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The Journal of Immunology, 2005, 175: 7965-7972.
Copyright © 2005 by The American Association of Immunologists

The Role of Low Molecular Weight Thiols in T Lymphocyte Proliferation and IL-2 Secretion1

Tanja Hadzic*,{ddagger},§, Ling Li{dagger},{ddagger}, Ningli Cheng*, Susan A. Walsh{dagger},{ddagger}, Douglas R. Spitz{dagger},{ddagger} and C. Michael Knudson2,*,{ddagger},§

* Department of Pathology, {dagger} Department of Radiation Oncology, {ddagger} Free Radical and Radiation Biology Program, and § Immunology Graduate Program, University of Iowa Roy J. and Lucille P. Carver College of Medicine, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. L-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and L-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular L-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or L-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The metabolism of hydroperoxides via the glutathione (GSH),3 GSH reductase, and GSH peroxidase pathway represents one of the major cellular defense mechanisms against oxidative stress (1). GSH is a tripeptide ({gamma}-glutamylcysteinylglycine) that cycles between its oxidized (GSSG) and its reduced (GSH) state to detoxify hydroperoxides in concert with GSH peroxidase using the reducing equivalents in NADPH and GSH reductase. In the absence of oxidative stress, 90–95% of GSH is in its reduced state (2, 3). In response to a stress, a cell undergoes enzymatic reactions that consume GSH by forming GSH conjugates with a great variety of electrophilic compounds or by forming GSSG. GSH content can be maintained through de novo synthesis or through the enzymatic reduction of its disulfide form, GSSG, using the glutathione reductase/NADPH system (4). De novo synthesis of GSH involves two enzymatically controlled reactions that use ATP and nonessential amino acids, glycine, cysteine, and glutamate (Glu) for production of GSH. In the first, rate-limiting reaction, Glu and cysteine are combined by the enzyme Glu cysteine ligase to form {gamma}-glutamylcysteine. L-buthionine-S,R-sulfoximine (BSO) is a specific and potent inhibitor of Glu cysteine ligase and thus GSH synthesis (5). In the second reaction, GSH synthetase catalyzes the formation of GSH by combining {gamma}-glutamylcysteine and glycine (3).

Numerous studies have implicated an important role for GSH in T cell proliferation (5). The 2-ME, reported to augment intracellular GSH levels, was shown to enhance proliferation of IL-2-induced PBL, large granular lymphocytes, and OKT-3 mAb-stimulated CD3+ T lymphocytes (6). Human T lymphocytes depleted of GSH using BSO were unable to proliferate in response to mitogenic lectins, which suggested a direct relationship between the proliferative response and GSH availability (7, 8). When human PBMC were cultured with BSO, their entry into S phase of the cell cycle was inhibited in a dose-dependent fashion (5). Exogenous GSH reversed the BSO-induced block on T cell proliferation (9). Yamauchi and Bloom (10) also demonstrated that IL-2-dependent cell line, NK3.3, failed to incorporate [3H]thymidine or to transition from G1 to S phase of the cell cycle in medium lacking the thiol-related compounds, L-cystine, and GSH, despite the presence of sufficient IL-2.

N-acetylcysteine (NAC) is a small m.w. thiol compound, and its deacetylation leads to increased levels of intracellular cysteine. Cysteine can be rate limiting in GSH synthesis, and NAC has been shown to increase GSH levels in some cell types (11). Treatment of murine splenic T cells with NAC markedly enhanced proliferation (12). However, the exact mechanism of NAC action on T cell proliferation has not yet been identified. It is known that in addition to its role as a precursor of GSH synthesis and supplier of cysteine, NAC can function as a thiol antioxidant due to its ability to scavenge reactive oxygen species, like hydrogen peroxide (H2O2) and hydroxyl radical (·OH) (13).

Cysteine exists in equilibrium between the reduced and oxidized form known as cystine. Cystine predominates in the extracellular environment, while cysteine predominates in the intracellular environment (14, 15). T lymphocytes are not able to take up cystine from the surrounding environment and convert it to cysteine. Instead, a recent study demonstrated that dendritic cells take up cystine, reduce it to cysteine, and release it into the extracellular space for uptake by T lymphocytes (16). When cystine uptake by APCs is inhibited by Glu, T lymphocytes are unable to proliferate (16). Thus, APCs modulate T cell proliferation by regulating the availability of thiols like cysteine and thioredoxin (14).

