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The Journal of Immunology, 2003, 171: 5107-5115.
Copyright © 2003 by The American Association of Immunologists

Intracellular Thiols Contribute to Th2 Function via a Positive Role in IL-4 Production 1

Martha M. Monick2,3,*, Lobelia Samavati2,*, Noah S. Butler*, Michael Mohning*, Linda S. Powers*, Timur Yarovinsky*, Douglas R. Spitz{dagger} and Gary W. Hunninghake*

* Department of Internal Medicine, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, and Veteran’s Administration Medical Center, Iowa City, IA 52242; and {dagger} Free Radical and Radiation Biology Program, Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A number of lung diseases, including many interstitial lung diseases and HIV infection, are associated with decreases in intracellular thiols. Altered Th1/Th2 T cell balance has also been associated with disease progression in many of the same diseases. IFN-{gamma} and IL-4 are critical effector cytokines of Th1 and Th2 cells, respectively. To determine the effect of thiols on the production of IFN-{gamma} and IL-4 by splenocytes, cells were incubated in the presence and the absence of N-acetylcysteine (NAC) and stimulated with {alpha}CD3 or {alpha}CD3 and IL-12. Augmenting intracellular soluble thiol pools (~2-fold) with 15 mM NAC blocked induction of IFN-{gamma} and increased production of IL-4 without causing significant changes in intracellular glutathione levels. The effect of NAC on IL-4 production was not linked to an increase in STAT6 phosphorylation, as STAT6 levels were decreased, nor did the increase in IL-4 occur with purified CD4 cells. We found that NAC increased splenocyte IL-4 production via an effect on APCs. We also found that NAC increased two IL-4 relevant transcription factors (AP-1) and NFATc. These studies suggest that increasing intracellular reduced thiol pools decreases IL-12 signaling and IFN-{gamma} production, while increasing IL-4 production. The sum of these effects may contribute to alterations in the balance between Th1 and Th2 responses in lung diseases associated alterations in intracellular thiol pools.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A number of lung diseases are associated with low levels of soluble thiols, including glutathione (GSH) 4 (1, 2, 3, 4). A likely mechanism for the reduction of GSH in the lungs of patients is an increased production of reactive oxygen species (ROS) that are generated as a part of the inflammatory component of these disorders. N-acetylcysteine (NAC) is a thiol that enters cells and can function as an antioxidant, as well as being deacetylated to augment cysteine pools and GSH synthesis. It has been shown that ROS play a pivotal role in numerous signaling pathways in physiologic and pathophysiologic settings. In T cells, several studies have suggested that T cell activation stimulates ROS generation (5, 6, 7).

Th cells differentiate into subsets, designated Th1 and Th2, which produce different cytokine profiles. The link between a specific Th phenotype is well established for a number of pulmonary diseases. For example, asthma is considered a Th2 disease, and sarcoidosis and hypersensitivity pneumonitis are considered Th1 diseases (8, 9, 10). The differentiation pathway for development of Th1 and Th2 subsets of T cells can be influenced by a number of factors, such as the dose and form of Ag, peptide Ag/TCR interactions (11), and costimulatory interactions between cell surface molecules, including CD28, CD 40, B7, ICAM-1, and LFA-1 (12, 13, 14). Both Th1- and Th2-specific cytokines promote the growth or differentiation of their own respective T cell subsets. It has been shown that IL-12 and IL-4 play a dominant role in differentiation of Th1 and Th2 cells, respectively (15, 16). Conditions (other than cytokines themselves) that predispose the development of Th1 vs Th2 responses are not entirely clear. In this study the possible role of redox regulation involving intracellular soluble thiols in the production of Th1/Th2-relevant cytokines, IFN-{gamma} and IL-4, was evaluated.

Differentiation of naive T cells into Th2 cells requires IL-4-mediated signals. An important signal downstream of IL-4 is STAT6 (17). STAT6 activation induces dimerization and translocation of STAT6 from the cytoplasm into the nucleus, where it binds DNA, leading to activation of transcription (18). STAT6 knockout mice exhibit a defect in Th2 response development, defective IgE class switching, and defects in Ag-induced airway eosinophilia (19, 20). Importantly, there are also studies which suggest that STAT6-independent Th2 differentiation can occur and that STAT6 can be activated independently of IL-4 and IL-13 receptor signaling (21, 22).

IL-12 exerts its effects on Th1 responses by signaling through STAT4. STAT4 contributes to IFN-{gamma} gene expression by direct interaction with DNA sequences in the IFN-{gamma} promoter (23). STAT4-deficient mice lack responses to IL-12 (20). It has been postulated that STAT4 activation is mandatory for multiple pathways that result in the induction of IFN-{gamma}.

