IL-4 gene expression up-regulated by mercury in rat mast cells: a role of oxidant stress in IL-4 transcription

Zhonglin Wu, David R. Turner1, and David B. G. Oliveira

Divisions of Renal Medicine and
1 Histopathology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK

Correspondence to: Z. Wu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the Brown Norway (BN) rat, chemical compounds [mercuric chloride (HgCl2), D-penicillamine or gold salts] induce a Th2-dominated autoimmune syndrome with tissue injury in the form of a vasculitis and arthritis. An early phase of vasculitis in the model occurs within 24 h of an injection of HgCl2, is {alpha}ß T cell independent and involves the mast cell. In addition, HgCl2 induces IL-4 mRNA in mast cells from BN rats. Our recent work has demonstrated that the balance of oxidative/antioxidative influences plays an important role in the modulation of mast cell function (degranulation) in chemically induced autoimmunity. The aim of this study was to determine, in mast cells, whether oxidative status influences IL-4 transcription and translation, which is required for the development of a Th2 response. Exposure of the mast cell line RBL-2H3 to HgCl2 enhanced both IL-4 mRNA and its promoter activity. Oxidative stress by hydrogen peroxide mimicked the effects of HgCl2 in enhancing IL-4 promoter activity. The enhancement of IL-4 gene expression by HgCl2 was significantly reduced by antioxidants (both sulphydryl and non-sulphydryl containing). The same pattern of regulation was also observed on IL-4 protein expression in the mast cells. These data suggest a novel mechanism of IL-4 transcriptional up-regulation by oxidative stress. Our results provide evidence to support our hypothesis that alterations in intracellular reactive oxygen species production modulate both IL-4 gene expression and mast cell function.

Keywords: IL-4, mast cells, oxidant stress


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ Th lymphocytes can be divided into at least two functionally distinct Th cell types, based on their cytokine secretion patterns and effector functions (1). Th1 cells produce IL-2, IFN-{gamma} and lymphotoxin, and are involved in cell-mediated immune responses and tissue injury in many organ-specific autoimmune diseases, whereas Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, and are responsible for T cell-dependent antibody responses and allergic responses (1,2). There is cross-regulation between these cellular subsets with IFN-{gamma} inhibiting Th2 cells and IL-4/IL-10 inhibiting Th1 cells (3).

The importance of the Th1/Th2 cell division focuses attention on the factors involved in determining whether an immune response will be principally of the Th1 or Th2 cell type. Differentiation of naive T cells towards either the Th1 or Th2 cell type is believed to be a consequence of several cellular influences, such as the cytokine environment; in particular, IL-4 is required for the development of a Th2 cell response (4). Although differentiated Th2 cells themselves produce this cytokine, which may then act in an autocrine or paracrine way to reinforce the differentiation process, the initial cellular source of IL-4 required for instigation of Th2 cell development is unknown. A rare subpopulation of T cells capable of producing IL-4 without prior exposure to this cytokine is one possibility (5). Another is the mast cell, which is known to produce an array of cytokines including IL-4 (6). It has been shown that CD4+ T cells alone are sufficient for an initial IgE response, but that non-CD4+ cells (probably mast cells or basophils) may augment a memory IgE response (7). The relationship between mast cells and Th2 cell activity therefore remains unclear.

We have previously investigated a possible role for the mast cell in a Th2-dominated model—chemically induced autoimmunity in the Brown Norway (BN) rat (810). Administration of mercuric chloride (HgCl2), D-penicillamine or gold salts to the BN rat induces an autoimmune syndrome characterized by the production of autoantibodies (1113), tissue injury in the form of a necrotizing vasculitis principally affecting the caecum, an inflammatory polyarthritis and marked increase of total serum IgE concentration (1416). This syndrome has been shown to be T cell dependent (17,18). The rise in serum IgE implicates Th2 cells, as, at least in the mouse, IL-4 is required for isotype switching to IgE (19). We have previously demonstrated that depletion of CD45RChi cells (a Th1 cell-like subset in the rat) aggravated the tissue injury (20), supporting Th2 cell involvement. Further evidence shows that the analogous syndrome induced by HgCl2 in susceptible stains of mice can be modulated by treatment with an antibody to IL-4 (21).