Thiols such as GSH and cysteine have not only been implicated in T cell proliferation, but also have been suggested to play an important role in the regulation of T cell function. Depletion of GSH in human T cells was shown to impair IL-2 production, which is known to stimulate T cell proliferation (11). NAC was found to increase IL-2 production in human peripheral blood T cells (17), as well as IL-6 production in Jurkat T cells (18). In vivo, GSH depletion in rats showed that GSH-deficient animals underwent a significantly decreased production of IL-6 and TNF-{alpha} (19). Furthermore, depletion of GSH was found to be associated with a reduced number of activated human T cells, as characterized by CD25 and CD69 surface markers, relative to cells with high GSH content (20).

Collectively, these data suggest that intracellular thiols regulate multiple signaling pathways in T lymphocytes. The purpose of the current study was to determine the mechanism by which GSH and other low m.w. thiols affect T cell proliferation. Direct measurements of low m.w. thiols in murine T lymphocytes treated with various combinations of BSO and exogenous thiols were performed. Our results indicate that neither GSH nor cysteine levels are required for T cell proliferation, but rather that GSH and cysteine are rate limiting only in the absence of other low m.w. reduced thiols. This suggests that reduced thiols, but not necessarily GSH, are required for T cell proliferation. In addition, depletion of intracellular thiols selectively impairs IL-2 secretion without affecting the expression of T cell activation surface markers or cell viability. Inhibition of T cell proliferation by BSO is completely overcome by exogenous IL-2. These results support the hypothesis that T cell proliferation is dependent on thiol-mediated regulation of IL-2 secretion, and suggest that redox regulation of IL-2 secretion is an obligatory step in T cell proliferative responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Unless otherwise specified, all reagents were obtained from Sigma-Aldrich. Stock solutions of 1 M NAC (catalog A9165) and 1 M L-cysteine (catalog C7352) were freshly prepared on the day of experiment. Stock solution of 0.1 M BSO (catalog A2515) was prepared fresh every month. Stock solution of 2-ME (catalog 0482) was prepared fresh every 4 mo (AMRESCO). FBS, L-glutamine, penicillin, and streptomycin were obtained from Invitrogen Life Technologies. Murine T cell enrichment mixture kit (catalog 13051) was obtained from StemCell Technologies. Anti-mouse CD3 Ab (clone 145-2C11; catalog 16-0031-85) was purchased from eBioscience. Anti-mouse CD3 FITC (catalog 553062), CD4 PE (catalog 09425B), CD25 FITC (catalog 102005S), and CD69 PE (catalog 104507S) were obtained from BD Pharmingen. Rat Ig FITC, rat Ig PE, and IL-2 neutralizing Ab (nAb; clone JES6-5H4) were generously provided by T. Waldschmidt (University of Iowa, Iowa City, IA). Viacount reagent (4000-0040) was purchased from Guava Technologies. TRIzol reagent (15596-026) was purchased from Invitrogen Life Technologies, and the iScrip cDNA Synthesis kit (170-8890) was obtained from Bio-Rad. TaqDNA polymerase (M0267S) was purchased from Biolabs.

Murine T cell separation

All animals were housed at the animal facilities at the University of Iowa, and all experimental procedures involving mice were approved by the University of Iowa Institutional Animal Care and Use Committee. Total splenic and purified murine T cells were prepared by negative selection, as previously described (21). In brief, spleen cells were prepared from C57BL/6 mice (Harlan Laboratories), incubated in RBC lysis buffer (10 mM Tris, 0.83% NH4Cl (pH 7.2)) for 7–9 min, and then washed twice with PBS solution. Mice examined were 8–20 wk old, and no differences were observed between the mice in this age range. Cells were sequentially treated with rat serum and a mixture of biotinylated mAbs to specific murine cell surface Ags (CD11b, CD45R, Ly-6G, TER119) for 15 min each at 4°C. Cells were pelleted, resuspended in 2% FCS-PBS, and then treated with an anti-biotin tetrameric Ab complex and a magnetic colloid for 15 min at 4°C. Cells were passed through a magnetic column tailored to highly enrich unlabeled T cells from suspension of murine spleen cells. For quality control, purified T cells were stained for CD3 (>95% stained positive).

Cell culture and treatments

Isolated splenic naive T cells were cultured in RPMI 1640 tissue culture medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium). Medium was used within 1 mo of the preparation due to decreased ability of untreated cells to proliferate efficiently in medium older than ~1 mo. T cell growth in old medium was normalized when 2-ME or NAC was added. This suggests that low levels of thiols present in complete medium are oxidized with storage and are critical for T cell proliferation. This most likely accounts for the routine use of 2-ME in most medium used for murine lymphocyte cultures. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. All cell culture experiments were conducted in 6-well (10 ml) and/or 96-well round-bottom plates (0.2 ml) coated with 1000 or 50 ng/well, respectively, anti-mouse CD3 Ab in a 50 mM Tris coating buffer (pH 9.5) for 2–4 h. Cell concentration was 0.5–1 x 106 cells/ml. Cells were treated with NAC, BSO, L-cysteine, and/or 2-ME at the time of CD3 activation.