The current study evaluated the effect of augmenting intracellular soluble thiol pools with NAC on the production of a Th1-specific cytokine (IFN-{gamma}) and a Th2-specific cytokine (IL-4) in stimulated ({alpha}CD3 or {alpha}CD3 plus IL-12) naive splenocytes. The effect of NAC did not appear to be due to an effect on total GSH levels, as NAC treatment increased intracellular soluble thiol pools (free NAC and cysteine) without augmenting total GSH content. In stimulated splenocytes, NAC blocked the induction of IFN-{gamma} by {alpha}CD3/IL-12. NAC blocked the activation of STAT4, consistent with the effect of NAC on IFN-{gamma} production. In contrast, increasing intracellular thiol pools with NAC increased the production of IL-4 after anti-CD3. This was not consistent with an effect on STAT6 activation (which was inhibited), but was consistent with increased nuclear localization and DNA binding of AP-1 and NFATc2. Purified CD4+ T cells did not alter cytokine production with NAC, suggesting that the NAC-dependent effect was localized in the APC populations. We evaluated splenocytes depleted of CD11b cells and treated with an anti-B7-2 (CD86) and found a block in IL-4 production and in the NAC effect. The obvious candidate for increased IL-4 production is alteration of IL-12. We used an IL-12 knockout animal and found that the NAC-induced increase in IL-4 production was not dependent on IL-12 expression. This study suggests that increasing intracellular thiol pools (free NAC and cysteine) can alter the Th1/Th2 balance by increasing IL-4 production and decreasing IFN-{gamma} production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Six- to 8-wk-old female BALB/c wild-type mice were purchased from Harlan (Indianapolis, IN). BALB/c IL-12p35-/- mice (C.129S1(B6)-Il12atm1Jm) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a pathogen-free environment at the Animal Care Facility at University of Iowa, and maintained on standard mouse chow and water ad libitum. All procedures used in this study were in compliance with Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Animals.

Abs and reagents

Recombinant murine IL-2 was purchased from Roche (Indianapolis, IN) and recombinant murine cytokines IL-4, IL-12, and IFN-{gamma} were purchased from R&D Systems (Minneapolis, MN). Low endotoxin and azide-free mAb against murine CD3{epsilon} was purchased from BD PharMingen (Los Angeles, CA). Monoclonal and polyclonal Abs against murine NFATc2, STAT6, and STAT4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-STAT4 Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Phospho-STAT6 Ab was obtained from Cell Signaling Technology (Beverly, CA). NAC was purchased from Sigma-Aldrich (St. Louis, MO). AP-1 and NF-{kappa}B oligonucleotides were from Promega (Madison, WI).

Cell culture

Spleens were aseptically removed from animals and then teased apart between the frosted edges of two glass slides. Cells were suspended in RBC lysis solution (1 mM KHCO3 and 15.5 mM NH4Cl), washed in 1x PBS, pelleted, filtered through a 70-µm pore size cell strainer (Falcon, Franklin Lakes, NJ), and resuspended in RMPI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine (Life Technologies), 50 µM 2-ME (Sigma-Aldrich) with or without stimulation at 37°C in 5% CO2 for the indicated time periods. Purified CD4+CD62L+ T cells were activated for 3 days in 12-well tissue culture plates precoated with 1 µg/ml {alpha}-CD3 and 1 µg/ml {alpha}-CD28.

Cell purifications and depletions

All cell purifications and depletions were conducted using protocols and reagents from Miltenyi Biotech (Auburn, CA). Single-cell suspensions were prepared from splenocytes depleted of RBC as described above. Briefly, CD4+CD62L+ T cells were isolated to >94% purity with positive selection using a combination of FITC anti-mouse L3T4 (GK1.5; BD Biosciences, San Diego, CA) and an anti-FITC Multisort Kit, followed by positive selection using anti-CD62L magnetic beads. CD11b+ cells were depleted from splenocyte suspensions using anti-CD11b+ magnetic beads. CD4+ T cells were depleted similarly using anti-CD4 magnetic beads.

ELISA

Murine IL-4, IL-2, IL-12p70, and IFN-{gamma} were detected in cell culture supernatants by use of ELISA DuoKits, all purchased from R&D Systems.

Isolation of nuclear extracts and EMSAs

The nuclear pellets were prepared by resuspending cells in 0.4 ml of lysis buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA), placing them on ice for 15 min, and then vigorously mixing after the addition of 25 µl of 10% Nonidet P-40. After a 30-s centrifugation (16,000 x g, 4°C), the pelleted nuclei were resuspended in 50 µl of extraction buffer (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, and 10% glycerol) and incubated on ice for 20 min. Nuclear extracts were stored at -70°C. The DNA binding reaction (EMSA) was performed at room temperature in a mixture containing 5 µg of nuclear proteins, 1 µg poly(d(I-C)), and 20,000 cpm of 32P-labeled, double-stranded oligonucleotide probe for 30 min. The samples were fractionated through a 5% polyacrylamide gel in 1x TBE (6.05 g/liter Tris base, 3.06 g/liter boric acid, and 0.37 g/liter EDTA-Na2·H2O). The sequence of the AP-1 nucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3' (AP-1), and the sequence for NF{kappa}B was 5'-AGTTGAGGGGACTTTCCCAGG-3'.

