo). Immunocytochemical characterization of cells containing intracellular TNF-{alpha} showed T cells as the only cell population synthesizing TNF-{alpha} (Fig. 1Go). TNF-{alpha}+ T cells (red) represent only a fraction of the total T cell population (green), as TSST-1 activates only the Vß8+ T cell subset. Flow cytometry analysis was performed to discriminate the cell population within PBMC producing TNF-{alpha}. A fraction of the CD4+ and CD8+ T cell populations stained positive for intracellular TNF-{alpha}, representing 4–5% of total lymphocytes in PBMC stimulated with TSST-1 (Fig. 2Go). However, the monocyte CD14+ cell population was negative for intracellular TNF-{alpha} by both, FACS and immunocytochemistry (Figs 1 and 2GoGo), therefore, LPS is not responsible for TNF-{alpha} secretion in this system. Control cells not stimulated with TSST-1 did not stain positive for TNF-{alpha} as expected. Among T cells, the CD4+ T cell fraction was the major producer of TNF-{alpha}. The enrichment for TNF-{alpha} in the CD4+ T cell fraction was ~6-fold on average. In this PBMC sample the proportion of CD4:CD8 is 4:1 whereas the ratio of double-positives CD4+TNF+:CD8+TNF+ is 14. From the above experiments we know that in PBMC cells stimulated with TSST-1, TNF-{alpha} is mainly produced by CD4+ T cells.



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Fig. 3. Kinetics of PBMC cell stimulation with TSST-1: inhibition by WN. Human PBMC were preincubated for 4 h with 10 nM WN and stimulated with TSST-1 (0.5 µg/ml). (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. Values shown are the mean ± SD of duplicate determinations. (B) After deproteinization, aliquots of the cellular RNA from each fraction were analyzed by RNase protection assays, using simultaneously an antisense TNF-{alpha} probe and an antisense GAPDH probe. Migration of the protected probes is indicated. (C) The gel from B was quantified on a molecular imager (BioRad). Results are plotted as the ratio of radioactivity in protected TNF-{alpha} bands to radioactivity in protected GAPDH bands (expressed in arbitrary units). WN (WORT) at 10 nM was freshly added every 5 h. The control vehicle (DMSO) was added to all samples not containing WN. Time of cell incubation is indicated in hours. One representative experiment of two is shown.

 


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Fig. 1. Confocal microscopy. Double immunofluorescence for TNF-{alpha} and cell membrane antigens on human PBMC after TSST-1 stimulation. PBMC were stimulated with TSST-1 for 5 h. Brefeldin A (10 µg/ml) was added for the last 2 h of culture. Samples were incubated with FITC-conjugated mAb directed against T lymphocyte (CD3) or monocyte (CD14) cell membrane antigens followed by a polyclonal antibody directed against intracellular TNF-{alpha} as described in Methods. Intracellular TNF-{alpha} was visualized after incubation with secondary TRITC-conjugated swine anti-rabbit Ig. A combined projection of two serial intermediate cell sections (x, y, z: 0.31 µm, 0.31 µm, 0.96 µm) was viewed by a confocal microscope. Intracellular TNF-{alpha} expression was only detected on T lymphocytes (arrows) after TSST-1 stimulation. Co-localization represents the projected images observed in CD and TNF- columns. Bar = 10 µm. One representative experiment of two is shown.

 


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Fig. 2. Characterization of the cell population synthesizing TNF-{alpha} by flow cytometry. PBMC were stimulated with TSST-1 as in Fig. 1Go. The cells were stained with antibodies specific for CD4, CD8 or CD14, fixed and stained for detection of intracellular TNF-{alpha}. Gated lymphocytes (LYM) on forward (FS) and side scatter (SS) are shown on the middle of the figure. Gated monocytes (MO) are shown on the bottom of the figure. Numbers inside the squares express the percentage of cells included. One representative experiment of three is shown.

