{gamma}-Glutamyl transpeptidase activity alters the T cell response to oxidative stress and Fas-induced apoptosis

Margaret L. Carlisle1, Miranda R. King1 and David R. Karp1

1 Simmons Arthritis Research Center, Department of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA

An abstract of the data was presented at the Annual Meeting of the American College of Rheumatology, November 2001.
Correspondence to: D. R. Karp; E-mail: david.karp{at}utsouthwestern.edu
Transmitting editor: A. Weiss


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The ectoenzyme {gamma}-glutamyl transpeptidase (GGT) is absent on resting naive peripheral blood T cells, highly expressed upon stimulation and intermediate on resting memory T cells. In other tissues, GGT is essential for the recapture of the antioxidant glutathione (GSH). T cells with different levels of GGT activity were examined for their ability to withstand oxidative stress. To create a model system that reflected the level of GGT seen on naive and memory T cells, Jurkat T cells were cloned by limiting dilution and their GGT expression analyzed. Jurkat expressing GGT at levels comparable to resting memory T cells have levels of intracellular reactive oxygen species (ROS) that are only 65% that seen in Jurkat that have low levels of GGT (similar to naive T cells). Treatment of the cells with H2O2 increases ROS in both cells, although the level seen in the GGThigh Jurkat is less than half that in the GGTlow variant. Despite protection from oxidative stress, the GGThigh Jurkat were found to be 2- to 3-fold more sensitive to Fas-induced apoptosis. The redox-regulated NF-{kappa}B pathway is activated in GGTlow cells, resulting in higher levels of cIAP-1/2 proteins that limit caspase activity. The GGTlow cells were found to have higher levels of NF-{kappa}B in the nucleus as well as lower levels of I{kappa}B-{alpha}. The GGTlow cells also express higher levels of the caspase inhibitors cIAP-1/2 and have lower levels of caspase activity. These findings suggest that GGT expression regulates ROS in T lymphocytes and modulates Fas-induced killing by altering NF-{kappa}B activity.

Keywords: apoptosis, human, T lymphocyte, transcription factor


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) are generated endogenously by all aerobic cells as byproducts of a number of metabolic reactions (1). Oxidative stress results from an imbalance in the pro-oxidant–anti-oxidant equilibrium during cell metabolism. While oxidative stress can damage cellular constituents and result in necrosis, the exposure of cells to low levels of ROS mimics several signaling events induced by extracellular stimuli. ROS have been shown to stimulate mitogen-activated protein kinases, cytosolic [Ca2+]i elevations, and the activation of p56lck and p72syk protein tyrosine kinases (25). Regulation of the intracellular redox environment seems to be critical for cellular proliferation, activation and cell survival (614).

The ability of oxidative stress to activate signal transduction and to induce tyrosine phosphorylation of multiple cellular proteins suggests that the intracellular oxidative state generated by excess ROS triggers distinct pathways that regulate the expression of certain transcription factors and genes (3,1519). Transcription factors known to be modulated by the intracellular redox state include NF-{kappa}B (20) and activating protein-1 (21,22). Different stimuli use oxidative stress conditions to activate NF-{kappa}B and induce gene expression (16,18,19,23). For example, pervanadate potentiates NF-{kappa}B activation via a Src family protein tyrosine kinase-dependent mechanism (24). Other forms of stress, such as exposure to UV light (3,24,25) and ionizing radiation, can generate ROS that activate Src family protein tyrosine kinases and subsequently activate NF-{kappa}B via an undefined signaling cascade.

Most of those studies have used chemical inducers of oxidative stress (e.g. H2O2) that mimic inflammation, but are often employed only for short-term exposures or at non-physiological concentrations. Another source of oxidative stress is via modulation of intracellular antioxidants, such as reduced glutathione (GSH) and reduced thioredoxin. GSH (L-{gamma}-glutamyl-L-cysteinylglycine) is the dominant, non-protein intracellular thiol, and plays an instrumental role in protection against oxidative stress and in the maintenance of the intracellular redox environment both by reacting directly with ROS, and as a substrate in the glutathione peroxidase-catalyzed detoxification of H2O2 and organic peroxides (26). Selected T cell functions can be potentiated in vivo by administration of GSH (27) and even a partial depletion of the intracellular GSH pool has significant consequences for a variety of lymphocyte functions (68,10,13,28), e.g. inhibition of cytotoxic T lymphocyte activity and inhibition of late activation events such as RNA/DNA synthesis.

The glutamyl cycle is important for the maintenance of intracellular concentrations of GSH, which are ~1000-fold greater than extracellular levels (29). In that cycle, {gamma}-glutamyl transpeptidase (GGT, EC 2.3.2.2, CD224) is a transmembrane ectoenzyme that is capable of initiating the breakdown of extracellular GSH by cleavage of the {gamma}-glutamyl bond. The metabolites can then be transported into the cell where a series of enzymatic reactions result in the de novo synthesis of intracellular GSH. We have previously reported that there is little or no GGT activity on resting peripheral blood T cells that phenotypically represent naive T cells, whereas GGT activity for resting memory T cells is markedly increased (30). Since GGT expression has been shown to facilitate cell survival under conditions of reduced cysteine availability, its regulated expression may affect many redox-regulated processes differently in naive and memory T cells. In this paper we show that GGT expression limits the production of intracellular ROS differently in naive and memory T cells. Moreover, activity of GGT in these cells alters the activity of NF-{kappa}B and regulates Fas-induced apoptosis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
The human T leukemic cell line Jurkat was obtained from the ATCC (Rockville, MD) and cultured in RPMI 1640 (Gibco/BRL, Gaithersburg, MD), 10% heat-inactivated FBS (Gibco/BRL), 2 mM L-glutamine, 10 mM HEPES and 10 µg/ml gentamicin. Jurkat cells were cloned by limiting dilution and their GGT expression analyzed by flow cytometry using mAb 3A8 [(30) gift from Dr V. Michael Holers, University of Colorado].