Western blotting

Western analysis for the presence of specific proteins or for phosphorylated forms of proteins was performed on nuclear proteins or whole cell sonicates and lysates from BALB/c splenocytes (24, 25). Nuclear protein (5–10 µg) or whole cell protein (20–50 µg) were mixed 1/1 with 2x sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromophenol blue, and 1.25 M Tris-HCl, pH 6.8; all from Sigma-Aldrich), fractionated on a 10% SDS-polyacrylamide gel, and run at 40 mA for 3 h. Proteins were transferred to nitrocellulose (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min at 20 V on a SemiDry Transfer Cell (Bio-Rad, Hercules, CA). The nitrocellulose was then blocked with 5% milk in TBST (Tris-buffered saline with 0.1% Tween 20) for 1 h, washed, and then incubated with primary Abs (diluted between 1/500 and 1/1000 in 5% milk in TBST) overnight at 4°C. The blots were washed four times with TBST and then incubated for 1 h with the appropriate HRP-conjugated anti-IgG Ab (Santa Cruz Biotechnology; diluted between 1/5,000 and 1/10,000 in 5% milk in TBST). Membranes were washed four times in TBST. Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus or ECL; Amersham Pharmacia Biotech). An autoradiograph was obtained, with exposure times of 10 s to 2 min. Equal loading of the blots is shown by either protein-specific blots (STATs) or total protein stain (NFATc2).

Measurement of intracellular NAC and total intracellular thiol levels

Total intracellular soluble small m.w. thiols (GSH, cysteine, {gamma}-glutamyl cysteine) and intracellular NAC levels were assayed following previous published protocols (26, 27, 28). Cell pellets were homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 1.34 mM diethylenetriaminepenta-acetic acid. NAC, cysteine, and {gamma}-glutamyl cysteine levels in cells were measured following derivatization with N-(1-pyrenyl)maleimide using a 15-cm C18 Reliasil column (Column Engineering, Ontario, Canada) coupled with HPLC with fluorescent detection (27). Total small m.w. soluble intracellular thiols were calculated as the sum of total GSH content determined spectrophotometrically (GSH and glutathione disulfide) plus cysteine and, where relevant, plus NAC, as determined by HPLC. No other soluble thiols reached detectable levels (100 fmol) using this system. The data are expressed as nanomoles of thiol per milligram of cellular protein as previously described (26, 27, 28).

Statistical analysis

Data were analyzed with PRISM software (GraphPad, San Diego, CA) using Student’s paired t test with p < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of naive splenocytes with soluble {alpha}CD3 or {alpha}CD3 plus IL-12 induces production of Th1/Th2 cytokines

Production of IL-4 and IFN-{gamma} by soluble {alpha}CD3 or {alpha}CD3 and IL-12 was analyzed by ELISA. Naive splenocytes were isolated from BALB/c mice and stimulated with 2.5 µg/ml of soluble {alpha}CD3 or soluble {alpha}CD3 plus 1 ng/ml of IL-12. Each mode of cell activation resulted in a peak of cytokine synthesis at 48 h. After completion of the experiments, supernatants were collected, and IL-4 or IFN-{gamma} was measured by ELISA. Soluble {alpha}CD3 alone stimulated significant production of IL-4 (Fig. 1). Addition of IL-12 as a stimulus slightly decreased the production of IL-4. To stimulate significant amounts of IFN-{gamma}, we needed to stimulate the cells with both {alpha}CD3 and IL-12 (Fig. 1). Previous studies have shown that a strong T cell stimulus via soluble {alpha}CD3 requires coengagement of accessory and costimulatory molecules, while plate-bound {alpha}CD3 initiates signal transduction by effectively cross-linking the TCR (29). In our system IFN-{gamma} was induced by immobilized {alpha}CD3 alone, but the IL-4 induction with immobilized {alpha}CD3 was significantly less (data not shown). Therefore, these studies use soluble {alpha}CD3 as a model for stimulation of a Th2 cytokine (IL-4) and soluble {alpha}CD3 plus IL-12 as a model for stimulation of a Th1 cytokine (IFN-{gamma}).



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  		FIGURE 1. Naive splenocytes produce IL-4 and IFN-{gamma} after stimulation with {alpha}CD3 and {alpha}CD3 plus IL-12. Naive splenocytes (2.5 million/ml) were exposed to either 2.5 µg/ml of soluble {alpha}CD3 or 2.5 µg/ml of {alpha}CD3 and 1 ng/ml of IL-12 or were left untreated for 48 h. Supernatant levels of IL-4 and IFN-{gamma} were determined by ELISA. The mean (±SEM) of six experiments are shown. p < 0.01.

