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Originally published In Press as doi:10.1074/jbc.M200808200 on March 15, 2002

J. Biol. Chem., Vol. 277, Issue 20, 17871-17876, May 17, 2002
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Protection of Mice from Allergen-induced Asthma by Selenite

PREVENTION OF EOSINOPHIL INFILTRATION BY INHIBITION OF NF-kappa B ACTIVATION*

Dae-won JeongDagger §, Min-Hyuk Yoo§, Tae Soo KimDagger , Jae-Hong KimDagger , and Ick Young KimDagger ||

From the Dagger  Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea and the  Department of Life Science, Kwangju Institute of Science and Technology, Kwang-Ju 500-712, Korea

Received for publication, January 25, 2002, and in revised form, March 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The potential anti-inflammatory effect of sodium selenite in a mouse model of asthma was investigated. Selenite was injected into the peritoneum of allergen (ovalbumin)-sensitized mice before allergen challenge. Ovalbumin challenge resulted in activation of the transcription factor NF-kappa B and an increase in the expression of cell adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, which are encoded by NF-kappa B-dependent genes) in lung tissue as well as in the recruitment of eosinophils to lung airways. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with selenite. Selenite administration also increased the activity of selenium-dependent glutathione peroxidase in lung tissue. Furthermore, supplementation of A549 human airway epithelial cell cultures with selenite increased glutathione peroxidase activity as well as inhibited both the generation of hydrogen peroxide and the activation of NF-kappa B induced by tumor necrosis factor alpha  in these cells. Selenite also reversed in vitro the activation of NF-kappa B induced by this cytokine in intact A549 cells. These results suggest that selenite regulates the activity of NF-kappa B by increasing the activity of glutathione peroxidase, thereby removing potential activators of NF-kappa B, and possibly also by direct oxidation of critical sulfhydryl groups of this transcription factor. These effects of selenite likely underlie its anti-inflammatory action in asthma.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Asthma is a chronic respiratory disease characterized by three major symptoms: airway hyper-reactivity, airway obstruction, and lung inflammation. Numerous cytokines (including interleukins 4, 5, 9, 10, and 13), the chemokine eotaxin, and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1),1 vascular cell adhesion molecule 1 (VCAM-1), and E-selectin contribute to the allergic inflammatory response associated with this condition (1-5). The expression of the genes for these various proteins in inflammation-related cells is induced as a result of activation of the transcription factor NF-kappa B (6-8) and is directly associated with the recruitment of eosinophils that is apparent early after pulmonary allergen challenge (9-11).

Dietary micronutrients and antioxidants such as vitamins A, C, and E, zinc, and selenium are implicated as determinants of the severity of bronchial asthma (12-14). Selenium is an essential biological trace element in mammals and is incorporated into selenoproteins in the form of selenocysteine, which is encoded in mRNA by the codon UGA (15). Mammalian selenoproteins include glutathione peroxidase (GPx), thioredoxin reductase, and thyroxine 5'-deiodinase (16-18). GPx is an antioxidant enzyme that scavenges H2O2 and organic hydroperoxides and whose expression level depends on selenium availability (16, 19-21).

The redox state of biological thiol groups regulates various intracellular signal transduction events in cells of the immune system. The activity of GPx is important for maintaining cellular thiol homeostasis. An increase in the amount of GPx in selenium-supplemented cells has been shown to inhibit both the activation of mitogen-activated protein kinases such as p38, JNK1 or JNK2, and ERK1 or ERK2 as well as the NF-kappa B-mediated signaling pathway (22-25).

Selenium has also been shown to prevent cancer in several animal models (26-30) as well as in humans (31). This chemopreventive effect is thought to result from modulation of cysteine residues in proteins by selenium. Selenium regulates the functions of many proteins by reacting with essential thiol groups to form S-Se-S (selenotrisulfide) or S-Se adducts (28). We and others have previously shown that proteins modulated as a result of thiol modification by selenium include the Na+- and K+-dependent ATPase, the glucocorticoid receptor, prostaglandin D synthase, the transcription factors AP-1 and NF-kappa B, the protease caspase-3, and the protein kinases Cdk2, protein kinase C, and JNK (32-40).