Preparation of separated peripheral blood T cell subsets
Peripheral blood was obtained from normal donors. Mononuclear cells were separated from venous blood by density centrifugation over Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). CD4+CD45RA T cells were prepared by immunomagnetic depletion using mAb to CD8 (clone OKT8), CD11b (clone LM2/1.6.11), CD14 (clone 63D3), CD16 (clone B73.1), CD19 (clone HD37), HLA-DR (clone L243) and CD45RA (clone 2H4). CD4+CD45RO T cells were prepared from peripheral blood mononuclear cells by immunomagnetic depletion using mAb to CD8, CD11b, CD14, CD16, CD19, HLA-DR and CD45RO (clone UCHL1). The anti-CD45RO and anti-CD95 (clone DX2) mAb were purchased from BD PharMingen (San Diego, CA); all other mAb were obtained through the ATCC.

GGT expression and enzymatic activity determination
Cells were stained with 3A8 and phycoerythrin-conjugated goat anti-mouse IgG, followed by flow cytometry. GGT enzymatic activity was determined by resuspending 1 x 106 cells in 1 ml of PBS containing 2.5 mM {gamma}-glutamyl p-nitroanilide and 60 mM glycyl-glycine. After 90 min at 37°C, the cells were centrifuged and the absorbance of the supernatant was read at 410 nm. The enzyme activity was expressed as nmol p-nitroaniline released/min/106 cells.

Intracellular thiol measurement
Intracellular thiols were measured by flow cytometry, as described (30). Briefly, cells were stained with either 100 µM N-ethyl maleimide (NEM; Sigma, St Louis, MO) and 40 µM monobromobimane (MBB; Molecular Probes, Eugene, OR) or MBB alone. Data were collected on a FACStar Plus (Becton Dickinson, San Jose, CA) equipped with dual lasers and controlled by CellQuest Plus (Becton Dickinson) software. MBB/thiol fluorescence was excited by the UV laser tuned to 320 nm and emission measured through a 450-nm bandpass filter. MBB/thiol fluorescence was measured on a logarithmic scale, with the amplifier set so that Fluoresbrite carboxy BB 6-µm microspheres (Polysciences, Warrington, PA) gave a mean fluorescence intensity of 2800.

Detection of intracellular ROS
Peripheral blood T cells or Jurkat clones were loaded with 10 µM dichlorofluorescin diacetate (DCF-DA; Molecular Probes) for 30 min before treatments to induce oxidative stress. After the indicated treatments, the cells were harvested into 2 ml of cold PBS, pelleted and resuspended in cold PBS. The cells were analyzed for changes in mean fluorescence intensity with a FACScan (Becton Dickinson). The amplifier gain was set by eye so that the mean fluorescence intensity of untreated cells was ~100.

Induction of apoptosis
The Fas-bearing Jurkat clones were cultured in RPMI 1640 and 10% dialyzed FBS for 2 h in the presence of 100 ng/ml of the apoptosis-inducing monoclonal anti-human CD95 antibody (clone CH11; Upstate Biotechnology, Lake Placid, NY). These cells were incubated in the absence or presence of the indicated concentrations of the GGT inhibitor acivicin, H2O2 and cysteinylglycine (Cys–Gly), all from Sigma.

Assessment of phosphatidylserine externalization
Jurkat cells were harvested and resuspended in 100 µl of FITC-conjugated Annexin V (Caltag, Burlingame, CA) at 1 µg/ml in binding buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2). After 10 min at room temperature, each sample was stained with propidium iodide (PI, 1 µg/ml). Apoptotic cells were identified as PI cells that bound Annexin V.

Assessment of internucleosomal DNA fragmentation by agarose gel electrophoresis
J.GGT.1 and J.GGT.2 cells were stimulated with anti-Fas mAb for 2 h, and then DNA was extracted from the cells, as in (31). The DNA samples were resolved on a 1% TAE horizontal agarose gel containing ethidium bromide and then viewed using an AlphaImager digital imaging system (Alpha Innotech, San Leandro, CA).