 
NAC inhibits stimulation-induced IFN-{gamma} production and enhances IL-4 production

To study the effects of increasing intracellular thiols on the production of Th1 vs Th2 cytokines, naive splenocytes were treated with NAC. Briefly, freshly isolated splenocytes were either pretreated with 15 mM NAC for 45 min or cultured in medium without NAC. After the preincubation period, splenocytes were treated with soluble {alpha}CD3 (2.5 µg/ml) or {alpha}CD3 plus IL-12 (1 ng/ml) for 48 h in the continued presence or absence of NAC. After completion of the experiments, the supernatants were evaluated for IL-4 and IFN-{gamma} proteins. The effect of NAC on soluble thiol pools was confirmed by measuring intracellular free NAC, total GSH content, and cysteine in treated and untreated cells. Intracellular NAC levels increased from undetectable levels to an average of 4.87 ± 2.15 nmol/mg protein. Treatment with NAC increased cysteine levels from 3.5 nmol/mg protein in untreated cells to 14.1 nmol/mg protein in NAC-treated cells. Interestingly, NAC treatment did not alter total GSH levels, with untreated cells demonstrating 18 nmol/mg protein compared with 20 nmol/mg protein in NAC-treated cells. This suggests that the NAC-dependent alterations shown in this study are due to an effect on soluble thiol levels that do no include alterations in total GSH content. In Fig. 2A, we show that NAC treatment of either the IL-4 relevant stimuli ({alpha}CD3) or the IL-12 relevant stimuli ({alpha}CD3 plus IL-12) increased total intracellular small m.w. soluble thiols (~2-fold). Fig. 2B demonstrates that NAC enhances IL-4 production in {alpha}CD3-treated splenocytes. This also occurred in the {alpha}CD3- plus IL-12-treated cells. In fact, the use of both stimuli with NAC overcame the slight decrease in IL-4 production seen with {alpha}CD3 plus IL-12 alone. In contrast, NAC inhibited {alpha}CD3- plus IL-12-induced IFN-{gamma} production (Fig. 2C). These data suggest that altering intracellular soluble thiols has opposite effects on production of a Th2 cytokine vs a Th1 cytokine. Therefore, increased thiol levels during stimulation appeared to increase IL-4 and decrease IFN-{gamma}.



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  		FIGURE 2. NAC decreases IFN-{gamma} production and increases IL-4 production. A, Intracellular thiol levels of naive splenocytes in the presence or the absence of NAC (15 mM) were measured using methods described in Materials and Methods. Total small m.w. intracellular thiols were calculated as the sum of total GSH plus cysteine plus, where relevant, NAC. B, IL-4 production was assessed in supernatants from naive splenocytes (2.5 million/ml) cultured in the presence and the absence of NAC (15 mM) and exposed to 2.5 µg/ml of soluble {alpha}CD3 or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 for 48 h. IL-4 was determined in culture supernatants by ELISA. C, IFN-{gamma} production was assessed in supernatants from naive splenocytes (2.5 million/ml) cultured in the presence and the absence of NAC (15 mM) and exposed to {alpha}CD3 or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 for 48 h. IFN-{gamma} was determined in culture supernatants by ELISA. The mean (±SEM) of six independent experiments are shown. p < 0.01.

 
APCs are required for the NAC effect on IL-4 and IFN-{gamma} production

In the set of experiments shown in Fig. 3, we fractionated splenocyte populations and evaluated IL-4 and IFN-{gamma} production. In Fig. 3A, CD4+CD62L+ T cells were isolated from whole splenocyte cell populations. Because these cells do not respond without a cofactor (provided by the APCs in the whole splenocyte cultures), they were stimulated with {alpha}CD3+{alpha}CD28 instead of {alpha}CD3 or {alpha}CD3 and IL-12. We found that in naive CD4+ T cells NAC had no effect on the amounts of IL-4 and IFN-{gamma} produced. In Fig. 3B, we examined the effect of depleting CD4+ cells. Depletion of CD4+ cells completely eliminated IL-4 production, while leaving significant IFN-{gamma} production. Having found that purified CD4+ cells did not respond to NAC, we next evaluated the effect of altering APCs in the mixed culture. Depletion of CD11b-expressing cells blocked IL-4 production and significantly decreased IFN-{gamma} production (Fig. 3C). Confirming a role for APCs in the production of both IL-4 and IFN-{gamma} in mixed splenocyte cultures, we found that addition of an Ab to B7-2 (CD86) inhibited both IL-4 and IFN-{gamma} production, and addition of NAC did not override that block (Fig. 3D). As a composite, the data presented in Fig. 3 suggest that the effect of NAC on IL-4 and IFN-{gamma} occurs at the level of the APCs present in the culture.