Surface marker staining

Isolated murine T cells and splenocytes, at 0.5–1 x 106 cells, were pelleted and suspended in 100 µl of the staining buffer (500 ml of PBS, 1% BSA, 0.01% NaN3) in the presence of 1 µg of rat IgG Fc (GJ Bk4) for 5 min. Then, cells were stained with 0.5 µg of anti-mouse CD3 FITC, CD25 FITC, or CD69 PE for 20 min on ice and away from light. Cells were washed and resuspended in 0.2 ml of the staining buffer before analysis on the FACSCalibur flow cytometer (BD Biosciences) using the CellQuest software. Rat Ig FITC, rat Ig PE, CD3{epsilon} FITC, and CD4 PE were used as the controls.

Cell proliferation assays

Following various treatments, T cells were pelleted and resuspended in propidium iodide (PI) staining solution containing 0.02 mg/ml RNase A, 0.05 mg/ml PI, 0.03% Nonidet P-40, and 0.10% sodium citrate (22). Cells were filtered using 105 µm polypropylene mesh sheet (Small Parts) to remove cell clumps. The percentage of cycling cells (percentage of S/G2/M) was determined by examining FL-2A histograms following doublet discrimination.

Cell viability assay

To measure viability, we used dye exclusion assay from Guava Technologies. First, 5 or 10 µl of cells was mixed with 45 or 40 µl of Guava ViaCount Reagent and allowed to stain for 5–10 min. Just before the placement in the Guava PCA cell analyzer (Guava Technologies), the cells were diluted with 150 µl of PBS and mixed, and the data were acquired. Each sample was analyzed in duplicate.

Measurement of intracellular GSH levels

After indicated treatment time, cells were harvested and washed once with PBS, and the pellets were frozen in liquid nitrogen and stored at –80°C before thiol analysis. Cell homogenates were prepared, and total (GSH + GSSG) and oxidized (GSSG) GSH were determined, as previously described (23). GSH and GSSG were distinguished by addition of 2 µl of 2-vinyl pyridine mixed 1:1 (v:v) with ethanol per 30 µl of sample, followed by incubation for 1 h and assay, as previously described (24). All biochemical determinations were normalized to the protein content of whole homogenates (25).

Measurement of intracellular thiol levels

HPLC (HPLC system; Shimadzu Scientific Instruments) with fluorescent detection was used in the analysis of intracellular low m.w. thiols, according to previously published assays (26). First, 0.1–50 pmol of standard thiols, including NAC, GSH, {gamma}GGC, and cysteine, was derivatized with 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho(2,1-b)pyran (ThioGlo-3) and resolved using a 15-cm C18 Reliasil column (Column Engineering) to generate standard curves. Total cellular protein extracts prepared from control, BSO-, 2-ME + BSO-, L-cysteine + BSO-, and NAC + BSO-treated cells were also treated with ThioGlo-3 and analyzed by HPLC. Cysteine levels were normalized to the protein content of whole cell homogenates (25).

Oxidation of low m.w. thiols

Low m.w. thiols, NAC, cysteine, and 2-ME, at 1 M concentrations, were treated with 2 M hydrogen peroxide or mock treated with water for 1 h. Then, 400 U/µl catalase was added to each sample for half an hour to remove excess hydrogen peroxide. An aliquot from each thiol was then treated with 0.6 mM 5',5'-dithio-bis(2-nitrobenzoic acid) to confirm oxidation of the thiol moiety. Complete oxidation of thiols was verified by the absence of 2-nitro-5 thio benzoic acid production, as measured by the spectrophotometer at 412 nm.

Measurement of mouse IL-2 and IL-6

IL-2 and IL-6 were measured by a sandwich ELISA (eBioscience) assay, according to the manufacturer’s instructions. The conversion of tetramethylbenzidine by HRP was detected by measuring the absorbance at 450 nm using the ELISA plate reader (Bio-Laboratory). Mouse rIL-2 and rIL-6 from their respective ELISA kits were used as standards. The sensitivity of the assay was 2 pg/ml for IL-2 and 4 pg/ml for IL-6.