Western blot analysis
Following treatments with the indicated combinations of 200 µM acivicin, 100 ng/ml Fas mAb, 10 µM Cys–Gly and 50 µM H2O2, Jurkat cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40, 0.5% DOC and 0.1% SDS), protease inhibitors (1 µM PMSF, 10 µg/ml aprotinin, 90 µM leupeptin and 73 µM pepstatin A) and 10 mM iodoacetimide on ice for 30 min. The protein content was measured by the method of Bradford. Proteins were resolved by SDS–PAGE gels and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). Western blots were performed with goat anti-caspase-3, rabbit anti-cIAP-1 and cIAP-2, rabbit anti-XIAP, mouse anti-human I{kappa}B-{alpha} or mouse anti-human NF-{kappa}B p65, followed by blotting with HRP-coupled anti-IgG secondary antibody, and detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). The antibodies against the inhibitor of apoptosis proteins were obtained from R & D Systems (Minneapolis, MN) and the anti-human NF-{kappa}B p65 mAb was purchased from BD Transduction Laboratories (San Diego, CA); the other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Colorimetric assay of caspase activity
J.GGT.1 or J.GGT.2 cells (3 x 106) were treated for 2 h with anti-CD95 mAb in the presence or absence of 10 µM Cys–Gly and then harvested. These cells were lysed for 10 min on ice in 50 µl of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl and 1% Triton X-100. The samples were incubated with 50 mM of the appropriate caspase substrate (caspase-3, Ac-DEVD-pNA; caspase-6, Ac-VEID-pNA; caspase-8, Ac-IETD-pNA; all from Alexis, Carlsbad, CA) in reaction buffer (100 mM HEPES, pH 7.4, 150 mM NaCl and 0.2% CHAPS) containing 5 mM DTT for 1 h at 37°C. The samples were plated in a 96-well microtiter plate and the optical density at 405 nm was determined using an ELISA plate reader.

NF-{kappa}B electrophoretic mobility shift assay
Jurkat cells (10 x 106) were treated with the indicated combination of 100 ng/ml anti-CD95 mAb, 10 µM Cys–Gly and 50 µM H2O2 for 2 h at 37°C. The cell pellet was resuspended in 100 µl of cell lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM EDTA, 0.25 mM DTT, 0.2 mM PMSF, 50 mM NaF, 1 mM NaVO4, 0.5 mM ß-glycerophosphate, 0.05% NP-40, 42.8 µg/ml leupeptin, 10 µg/ml aprotinin and 48.6 µg/ml pepstatin A) on ice for 10 min. The nuclear pellets were resuspended in 50 µl of ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and 0.5 mM PMSF) on ice for 20 min. The nuclear extract was then centrifuged for 5 min at 4°C and snap frozen. The protein content was measured by the method of Bradford. Electrophoretic mobility shift assays were performed by incubating 20 µg of nuclear extract with 60,000 c.p.m. of 32P end-labeled probes in a 20 µl final volume of binding buffer containing 10 mM HEPES, pH 7.9, 60 mM KCl, 25 mM MgCl2, 0.5 mM EDTA, 2.5 mM DTT, 12% glycerol and 4 µg of poly(dI–dC)·poly(dI–dC) for 15 min at room temperature. The following coding strand templates and probe designations were used in this study: 5'-CAA CGG CAG GGG AAT TCC CCT CTC CTT-3' ({kappa}B-PD) and 5'-CAA CGG CAG ATC TAT CTC CCT CTC CTT-3' ({kappa}B-MT) (32).

Statistics
All experiments were performed at least twice and data reported are means ± SE. Statistical analyses were performed using the two-tailed Student’s t-test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Jurkat T cells model GGT expression and GGT enzymatic activity of resting memory and naive peripheral blood T cells
We previously determined the expression of GGT on subsets of human peripheral blood T cells by two- and three-color flow cytometry (30). GGT expression is present at a very low level on the majority of CD4+ and CD8+ T cells. Within the resting CD4+ T cell population, expression of GGT is heterogeneous. There is little, if any, GGT expressed by CD45RA+ cells. CD45RO+ cells express low, but enzymatically significant levels of GGT. Since catabolism of GSH by GGT+ cells could effect GGT cells in a mixed culture, immunomagnetic separation was used to purify resting naive and memory T cells. Figure 1(A) shows the expression of GGT on CD4+ T cells isolated on the basis of CD45 isoform expression. Negatively selected peripheral blood T cell subpopulations were at least 95% CD3+ and CD4+, and the memory T cells were >95% CD45RO+, while the naive T cells were typically 99% CD45RA+. As expected, CD45RA+ cells (isolated by depletion of CD45RO+ cells) do not express GGT, while CD45RO+ cells do. Table 1 shows the GGT enzymatic activity for freshly isolated CD45RA+CD4+CD3+ and CD45RO+CD4+CD3+ peripheral blood mononuclear cells. There was little or no observed enzymatic activity on the resting T cells that phenotypically represented naive T cells. GGT enzymatic activity for resting memory T cells was ~10- to 15-fold that measured for naive T cells.



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Fig. 1. (A) Surface expression of GGT antigen. CD4+CD45RA+ and CD4+CD45RO+ peripheral blood T cells and Jurkat clones J.GGT.1 and J.GGT.2 were stained with 3A8, and analyzed by flow cytometry. GGT staining is shown in solid lines and an isotype control is shown in dotted lines. (B) Effect of increased GGT enzymatic activity on ROS in CD4+CD45RO+ peripheral blood T cells as compared to CD4+CD45RA+ T cells. Purified CD4+CD45RO+ and CD4+CD45RA+ peripheral blood T cells were cultured for 2 h with or without 200 µM acivicin and exposed to 25 µM H2O2 for 1 h. Intracellular ROS were determined by flow cytometry. In each case, two experiments were performed in triplicate and values shown are means ± SEM from a representative experiment. (C) Intracellular thiol levels for Jurkat clones. J.GGT.1 and J.GGT.2 cells were stained with 40 µM MBB. Background staining was determined by pretreating controls with 100 µM NEM. Two experiments were performed in duplicate and values shown are means ± SEM from a representative experiment. *P < 0.05 versus J.GGT.2.

