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Pre-Exposure to Oxidative Stress Decreases the Nuclear Factor-B-Dependent Transcription in T Lymphocytes¹

Department of Microbiology and Immunology, University of Tampere Medical School, Tampere, Finland

	Abstract

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Reactive oxygen species (ROS) are used as signaling molecules in T cell activation. One of the main targets of ROS is the transcription factor nuclear factor-B (NF-B). NF-B-dependent transcription is inhibited by antioxidants, and the activation is induced or potentiated by ROS. However, chronic oxidative stress is known to reduce the activation of T cells and NF-B. To analyze these phenomena in more detail, we have exposed Jurkat T cells in vitro to oxidative stress (H₂O₂) at various times before or simultaneously with signals known to activate NF-B (phorbol dibutyrate (PDBu) and TNF). Simultaneously applied H₂O₂ strongly potentiated the PDBu- or TNF-induced transcriptional activity of NF-B. In contrast to this, H₂O₂ given 3 to 20 h before the activating signal reduced NF-B-dependent transcriptional activity. This was not due to the oxidation-induced modification of NF-B; cytoplasmic NF-B was able to bind to DNA after dissociation from IB by detergent treatment. H₂O₂ pre-exposure effectively inhibited the PDBu- or TNF-induced phosphorylation and degradation of IB, but H₂O₂ given simultaneously with PDBu or TNF enhanced the degradation. Oxidative stress was also followed by a strongly decreased ability to form intracellular ROS. Taken together, these data indicate that IB phosphorylation is the target of action of ROS, and as the ROS-forming capacity is weaker after chronic oxidative stress, IB is not effectively phosphorylated and degraded, thus leading to decreased NF-B-dependent transcription.

	Introduction

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Lymphocytes are exposed to reactive oxygen species (ROS)³ derived from activated macrophages and neutrophils during an inflammatory response. In addition to these exogenously derived ROS, it is now becoming more and more evident that ROS are produced within lymphocytes and are used as signaling molecules. Triggering of T cells via the TCR increases intracellular ROS levels, and antioxidants effectively down-regulate the early activation events in these cells (1, 2, 3). ROS activate several signaling systems within T lymphocytes, e.g., elevation of intracellular calcium, activation of protein kinase C, increased tyrosine phosphorylation, and activation of the Ras pathway (4, 5, 6, 7, 8). One of the more downstream targets of action of ROS is clearly the NF-B transcription factor, which is involved in the transcriptional regulation of several genes activated during immune and inflammatory responses (1, 2, 9). NF-B consists of Rel family proteins. These proteins are kept in cytoplasm in an inactive form by the IB family inhibitors. Cellular activation leads to phosphorylation, ubiquitination, and subsequent proteolytic degradation of IB, thus allowing the NF-B proteins to migrate to the nucleus where they bind to DNA, usually as dimers. The ability to control transcription depends on the composition of the NF-B complex as well as on the phosphorylation status of the NF-B proteins. In only a few cell types does ROS induce a sufficient signal to activate NF-B, but NF-B activation induced by several activators is uniformly inhibited by various antioxidants (10, 11). The exact localization of this redox-controlled step is not known.

During chronic infections or autoimmune diseases, lymphocytes are exposed to long-standing oxidative stress, and this is often associated with decreased T lymphocyte functions. Flescher et al. (12) have shown that normal peripheral blood T cells exposed to oxidative stress for 2 days in vitro have a decreased capacity to activate NF-B after TCR-mediated stimulation. To further analyze the difference between acute and chronic oxidative stress on NF-B activation, we now have pre-exposed Jurkat T lymphoma cells to hydrogen peroxide (which is an insufficient signal to activate NF-B) and analyzed its effect on the inducibility of NF-B-dependent transcription.

	Materials and Methods

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Cell cultures

Jurkat T lymphoma cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium (Life Technologies, Paisley, Scotland) containing 10% FCS (Life Technologies), 10 mM HEPES buffer, 2 mM L-glutamine, and antibiotics. During exponential growth, the cells were stimulated, at 10⁶ cells/ml, with either 100 ng/ml of phorbol dibutyrate (PDBu; Sigma Chemical Co., St. Louis, MO) or 20 ng/ml of TNF (recombinant human TNF, Genzyme Corp., Cambridge, MA) in the presence or the absence of H₂O₂ (0.1 mM) as described in Results.