RT-PCR

Total cellular RNA was prepared from mouse T cells using TRIzol reagent, according to the manufacturer’s directions. cDNA used for RT-PCR analysis was then synthesized from total cellular RNA using iScript reverse transcriptase. PCR amplifications were then performed. cDNA was subjected to PCR amplification using primers for mouse IL-2 (sense, 5'-TCGCATCCTGTGTCACATTGACAC-3'; antisense, 3'-GGCACTCAAATGTGTTGTCAGAGC-5'; 0.5 µM, 450-bp product) and mouse {beta}-actin (sense, 5'-GTGGGGCCGCTCTAGGCACCAA-3'; antisense, 3'-CTCTTTGATGTCACGCACGATTTC-5'; 0.25 µM, 540-bp product). After an initial denaturation for 5 min at 94°C, cDNA was amplified in a final volume of 25 µl with 1.25 U of TaqDNA polymerase using the manufacturer’s buffer and 1.5 mM MgCl2. Amplification consisted of 22 cycles of 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. PCR products were separated by 2% agarose gel electrophoresis, and bands were visualized with ethidium bromide and UV translumination. Gels were photographed, and bands were analyzed by computerized densitometry using an imaging system from Ultraviolet Products.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GSH is not rate limiting for T cell proliferation in the presence of exogenous low m.w. thiols

Numerous studies implicate GSH as a critical regulator of T cell proliferation. Consistent with this hypothesis, we find that BSO markedly suppressed T cell proliferation while decreasing GSH levels (Fig. 1, A and B). Also, as previously reported, NAC markedly increased T cell proliferation (Fig. 1B). As NAC increases intracellular cysteine levels, it is frequently proposed to act through increased GSH levels, as cysteine can be rate limiting in GSH synthesis. However, NAC failed to increase GSH levels despite a marked increase in proliferation (Fig. 1, A and B). This suggests that NAC effects on T cells are independent of GSH. This prompted us to examine T cells simultaneously treated with both NAC and BSO. As BSO acts at a step downstream of cysteine, GSH levels were reduced by ~84% under these conditions (Fig. 1A). Surprisingly, despite this reduction in GSH levels, proliferation was increased in the BSO + NAC-treated cells to the same extent as NAC alone (Fig. 1B). In most samples, GSSG levels were undetectable independent of treatment (data not shown). The differences in proliferation could not be explained by alterations in cell viability, as NAC and/or BSO only mildly affected cell death (Fig. 1C).



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  		FIGURE 1. T cell proliferation is independent of GSH levels. Pure murine T cells from C57BL/6 mice were activated with anti-CD3 and treated with 20 mM NAC, 1 mM BSO, combination of NAC + BSO, as indicated, or left untreated. After 48 h, cells were analyzed for: A, total GSH levels using the thiol assay; B, proliferation using PI staining; or C, cell viability using the Guava PCA cell analyzer. Each filled diamond represents an independent experiment, and the mean ± SD of each group is indicated to the left of the symbols.

 
Because GSH levels are not rate limiting for T cell proliferation in the presence of NAC, the ability of 2-ME or L-cysteine to rescue cells from BSO-induced block of proliferation was examined. Under these various conditions, both proliferation and GSH levels of T cells were determined. As observed for NAC, both 2-ME and L-cysteine were able to reverse the inhibitory effect of BSO on T cell proliferation without restoring GSH levels (Fig. 2). In fact, under each of these conditions, the level of proliferation was increased by the presence of the thiol relative to the cells treated with medium alone in the absence of BSO.



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  		FIGURE 2. Low m.w. thiols enhance T cell proliferation in the presence of BSO. Isolated splenic T cells from wild-type mice were treated with 20 mM NAC, 50 µM 2-ME, and 10 mM L-cysteine (L-cyst) in the presence of 1 mM BSO, as indicated, during anti-CD3 activation for 48 h. A, Cell proliferation was determined using PI staining for DNA content, and B, GSH levels using the thiol assay. The graphs represent the mean ± SD of duplicate samples, and the data are representative of at least three separate experiments.

 
BSO inhibition of T lymphocytes is more profound in the absence of accessory cells

Accessory cells have been suggested to control T lymphocyte proliferation by maintaining cysteine-rich extracellular environment. Accessory cells take up cystine, reduce it to cysteine, and release it into the extracellular space, where it is available to T lymphocytes (14). The uptake system used by the accessory cells depends on high intracellular concentrations of Glu to drive the uptake of cystine. Thus, cystine uptake by accessory cells is competitively inhibited by Glu (16). Because BSO inhibition of T lymphocytes was more profound in the absence of accessory cells (Fig. 3), we tested the effect of Glu on proliferation of splenocytes and pure T cells in the absence or presence of BSO. Although Glu and BSO only partially inhibited splenic T cell proliferation, the combination treatment decreased proliferation by 81%. Consistent with Glu acting exclusively on accessory cells to block cysteine release, Glu alone had no effect on proliferation of pure T cells. Both 2-ME and NAC overcame the inhibition by BSO + Glu of both pure T cells and total splenocytes (Fig. 3).