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  		FIGURE 3. APCs are required for the NAC effect on IL-4 and IFN-{gamma} production. A, CD4+CD62L+ T cells were isolated from mixed splenocyte populations according to procedures detailed in Materials and Methods. Cells (0.5 million/ml) were cultured with and without NAC (15 mM) and with and without {alpha}CD3 and {alpha}CD28 for 72 h. IL-4 and IFN-{gamma} levels were determined in culture supernatants by ELISA. B, Splenocytes were depleted of CD4+ cells, cultured at 2.5 million/ml with and without NAC (15 mM), and exposed to 2.5 µg/ml of soluble {alpha}CD3 or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 for 48 h. IL-4 and IFN-{gamma} were analyzed as described above. C, Splenocytes were depleted of CD11b+ cells, cultured at 2.5 million/ml with and without NAC (15 mM), and exposed to 2.5 µg/ml of soluble {alpha}CD3 or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 for 48 h. IL-4 and IFN-{gamma} were analyzed as described above. D, Naive splenocytes (2.5 million/ml) cultured in the presence and the absence of NAC (15 mM) and exposed to 2.5 µg/ml of soluble {alpha}CD3 (IL-4) or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 (IFN-{gamma}) with the further addition in some groups of {alpha}CD86 (B7-2) for 48 h. IL-4 and IFN-{gamma} were analyzed as described above.

 
NAC-induced increases in IL-4 production are not due to effects on IL-12

Because of the demonstrated link between IL-12 and IL-4 inhibition (30, 31) and the observation that NAC breaks the IL-12 disulfide bonds resulting in a loss of the p40/p35 heterodimer (32), we wanted to evaluate the role of IL-12 in the NAC-induced increases in IL-4. Splenocyte cultures from IL-12-/- animals were incubated with our standard stimuli ({alpha}CD3 or {alpha}CD3 plus IL-12) and evaluated for IL-4 production. Fig. 4 demonstrates that alterations in IL-12 levels are not required for the increase in IL-4 production by NAC.



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  		FIGURE 4. NAC-induced increases in IL-4 production are not due to effects on IL-12. Splenocytes were isolated from BALB/c IL-12p35-/- mice, cultured at 2.5 million/ml, and exposed to 2.5 µg/ml of soluble {alpha}CD3 or 2.5 µg/ml of soluble {alpha}CD3 and 1 ng/ml of IL-12 for 48 h. IL-4 was analyzed as described above. The data are representative of two separate experiments.

 
NAC inhibits IL-12-mediated STAT4 activation

To assess the effect of {alpha}CD3 plus IL-12 on STAT4 activation, naive splenocytes were stimulated with {alpha}CD3 (2.5 µg/ml) and IL-12 (1 ng/ml) for various time periods. Whole cell protein was isolated, and STAT4 activity was analyzed using a phospho-specific STAT4 Ab. Fig. 5A demonstrates that IL-12 activates STAT4. To evaluate the effect of NAC on STAT4 activation, freshly isolated splenocytes were stimulated with {alpha}CD3 plus IL-12 in the presence or the absence of NAC (15 mM) for different time periods. Whole cell proteins were isolated, and phosphorylated STAT4 was measured. Fig. 5B demonstrates that NAC partially inhibits IL-12-mediated STAT4 activation. This suggests that one-way increased thiol levels down-regulate IFN-{gamma} production is via inhibition of {alpha}CD3- plus IL-12-induced STAT4 activation.



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  		FIGURE 5. STAT4 activation by {alpha}CD3 plus IL-12 is blocked by NAC pretreatment. A, Naive splenocytes (2.5 million/ml) were cultured in the presence of soluble {alpha}CD3 (2.5 µg/ml) and IL-12 (1 ng/ml) for different time periods (0, 5, 15, and 30 min and 1 and 2 h). Whole cell extracts were prepared and 20 µg of proteins were subjected to SDS-PAGE and Western blot analysis. The blots were analyzed for activated STAT4 using a phospho-specific STAT4 Ab. Ab concentrations of 1/1,000 (primary) and 1/10,000 (secondary) were used. Immunoreactive bands were visualized using chemiluminescence and exposure times of 30 s to 5 min. Equal loading of gels is demonstrated by stripping and reprobing blots for total STAT4. B, Naive splenocytes were cultured as described above in the presence and the absence of NAC (15 mM). Whole cell extracts were analyzed for phosphorylated STAT4 as described above. The figure is representative of at least four independent experiments. The graph depicts densitometry performed on immunoreactive bands and expressed as the fold increase (experimental value/control value) in arbitrary units.