In remains unclear, however, whether a low dietary intake of selenium and reduced GPx activity are associated with an increased risk of asthmatic inflammation. Given that the activities of GPx and NF-kappa B are determined by selenium availability, we have now investigated the effects of the activity levels of these proteins on allergen-induced asthmatic reactions both in selenite-treated mice and in selenite-supplemented airway cells. We now show that the increased GPx activity in selenite-treated mice is associated with inhibition of allergen-induced NF-kappa B activation as well as of consequent inflammatory gene expression and eosinophil recruitment to the airways. In addition, NF-kappa B activity in A549 airway epithelial cells supplemented with selenite was directly inhibited as a result of thiol modification of this transcription factor. Furthermore, selenite-supplemented A549 cells were shown to scavenge reactive oxygen intermediates (ROI), such as H2O2, that contribute to NF-kappa B activation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- The human airway epithelial cell line A549 was cultured under a humidified atmosphere of 5% CO2 at 37 °C in Ham's F-12 medium supplemented with 10% of fetal bovine serum, 100 units/ml of penicillin G, 100 units/ml of streptomycin, and 0.25 µg/ml of amphotericin.

Sensitization and Challenge of Mice-- The sensitization and challenge of mice were achieved by a modified version of the method described by Kanehiro et al. (4). In brief, mice (6 weeks of age) were injected intraperitoneally with 500 µg of ovalbumin (Sigma) and 2 mg of aluminum (Pierce). A second intraperitoneal injection of 20 µg of ovalbumin adsorbed to aluminum hydroperoxide gel was administered 10 days after the first injection. After an additional 10 days, the mice were exposed to an aerosol of 1% ovalbumin in phosphate-buffered saline (PBS) for 30 min at 1-day intervals for 3 days. Two days after the final exposure to the aerosol of 1% ovalbumin, the animals were challenged with a nebula of 10% ovalbumin in PBS. Nebulization was performed in a plastic chamber connected to an ultrasonic nebulizer (Omron, Vernon Hills, IL) that allows entry of an ovalbumin aerosol. Sodium selenite (50 µg) or N-acetylcysteine (3 mg) was injected into the peritoneum of mice 1 and 24 h before ovalbumin challenge.

Lung Histology and BAL Fluid Collection-- Lungs and bronchoalveolar lavage (BAL) fluid were removed and collected, respectively, from mice 48 h after ovalbumin challenge. Sections of lung tissue and BAL fluid were stained with hematoxylin-eosin (Sigma) and Diff-Quick (Merck), respectively, for examination of histology and eosinophil recruitment as described previously (10).

Preparation of Nuclear Extracts from Lung and A549 Cells-- Nuclear extracts were prepared as described previously (38), with modifications. A549 cells were washed with PBS, scraped into lysis buffer (10 mM Hepes-NaOH, pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptin), and maintained on ice for 15 min before the addition of Nonidet P-40 to a final concentration of 0.3%. After centrifugation of the cell lysate at 10,000 × g for 1 min at 4 °C, the nuclear pellet was resuspended in nuclear extraction buffer (20 mM Hepes-NaOH, pH 7.9, 0.5 mM EGTA, 0.5 mM EDTA, 420 mM NaCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptin) and incubated on ice for 30 min with intermittent mixing. The nuclear extract (supernatant) was then collected after centrifugation at 12,000 × g for 5 min at 4 °C. For preparation of a nuclear extract from lung tissue, the lungs were washed twice with PBS to remove blood and then homogenized vigorously with a pestle in lysis buffer containing 0.3% Nonidet P-40. The homogenate was then processed further as described for the A549 cell lysate.