In the BN rat model, significant vasculitis occurs in the caecum within 24 h of an injection of HgCl2 (22,23), a time at which there is no significant T cell infiltrate, although vasculitis peaks at ~2 weeks after the administration of HgCl2, at the same time as the serological response. Our recent work has demonstrated that the mast cell plays a role in the early phase, since the early vasculitis is {alpha}ß T cell independent, is accompanied by in vivo evidence of mast cell degranulation and can be ameliorated by treatment with mast cell stabilizers (10). Our previous studies have also shown that IL-4 mRNA expression was up-regulated in the caecum within 24 h of treatment with HgCl2 (22), and that in vitro HgCl2 significantly enhanced degranulation and induced IL-4 mRNA transcripts in mast cells from BN rats (8).

We have recently shown that: (i) the compounds which induce an autoimmune syndrome in BN rats generate reactive oxygen species (ROS) in mast cells of BN rats (9) and in the mast cell line RBL-2H3 (unpublished observation); (ii) direct oxidative stress mimics the effect of these compounds on degranulation in mast cells; and (iii) modulation of ROS production/redox balance within mast cells modulates the effects of these compounds on mast cell function (degranulation) (9). These results support the hypothesis that the balance of oxidative/antioxidative influences plays an important role in the modulation of mast cell function, especially in the context of chemically induced autoimmunity. Based on these results, we propose as a unifying hypothesis that alterations in intracellular ROS production modulate IL-4 mRNA transcription, in a similar way to that found with mast cell degranulation.

The aims of this work, therefore, were to determine whether HgCl2 can induce or enhance IL-4 expression in the mast cell line RBL-2H3 and, if so, to test our hypothesis by determining whether oxidative stress and antioxidatives would modulate IL-4 gene expression. For comparison and monitoring of RT-PCR analysis, spleen cells from BN rats were also examined for HgCl2 stimulation and antioxidant inhibition in this study.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture and reagents
The rat mast cell line RBL-2H3 was purchased from ATCC (Rockville, MD). The cells were maintained in Eagle's minimum essential medium supplemented with 15% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml of streptomycin (Gibco/BRL Life Technologies, Paisley, UK). The cells were removed twice a week by trypsinization and passaged to new flasks at a cell density of 2x106 cells/20 ml of medium. Spleens were removed from male BN rats (10–12 weeks old) and teased apart in RPMI 1640 medium (Gibco/BRL Life Technologies). After incubation in Boyle's solution to lyse red blood cells, total spleen cells (1–5x107) were cultured for 2 and 4 h with or without HgCl2 or other compounds. The viability of the cells was monitored before and after experiments using Trypan blue exclusion.

HgCl2, hydrogen peroxide (H2O2), glutathione ethyl ester (reduced GSH), N-acetyl-L-cysteine (NAC) and desferrioxamine mesylate were purchased from Sigma (St Louis, MO), and pyruvate from Boehringer Mannheim (Lewes, UK). All the compounds were dissolved or diluted in PBS, and the pH of reduced GSH and NAC solution was adjusted to 7.4 before use. Mouse anti-rat IL-4 mAb was purchased from Serotec (Oxford, UK).

RT-PCR
RNA was prepared by a method adapted from Chomczynski and Sacci (24). Briefly, total RNA was isolated by treatment of washed cells with guanidium lysis buffer followed by extraction with phenol–chloroform. cDNA was synthesized from ~5 µg of total RNA using a cDNA cycle kit (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations.

For PCR, cDNA was made up with 25 pmol of each PCR primer, 200 µM concentration of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Little Chalfont, UK), 1xreaction buffer and 5 U Taq Polymerase (Appligene-Oncor, Ilkirch, France) for ß-actin amplification or 7.5 U AmpliTaq Gold (PE Applied Biosystem, Foster City, CA) with 1 mM MgCl2 for IL-4 amplification. The forward and reverse primers and the expected PCR product sizes (in parentheses) are as follows: ß-actin 5'-ATGCCATCCTGCGTCTGGACCTGGC-3' and 5'-AGCATTTGCGGTGCA CGATGGAGGG-3' (607 bp); IL-4 5'-TGATGGGTCTCAGCCCCCACCTTGC-3' and 5'-CTTTCAGTGTTGTGAGCGTGGACTC-3' (378 bp). The amount of starting material (cDNA) and the number of PCR cycles were selected so that each product was within the exponential phase. A negative control (without cDNA) was used to detect any false-positive product. PCR was performed in a DNA thermal cycler (PHC-3; Techne, Cambridge, UK) with 25–40 cycles of 94°C (1 min), 60°C (2 min) and 72°C (3 min). Pre-PCR incubation at 95°C for 10 min was performed for amplification of IL-4 cDNA.