 

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Table 1. GGT enzymatic activity of peripheral blood t cell subpopulations and Jurkat clones
 
The mean level of intracellular thiol (largely GSH) in CD4+CD45RO+ T cells is 14% greater than that of CD4+CD45RA+ T cells (30). This observation supports the hypothesis that up-regulation of GGT on memory T cells provides these cells with a mechanism for more efficient recapture of released GSH. As a result, the increased GGT activity on CD45RO+ T cells should be better able to blunt the production of intracellular ROS. To test the response of memory and naive T cells to an oxidative stress in vitro, the CD45RA+ and CD45RO+ T cells were cultured for 2 h with or without 200 µM acivicin in order to inhibit GGT enzymatic activity. The cells were then exposed to 25 µM H2O2 for 1 h. Inhibition of the GGT activity of the CD45RO+ T cells caused the level of intracellular ROS to increase to the level of intracellular ROS observed for CD45RA+ T cells (Fig. 1B). Thus, the increased expression of GGT on CD4+ memory (CD45RO+) T cells confers an increased ability to blunt the response to oxidative stress compared to CD4+ naive (CD45RA+) T cells.

To create a model system that reflected the level of GGT seen on naive and memory T cells, we cloned Jurkat cells by limiting dilution and analyzed their GGT expression by flow cytometry using the mAb 3A8. Unselected populations of Jurkat have heterogeneous GGT expression (not shown). Several clones were obtained that expressed GGT levels close to memory and naive peripheral blood T cells. The level of GGT expression on representative low (J.GGT.1) or high (J.GGT.2) expressing clones is shown in Fig. 1(A). The level of GGT enzymatic activity for subpopulations of peripheral blood T cells and the Jurkat clones parallels the expression of GGT antigen by flow cytometry (Table 1). We examined the differences in intracellular thiol (largely GSH) in our Jurkat clones by flow cytometry using the GSH-reactive dye, MMB. The results of a representative analysis are shown in Fig. 1(C). The mean level of intracellular thiol (GSH) in the J.GGT.2 cells was ~30% greater than that of the J.GGT.1, consistent with the published finding for peripheral blood T cells (30) and supporting the hypothesis that GGT participates in the recapture of GSH by T cells. Furthermore, Cys–Gly, a product of GGT-catalyzed GSH degradation and a source of cysteine for intracellular GSH synthesis (33,34), increases intracellular GSH levels in Ramos transfectants (35).

Increased GGT activity protects Jurkat T cells from oxidative stress
As with naive and memory peripheral blood T cells, the varied expression of GGT on Jurkat clones should lead to different abilities to withstand oxidative stress. Figure 2(A) shows that the level of intracellular ROS in J.GGT.1 cells is >50% higher than that seen in J.GGT.2. Treatment of the cells with H2O2 increases ROS in both cells, although the level seen in the J.GGT.1 cells is more than twice the level in the GGThigh variant. To confirm that GGT activity explained this observation, the two Jurkat clones were labeled with DCF-DA and then exposed to 25 µM H2O2 in the presence or absence of 10 µM Cys–Gly. Treatment with Cys–Gly decreased the level of ROS and reversed the effects of lower GGT enzymatic activity (Fig. 2B). This suggests that the ability of Jurkat cells to combat oxidative stress is controlled, in part, by the GGT expressed on their cell surfaces. Next, the effect of inhibiting GGT activity was studied. The J.GGT.1 and J.GGT.2 cells were cultured for 2 h with or without the GGT inhibitor acivicin. The cells were then exposed to peroxide along with 10 µM GSH, 10 µM Cys–Gly or neither. Inhibition of GGT activity on the J.GGT.2 cells caused the level of intracellular ROS to increase to that observed for J. GGT.1 cells that were not treated with acivicin (Fig. 2C and D). In the absence of acivicin, ROS decreased by ~50% when the stressed J.GGT.2 cells were given GSH. However, when GGT activity was inhibited, GSH was unable to blunt the peroxide-induced rise in ROS in J.GGT.2 cells. The effect of acivicin on J.GGT.2 cells is reversed by Cys–Gly. In the absence of acivicin, J.GGT.1 cells given Cys–Gly have the same level of ROS as seen in J.GGT.2 cells. There is still a difference in DCF fluorescence between the J.GGT.1 cells and J.GGT.2 cells after treatment with acivicin, suggesting that short-term inhibition of the ability to metabolize extracellular GSH does not totally compensate for the higher intracellular thiol level in the cells at the start of the experiment (see Fig. 1). Therefore, the effects of acivicin and Cys–Gly on ROS levels in the Jurkat clones confirm that increased GGT expression on the cell surface, and the associated increased GGT enzymatic activity, allows cells to better recapture GSH and, thereby, adjust to alterations to their redox environments.



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Fig. 2. Effect of increased GGT enzymatic activity on ROS in Jurkat clones. (A) J.GGT.1 and J.GGT.2 clones were labeled with DCF-DA for 30 min. Intracellular ROS were determined by flow cytometry. *P < 0.0001 versus J.GGT.2. (B) Cells were cultured with 25 µM H2O2 for 1 h in the presence or absence of 10 µM Cys–Gly. {dagger}Not statistically different from untreated J.GGT.2. *P < 0.0001 versus untreated J.GGT.2. **P < 0.0001 versus Cys–Gly-treated J.GGT.2. (C and D) J.GGT.1 and J.GGT.2 cells were cultured for 2 h with or without 200 µM of the GGT inhibitor acivicin. As indicated, cells were then exposed to 25 µM H2O2 and simultaneously treated with 10 µM GSH, 10 µM Cys–Gly or neither. P < 0.01 for differences between J.GGT.1 and J.GGT.2 for all conditions. In each case, three experiments were performed in triplicate and values shown are means ± SEM from a representative experiment.