Electrophoretic Mobility Shift Assay Analysis-- Electrophoretic mobility shift assay (EMSA) analysis was performed with nuclear extracts and a double-stranded oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Santa Cruz Biotechnology) containing a consensus NF-kappa B binding site (underlined); the probe was end-labeled with [gamma -32P]ATP by T4 polynucleotide kinase (Takara, Japan) and was purified by chromatography on a spin column prepared with Sephadex G-25 (Sigma). The EMSA buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5% glycerol, 50 mM NaCl, 1 mM MgCl2, 0.1 mM GTP, 0.1 mg/ml bovine serum albumin, and 50 µg/ml poly(dI-dC)) containing 5 µg of nuclear extract protein was mixed with the 32P-labeled oligonucleotide probe (30 fmol) and then incubated (final volume, 20 µl) at room temperature for 30 min. For competition experiments, 3 pmol of unlabeled oligonucleotide probe or of a mutant duplex (5'-AGTTGAGGCGACTTTCCCAGGC-3'; Santa Cruz Biotechnology) were added to the reaction mixture 10 min before exposure to 30 fmol of the labeled probe. DNA-protein complexes were separated by electrophoresis through a 6% polyacrylamide gel under nondenaturing conditions, after which the gel was dried and exposed to Kodak X-Omat film.

Immunoblot Analysis-- The lungs were washed twice with PBS and then homogenized with a pestle in 500 µl of a solution containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% Nonidet P-40. The homogenate was centrifuged at 12,000 × g for 10 min, and the resulting supernatant was collected as cytosolic extract of lung tissue. The extract (40 µg of protein) was mixed with sample buffer, boiled for 5 min, and fractionated by SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to an Immobilon-P membrane (Millipore) and probed with a mouse monoclonal antibody to ICAM-1, rabbit polyclonal antibodies to VCAM-1, or a mouse monoclonal antibody to E-selectin (Santa Cruz Biotechnology). Immune complexes were detected with appropriate secondary antibodies and enhanced chemiluminescence reagents (Amersham Biosciences).

Assay of GPx Activity-- A549 cells (5 × 104) were seeded onto 60-mm dishes and cultured for 24 h. They were then incubated first for 7 days in medium supplemented with 10% dialyzed fetal bovine serum and then for 3 days in the presence of 100 nM selenite, as described previously (25). Cytosolic extracts were prepared by scraping the cells into a solution containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% Nonidet P-40 and passing them several times through a 26-gauge needle attached to a syringe, followed by centrifugation at 12,000 × g for 10 min. Cytosolic extract (40 µg of protein) prepared from A549 cells or from lung tissue (as described for immunoblot analysis) was incubated at room temperature for 2 min in 1 ml of a reaction mixture containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM reduced glutathione, 200 µM NADPH, and 1 unit of glutathione reductase (Sigma), after which the reaction was initiated by the addition of tert-butyl hydroperoxide to a final concentration of 35 µM. GPx activity was monitored by measuring the decrease in absorbance at 340 nm, reflecting the oxidation of NADPH.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibition by Selenite of Airway Obstruction and Eosinophil Infiltration of the Lung-- The effect of selenite on airway inflammation was examined by intraperitoneal injection of this compound 1 and 24 h before challenge of sensitized mice with ovalbumin. Histological analysis of lung sections stained with hematoxylin-eosin revealed that ovalbumin challenge of mice resulted in an increase in muscle mass and airway obstruction, compared with control sensitized mice that were challenged with PBS (Fig. 1). These effects of ovalbumin challenge were prevented by pretreatment with selenite. Pretreatment of mice with N-acetylcysteine (NAC), a glutathione precursor and ROI scavenger with antioxidant activity, also greatly reduced the effects of ovalbumin challenge. Examination of BAL fluid collected from mice 48 h after ovalbumin challenge revealed a marked increase in the number of eosinophils (Fig. 2). Thus, whereas the number of eosinophils in BAL fluid from naive control mice or from PBS-challenged sensitized mice was less than 1% of total cells, that in BAL fluid from the ovalbumin-challenged mice was ~47%. Again, prior administration of selenite or NAC greatly reduced the extent of eosinophil infiltration into the airways of ovalbumin-challenged mice.