The PCR products were separated in 1.5% agarose gels containing ethidium bromide and photographed. A 100 bp DNA ladder (Gibco/BRL Life Technologies) was included for reference. Densitometric analysis of the bands was carried out using the gel Imager Program (Image 7500; UVP, Cambridge, UK). Results are expressed as the ratio of the intensity of the band for IL-4 to the intensity of the band for ß-actin. The specificity of the PCR products was further checked by DNA sequencing using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit and ABI 373A DNA sequencer (Applied Biosystem, Perkin-Elmer, Foster City, CA). The sequence data were analysed by Sequence Navigator and Auto Assembler software.

Plasmid construction
The IL-4 promoter of the BN rat was cloned into pGEM-T vector (Promega, Madison, WI) and sequenced (kindly provided by Dr K. Gillespie, Bristol). The promoter region (between –726 and +26) was then amplified by PCR using Vent polymerase (New England Biolabs, Beverly, MA) with the forward primer 5'-GTAGCTCTCGAGTGCAGAGATACACACATCC-3' (incorporated XhoI site underlined) and the reverse primer 5'-GACCAAAAGCTTCTAACAATGCAATGGTGAC-3' (incorporated HindIII site underlined). The amplified 732 bp fragment was digested with the relevant restriction enzymes, purified and inserted into the corresponding sites of the pGL3-basic vector upstream of the luciferase gene (Promega). The construct was further confirmed by sequencing between the two sites, and purified by the EndoFree plasmid kit (Qiagen, Hilden, Germany). The ß-galactosidase reporter construct incorporating the rat actin promoter was a gift of Dr S. Goodbourn (Department of Biochemistry, St George's Hospital Medical School) (25). Expression of actin and G6PDH genes has been tested in this experimental set-up, and shown to be equivalent and unaffected by HgCl2, oxidant or antioxidants.

Transient transfection assays
Transient transfection was carried out using SuperFect transfection reagent (Qiagen). RBL-2H3 cells (8x105 cell/dish) were plated in a 60-mm dish 1 day before transfection. The cells were first co-transfected with 2.5 µg of the IL-4 promoter construct and 2.5 µg of the actin control plasmid by incubation with the reagent–DNA complexes for 1 h at 37°C, then washed and maintained in fresh medium. After 40 h incubation, the cells were pretreated for 1 h in the presence or absence of 2 mM GSH, followed in some cases by washing twice with PBS. The cells were then incubated with or without HgCl2 or H2O2 for 6 h.

For reporter gene assays the cells were lysed in 250 µl lysis buffer containing 25 mM Tris–phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol and 1% Triton X-100, and cell debris was pelleted. The luciferase activity (determined by the IL-4 promoter) in the cell lysates was determined by a luminometer (LB9501; Berthold, Bad Wildbad, Germany), while ß-galactosidase activity (determined by the actin promoter) was measured by a spectrophotometer after incubation of the cell lysates with substrate solution (Promega). The values of luciferase activity obtained were normalized against ß-galactosidase activity.

Immunohistochemistry
Cytospin preparations of RBL-2H3 cells were fixed in alcohol and endogenous peroxidase production blocked with methanol/H2O2. Antigen retrieval was effected by microwave treatment in citrate buffer. The mouse mAb against rat IL-4 at 1:100 dilution using 1% BSA was applied to the cells and a Doko Envision kit (monoclonal) provided enhancement and the visual marker. The slides were lightly counterstained with Mayes haematoxylin.

Statistics
Summary statistics represent mean ± SEM. The percentage enhancement or, where appropriate, the percentage inhibition was used to analyse the difference in IL-4 mRNA expression. Data were analysed using the computer program Arcus Pro Stat. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
HgCl2 up-regulates IL-4 mRNA synthesis in RBL-2H3 cells
To determine whether HgCl2 induces or enhances IL-4 gene expression in the rat mast cell line RBL-2H3, both RT-PCR analysis and transient transfection assays with the IL-4 promoter were performed. Preliminary experiments (data not shown) optimized the concentration of HgCl2 to produce a range of concentrations that had stimulatory effect without influencing the viability of the cells. Within this range, HgCl2 enhanced the baseline expression of IL-4 mRNA in a dose-dependent manner in RBL-2H3 cells (Fig. 1A and BGo). The PCR products for IL-4 and ß-actin were ~400 or 600 bp in size as shown in agarose gels respectively, and their specificity was confirmed by DNA sequencing (data not shown). A concentration of HgCl2 at 1–2x10–5 M was then used in the majority of the following experiments. The concentrations of HgCl2 (as well as GSH) used in this study are comparable to those used in previous work (9,26,27). Time-course experiments showed that up-regulation of IL-4 mRNA was maximal at 2–4 h, decreasing by 6 h and almost back to baseline by 24 h (Fig. 1CGo), again comparable to previous work (28).