Transfection, luciferase, and ß-galactosidase assays

Jurkat cells were transfected using the DEAE-dextran method. Cells (10 x 10⁶/ml) in RPMI 1640 were suspended with 250 µg of DEAE-dextran (Pharmacia Fine Chemicals, Uppsala, Sweden), 50 mM Tris-HCl (pH 7.5), 3 µg of NF-B-Luc plasmid p-55IgLuc, and 3 µg of ß-galactosidase control plasmid (13). p-55IgLuc contains three tandem Ig NF-B motifs driving a minimal (-55 to +19) human IFN-ß promoter (14). These plasmids were provided by Prof. K. Saksela (Institute of Medical Technology, Tampere, Finland). The amount of transfected DNA was equalized to 20 µg using herring sperm DNA (Sigma Chemical Co.). Samples were incubated at 37°C for 90 min. After incubation the samples were treated for 2 to 3 min with DMSO (final DMSO volume, 10%) and suspended in culture medium. Transfected cells were stimulated 24 h after the transfection. Luciferase activity was measured by using a commercial luciferase assay system (Promega, Madison, WI). ß-Galactosidase activity was measured in luciferase assay lysates using the following procedure: 50 µl of cell lysate, 5 µl of 10x lacZ buffer (10x lacZ = 500 mM NaCl, 100 mM MgCl₂, and 100 mM ß-ME), and 50 µl of 10 mM ONPG (Sigma). Samples were incubated at 37°C for 1 h, and the reaction volume was adjusted to 1 ml with H₂O. ß-Galactosidase activity was measured spectrophotometrically (OD = 420 nm).

Measurement of intracellular ROS

2'7'-Dichlorofluorescin diacetate (DCFH-DA; Molecular Probes, Inc., Eugene, OR) is a stable, nonfluorescent, cell-diffusible dye (15). Intracellular esterases cleave the acetyl groups from the molecule to produce nonfluorescent DCFH. This is trapped inside the cell, and in the presence of ROS, DCFH is further modified to fluorescent DCFH, which can be detected by flow cytometry. Cells were preloaded with 5 µM DCFH-DA at 37°C for 15 min and stimulated as indicated. Ten thousand individual data points were collected for each sample point using a Becton Dickinson FACScan flow cytometer (Mountain View, CA). The data are expressed as the mean fluorescence intensity. The mean fluorescence of unstimulated samples was subtracted from that of the stimulated ones at each data point.

Western blotting

The cytoplasmic protein fractions were prepared as previously described (16). Proteins (5 µg) were analyzed in 10% SDS-PAGE and transferred to Immobilon-P (PVDF) membranes (Millipore Corp., Bedford, MA). Membranes were incubated overnight at 4°C with anti-IB (1/1000) Ab (Santa Cruz Technology, Santa Cruz, CA) followed by horseradish peroxidase-conjugated swine anti-rabbit Ig (1/2000; Dako, Clostrun, Denmark) for 1 h at room temperature. To detect the mobility shift of phosphorylated IB, the cells were lysed with lysis buffer (New England Biolabs, Inc., Beverly, MA) containing phosphatase inhibitors. Proteins (50 µg) were fractioned in 15% SDS-PAGE and transferred to Immobilon-P (PVDF) membranes (Millipore). Membranes were incubated overnight at 4°C with anti-IB (1/1000) Ab (Santa Cruz) followed by biotin-conjugated anti-rabbit Abs (1/3000; Dako) for 30 min at room temperature and streptavidin-biotinylated horseradish peroxidase complex (1/5000; Amersham, Aylesbury, U.K.) for 20 min at room temperature. Signals were visualized using enhanced chemiluminescence system (ECL) according to the manufacturer’s recommendation (Amersham). Results were quantitated using densitometric scanning. The equal loading of proteins was verified by using Coomassie brilliant blue R staining according to the manufacturer of the PVDF membranes.

	Results

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Hydrogen peroxide pre-exposure decreases the transcriptional activity of NF-B

To examine the effect of hydrogen peroxide pre-exposure on the induction of NF-B-dependent transcription, the cells were transfected with a luciferase reporter plasmid (p-55IgLuc) containing three repeats of NF-B binding sites in front of a human IFN-ß promoter (14). To control the efficiency of transfection and the viability of the transfected cells, a ß-galactosidase plasmid was cotransfected together with the p-55IgLuc plasmid. The cells were treated with 0.1 mM H₂O₂ for either 3 or 20 h, and the cultures were stimulated with either TNF (20 ng/ml) or PDBu (100 ng/ml). Four hours thereafter the cells were lysed, and both luciferase and galactosidase activities were measured as described in Materials and Methods. The data shown in Figure 1 demonstrate that H₂O₂ pre-exposure clearly reduced both the TNF- and PDBu-induced NF-B-dependent transcription, while simultaneously applied H₂O₂ had an increasing effect.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. The effect of timing of H2O2 addition on NF-B-dependent transcriptional activity. Jurkat T cells were transfected with the p-55IgLuc plasmid (containing two NF-B binding sites in front of the IFN-ß promoter) together with a ß-galactosidase plasmid using the DEAE-dextran method. H2O2 (0.1 mM) was added to cultures either 20 or 3 h before or simultaneously (0 h) with TNF (20 ng/ml) or PDBu (100 ng/ml). After 4 h the cells were lysed, and the luciferase and galactosidase activities were measured. The luciferase activities were corrected according to the galactosidase activity of the same group. The data shown are expressed as the percent increase in the stimulation index (luciferase activity in the stimulated cells/that in the nonstimulated cells; mean of six independent experiments). The actual stimulation index of the control group varied between 13 and 30 in the case of PDBu and between 11 and 25 in the case of TNF.