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  		FIGURE 3. Cotreatment with Glu and BSO inhibits splenocyte proliferation. Murine splenocytes and pure T cells were activated with anti-CD3 and left untreated or treated with 1 mM BSO, 2 mM Glu, Glu + BSO, NAC + Glu + BSO, or 2-ME + Glu + BSO. After 48 h, cells were stained with PI and assayed for proliferation. The graph represents the mean ± SD of duplicate samples.

 
Reduced thiols are absolutely required for T cell proliferation

To confirm the importance of the oxidation state of these thiols, the effect of both reduced and oxidized forms of 2-ME, L-cysteine, and NAC on T cell proliferation was determined. When 2-ME, NAC, and L-cysteine were oxidized with excess H2O2, they could not reverse the BSO block on proliferation. Furthermore, oxidized thiols did not affect the proliferation of untreated murine T cells, showing the oxidized thiols, following treatment with catalase, did not contain residual toxic levels of H2O2 (Fig. 4).



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  		FIGURE 4. Reduced low m.w. thiols, but not oxidized thiols, enhance T cell proliferation. Murine splenic T cells were activated with anti-CD3 for 48 h while left untreated (no drug) or treated with reduced or oxidized (oxid) thiols (50 µM 2-ME, 10 mM L-cyst, and 20 mM NAC, as indicated) in the presence or absence of BSO. Cell proliferation was measured by PI staining for DNA content. The graph represents the mean of two independent experiments (±SD) of duplicate samples.

 
Cysteine is not rate limiting for T cell proliferation in the presence of 2-ME

Cysteine, derived from APCs in vivo, has been suggested to be rate limiting for lymphocyte proliferation (16). We reasoned that cysteine may be the critical, rate-limiting thiol in these pure lymphocyte cultures. When treated with NAC, 2-ME, and exogenous L-cysteine in the presence of BSO, T cells underwent enhanced proliferation (Fig. 5). Therefore, we examined the effect of BSO in combination with NAC, 2-ME, and L-cysteine on intracellular cysteine levels 48 h after CD3 activation. Consistent with this reasoning, cysteine levels decreased in the presence of BSO and were markedly enhanced by cotreatment with NAC and L-cysteine. However, 2-ME + BSO treatment resulted in intracellular cysteine levels comparable to BSO treatment alone (Fig. 5). In this experiment, 2-ME completely restored, and even enhanced, proliferation of BSO-treated T cells. Thus, under these conditions, 2-ME rescued proliferation without increasing intracellular cysteine levels. We conclude that L-cysteine, like GSH, is not singularly required for T cell proliferation, but there appears to be an absolute requirement for small m.w. reduced thiols.



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  		FIGURE 5. T cell proliferation does not correlate with cysteine levels. Murine T cells were activated with anti-CD3 while being treated with 20 mM NAC and 50 µM 2-ME in the presence of 1 mM BSO for 48 h. T cell proliferation was measured using PI staining for DNA content. Cysteine levels were measured using HPLC method and normalized to protein (mg) levels. The graph represents the mean ± SD of duplicate samples.

 
BSO treatment of T cells blocks IL-2 and IL-6 secretion without affecting T cell surface marker expression

Following CD3 stimulation, numerous events occur before the start of T cell proliferation, including cytokine secretion and surface receptor up-regulation. We investigated these downstream events of CD3 stimulation in an attempt to identify the steps affected by BSO inhibition and reversed by reduced thiols. Using flow cytometry, the expression of IL-2R{alpha} (CD25) and the activation inducer molecule (CD69) was examined in the presence of BSO and/or NAC. Before activation, T cells did not express either CD25 or CD69. Treatment of cells with BSO had no effect on the expression of CD25 and CD69 in activated T cells. Addition of NAC alone or NAC + BSO markedly enhanced the expression of both CD25 and CD69 (Fig. 6).



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  		FIGURE 6. BSO-treated T cells have a normal CD25 and CD69 expression. Murine T cells were purified and activated with immobilized anti-CD3 for 48 h. Cells were pelleted and stained for CD25 FITC and CD69 PE, as described in Materials and Methods. Cells were pelleted again and fixed for later analysis by FACS. Representative contour plots are shown, and the percentage of cells positive for both CD25 and CD69 is indicated.