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Fig. 1. RT-PCR analysis of IL-4 mRNA expression in RBL-2H3 mast cells treated with HgCl2. (A) The mast cells were treated without (lane 1) or with HgCl2 2x10–5, 1.5x10–5, 10–5, 10–6 or 10–7 M (lanes 2–6) for 4 h. M indicates DNA size markers, product sizes are on the right side. C indicates the reaction control (without cDNA). (B) Results from (A) are expressed as the ratio of the intensity of the band for IL-4 to the intensity of the band for ß-actin and are representative of two independent experiments. (C) The mast cells were treated without or with HgCl2 (10–5 M) for 2, 4, 6 or 24 h.

 
To clarify that the elevated level of IL-4 mRNA caused by HgCl2 treatment is due to increased synthesis rather than a decreased breakdown rate, transient transfection assays were performed. RBL-2H3 cells were transfected with either the pGL3 basic plasmid or the construct that contained the IL-4 promoter in pGL3. Approximately 100% increase in relative luciferase activity was observed in transfection with the construct containing the IL-4 promoter as compared with pGL3 basic alone (data not shown), indicating constitutive expression of the IL-4 gene in the cells. Upon treatment with HgCl2 (Fig. 2Go), significant increases of luciferase activity were recorded (P = 0.0308 two-tailed paired t-test).



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Fig. 2. Effect of HgCl2 on activation of IL-4 promoter activity in RBL-2H3 cells. The cells were co-transfected with pGL3-IL-4 promoter–luciferase construct and rat actin promoter–ß-galactosidase construct. After a 40 h incubation, the cells were cultured for 6 h in the absence and in the presence of HgCl2 (10–5 M). Luciferase activities in cell lysates were determined by a luminometer and normalized with ß-galactosidase activity of the lysates. The experiment was carried out in triplicate. Values are shown as mean ± SEM. *P < 0.05 compared with control group. Similar results were obtained from three separate experiments.

 
In agreement with the above results and those from our previous studies on BN rats (22), IL-4 mRNA was readily detectable in spleen cells from BN rats and was up-regulated in the presence of HgCl2 (data shown below).

Oxidative stress influences IL-4 promoter activity in RBL-2H3 cells
To test the hypothesis that ROS may cause up-regulation of mast cell IL-4 transcription, RBL-2H3 cells were exposed to H2O2 and IL-4 mRNA, and its promoter activity was analysed. Concentrations of H2O2 between 10–7 and 10–9 M modestly but significantly enhanced IL-4 mRNA expression (Fig. 3Go, Kruskal–Wallis H-test P = 0.0114). In transient transfection assays, RBL-2H3 cells treated with different concentrations of H2O2 (range 10–6 to 10–8 M) displayed a modest but consistent augmentation in luciferase activity, maximal at 10–8 M H2O2 (representative example shown in Fig. 4Go). The increase in luciferase activity was significant (P = 0.006, n = 5, t-test corrected for multiple comparisons at the different concentrations) at the concentration of 10–8 M H2O2 using pooled data from two separate experiments. The fact that H2O2 enhanced IL-4 promoter activity in a way similar to that seen with HgCl2 indicated a possible role of ROS in up-regulation of mast cell IL-4 by HgCl2. This question was addressed by exposing the cells to HgCl2 with or without redox-active compounds.



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Fig. 3. RT-PCR analysis of IL-4 mRNA expression in RBL-2H3 mast cells treated with H2O2. The mast cells were incubated for 4 h in the absence and in the presence of H2O2 at the indicated concentrations. Results are expressed as the percentage enhancement of IL-4 mRNA from four separate experiments. *P < 0.05 compared with control group.