 
Jurkat T cells with higher levels of GGT activity are more sensitive to apoptosis
The normal reducing environment required for cellular integrity is provided by GSH. By exceeding intracellular reducing capacity, oxidative stress can induce programmed cell death [reviewed in (36) (37)]. Multiple apoptotic mechanisms, such as Fas (CD95)-mediated apoptosis and activation-induced cell death, are regulated by GSH levels, redox potential or ROS (3841). However, oxidative stress has been reported to have both anti-apoptotic and pro-apoptotic effects (16,42). Based on this information, the role of GGT expression in apoptosis of Jurkat cells was investigated.

Jurkat clones were stimulated with 100 ng/ml of anti-Fas mAb in the presence or absence of 50 µM H2O2 for 2 h. A subset of the cells was also treated with 10 µM Cys–Gly. Apoptosis was analyzed by staining with FITC-conjugated Annexin V and PI. While the J.GGT.2 cells were protected from oxidative stress, they were found to be 2- to 3-fold more sensitive to Fas-induced apoptosis (Fig. 3A). The two Jurkat clones were stained with anti-Fas (Fig. 3D). The density of CD95 was the same on both cell types. Thus the differences in apoptosis were not merely due to differences in initial signaling potential. Anti-Fas treatment by itself did not alter the intracellular ROS level as measured by DCF, although CD95 cross-linking did cause a 28% increase in ROS of cells also treated with H2O2 (data not shown). Treatment with Cys–Gly reversed the anti-apoptotic effects of lower GGT activity. The apoptosis results with the J.GGT.1 and J.GGT.2 cells were confirmed with an additional panel of single-cell clones, selected on the basis of their GGT expression (Table 2). With greater concentrations of CH11 or prolonged treatment with the mAb (>4 h) the differences in the percent of Annexin V+ cells were less pronounced (data not shown). Therefore, the effect of GGT activity is to make cells more susceptible to brief or low-level stimulation through CD95.



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Fig. 3. Effect of increased GGT enzymatic activity on the susceptibility of Jurkat clones to apoptosis. (A) J.GGT.1 and J.GGT.2 cells were activated with 100 ng/ml CH11, an anti-CD95 mAb, for 2 h. Subsets of those cells were simultaneously cultured with the indicated combinations of 50 µM H2O2 and 10 µM Cys–Gly. The cells were then stained with FITC-conjugated Annexin V and PI, and analyzed by flow cytometry. (B) J.GGT.1 and J.GGT.2 cells were cultured in the presence or absence of 100 ng/ml CH11 for 2 h. DNA was extracted from the cells and electrophoresed on a 1% TAE horizontal agarose gel containing ethidium bromide. (C) Jurkat clones were cultured for 2 h with or without 200 µM acivicin. Following that incubation, the cells were stimulated with 100 ng/ml CH11 and simultaneously subjected to 50 µM H2O2 for 2 h. A subset of the cells was treated with 10 µM Cys–Gly. In each case, three experiments were performed in triplicate and values shown are means ± SEM from a representative experiment. (D) The Jurkat clones were stained with anti-CD95 and goat anti-mouse IgG–phycoerythrin secondary antibody and analyzed by flow cytometry.

 

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Table 2. Anti-Fas- and H2O2-induced apoptosis by Jurkat clones
 
It has been shown that oligonucleosomal cleavage of DNA occurs in the majority of Jurkat cells very soon after anti-Fas exposure (43). To test this, cytoplasmic DNA from J.GGT.1 and J.GGT.2 was analyzed by agarose gel electrophoresis. The internucleosomal cleavage of DNA was much greater in the J.GGT.2 cells than in the J.GGT.1 cells after a 2-h exposure to CH11 (Fig. 3B). There was no DNA cleavage in cells treated with H2O2 alone nor did peroxide appear to change the relative levels of fragmented DNA in the two cell types (data not shown).

To confirm that GGT activity was responsible for these findings, cells were pretreated with 200 µM acivicin for 2 h and then exposed to 100 ng/ml anti-Fas mAb, 50 µM H2O2 and 10 µM Cys–Gly, as indicated, for an additional 2 h. Acivicin treatment caused the sensitivity of the J.GGT.2 cells to Fas-induced apoptosis to decrease to the level observed for the J.GGT.1 cells (Fig. 3C). Treatment with 10 µM Cys–Gly alone or in combination with acivicin did not significantly affect the degree of apoptosis by the J.GGT.2 clone but increased the degree of apoptosis by the J.GGT.1 cells to a level comparable to that of the J.GGT.2 cells. The effect of acivicin treatment was most profound when the cells were subjected to exogenous oxidative stress from H2O2. Acivicin treatment of the J.GGT.2 clone resulted in an ~13% decrease in Fas-induced apoptosis in the absence of H2O2 (data not shown).

Jurkat T cells with higher levels of GGT activity have more caspase activity
Diverse stimuli that cause apoptosis, including ligation of Fas, transmit their death signals through activation of caspases. We investigated the activity of selected caspases in the Jurkat clones expressing different levels of GGT. Activation of CD95 with the agonistic mAb CH11 resulted in an increase in the activity of the caspase-3, -6 and -8 (Fig. 4A). The Fas-induced J.GGT.2 cells consistently have more caspase activity than do similarly treated J.GGT.1 cells. Simultaneous incubation with Cys–Gly increases the level of caspase-3 and -8 activity in J.GGT.1 cells to that observed in J.GGT.2 (Fig. 4B), suggesting that modulation of intracellular ROS associated with GGT activity can alter caspase activity.