 
NAC inhibits {alpha}CD3-mediated STAT6 activation

Binding of IL-4 to its receptor induces activation of STAT6 and contributes to further IL-4 production (33). Some studies suggest that STAT6 can be activated with stimuli other than IL-4 (22, 34). To assess the role of {alpha}CD3 treatment on STAT6 activation, the following experiment was performed. Naive splenocytes were freshly isolated from BALB/c mice and treated with 2.5 µg/ml of {alpha}CD3 for various time periods. Whole cell proteins were then isolated, and STAT6 activation was evaluated using an Ab specific for the phosphorylated form of STAT6. These experiments show that STAT6 is phosphorylated in response to soluble {alpha}CD3 (Fig. 6A). To confirm that the induction of STAT6 activation was solely due to {alpha}CD3-mediated activation and was not due to the presence of IL-4, these experiments were repeated in the presence of IL-4-neutralizing Ab (10 µl/ml). IL-4-neutralizing Ab did not alter the aCD3-induced STAT6 phosphorylation (data not shown). These observations suggest that TCR ligation alone can induce STAT6 activation. Having found that {alpha}CD3 induced STAT6 activation, the effect of increased intracellular thiols on STAT6 activation was evaluated next. Naive splenocytes were treated with and without NAC, followed by {alpha}CD3 (2.5 µg/ml) for various time periods. Whole cell proteins were isolated, and STAT6 activity was assessed using a phosphorylation-specific STAT6 Ab. Fig. 6B shows that NAC inhibits {alpha}CD3-mediated STAT6 activation. NAC also inhibited activation of STAT6 by IL-4 plus IL-2 (data not shown). These data show that NAC inhibits STAT6 activation, suggesting that the NAC increases IL-4 production via a STAT6-independent pathway.



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  		FIGURE 6. STAT6 activation by {alpha}CD3 is blocked by NAC pretreatment. A, Naive splenocytes (2.5 million/ml) were cultured in the presence of soluble {alpha}CD3 (2.5 µg/ml) for different time periods (0, 5, 15, and 30 and 1 and 2 h). Whole cell extracts were prepared, and 20 µg of protein was subjected to SDS-PAGE and Western blot analysis. The blots were analyzed for activated STAT6 using a phospho-specific STAT6 Ab. Ab concentrations of 1/1,000 (primary) and 1/10,000 (secondary) were used. Immunoreactive bands were visualized using chemiluminescence and exposure times of 30 s to 5 min. Equal loading of gels was demonstrated using with Ponceau S total protein stain. B, Naive splenocytes were cultured as described above in the presence and the absence of NAC (15 mM). Whole cell extracts were analyzed for phosphorylated STAT6 as described above. The figure is representative of at least four independent experiments. The graph depicts densitometry performed on immunoreactive bands and expressed as the fold increase (experimental value/control value) in arbitrary units.

 
NAC increases nuclear translocation of IL-4 relevant transcription factors AP-1 and NFATc2

Because STAT6 did not appear to be critical for increased IL-4 production by {alpha}CD3 with NAC, the effect of NAC on other transcription factors that might enhance IL-4 expression was evaluated. The NFAT proteins are a family of transcription factors (NFATc1, NFATc2, and NFATc3) that are present in both Th1 and Th2 cells. In Th2 cells, NFAT proteins have been shown to act cooperatively with additional transcription factors, such as AP-1, and NF{kappa}B, to enhance IL-4 gene expression (16). To evaluate the effect of NAC on the nuclear expression of these transcription factors, untreated or {alpha}CD3-treated naive splenocytes were cultured in the presence or the absence of 15 mM NAC for 45 min. Subsequently, nuclear extracts were prepared and used to evaluate AP-1 DNA binding and NFATc2 nuclear localization. Fig. 7A clearly demonstrates that NAC alone strongly up-regulates AP-1 DNA binding, and that {alpha}CD3 plus NAC treatment resulted in AP-1 levels greater than {alpha}CD3 alone. These results are in agreement with previous observations that antioxidants enhance AP-1 binding (35). The NAC effect did not require {alpha}CD3, suggesting that NAC provides a ready pool of AP-1 to cooperate with other stimuli-specific effects. NFATc2 has been directly linked to IL-4 production in a recent study by Rengarajan et al. (36). The effect of NAC on NFATc2 was evaluated by treating naive splenocytes with {alpha}CD3 with and without NAC for various time points, followed by isolation of nuclear protein. Fig. 7B demonstrates that soluble {alpha}CD3 triggers an early activation of NFATc2 and that this activation is enhanced in the presence of NAC. In comparison with the effects of NAC alone on AP-1 binding, NAC alone had no effect on NFATc2 nuclear localization (data not shown). Although {alpha}CD3 increased NF{kappa}B, there was no effect of NAC on NF{kappa}B DNA binding (data not shown). These data suggest that increasing intracellular small m.w. soluble thiols activates at least two of the transcription factors (AP-1 and NFATc2) involved in IL-4 transcription, independent of increased levels of total GSH.