 
Inhibition of PI 3-K decreases TNF-{alpha} secretion at a post-transcriptional level
The PI 3-K inhibitor WN (20,21) has been shown to partially inhibit TCR signaling and also to block CD28 co-stimulation of T cells (27). We tested whether WN is able to inhibit TNF-{alpha} secretion when PBMC are stimulated with TSST-1. Preincubation of the cells with 10 nM WN significantly inhibited (50%) the secretion of TNF-{alpha} (Fig. 3AGo). Then, we measured the mRNA of TNF-{alpha} in the same cellular samples by RNase protection assay (Fig 3BGo). The quantification of the bands corresponding to TNF-{alpha} and normalization with GAPDH showed that WN did not change the expression of the mRNA for TNF-{alpha} (Fig. 3CGo). This suggested that WN was inhibiting a translational or post-translational step during TNF-{alpha} synthesis. The GAPDH mRNA is used as an internal control for each sample, as the expression of this gene is constant until the 11 h time point; afterwards it is up-regulated, probably due to the metabolically active state of the PBMC. As expected, WN inhibited the percentage of intracellular TNF-{alpha}+ cells in both CD4+ and CD8+ T cells (Fig. 2Go).

The inhibitory effect of WN upon TNF-{alpha} secretion increased with concentration up to 100 nM reaching a 60% inhibition (Fig. 4AGo). In the same samples, the mRNA ratio for TNF-{alpha}:GAPDH did not change upon WN treatment (Fig. 4B and CGo). Similar results were found with the unrelated PI 3-K inhibitor LY 294002 (Fig. 5Go), which inhibits PI-3 kinase activity by a different mechanism (22) . Its use at a concentration of 1.4 µM—the median inhibitory concentration (IC50) of this compound in intact cells (22) —decreased the secretion of TNF-{alpha} in supernatant ~30% (Fig. 5AGo), but was not inhibitory for the mRNA of TNF-{alpha} (Fig. 5B and CGo). Preincubation of PBMC with LY 294002 at a higher concentration (5.0 µM) completely inhibited the secretion of TNF-{alpha}, whereas there remained 60% of the mRNA for TNF-{alpha} (Fig. 6Go). Therefore, LY 294002, similar to the results obtained with the treatment of PBMC with WN, inhibits TNF-{alpha} production also at a translational or post-translational step. These results suggest that PI 3-K is involved in the translational or post-translational control of TNF-{alpha} expression.



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Fig. 4. Inhibition of TNF-{alpha} secretion by WN: dose–response. PBMC were preincubated for 4 h with different concentrations of WN and stimulated with TSST-1 for 5 h. (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. (B) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography as indicated in Fig. 3Go. (C) Radioactivity in protected TNF-{alpha} bands in (B), normalized to protected GAPDH bands (expressed in arbitrary units). One representative experiment of three is shown.

 


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Fig. 5. LY294002 inhibits TNF-{alpha} secretion. PBMC were preincubated for 1 h with LY294002 at 1.4 µM and stimulated with TSST-1 for 5 h. (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. (B) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography. Arrowheads indicate the position of protected probes; MW = mol. wt markers. (C) Results from (B) are plotted as the ratio of radioactivity in protected TNF-{alpha} bands to radioactivity in protected GAPDH bands (expressed in arbitrary units). Cells not treated with LY 294002 were incubated with vehicle (DMSO). One representative experiment of two is shown.

 


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Fig. 6. Kinetics of the inhibitory effect of LY 294002 on the expression of TNF-{alpha}. PBMC were preincubated for 1 h with LY 294002 at 5.0 µM and stimulated with TSST-1 during several hours. (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. (B) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography. Arrowheads indicate the position of protected probes. (C) Results from (B) are plotted as the ratio of radioactivity in protected TNF-{alpha} bands to radioactivity in protected GAPDH bands (expressed in arbitrary units). Cells not treated with LY 294002 were incubated with vehicle (DMSO).