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Fig. 4. Effect of increased GGT enzymatic activity on caspase activity during Fas-induced apoptosis. (A) J.GGT.1 and J.GGT.2 cells (3 x 106) were treated with 0 or 100 ng/ml CH11 for 2 h. Cell extracts were incubated with Ac-DEVD-pNA, Ac-VEID-pNA or Ac-IETD-pNA, caspase-3, -6 and -8 substrates respectively. (B) Cells were treated and analyzed as in (A) except that a subset of cells exposed to CH11 was also simultaneously subjected to 10 µM Cys–Gly, and only caspase-3 and -8 activity were examined. Two experiments were performed in triplicate and values shown are means ± SEM from a representative experiment.

 
Acivicin and Cys–Gly treatment decreases the NF-{kappa}B activity in GGTlow Jurkat to the levels seen for comparably treated GGThigh cells
ROS have been reported to strongly activate the transcription factor NF-{kappa}B in lymphocytes or in various transformed cell lines (16,44). Since increased GGT activity leads to decreased intracellular ROS, we performed gel shift assays to determine whether different levels of GGT activity could modulate the induction of this transcription factor in Jurkat cells. Analysis of nuclear extracts from Jurkat clones shows that NF-{kappa}B activity in J.GGT.1 cells is 4- to 5-fold greater than the activity observed in J.GGT.2 cells (Fig. 5A, lanes 1 and 2). To rule out the possibility that J.GGT.2 cells had less NF-{kappa}B overall, Western blotting was performed on cytoplasmic extracts (Fig. 5B). In untreated Jurkat cells, the amount of NF-{kappa}B p65 in the cytoplasm of the J.GGT.2 cells is greater than that within the cytoplasm of the J.GGT.1 cells. This suggests that the greater oxidative stress in the J.GGT.1 cells causes more tonic NF-{kappa}B activation and nuclear translocation. Furthermore, Western blot analyses of I{kappa}B-{alpha} content of the cytoplasm of Jurkat clones revealed 4- to 5-fold more of this inhibitory protein in the untreated J.GGT.2 cells than in J.GGT.1 (not shown). Thus, the higher level of intracellular ROS in these cells appears to augment activation of kinases that lead to phosphorylation and degradation of I{kappa}B-{alpha}.



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Fig. 5. (A) Effect of increased GGT activity on NF-{kappa}B nuclear translocation in Jurkat clones. NF-{kappa}B-binding activity was tested by gel shift assay, using either a [32P]{kappa}B oligonucleotide (lanes 1 and 2) or a 32P probe in which the {kappa}B binding site has been mutated (lanes 3 and 4). (B) Effect of increased GGT activity on cytosolic NF-{kappa}B p65 in Jurkat clones. Cytosolic protein from untreated J.GGT.1 and J.GGT.2 cells was analyzed by Western blot using mouse anti-NF-{kappa}B p65 mAb. (C) Effect of increased GGT activity on NF-{kappa}B nuclear translocation in acutely stressed Jurkat clones. J.GGT.1 and J.GGT.2 cells were cultured for 2 h with or without 200 µM of the GGT inhibitor acivicin. Subsets of the cells were then treated with 50 µM H2O2 or 10 µM Cys–Gly for 2 h, as indicated. Gel shift analysis was performed using a [32P]{kappa}B oligonucleotide. (D) Effect of increased GGT activity on I{kappa}B-{alpha} levels in acutely stressed Jurkat clones during Fas-induced apoptosis. J.GGT.1 and J.GGT.2 cells were pretreated with acivicin as in (C). Subsets of the cells were then treated with 100 ng/ml CH11, 50 µM H2O2 or 10 µM Cys–Gly for 2 h, as indicated and Western blotting was performed with mouse anti-I{kappa}B-{alpha} mAb. The data shown are from one representative experiment of three performed with identical results. H, GGThigh Jurkat clone (J.GGT.2); L, GGTlow Jurkat clone (J.GGT.1).

 
To test whether GGT enzymatic activity explained the observed differences in NF-{kappa}B activity in the Jurkat clones, we pretreated a subset of the J.GGT.1 and J.GGT.2 cells with 200 µM acivicin. All of the cells were then exposed to oxidative stress (50 µM H2O2) for 2 h in the presence or absence of Cys–Gly (10 µM) and nuclear extracts from these cells were analyzed by electrophoretic mobility shift assay. Treatment with anti-Fas alone led to a the NF-{kappa}B activation detected in the nuclear extracts of acivicin-treated J.GGT.2 cells increased to a level equivalent to that seen for J.GGT.1 cells. Acivicin treatment does not affect the intensity of the activated NF-{kappa}B complex for the J.GGT.1 Jurkat beyond that seen for cells receiving H2O2 alone. Exposure to exogenous Cys–Gly inhibited ROS-mediated NF-{kappa}B activation for J.GGT.1 cells treated with H2O2 alone or in combination with acivicin (Fig. 5C, lanes 8 and 10). These data, taken together, indicate that changes in GGT activity alter the intracellular redox environment and thus modulate NF-{kappa}B activation.