 


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Fig. 4. Effect of H2O2 on activation of IL-4 promoter activity in RBL-2H3 cells. After transfection and incubation (described in the legend of Fig. 2Go), the cells were incubated for 6 h without or with the indicated concentrations of H2O2. Luciferase activities were normalized with ß-galactosidase activity of cell lysates. Results are representative of three independent experiments. Values are shown as mean ± SEM. *P < 0.05 compared with control group.

 
Antioxidants decrease HgCl2-enhanced up-regulation of IL-4 mRNA
The increase in the amount of IL-4 mRNA transcript in splenocytes from BN rats treated with HgCl2 for 4 h, but not for 2 h (Fig. 5Go), was decreased by co-incubation with GSH 2x10–3 M (using percentage increase and inhibition with respect to controls from four independent experiments, Mann–Whitney U-test P = 0.0139). NAC, a precursor of intracellular GSH, was also used on spleen cells of BN rats and showed a similar inhibition of HgCl2-enhanced IL-4 mRNA transcripts (data not shown).



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Fig. 5. GSH inhibits HgCl2-induced up-regulation of IL-4 mRNA expression in spleen cells of BN rats. RT-PCR was performed using splenocytes incubated for 2 and 4 h in the absence of HgCl2 and in the presence of HgCl2 (10–5 M) ± GSH (2x10–3 M). Results are expressed as the ratio of the intensity of the band for IL-4 to the intensity of the band for ß-actin and are representative of four independent experiments.

 
Since GSH contains free sulphydryl groups which might react with HgCl2 extracellularly, the effect of GSH on HgCl2-enhanced IL-4 gene expression was further determined by introducing washing steps prior to adding HgCl2. Following 40 h transfection of IL-4 promoter constructs, RBL-2H3 cells were incubated with or without GSH (2x10–3 M) for 1 h and then washed twice with PBS. The washed cells were cultured in the presence or absence of HgCl2 (10–5 M) for 6 h. The relative luciferase activity and the percentage enhancement, shown in Fig. 6Go, was calculated by subtracting the luciferase activity in HgCl2 untreated cells from the activity in HgCl2-treated ones and 100% enhancement was defined as that of the HgCl2-treated cells without GSH. The results shown in Fig. 6Go demonstrate a significant inhibition of HgCl2-enhanced promoter activity down to 38% (GSH) or 51% (GSH, wash). The IL-4 promoter activity was significantly reduced (P = 0.0235, Newman and Keuls test) in the mast cells by GSH even when the cells were washed twice before being stimulated with HgCl2. However, no differences were observed between cells treated with or without washing steps (P = 0.4734).



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Fig. 6. GSH decreases HgCl2-induced up-regulation of IL-4 promoter activity in RBL-2H3 cells. After transfection and incubation (described in the legend of Fig. 2Go), RBL-2H3 cells were incubated with or without GSH (2x10–3 M) for 1 h and then washed twice with PBS. The washed cells were cultured in the presence or absence of HgCl2 (10–5 M) for 6 h. The relative luciferase activity data and the percentage enhancement shown here were calculated by subtracting the luciferase activity in HgCl2 untreated cells from the activity in HgCl2-treated ones and 100% enhancement was defined as that of the HgCl2-treated cells without GSH. Data are shown as mean ± SEM of measurements from triplicates and are representative of two independent experiments. *P < 0.05 compared with control group (cells treated with HgCl2 only).

 
To investigate if antioxidants, in addition to GSH and NAC, similarly reduce IL-4 transcripts, the effects of non-sulphydryl-containing antioxidants, the iron chelator desferrioxamine and the H2O2 scavenger pyruvate (9,29), were determined. As shown in Fig. 7Go, treatment of the RBL-2H3 cells with either desferrioxamine or pyruvate significantly decreased HgCl2-enhanced IL-4 mRNA expression (Kruskal–Wallis P = 0.0016).



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Fig. 7. Antioxidants desferrioxamine mesylate and pyruvate decrease HgCl2-induced up-regulation of IL-4 mRNA expression in RBL-2H3 cells. The mast cells were incubated for 16 h in the absence or in the presence of 10–4 M desferrioxamine (Desf.) or 5x10–2 M of pyruvate (Pyr.) and then exposed to HgCl2 (10–5 M) for 2 or 4 h. The same amounts of the antioxidants were also added at –2 and 0 h before the addition of HgCl2. Data pooled from four separate experiments and expressed as the ratio of the intensity of the band for IL-4 to the intensity of the band for ß-actin. *P < 0.05 compared with HgCl2 group (cells treated with HgCl2 only).