 
WN inhibits TNF-{alpha} secretion in a T cell line
It could be considered that the superantigen TSST-1 did activate a non-T cell type to produce a cofactor (i.e. co-stimulatory protein) acting upon T cells that enables superantigen-activated T cells to produce TNF-{alpha}. This situation cannot be excluded from the experiments employing PBMC. Therefore, similar experiments were performed using T cell lines to verify the cellular locus of the effect of PI 3-K inhibitors.

It has previously been shown that human T cells specific for TSST-1 are able to proliferate when incubated with this superantigen in the absence of accessory cells, due to the synergic action of signals delivered by the TCR and MHC class II molecules (28). Figure 7Go shows that, in the absence of accessory cells, TSST-1 was able to induce TNF-{alpha} synthesis in a CD4+ MHC class II+ human allogeneic T cell line. As previously shown for PBMC, the secretion of TNF-{alpha} in the supernatant could be inhibited by preincubation of the allogeneic T cell line with WN. However, WN, did not decrease the expression of the mRNA for TNF-{alpha} (Fig. 7B and CGo), confirming the results observed in the PBMC system.



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Fig. 7. WN inhibits TNF-{alpha} secretion in a T cell line. An allogeneic T cell line was prepared as described in Methods. Where indicated, 8.0x106 T cells were preincubated for 4 h with 20 nM WN and stimulated with 0.5 µg/ml TSST-1 for 5 h. (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. (B) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography as indicated in Fig. 3Go. (C) Radioactivity in protected TNF-{alpha} bands in (B), normalized to protected GAPDH bands (expressed in arbitrary units). The control vehicle (DMSO) was added to all samples not containing WN. One representative experiment of two is shown.

 
Inhibition of PI 3-K decreases translation of TNF-{alpha}
We checked whether the inhibition of TNF-{alpha} secretion by WN is controlled at the level of protein synthesis. WN elicited a marked effect on TNF-{alpha} biosynthesis as measured by the incorporation of [35S]methionine into immunoprecipitable material (Fig. 8AGo). PBMC incubated in the presence of WN incorporated as much [35S]methionine into HLA class I molecules, used as internal control, as did PBMC incubated without WN (Fig. 8AGo). The same conditions resulted in a 6-fold decrease in incorporation into TNF-{alpha} (Fig. 8BGo) upon normalization to the total protein content. The effect of WN in total protein synthesis (data not shown) was negligible. The mRNA levels of TNF-{alpha} did not change significantly (0.8-fold) by effect of WN treatment (Fig. 8C and DGo).



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Fig. 8. Rate of synthesis of TNF-{alpha}. PBMC cells were stimulated with TSST-1 for 3 h in the presence or absence of WN. Cells were starved in a methionine-free medium for 30 min and pulse labeled with [35S]methionine for 20 min before harvesting. Cells were lysed and immunoprecipitated sequentially with the anti-TNF-{alpha} and the anti-MHC class I antibodies. Labeled TNF-{alpha} was visualized by fluorography after PAGE. (A) Immunoprecipitation with anti-TNF-{alpha} antibodies (left) or with anti-MHC class I antibodies (right). Arrows indicate the position of TNF-{alpha} and MHC class I proteins. (B) The radioactivity in the immunoprecipitated bands in (A) was quantified and data are expressed in arbitrary units. (C) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography. Arrows indicate the position of protected probes. (D) Radioactivity in protected TNF-{alpha} and GAPDH bands in (C) was quantified as in Fig. 3Go. Data are presented as the ratio TNF-{alpha}:GAPDH (expressed in arbitrary units). TSST-1 = 500 ng/ml; wort = 10 nM WN; MW = mol. wt markers. One representative experiment of three is shown.

 
Therefore, pharmacological inhibition of PI 3-K specifically inhibits the protein synthesis of TNF-{alpha} suggesting that PI 3-K is implicated, at least partially, in the signaling pathway controlling translation of this cytokine.