The NF-{kappa}B nuclear translocation results were substantiated by examining the level of I{kappa}B-{alpha} using Western blot analyses. Peroxide treatment alone did not alter the ratio of I{kappa}B-{alpha} present in CH11-treated J.GGT.1 and J.GGT.2 cells (Fig. 5D, cf. lanes 3 and 4 to lanes 1 and 2). When the cells were exposed to acivicin and H2O2 together, there was an increase in the degradation of I{kappa}B-{alpha} in the J.GGT.2 cells. The I{kappa}B-{alpha} protein in J.GGT.2 cells exposed to CH11, H2O2 and acivicin was degraded to a level equivalent to that seen in J.GGT.1 cells (Fig. 5D, lanes 5 and 6). Exposure to exogenous Cys–Gly inhibited ROS-mediated I{kappa}B-{alpha} degradation for CH11-stimulated J.GGT.1 cells treated with H2O2 alone or in combination with acivicin (Fig. 5D, lanes 8 and 10). CH11 treatment alone did not lead to I{kappa}B activation/degradation under these conditions (Fig. 5, lanes 1 and 2) nor did CH11 alone lead to nuclear translocation of NF-{kappa}B (data not shown). Thus, the differences seen between J.GGT.1 and J.GGT.2 are attributable to their different responses to oxidative stress and not to different susceptibility to anti-Fas.

cIAP-1 levels increase in GGTlow Jurkat T cells with Fas-induced apoptosis
Caspase activation is tightly regulated by interactions with inhibitor-of-apoptosis proteins (IAP). At least five mammalian IAP have been described (4547). XIAP, cIAP-1 and cIAP-2 bind to and inhibit active caspase-3 and -7 at subnanomolar (XIAP) to submicromolar (cIAP-1/2) concentrations (48,49). Since XIAP, cIAP-1 and cIAP-2 are induced by NF-{kappa}B, it seemed possible that one or more of these inhibitory proteins might be up-regulated by increased ROS in T cells with low levels of GGT activity. There are no discernable differences in the levels of XIAP protein in the whole-cell lysates prepared from the J.GGT.1 and J.GGT.2 cells (not shown). Similarly, Western blots performed on cell lysates of untreated J.GGT.1 and J.GGT.2 show that the cIAP-1/2 levels are equivalent in spite of differences in intracellular ROS resulting from chronic oxidative stress (Fig. 6, lanes 1 and 2). J.GGT.1 cells activated by CH11 alone or with peroxide contain more cIAP-1 protein than do similarly treated J.GGT.2 cells (Fig. 6, lanes 3–6). The level of cIAP-1 in CH11-stimulated J.GGT.1 and J.GGT.2 clones receiving acute oxidative stress (50 µM H2O2) (Fig. 6, lanes 5 and 6) is greater than in the cells receiving CH11 treatment only (Fig. 6, lanes 3 and 4). In the presence of acivicin and H2O2, the level of cIAP-1 in J.GGT.2 cells was equivalent to that in J.GGT.1 (Fig. 6, lanes 7 and 8). By decreasing the ROS within the J.GGT.1 cells, exposure to exogenous Cys–Gly prevented the NF-{kappa}B-induced increase in cIAP-1 for CH11-stimulated J.GGT.1 cells (Fig. 6, lanes 10 and 12). These findings suggest that expression of GGT alters the intracellular redox environment, leading to regulation of NF-{kappa}B activity. Caspase activity is limited in these cells, in part, through the up-regulation of cIAP-1.



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Fig. 6. Effect of increased GGT activity on cIAP-1/2 protein in Jurkat clones. J.GGT.1 and J.GGT.2 cells were cultured for 2 h with or without 200 µM acivicin. The cells were then treated with 50 µM H2O2 and simultaneously stimulated with 100 ng/ml CH11. A subset of the cells was also treated with 10 µM Cys–Gly. Cytoplasmic protein was analyzed by Western blot using rabbit anti-cIAP-1 and anti-cIAP-2 antibodies. The data shown are from one representative experiment of three performed with identical results. H, GGThigh Jurkat clone (J.GGT.2); L, GGTlow Jurkat clone (J.GGT.1).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we present evidence that there is a clear relationship between the level of GGT activity and the functional consequences of oxidative stress in T cells. Increased GGT expression on T cells provides these cells with a mechanism for more efficient recapture of released GSH (30). Although differences in responses to oxidative stress have been reported for malignant T cell lines and, to a lesser extent, for normal, peripheral blood T cells of varying differentiation stages (50), those results did not attribute the varied responses to differences in GGT activity levels of the cells. Our data indicate that the activity of GGT on the cell serves to limit the production of ROS in response to oxidative stress. In the presence of increasing oxidative stress, treatment with acivicin, an irreversible inhibitor of GGT enzymatic activity, increases the level of intracellular ROS. Treatment with 10 µM Cys–Gly was sufficient to reverse the effects of decreased GGT activity seen in untreated J.GGT.1, or naive peripheral blood T cells, or as the result of acivicin treatment of J.GGT.2 or memory T cells. Thus, the ability of normal and transformed cells to combat oxidative stress is controlled, in part, by the GGT expressed on their cell surfaces.