 
Oxidative stress and antioxidatives modulate IL-4 protein expression
To examine protein expression of IL-4 in RBL-2H3 cells, immunoperoxidase staining with anti-rat IL-4 antibody was performed. Basal expression of IL-4 was detected in RBL-2H3 cells (Fig. 8AGo) and the level of the protein was enhanced after HgCl2 or H2O2 treatment for 2 days (Fig. 8B and CGo). The HgCl2-enhanced IL-4 protein expression was reduced by co-incubation with GSH 2x10–3 M (Fig. 8DGo) or NAC (data not shown).






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Fig. 8. Immunoperoxidase staining with IL-4 mAb. The RBL-2H3 cells were cultured for 48 h in the absence (A) or presence of HgCl2 at 10–5 M (B), or presence of H2O2 at 10–7 M (C), or presence of HgCl2 at 10–5 M and GSH at 2x10–3 M (D) (original magnification x400).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study demonstrates that HgCl2, a compound inducing autoimmunity in BN rats in vivo, enhances both IL-4 gene and protein expression in a mast cell line RBL-2H3 in vitro, that oxidative stress by H2O2 mimics the effects of HgCl2 in enhancing IL-4 promoter activity and translation, and that antioxidants diminish the HgCl2-induced enhancement of IL-4. Our results provide evidence to support our hypothesis that alterations in intracellular ROS production modulate both IL-4 gene expression and mast cell function (9).

Our data from the mast cell line have confirmed our previous work in BN rat peritoneal mast cells showing an enhancing effect of HgCl2 on IL-4 mRNA (8). In general, isolation and purification of large numbers of normal mast cells are always difficult, while results from unpurified mast cells are often questioned on the basis of contamination with other cells. The availability of large numbers of RBL-2H3 cells made it possible to perform the transient transfection assay in the present study and to further investigate the transcriptional regulation of the IL-4 gene in mast cells. The results from the transient transfection in RBL-2H3 cells demonstrated that the enhancing effect of HgCl2 on IL-4 mRNA was mediated by activation of the IL-4 promoter of the mast cells, rather than mediated by stabilization of IL-4 mRNA. Therefore, all the results strongly support the up-regulatory effect of HgCl2 on IL-4 gene expression in rat mast cells. Moreover, our current data, together with previous results (22,28), suggest similar effects of HgCl2 on up-regulating IL-4 mRNA expression in both mast cells and spleen cells (which are predominately lymphocytes).

Oxidative stress is involved in many diseases including inflammation and autoimmune disease (30,31). H2O2 is one of the predominant oxidants produced by cells such as eosinophils and neutrophils, and, intracellularly, is a central mediator to which other oxidants may convert (3234). We have previously shown that exposure of peritoneal mast cells and the RBL-2H3 mast cell line to HgCl2, gold compounds or D-penicillamine induces the intracellular production of ROS, and that direct oxidative stress in the form of H2O2 also causes sensitization for serotonin release in a similar way to HgCl2 (9). Other recent work has revealed that in vitro exposure to HgCl2 produced a concentration-dependent increase of oxidants in different regions of the rat brain and that in vivo exposure to mercury produced a significant decrease of the antioxidant enzyme superoxide dismutase (35). Our data in the current study have suggested that oxidant stress by H2O2 may enhance IL-4 expression in RBL-2H3 cells, which closely resembles the effect of HgCl2. Very low concentrations of H2O2 have been used in the transient transfection assay, which are more likely to be physiologically relevant and to have no effect on the viability of the cells (36). Interestingly, the concentration of H2O2 (10–8 M) which produced the highest IL-4 promoter activity is the concentration which caused the greatest enhancement of serotonin release in peritoneal mast cells (9), suggesting a similar effect both on IL-4 promoter activity and mast cell function.