 
Because BSO did not affect the surface expression of CD25 or CD69, the effect of BSO on cytokine secretion was examined. Forty-eight hours after activation, murine T cell supernatants were collected and analyzed for IL-2 and IL-6 secretion using ELISA. We found that NAC induced and BSO impaired the secretion of IL-2 (Fig. 7A). Secretion of IL-2 in NAC + BSO-treated cells was comparable to NAC-treated cells. In the presence of NAC, IL-2-secreted levels increased by 53%, while in the presence of BSO IL-2 levels dropped by 95%. BSO was also effective in decreasing the IL-6 levels, as evidenced by the 78% drop of this cytokine (Fig. 7B). NAC was able to reverse the BSO inhibition of IL-6 secretion, but NAC alone had no effect on IL-6 levels (Fig. 7B).



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  		FIGURE 7. BSO impairs T cell secretion of IL-2 and IL-6. A and B, Murine T cells were left untreated (no drug) or treated with 20 mM NAC, 1 mM BSO, or NAC + BSO. IL-2 and IL-6 were measured using ELISA. The graph represents the mean ± SD of duplicate samples. C, Murine T cells were activated with anti-CD3 and left untreated for 4 and 24 h or treated with 1 mM BSO for 4, 8, 12, 18, and 24 h. A sample with no cDNA was used as a negative control. mRNA levels for IL-2 and {beta}-actin were determined using RT-PCR. The data are representative of two independent experiments.

 
Following CD3 activation, IL-2 mRNA levels increase within 2–4 h (27). Because BSO decreases GSH by inhibiting de novo synthesis, we reasoned the initial response to CD3 may be intact and BSO may inhibit IL-2 in a time-dependent manner. Therefore, the effect of BSO on IL-2 mRNA levels was examined using RT-PCR at various times following CD3 activation. As expected, control cells demonstrated IL-2 mRNA at both 4 and 24 h. However, in the presence of BSO, IL-2 mRNA levels were normal at 4 h and decreased in a time-dependent manner with undetectable levels at 18 and 24 h (Fig. 7C). These results demonstrate that BSO regulates IL-2 through changes in mRNA levels.

Exogenous IL-2 rescues T cell proliferation from BSO-induced block

To test the possibility that increases in proliferation of T cells treated with NAC were due to increased IL-2 secretion, we measured proliferation by PI staining in the presence of exogenous murine IL-2 or IL-2 + NAC. Murine T cells exposed up to 20 ng/ml exogenous IL-2 failed to undergo increased proliferation compared with the control cells. To test whether exogenous IL-2 or IL-6 could overcome BSO-induced block of T cell proliferation, T cells were treated with or without BSO for 48 h in the presence of IL-2 and IL-6. Surprisingly, exogenous IL-2, but not IL-6 (data not shown), completely rescued T cell proliferation in the presence of BSO (Fig. 8A). When GSH levels were examined in these same samples, we found that GSH levels in IL-2 + BSO-treated cells were as low as in cells cultured with BSO, indicating that IL-2 had no effect on GSH levels (Fig. 8B).



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  		FIGURE 8. Exogenous IL-2 restores T cell proliferation in BSO-treated cells. Anti-CD3-activated murine T cells were cultured with 20 ng/ml IL-2, 1 mM BSO, and IL-2 + BSO, or left untreated (no drug). After 48 h, cells were assayed for: A, proliferation by PI, and B, total GSH by thiol assay. The graph represents the mean ± SD of duplicate samples.

 
Because murine T lymphocytes were exquisitely sensitive to changes in the extracellular thiol levels, we determined whether nAb blocked the effect of exogenous IL-2. IL-2 nAb had little effect on T cell proliferation in the absence of BSO (Fig. 9). As before, IL-2 completely rescued the block in proliferation by BSO, and this effect was completely neutralized by nAb. These results suggest that BSO has two effects. BSO decreases IL-2 production and sensitizes cells to nAb by increasing cellular requirement for IL-2.