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  		FIGURE 7. NAC increases DNA binding of AP-1 and nuclear localization of NFATc2. A, AP-1 activation in splenocytes (2.5 million/ml) after treatment with NAC (15 mM) and anti-CD3 (2.5 µg/ml) for different time points. Nuclear extracts were isolated, and 5 µg of protein was mixed with 32P-labeled AP-1-specific oligonucleotides and separated on a 10% polyacrylamide gel. Arrowheads indicate the positions of the specific DNA protein complexes. The gel shift is a representative of three independent experiments. B, NFATc2 nuclear localization in presence and the absence of NAC (15 mM). Naive splenocytes were exposed to 2.5 µg/ml of soluble anti-CD3 for different time periods or were left untreated. Five micrograms of nuclear proteins were subjected to SDS-PAGE, and standard Western analysis was performed. The blots were analyzed for NFATc2 using Ab against total NFATc2. A primary Ab concentration of 1/500 and a secondary Ab of 1/5000 were used. Immunoreactive bands were visualized using chemiluminescence. Equal loading of gels was demonstrated using with Ponceau S total protein stain. The gel is a representative of six independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study the effects of NAC on induction of a marker Th1 cytokine (IFN-{gamma}) and a marker Th2 cytokine (IL-4) were evaluated. We found that increasing intracellular soluble thiols with NAC significantly inhibited IFN-{gamma} production in splenocytes following treatment with {alpha}CD3 plus IL-12. In contrast, NAC significantly increased IL-4 production by {alpha}CD3 (Fig. 8). Both of these events (altered cytokine production) occurred in the setting of increased thiols (NAC and cysteine) with no changes in total GSH content. The effects of NAC on IL-4 production required APCs and did not occur if purified CD4+CD62L+ T cells were stimulated. IL-12 modulation (including a possible extracellular break in the IL-12 disulfide bonds) was not responsible for the effect of NAC on IL-4 production because it occurred in cells from an IL-12 knockout animal. Other possible mechanisms for the alterations in cytokine production include changes in signaling pathways and altered expression or activation of transcription factors. Consistent with an effect of NAC on IL-12 signaling, a decrease in STAT4 activation in the presence of NAC was detected. STAT6 can play the same positive role in IL-4 production that STAT4 does in IFN-{gamma} production. Interestingly, {alpha}CD3 activates STAT6 to a similar extent as does the classic Th2 stimulus, IL-4 plus IL-2. In contrast to the coordinated response to NAC of STAT4 and IFN-{gamma} production, NAC increased IL-4 production and inhibited STAT6 activation. Because our results indicate that the NAC-induced increases in IL-4 are independent of STAT6, other transcription factors relevant to IL-4 expression were evaluated. Both AP-1 and NFATc2 have been positively linked to IL-4 production (16, 36, 37). When we evaluated the effect of NAC on AP-1 DNA binding and NFATc2 nuclear translocation, we found that NAC caused a significant increase in the activation of both transcription factors. These data suggest that NAC promotes IL-4 synthesis in a STAT6-independent manner, potentially via an effect on other transcription factors, and that NAC decreases IFN-{gamma} production via inhibition of IL-12-mediated STAT4 activation. These observations are consistent with our hypothesis that an increase in intracellular soluble thiols affects the Th1/Th2 balance in favor of Th2 expression, rather than Th1 expression. The effects of NAC appear to be independent of changes in total GSH content.



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  		FIGURE 8. Increasing the concentration of intracellular small m.w. thiols inhibits IFN-{gamma} production and increases IL-4 production. This diagram depicts NAC effects on IL-12 and IL-4 production with the relative effects on STAT6 and STAT4.

 
A number of studies have indicated that cellular redox status plays a critical role in signal transduction in multiple physiologic and pathophysiologic conditions. Our data suggest that the altered cytokine effects in our mixed cultures are due to an effect on APCs in the cell population. During the inflammatory response, APCs are exposed to increased levels of intra- and extracellular ROS. This initial increased level of ROS has an important effect on the subsequent gene expression. ROS may modulate critical molecules of APC signaling pathways via redox regulation of sensitive (sulfhydryl groups) signaling molecules (38). GSH is a major intracellular redox buffer and plays a substantial role in protecting cells against oxidative injury. Cysteine (Cys) is a rate-limiting precursor for GSH synthesis, but also has thiol antioxidant activity in its own right. Low m.w. thiols such as 2-ME and NAC have been shown to increase resistance to oxidative stress-mediated apoptosis, increase synthesis of IL-2, and enhance proliferation (39). In this study the demonstrated effects of NAC treatment are due to alterations in intracellular NAC and cysteine levels and not to increases in total GSH. We have not yet determined the mechanism behind the APC-dependent alterations produced by NAC. We have shown that the effect of NAC is not due to an effect on IL-12 (either production in the case of {alpha}CD3 alone or dissolution of the disulfide bonds in the systems that include added IL-12). It will be of interest to define the NAC-dependent APC changes that lead to an increased production of IL-4 by CD4+ cells.

Some studies in other systems have found opposite results from our data. P. Jeannin (unpublished observations) has shown in human T cells that NAC increases IL-2 expression, decreases IL-4 expression, and has no effect on IFN-{gamma} expression. In contrast to their study we found increased IL-4 production in NAC-treated cells. The opposite effects seen in the two systems are possibly explained by the fact that the as yet undetermined APC effect in our system seems to override T cell-specific responses.