GSH is a thiol antioxidant which is capable of increasing T cell proliferation and IL-2 production (27,37), and decreasing oxidative stress-induced apoptosis (38). NAC is a precursor of intracellular GSH (39). It has been shown in this report that these antioxidants can inhibit HgCl2-enhanced IL-4 gene expression and that the effect of inhibition may be due to intracellular events rather than the extracellular binding of Hg2+ ions. Desferrioxamine is an iron chelator which inhibits the Fenton reaction and hence prevents H2O2 from converting into the highly reactive hydroxyl radical (40). Pyruvate is capable of reducing H2O2 to water while it concomitantly undergoes non-enzymatic decarboxylation at the 1-carbon position (29). The fact that both the sulphydryl- and non-sulphydryl-containing antioxidants effectively reduce the HgCl2-enhanced IL-4 gene expression supports our hypothesis that the balance of oxidative/antioxidative influences may play an important role in the modulation of both IL-4 transcription and mast cell function. Our report is consistent with the observation that GSH and NAC decrease phorbol myristate acetate plus ionomycin- or concanavalin A-induced IL-4 production in T cells, and IL-4-induced IgE and IgG4 production in vitro, and IgE and IgG1 in vivo in a mouse model (27), although the generation of intracellular ROS and the effect of direct oxidative stress on the T cells were not analysed in that study. Studies on whether antioxidants are able to inhibit HgCl2-induced early vasculitis and up-regulation of IL-4 in mast cells in vivo are ongoing in this laboratory.

Factors and mechanisms regulating oxidant response in eukaryotic cells remain to be understood. NF-{kappa}B is one inducible transcription factor activated by H2O2 treatment of cultured cells (41). Many inducers of NF-{kappa}B seems to depend upon the production of ROS, as is evident from the inhibitory effect of antioxidants on its induction (42,43). HgCl2 has been shown to activate NF-{kappa}B in the RBL-2H3 cells in a transient transfection assay (our unpublished observation), but further studies are required to clarify the mechanism of the activation of NF-{kappa}B by HgCl2.

Other work has shown that mercury-induced IL-4 gene expression in T cells is associated with protein kinase C activation and L-type calcium channels (26). However, the transcriptional mechanisms of IL-4 gene expression following HgCl2 stimulation are undetermined (44). Transcription factors such as NF-AT and AP-1, and signal transducer and activator of transcription (STAT) 6, are possible candidates for involvement in IL-4 gene regulation. Studies on antioxidants regulating IgE isotype switching have suggested that coordination between STAT 6 and NF-{kappa}B is required for induction of germline C{varepsilon} transcription by IL-4 in human B cell lines (45). Although there is no evidence showing an NF-{kappa}B binding site within the rat IL-4 promoter region, all five NF-AT family members are distantly related to the NF-{kappa}B family (44). Therefore, further studies on transcriptional regulation of IL-4 and NF-{kappa}B will be necessary for a full understanding of the molecular mechanisms of HgCl2-enhanced IL-4 gene expression in mast cells.

Although we observed similar induction of IL-4 transcription in both RBL-2H3 mast cells and splenocytes from BN rats in vitro, in vivo IL-4 gene expression in the spleen of HgCl2-treated BN rats peaked 10 days after the start of HgCl2; this may represent activation of Th2-type CD4+ cells (22,46). In contrast, an early peak of IL-4 expression in the caecum caused by HgCl2 treatment in vivo occurs at 2 days when lymphocyte infiltration of the tissue is not prominent and may represent IL-4 transcription by resident mast cells (22). A speculative possibility is that production of IL-4 by mast cells upon HgCl2 stimulation could play a role in initiating and/or augmenting the subsequent Th2 cell development in the BN model.

In conclusion, we have provided evidence to support the hypothesis that oxidative stress plays a role in the up-regulation of IL-4 gene expression in rat mast cells and splenocytes. This could be directly relevant to the pathogenesis of HgCl2-induced autoimmunity in the BN rat and, more generally, suggests that oxidative stress may favour a Th2-dominated state in predisposed individuals.


    Acknowledgments
 
We wish to thank Dr K. Gillespie (Southmead Hospital, Bristol) for providing IL-4 promoter clone, Dr S. Goodbourn (Department of Biochemistry) for the actin construct and access to the Luminometer, and Dr A. Clarke (Department of Haematology) for the densitometric analysis. This work was supported by a Wellcome Trust Project Grant.


    Abbreviations
 
BN Brown Norway
GSH glutathione ethyl ester
H2O2 hydrogen peroxide
HgCl2 mercuric chloride
NAC N-acetyl-L-cysteine
ROS reactive oxygen species
STAT signal transducer and activator of transcription

    Notes
 
Transmitting editor: M. Feldmann

Received 13 April 2000, accepted 27 November 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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