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  		FIGURE 9. Neutralized IL-2 cannot overcome BSO-induced inhibition of T cell proliferation. Anti-CD3-stimulated murine T cells were left untreated (no drug) or treated with 20 ng/ml IL-2, 1 mM BSO, and IL-2 + BSO in the presence or absence of IL-2 nAb (4 µg/ml), as indicated, for 48 h. Cell proliferation was determined using the PI staining for DNA content. The graph represents the mean ± SD of duplicate samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, the role of GSH and other low m.w. thiols in T cell proliferation was dissected through the use of small m.w. antioxidants, inhibitors of GSH synthesis, and measurement of intracellular thiols. GSH has been suggested to regulate lymphocyte proliferation based on the observations that BSO-induced depletion of GSH results in the inhibition of the T cell proliferative response (4, 5, 7, 19, 28). Our study demonstrated that GSH is rate limiting for T cell proliferation only in the absence of exogenous small m.w. thiols. Interestingly, Yim et al. (11) have shown previously that NAC increases GSH levels and stimulates T proliferation of murine splenocytes. In addition, NAC partially restores GSH levels and proliferation in cells treated with BSO. Importantly, these studies did not use purified T cells, and proliferation was examined 72 h after activation. Both studies demonstrate that NAC protects from GSH depletion in BSO-treated cells, most likely reflecting decreased consumption of GSH in cells treated with NAC.

In this study, we also measured levels of intracellular cysteine, as cysteine has been shown to be rate limiting for T cell growth and proliferation after Ag receptor engagement (29). T cells require exogenous cysteine, as they are unable to take up and reduce extracellular cystine (14). APCs are proposed to regulate T cell proliferative response by increasing the availability of cysteine, which can be used by T cells (16). This has led to the suggestion that cysteine may be the rate-limiting thiol in T lymphocytes. Consistent with this possibility, in our study, cysteine levels were increased by NAC and reduced when T cell proliferation was inhibited with BSO. However, treatment of T cells with 2-ME + BSO resulted in enhanced proliferation, while both GSH and cysteine levels were reduced. This result demonstrated that, in vitro, cysteine is not rate limiting for T cell proliferation in the presence of 2-ME and BSO. These results suggest that GSH, cysteine, and other low m.w. thiols perform a redundant role in T cell proliferation.

Under the conditions described in this work, T cell proliferation is markedly inhibited by BSO without significant increased cell death. These results suggest that selective signaling pathways may be inhibited under these conditions. Consistent with this, T cells express normal surface levels of CD25 and CD69 under thiol-limited conditions. Although T cells express normal levels of surface molecules under thiol-limited conditions, it is possible that signaling through those molecules is inhibited. A study by Nambiar et al. (30) showed that heat shock-induced oxidative stress down-regulated TCR/CD3-mediated Ca2+ signaling and that NAC significantly reversed this down-regulation. In our system, BSO treatment of T cells might also be inhibiting Ca2+-dependent up-regulation of IL-2, which NAC or exogenous IL-2 could reverse. This possibility is also consistent with the work of Petrov and Lijnen (31), who demonstrated the ability of exogenous IL-2 to restore the proliferative response of lymphocytes treated with calcium antagonists. The finding that lymphocytes treated with calcium antagonists also had normal expression of IL-2Rs, but decreased production of IL-2, further supports the hypothesis that calcium-signaling pathways may be thiol sensitive.

Although surface expression of T cell activation markers was unaffected, under thiol-limited conditions, secretion of IL-2 was markedly reduced. The important role of IL-2 as both an autocrine and paracrine T cell growth factor is well established. Cells isolated from thymus, spleen, and lymph nodes of IL-2-deficient mice mount a very weak response to Con A or anti-CD3 in vitro (32). Surprisingly, in our system, exogenous IL-2 completely rescued T cell proliferation under thiol-limited conditions, suggesting that IL-2 signaling is particularly sensitive to alterations in thiol levels within T cells. Our findings are consistent with those of Iwata et al. (33), who showed that IL-2 stimulated proliferation of human PBMCs grown in medium free of both cystine and GSH. In contrast, another study demonstrated that, although BSO blocked IL-2 production, exogenous IL-2 was not able to overcome the proliferative block by BSO (9). However, this latter study used human T cells that had been pretreated with BSO for 16 h before activation. Pretreatment of murine T cells results in extensive cell death, which makes a direct comparison impossible. Therefore, whether the differences with our studies are due to species variations (human vs mouse) or experimental design changes (pretreatment or not) is not known.