Effect of hydrogen peroxide pre-exposure on IB

Nuclear localization of NF-B proteins is controlled by inhibitor proteins, of which IB is the most prominent. To test whether the decreased transcriptional activation capacity of NF-B after chronic oxidative stress is due to the decreased degradation of IB, the cytoplasmic extracts were analyzed in SDS-PAGE followed by immunoblotting with anti-IB. As previously shown (17, 18), stimulation of cells with TNF and PDBu induced the degradation and reformation of IB (Fig. 2A). Stimulation of cells together with PDBu and H₂O₂ or with TNF and H₂O₂ induced a clear degradation that was more long lasting in TNF- plus H₂O₂-stimulated cells than after TNF stimulation. When the cells were first treated with H₂O₂ for 3 h and then stimulated with PDBu or TNF, clear degradation was not detected. H₂O₂ alone did not induce IB-degradation (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Effect of H2O2 pre-exposure on IB. A, Jurkat cells were stimulated with PDBu (100 ng/ml), TNF (20 ng/ml), or PDBu/TNF plus H2O2 (0.1 mM), or cells were first incubated for 3 h with 0.1 mM H2O2 and then stimulated with PDBu/TNF. B, Jurkat cells were stimulated with 0.1 mM H2O2. Cells were collected at the time points indicated. Cytoplasmic extracts (5 µg) were fractioned with 10% SDS-PAGE and transferred onto PVDF membranes. C, To analyze the phosphorylation of IB the cells were stimulated with TNF for 5 min or first incubated for 3 h with 0.1 mM H2O2 and then stimulated with TNF. Total cellular extracts (50 µg) were fractioned with 15% SDS-PAGE and transferred onto PVDF membranes. Immunodetections were performed using anti-IB (1/1000) Ab. Arrowheads indicate the position of IB, and an arrow in C points the phosphorylated IB. The data shown are from one representative experiment of four performed.

Effect of hydrogen peroxide pre-exposure on the induction of intracellular ROS formation

As the NF-B activators are also potent inducers of intracellular ROS formation, we analyzed intracellular ROS levels using the ROS-reactive fluorochorome DCFH. In normal Jurkat cells H₂O₂ induced a strong ROS formation that had begun to increase by 5 min after the stimulation (Fig. 3). These levels declined to the baseline after 1 h. PDBu in untreated cells caused a much smaller response (note the different y-axis scale) that peaked 30 to 45 min after the stimulation (Fig. 4). In contrast to this, in the H₂O₂-preexposed (for 3 h) cells, PDBu had almost no effect. TNF as the inducer behaved in the same way as PDBu, but the kinetics of ROS formation were faster, and the down-regulative effect was not as strong (Fig. 5). Thus, it may be concluded that H₂O₂ pre-exposure had strongly decreased the ROS forming capacity of PDBu and TNF.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. The effect of H2O2 on intracellular ROS levels in Jurkat cells. Jurkat cells were preloaded with DCFH-DA and exposed to 0.1 mM H2O2. At different time points thereafter, the intracellular fluorescence was quantitated by flow cytometry. The data shown are expressed as an increase in mean fluorescence intensity (the mean fluorescence of unstimulated samples was subtracted from that of the stimulated samples at each time point). The data shown are from one representative experiment of three performed.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. The effect of H2O2 pre-exposure on the PDBu-induced intracellular ROS levels. Jurkat cells were preloaded with DCFH-DA, exposed to 0.1 mM H2O2 for 3 h, and stimulated with 100 ng/ml of PDBu. At different time points thereafter, the intracellular fluorescence intensity was quantitated by flow cytometry. The data shown are expressed as an increase in mean fluorescence intensity (the mean fluorescence of unstimulated samples was subtracted from that of the stimulated samples at each time point). The data shown are from one representative experiment of three performed.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. The effect of H2O2 pre-exposure on TNF-induced intracellular ROS levels. Jurkat cells were preloaded with DCFH, exposed to 0.1 mM H2O2, and stimulated with 20 ng/ml of TNF. At different time points thereafter, the fluorescence intensity was quantitated by flow cytometry. The data shown are expressed as an increase in mean fluorescence intensity (the mean fluorescence of unstimulated samples was subtracted from that of the stimulated samples at each time point). The data shown are from one representative experiment of three performed.