STAT proteins play a fundamental role in relaying intracellular signals elicited by cytokines (20). STAT4 is crucial for Th1 responses, and IL-12 and IL-18 have been shown to play a central role in cell-mediated immunity via activation of STAT4 (40). STAT4-deficient mice have impaired responses to IL-12 and lack Th1 differentiation (20). STAT4 activation has been shown to contribute to IFN-{gamma} production (40). Our data show that {alpha}CD3 alone did not activate STAT4; however, in the presence of IL-12, {alpha}CD3 activated STAT4, and this was associated with a 9-fold greater induction of IFN-{gamma}. Our results demonstrate that altering the intracellular soluble thiol pools decreased both STAT4 activation and IFN-{gamma} production, suggesting that these pathways are under redox regulation. Our data do not address the issue of which cell population is the source of the altered STATs, although we believe that it is probably the T cells.

STAT6 is activated upon exposure of T cells to IL-4 and IL-13 (14, 15). However, there are some reports suggesting that STAT6 can be activated by stimuli other than IL-4 and IL-13, including costimulation by CD28 and IL-2 (22). Although a direct role for STAT6 DNA binding in the IL-4 promoter has been postulated, several studies clearly demonstrate STAT6-independent IL-4 production (21, 41). These data suggest that STAT6 activation is not essential for IL-4 transcription, which is consistent with our data showing a divergent effect of NAC on STAT4 activation and IL-4 production. In our studies {alpha}CD3 stimulation of naive splenocytes activated STAT6. This was inhibited by NAC, and the inhibition did not decrease IL-4 production. IL-4 production, in fact, was increased by NAC pretreatment.

AP-1 is a transcription factor that positively regulates several cytokine genes, including IL-4. Several studies have indicated that cellular redox status affects AP-1 activation (42, 43, 44, 45). Further, DNA binding of the AP-1 complex requires that specific cysteine residues become reduced. In our study we showed that NAC strongly enhanced AP-1 DNA binding. The effect of NAC on AP-1 DNA binding is in agreement with previous studies and may explain in part the up-regulation of IL-4 by NAC. The calcium-regulated NFATc2 protein is located in the cytoplasm in resting cells. After Ag stimulation, NFATc2 is dephosphorylated by the phosphatase calcineurin and rapidly translocated into the nucleus, where it interacts with other transcription factors to induce cytokine gene expression (46). Although knockout studies have suggested a negative role for NFATc2 in IL-4 production (36), more recent studies have demonstrated a clear positive link between NFATc2 and IL-4 transcription (47). Calcineurin is a serine/threonine phosphatase that contains iron and zinc at its active site. It has been shown that calcineurin is sensitive to oxidative stress and may be modulated by the intracellular redox potential (48). In general, thiol antioxidants, such as NAC, GSH, and cysteine, appear to activate calcineurin, while oxidants, such as hydrogen peroxide, inactivate this phosphatase (49). These studies together with our data suggest an enhanced activation of NFATc2 in NAC-pretreated cells, which may explain in part the enhanced expression of IL-4.

In summary, the novel observations of this study are that alteration of intracellular small m.w. soluble thiol levels with NAC inhibits IFN-{gamma} production and increases IL-4 production independently of increases in GSH content. These alterations in cytokine production by mixed cell populations required the presence of APCs and did not occur in purified CD4+CD62L T cells. We found that both STAT6 and STAT4 activation appear to be subject to redox regulation by thiols. Inhibition of STAT4 activation was associated with significant down-regulation of IFN-{gamma} production. In contrast, inhibition of STAT6 did not decrease the expression of IL-4. The increased IL-4 production in the presence of NAC is consistent with the positive effect of NAC on AP-1 and NFATc2. These observations suggest that changing the intracellular soluble thiol pools may influence the Th1 and Th2 balance and contribute to the induction of particular cytokine profiles.


   Acknowledgments
 
We thank Susan A. Walsh for expert technical assistance with the measurement of cellular thiols.


   Footnotes
 
1 This work was supported by a Veterans Affairs Merit Review Grant (to G.W.H.); National Institutes of Health Grants HL60316 and ES09607 (to G.W.H.) and HL51469 and CA66081 (to D.R.S.), Environmental Protection Agency Grant R826711 (to G.W.H.), and Grant RR00059 from the General Clinical Research Centers Program, National Center for Research Resources, National Institutes of Health. Back

2 M.M.M. and L.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Martha Monick, Department of Internal Medicine, Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa, 100 EMRB, Iowa City, IA 52242. E-mail address: martha-monick{at}uiowa.edu Back

4 Abbreviations: GSH, glutathione; NAC, N-acetylcysteine; ROS, reactive oxygen species. Back

Received for publication March 4, 2003. Accepted for publication September 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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