Due to the role that PI 3-K plays in both the activation of p70S6K in several cell types (15,16,29) and inactivation of the translational repressor 4EBP1 (18,19,20), we looked for an involvement of p70S6K or 4EBP1 in control of TNF-{alpha} translation in activated T cells. The activation of p70S6K and the phosphorylation of 4EBP1 are inhibitable with rapamycin (18,19,3033). We tested the effect of rapamycin on TNF-{alpha} expression. With some exceptions, rapamycin did not significantly inhibit the expression of TNF-{alpha} (Fig. 9Go), suggesting that there is no contribution of p70S6K and 4EBP1 to the translational regulation of TNF-{alpha} in activated T lymphocytes.



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Fig. 9. Rapamycin does not inhibit TNF-{alpha} expression. PBMC were preincubated during 4 h with different concentrations of rapamycin and stimulated with TSST-1 for 5 h. Determination of TNF-{alpha} concentration in cell supernatants by ELISA. Cells not treated with rapamycin were incubated with vehicle (DMSO). One representative experiment of four is shown.

 
Inhibition of p38 MAPK inhibits the expression of TNF-{alpha} post-transcriptionally
The expression of TNF-{alpha} in monocytes stimulated with LPS has been suggested to be under control of p38 MAPK by a translational mechanism (4). To examine whether the p38 MAPK pathway mediates TNF-{alpha} translation in T lymphocytes induced by superantigen, we analyzed the effects of SB 203580, an inhibitor of p38 MAPK. SB 203580 directly inhibits p38 MAPK catalytic activity (IC50 of 0.6 µM) without influencing Erk, c-jun N-terminal kinase and Erk 6 activities (34). Figure 10Go(A) shows that SB 203580 inhibited the expression of TNF-{alpha} in supernatant but not the expression of the mRNA for TNF-{alpha} (Fig. 10B and CGo). The inhibitory effect was already significant (30%) with 1 µM SB 203580 and by increasing its concentration to 10 µM the inhibition of TNF-{alpha} expression reached 60%. WN added in combination with SB 203580 had an additive effect on its inhibition of TNF-{alpha} secretion (Fig. 10A and CGo).



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Fig. 10. SB 203580 inhibits TNF-{alpha} secretion. PBMC were preincubated for 1 h with different concentrations of SB 203580 and stimulated with TSST-1 for 5 h. (A) Determination of TNF-{alpha} concentration in cell supernatants by ELISA. (B) mRNA levels for TNF-{alpha} and GAPDH in the experiment shown in (A) were determined by RNase protection analysis and autoradiography as indicated in Fig. 3Go. Arrowheads indicate the position of protected probes hybridized to the TNF-{alpha} and GAPDH mRNAs. (C) Radioactivity in protected TNF-{alpha} bands in (B), normalized to protected GAPDH bands (expressed in arbitrary units). Cells not treated with inhibitors were incubated with vehicle (DMSO). One representative experiment of three is shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We report here that in superantigen-stimulated CD4+ T cells, the PI 3-K inhibitors WN and LY 294002 inhibit TNF-{alpha} biosynthesis, with control occurring at the level of translation of mRNA. The effect of these PI 3-K inhibitors is selective for TNF-{alpha}, since HLA class I molecules (Fig. 8Go) or total protein synthesis (data not shown) were not affected. This suggests that control of TNF-{alpha} biosynthesis in antigen-stimulated T cells is exercised, at least partially, by signaling through PI 3-K. Based on the inhibitory effect of WN, PI 3-K has also been reported to be involved in the signaling pathway controlling secretion of IL-2 in resting and activated human T cells (2325). However, the PI 3-K-controlled step during IL-2 synthesis and secretion was not reported in those studies.