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  		Fig. 1.   Anti-inflammatory effect of selenite in ovalbumin-challenged mice. Sensitized mice were challenged with PBS or with ovalbumin (OVA), 48 h after which lung tissue was subjected to histological analysis by staining with hematoxylin-eosin (upper panels). Sensitized mice that were pretreated with 2.5 mg/Kg of selenite (Se) or with 150 mg/Kg of NAC before ovalbumin challenge were similarly analyzed (lower panels). The arrows indicate lung obstruction. Magnification was ×50.


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  		Fig. 2.   Inhibition by selenite of eosinophil recruitment to airways. A, BAL fluid was collected 48 h after challenge from mice treated as described in the legend to Fig. 1. The fluid was stained with Diff-Quick for microscopic detection of eosinophils (arrows). Magnification was ×200. B, the number of eosinophils in BAL fluid of mice treated as in A was determined as a percentage of the total cells. Naive mice were also examined as a control. The data are the means ± S.E. of values obtained from seven animals/group. *, p < 0.0001 versus control (Student's t test). OVA, ovalbumin; Se, selenite.

Inhibition by Selenite of NF-kappa B Activation in the Lung-- Given that activation of NF-kappa B is required for the expression of inflammatory mediator genes that occurs early during lung inflammation (9-11), we next examined the possible effect of selenite on the activation of this transcription factor in the lungs of ovalbumin-challenged mice. EMSA analysis revealed that ovalbumin challenge of sensitized mice resulted in a marked increase in the DNA binding activity of NF-kappa B in nuclear extracts prepared from the lungs 48 h after challenge (Fig. 3). This effect of ovalbumin challenge was greatly reduced in mice that had been pretreated with selenite or with NAC.


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  		Fig. 3.   Inhibition by selenite of the activation of NF-kappa B in lung tissue. The lungs were removed 48 h after challenge from mice treated as described in the legend to Fig. 1. Nuclear extracts were then prepared from the tissue and subjected to EMSA analysis of the DNA binding activity of NF-kappa B. Nuclear extract prepared from the lung tissue of a naive control mouse was similarly analyzed. The arrow indicates the specific DNA-probe complex. OVA, ovalbumin; Se, selenite.

Inhibition by Selenite of Inflammatory Mediator Gene Expression in the Lung-- Cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin are expressed during asthmatic inflammation, and the genes that encode these proteins are regulated by NF-kappa B. We therefore examined the expression of adhesion proteins in cytosolic extracts prepared from the lungs of mice challenged with ovalbumin. Immunoblot analysis revealed that the abundance of ICAM-1, VCAM-1, and E-selectin in the lungs of ovalbumin-sensitized challenged mice was markedly increased compared with that in the lungs of naive control mice (Fig. 4). The expression of all three proteins was greatly inhibited by treatment of mice with selenite or with NAC before ovalbumin challenge. It has been well known that IgE is produced and secreted from B cells stimulated by cytokine during lung inflammation (41). Therefore, when horseradish peroxidase-conjugated mouse anti-IgG monoclonal antibody was used to probe a mouse anti-ICAM-1 or a mouse anti-E-selectin monoclonal antibody, heavy chain (molecular mass = approx 55 kDa) of IgE originated from the lung of ovalbumin-challenged mice was detected as shown in Fig. 4 (A and C).


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  		Fig. 4.   Immunoblot analysis of the effects of selenite treatment on the expression of ICAM-1 (A), VCAM-1 (B), and E-selectin (C) in the lungs of ovalbumin-challenged mice. Sensitized mice were pretreated (or not) with selenite (Se) or NAC before ovalbumin (OVA) challenge. The cell extracts were prepared from the lungs of the experimental mice 48 h after challenge as well as from the lung tissue of a naive control mouse. The extracts were subjected to immunoblot analysis with antibodies to ICAM-1, VCAM-1, or E-selectin; the blots were reprobed with antibodies to beta -actin to confirm consistent application of samples. The positions of molecular size standards (in kDa) are indicated on the left.