Based on our findings, we can propose two models that would explain the relationship between IL-2, thiols, and T cell proliferation. In one model, the reduction in proliferation is directly related to decreased IL-2 secretion. Alternatively, the reduction in thiol pools may have two effects. In addition to decreased IL-2 secretion, BSO may render T cells more dependent on IL-2 for proliferation. In this case, T cells would require IL-2 only if thiol pools are limited. Our finding that neither IL-2 nAb nor exogenous IL-2 affected T cell proliferation under nonthiol-limited conditions supports this alternative model. The correlation among low levels of GSH, low IL-2 production, and low proliferation has been reported previously in T cells isolated from the synovial joints of patients with rheumatoid arthritis (34). This finding seemed to suggest that thiol-mediated regulation of IL-2 is responsible for the inhibitory effect of BSO and the stimulating effect of NAC on T cells. Indeed, our studies using RT-PCR show that BSO markedly reduced IL-2 mRNA levels at 24 h (Fig. 7C), demonstrating that BSO regulates IL-2 at the transcriptional level. However, the definitive mechanism by which NAC exerts its effects on T cells has not yet been identified, although NAC might regulate T cells at the transcriptional level like BSO.

Three major transcription factors are activated in T cells in response to CD3 stimulation, including NF-AT, AP-1, and NF-{kappa}B. These transcription factors have been shown to play a critical role in IL-2 gene transcription (27). In addition, NF-{kappa}B and AP-1 have shown to be the targets of CD28 costimulation, which caused a pronounced increase in IL-2 mRNA stability (35). It would be of interest to determine whether BSO inhibits IL-2 by blocking these transcription factors or by reducing the IL-2 mRNA stability. Transcription factors, NF-{kappa}B and AP-1, are also considered stress response transcription factors that regulate the expression of various downstream target genes known to be involved in cellular antioxidant defense mechanisms (36). The activity of these transcription factors can be modulated by redox status in two ways: induction and translocation of the transcription factor and transcription factor DNA-binding activity (37, 38, 39). The redox signaling protein thioredoxin (Trx) regulates both of these processes. Trx has been shown to be a critical regulator of T cell function and proliferation. Trx is a 12-kDa ubiquitous, multifunctional protein with a redox-active site (-Cys-Gly-Pro-Cys-) that is highly conserved in both prokaryotic and eukaryotic genomes (38, 40, 41, 42). It has been reported that Trx translocates from the cytosol into the nucleus in response to various stresses, where it regulates the DNA-binding activity of proteins, such as AP-1 and NF-{kappa}B (43). Trx also functions extracellularly as one of the key regulators of cell signaling. In activated lymphocytes, Trx is secreted via a nonclassic pathway, but its exact export route has not yet been identified (44).

The various functions of Trx as a redox signaling protein could explain the stimulating effects of NAC on T lymphocytes. It is possible that NAC modulates Trx activity to enhance T cell proliferative response and secretion of IL-2. Trx has already been shown to enhance the expression of low affinity IL-2R, IL-2R/p55 (45), and to have cocytokine activity by synergizing with IL-2 to enhance the proliferative response of murine T lymphocytes (46). We hypothesize that NAC alters the levels or redox status of Trx to increase T cell proliferation and IL-2 production. Trx could exert its effects by increasing the expression of IL-2Rs and modulating the redox status of transcription factors associated with T cell activation. It would be of interest to determine whether NAC indeed acts through the Trx pathway by examining Trx levels, activity, and oxidation state in NAC-treated lymphocytes.

In summary, the findings described in the present study suggest that intracellular thiols regulate selective signaling pathways in T lymphocytes. Our results support the hypothesis that T cell proliferation and IL-2 production are mediated by small m.w. thiols, and suggest that redox regulation of IL-2 secretion is an obligatory step in T cell proliferative responses. Identification of the specific thiol-sensitive pathways that regulate T cell proliferation and IL-2 secretion may provide a novel target for immunoregulation of T cells.


   Acknowledgments
 
We thank Dr. Morris Dailey for helpful advice on the effects of antioxidants on T lymphocyte function; Teresa Dulling for making the flow cytometry facility available; Kelly Andringa and Mitchell Coleman for support in preparing thiol assays and HPLC; and Chris van de Wetering and Matthew Kemp in the Knudson laboratory for their support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants RO1CA88967 (to C.M.K.), PO1CA066081 (to D.R.S.), and RO1CA100045 (to D.R.S.). Back

2 Address correspondence and reprint requests to Dr. C. Michael Knudson, Department of Pathology, University of Iowa Roy J. and Lucille P. Carver College of Medicine, 200 Hawkins Drive, 1171 ML, Iowa City, IA 52242. E-mail address: c-knudson{at}uiowa.edu Back

3 Abbreviations used in this paper: GSH, glutathione; BSO, L-buthionine-S,R-sulfoximine; Glu, glutamate; GSSG, glutathione disulfide; nAb, neutralizing Ab; NAC, N-acetylcysteine; PI, propidium iodide; Trx, thioredoxin. Back

Received for publication May 24, 2005. Accepted for publication September 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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