Although increased GGT activity helps cells control ROS production, it is the decreased metabolism of extracellular GSH that unexpectedly decreases the susceptibility of Jurkat to undergo programmed cell death. Many of the chemical and physical stimuli that elicit apoptosis generate ROS, such as H2O2 and hydroxyls (51), which at low levels modulate apoptotic signal transduction pathways (52). Our findings show that the redox regulated NF-{kappa}B pathway can be induced when cells with reduced GGT enzymatic activity are pretreated with mild oxidative stress. This can be attributed to the antioxidant role of GGT in T cells. Cells use their intracellular GSH reserves to blunt the intracellular ROS introduced via the peroxide treatment. Fas-induced apoptosis of Jurkat T cells has been reported to result in a rapid and specific export of GSH (40), and, as a consequence, cells with decreased GGT expression are less capable of replacing that cellular GSH due to decreased cleavage of extracellular GSH and lower levels of key precursors for de novo GSH synthesis. Cytoplasmic GSH levels are determined largely by the availability of its precursor, L-cysteine, and the most significant source of L-cysteine in blood plasma is L-cystine. In contrast to most cells, T lymphocytes have a very low transport activity for L-cystine and hence a low baseline supply of the GSH precursor (53,54). Therefore, T cells will be highly susceptible to changes in intracellular redox state resulting from any reductions in GGT enzymatic activity. Oxidative stress ultimately leads to increased intracellular ROS, through which cells with low GGT activity can trigger signaling pathways and activate transcription factors such as NF-{kappa}B.

The target molecules subject to redox regulation during NF-{kappa}B activation are still unknown, but direct activation of NF-{kappa}B by ROS is unlikely since oxidation of a conserved cysteine residue in the DNA-binding domain of NF-{kappa}B actually results in a loss of DNA-binding activity (55,56). Degradation of I{kappa}B-{alpha} is preceded by phosphorylation on Ser32 and 36. NF-{kappa}B activation induced by such proinflammatory cytokines as IL-1ß and TNF-{alpha} involves rapid proteolysis of the associated I{kappa}B-{alpha} by the phosphorylation-dependent ubiquitination-proteasome pathway (20). H2O2-mediated NF-{kappa}B activation correlates with a slow, sustained degradation of I{kappa}B-{alpha} and does not involve the phosphorylation of Ser32 and 36 residues (57,58). Although the C-terminal Pro–Glu–Ser–Thr (PEST) sequence of I{kappa}B-{alpha} has been shown not to be required for signal-induced degradation, the Tyr42 residue and the serine/threonine residues within the PEST sequence were critical for inducible degradation of I{kappa}B-{alpha} by H2O2. It has been suggested that casein kinase II is responsible for the ROS-augmented phosphorylation of the I{kappa}B PEST domain. However, there are several other protein kinases that have been reported to be activated by H2O2 and other inducers of oxidative stress, including the mitogen-activated protein kinase Jun kinases, ZAP-70 tyrosine kinase, p72syk-related kinases and members of the Src family of tyrosine kinases (Lck and Fyn) (2,3,25,5963).

Lahdenpohja et al. have demonstrated that stimulation of naive peripheral blood T cells (CD45RA+) with anti-CD3/anti-CD28 mAb leads to enhanced DNA binding of NF-{kappa}B (64). The nuclear translocation of c-Rel protein was increased in CD45RA+ cells, but no such increase was seen for memory cells (CD45RO+) (64). Following stimulation, both the naive and memory cells showed a prolonged degradation of I{kappa}B-{alpha}, correlating with the enhanced c-Rel nuclear translocation, but it was more long lasting in the naive cells (64). Costello et al. have shown that intracellular thiol levels are decreased following CD28 co-stimulation, suggesting that such co-stimulation alters the intracellular redox environment (65) in a pro-oxidant direction. Our previous studies showed that GGT is highly expressed on resting CD4+ memory T cells, whereas CD4+ naive T cells express little, if any, GGT (30). Contrary to the findings of Lahdenpohja et al. (50), data shown in this report demonstrate that CD45RA+ cells have significantly more intracellular ROS than CD45RO+ cells, suggesting that the level of expression of antigenic GGT on resting memory T cells is important for the recapture of GSH and, subsequently, for the detoxification of intracellular peroxides. It is possible that memory T cells use their surface GGT to manage oxidative stress resulting from CD28 stimulation, or exogenous H2O2 induction, leading to the variations in NF-{kappa}B activity observed.

Our results indicate that the metabolism of extracellular GSH by GGT is important in T cell signaling and the activation of transcription factors. These findings suggest that up-regulation of GGT on subsets of T lymphocytes may promote apoptotic cell death. This may be one way of promoting the elimination of memory/effector T cells at the end of an immune response. Since the redox-controlled transcription factor NF-{kappa}B is used extensively for signaling and gene induction in immune cells, it is likely that GGT activity will be important in the modulation of T cell functions other than apoptosis. The regulated expression of GGT observed at various stages of T cell differentiation indicates the possibility for distinct roles for this enzyme in different aspects of T cell physiology. Studies are in progress to better define the mechanisms by which this enzyme may effect its role within immune responses.


    Acknowledgements
 
The authors wish to thank Dr V. Michael Holers for 3A8 antibody and critical reading of the manuscript. This work was supported by PHS grant AI42772 (D. R. K.).


    Abbreviations
 
DCF-DA—dichlorofluorescin diacetate

GGT—{gamma}-glutamyl transpeptidase

GSH—reduced glutathione

IAP—inhibitor-of-apoptosis protein

MBB—monobromobimane

NEM—N-ethyl maleimide

PEST—Pro–Glu–Ser–Thr

PI—propidium iodide

ROS—reactive oxygen species


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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