	Discussion

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Cells use various mechanisms to neutralize the effects of oxidative stress (25). Glutathione (GSH; L--glutamyl-L-cysteinyl-glycine) is probably the most important intracellular antioxidant. GSH reduces peroxides and is thus converted to the oxidized form, GSH disulfide. Activation of T cells via the TCR is known to decrease the GSH/GSH disulfide ratio (26). Thioredoxin (Trx) is a protein with two redox-active sulfhydryl groups, and it probably plays a significant role in the antioxidative response of T lymphocytes. Trx can both scavenge harmful ROS and regenerate enzymes whose critical cysteine residues have been oxidized. In Jurkat T cells hydrogen peroxide is an efficient inducer of Trx expression (27). Manganese-containing superoxide dismutase also contributes to cellular protection from oxygen toxicity, and its expression can be induced with ROS-forming cytokines such as TNF (28). The data shown in this report demonstrate that hydrogen peroxide induces in Jurkat cells an adaptive response that down-regulates ROS formation after a secondary stimulation. We do not know yet which of the mechanisms mentioned above is responsible for it. In the present experiments we analyzed the effect of this adaptation on regulation of the NF-B transcription factor, but it is probable that several T cell functions (both NF-B dependent and independent) are affected. For example, it could be expected that this adaptation would protect T cells from ROS-induced apoptotic cell death.

It has previously been shown that cells overexpressing catalase are unable to activate NF-B, but, in contrast, overexpression of Cu/Zn-dependent superoxide dismutase, which enhances the production of H₂O₂ from superoxide, potentiated NF-B activation (29). It has also been shown that the phosphorylation and degradation of IB are inhibited when GSH peroxidase is overexpressed (30). In this report we have shown that chronic oxidative stress reduced the intracellular ROS levels and inhibited the phosphorylation and degradation of IB. Chronic oxidative stress could enhance the function of detoxifiant enzymes, which could inhibit the new ROS formation induced by a second stimulus.

We have previously reported that naive T cells (CD45RA⁺) exposed to H₂O₂ demonstrate higher NF-B nuclear translocation than T cells of the activated/memory (CD45RO⁺) phenotype (31). As the maintenance of immunologic memory probably requires continuous or repeated antigenic contact (reviewed in Refs. 32 and 33), we hypothesized that memory cells are, consequently, repeatedly exposed to oxidative stress (delivered both from the TCR/CD28-mediated signals and from extracellular sources), and this would then modify their antioxidative capacity. The data reported here support this hypothesis, but it should be noted that the Jurkat cells used in the present studies are continuously proliferating, malignant cells, and although they are widely used as a model in T cell signaling studies, they do not necessarily behave in the same way as normal, resting T lymphocytes.

The data shown here might also explain some previously published discrepant observations. Shatrov et al. (34) observed that HIV gp120, which binds to the CD4 molecule on the T cell surface, is a strong inducer of intracellular ROS and consequently is able to augment the TNF-induced NF-B activation when given simultaneously with TNF. Jabado et al. (35) have shown that pretreatment with gp120 resulted in inhibition of phorbol ester-induced NF-B activity. Thus, it is likely that ROS induced by the gp120-CD4 interaction have a similar timing-dependent effect on NF-B activation as the exogenously added H₂O₂ used in our experiments. Moreover, it could be speculated that T cells activated by gp120 alone would be hyporeactive to a subsequent TCR/CD28 stimulus, thus providing one explanation for the T cell deficiency associated with HIV infection.

	Acknowledgments

 
The authors thank Ms. Mervi Tiilikainen for expert technical assistance.

	Footnotes

 
¹ This work was supported by grants from the Academy of Finland, the Research Foundation of Orion Corp. (to N.L.), the Scientific and Cultural Fund of Tampere City (to N.L.), and the Research Fund of the Tampere University Hospital (to N.L.).

² Address correspondence and reprint requests to Dr. N. Lahdenpohja, University of Tampere Medical School, POB 607, 33101 Tampere, Finland. E-mail address:

³ Abbreviations used in this paper: ROS, reactive oxygen species; NF-B, nuclear factor-B; PDBu, phorbol dibutyrate; DCFH-DA, 2'7'-dichlorofluorescein diacetate; PVDF, polyvinylidene difluoride; GSH, glutathione; Trx, thioredoxin.

Received for publication May 5, 1997. Accepted for publication October 22, 1997.

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