PI 3-K is required in T cells for mitogenic activation of the protein kinases PDK1 and PKB, which in turn activate p70S6K (16). p70S6K plays a central role in selective translational up-regulation of a class of mRNAs containing a 5' oligopyrimidine tract at their transcriptional start site (35). It had been suggested that an upstream component in the pathway leading to p70S6K activation could also lead to phosphorylation and inactivation of the translational repressor 4EBP1 (31). PKB activity seems to be required and sufficient for inactivation of 4EBP1 in human embryonic kidney 293 cells (19). The activation of p70S6K and the phosphorylation of 4EBP1 are inhibitable with rapamycin (18,19,3033) . We therefore tested the effect of rapamycin on TNF-{alpha} expression. With some exceptions, we have not found a significant inhibition of TNF-{alpha} by rapamycin (Fig. 9Go), suggesting that there is no contribution of mTOR and, consequently, p70S6K and 4EBP1 to the translational regulation of TNF-{alpha} in T lymphocytes. Therefore, the signals generated by PI 3-K activity controlling translation of TNF-{alpha} probably follow another signaling pathway different from p70S6K. PI 3-K also has functions that are cell-type specific. For example, WN inhibited the IL-2-induced Erk activation in T cells (36), and a PI 3-K-dependent pathway regulates Erk activation in T cells and other hemopoietic cells (16,37) . This indicates that in T cells, PI 3-K could regulate Erk and, consequently, Mnk1 and eIF-4E activities (see below).

The expression of TNF-{alpha} in the human monocytic cell line THP-1 has been shown to be under translational control of p38 MAPK (4). Both Erk and p38 MAPK activate the kinase Mnk1 in vitro and in vivo (5,6). Mnk1 has been described to phosphorylate the translation initiation factor eIF-4E in vitro and in vivo (68). This indicates an important link between MAPK activation and translational initiation (68). We have found that by blocking p38 MAPK activity with the specific inhibitor SB 203580 (4,34), the secretion of TNF-{alpha} by T cells is dampened, without affecting the steady-state level of its mRNA (Fig. 10Go). This implicates p38 MAPK in the control of TNF-{alpha} biosynthesis also in T cells and raises the question of the role of PI 3-K in p38 MAPK activation. By blocking simultaneously both signaling pathways with WN and SB 203580, we observed an additive effect on the inhibition of TNF-{alpha} secretion (Fig. 10Go). This could suggest that PI 3-K and p38 MAPK signaling pathways are acting on TNF-{alpha} translation independently of each other. However, the inhibition on TNF-{alpha} secretion by using both inhibitors, WN and SB 203580, was incomplete, indicating that other signaling pathways controlling translation of TNF-{alpha} in T cells were involved. In relationship to this, the signaling pathways ras/raf-1 and SAPK/JNK have been described to be involved in derepression of the translational blockade, imposed by the TNF-{alpha} 3'-untranslated region in RAW mouse macrophages stimulated with LPS (38,39).

Further work will be necessary to characterize the relationship between the signaling pathways involved in control of TNF-{alpha} translation in T cells.


    Acknowledgments
 
This research was supported by grant SAF97-0111 from the CICYT (to E. E.), grant ERB 4061 PL 97-0701 from the European Community (to E. E.), and grants to N. F. from CIRIT, to M. B. from Dirección General de Enseñanza Superior e Investigación Científica, Ministerio de Educación y Cultura, to M. R. from Fundación Gran Mariscal de Ayacucho (Venezuela) and to C. P.-M. from University of Mérida (Venezuela). We thank Mr Enric Esplugues by the excellent artwork, Ms Susanna Castel, Ms Elisenda Coll and Mr Jaume Comas (`Serveis CientificoTecnics de la Universitat de Barcelona', Barcelona, Spain) for their expert assistance in confocal microscope and flow cytometry analysis, and Drs Pilar Lauzurica, Simon Mackenzie and Markus Nabholz for helpful suggestions and critical reading of the manuscript.


    Abbreviations
 
EBVEpstein–Barr virus
Erkextracellular signal-regulated kinase
LPSlipopolysaccharide
MAPKmitogen-activated protein kinase
PBMCperipheral blood mononuclear cell
PEphycoerythrin
PI 3-Kphosphatidylinositol 3-kinase
PKBprotein kinase B
TNFtumor necrosis factor
TSSTtoxic shock syndrome toxin
WNwortmannin

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: C. Terhorst

Received 24 November 1998, accepted 26 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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