Inhibition by Selenite of TNF-alpha -induced NF-kappa B Activation in A549 Cells-- To examine the possible effect of selenite on the DNA binding activity of NF-kappa B in vitro, we exposed A549 airway epithelial cells to TNF-alpha for 1 h to induce NF-kappa B activation and then subjected nuclear extracts prepared from the cells to EMSA analysis. Incubation of the nuclear extract with 3 µM selenite for 10 min before exposure to the 32P-labeled probe reversed the increase in the DNA binding activity of NF-kappa B induced by TNF-alpha (Fig. 5A). The DNA binding activity of NF-kappa B lost after treatment of nuclear extract with 10 µM selenite was completely recovered by subsequent exposure to 2 mM DTT. These results suggest that selenite is able to inhibit NF-kappa B activity in vitro by direct modification of thiol groups. The addition of 0.5 mM DTT was necessary for the full reduction of NF-kappa B but was not sufficient to recover NF-kappa B inactivated by selenite.


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  		Fig. 5.   Effects of selenite on the DNA binding activity of NF-kappa B in nuclear extracts of A549 cells. A, effect of selenite (Se) in vitro. The cells were incubated for 1 h with recombinant human TNF-alpha (Pepro Tech) at a concentration of 40 ng/ml, after which the nuclear extracts were prepared. The extracts were incubated for 10 min at room temperature in the absence or presence of 3 or 10 µM selenite before the addition of binding buffer containing 0.5 or 2 mM DTT and 32P-labeled kappa B oligonucleotide for EMSA analysis. The asterisk indicates that the nuclear extract was exposed to DTT before treatment with selenite. B, effect of selenite in vivo. The cells were incubated (or not) in the presence of 5 µM selenite for 6, 12, or 24 h before exposure to TNF-alpha (40 ng/ml) for 1 h. The nuclear extracts were then subjected to EMSA analysis with or without prior incubation in the presence of a 100-fold molar excess of unlabeled wild-type (WT) or mutant (Mut) kappa B oligonucleotide. The positions of free probe and NF-kappa B-DNA complexes are indicated.

To determine the effect of selenite treatment in vivo on the DNA binding activity of NF-kappa B, we incubated A549 cells with 5 µM selenite for 6, 12, or 24 h before exposure of the cells to TNF-alpha for 1 h. The increase in the DNA binding activity of NF-kappa B induced by TNF-alpha was inhibited in a time-dependent manner by pretreatment of the cells with selenite (Fig. 5B). The specificity of the observed DNA binding activity was revealed by its sensitivity to competition by a 100-fold excess of unlabeled kappa B oligonucleotide and by its resistance to the addition of a mutant oligonucleotide.

Increase in GPx Activity in Selenite-supplemented A549 Cells and Selenite-treated Mice-- Exposure of cultured cells to selenite has been shown to result in an increase in GPx activity capable of scavenging H2O2 (19-21). Incubation of A549 cells with 100 nM selenite for 3 days induced an approximately 3.3-fold increase in GPx activity measured in cell extracts (Fig. 6A). The GPx activity in the lung tissue of mice treated with selenite before ovalbumin challenge was also about twice that in the lungs either of mice not treated with selenite before challenge or of naive control mice (Fig. 6B). Immunoblot analysis revealed that the increases in GPx activity in both A549 cells and mouse lung induced by selenite were accompanied by increases in the abundance of GPx protein (Fig. 6).


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  		Fig. 6.   Effects of selenite on GPx activity in A549 cells (A) and mouse lung (B). A, A549 cells were maintained for 7 days in culture medium containing 10% dialyzed fetal bovine serum and were then incubated for 3 days in the absence (Control) or presence of 100 nM selenite (Se). Cytosolic extracts were then prepared and assayed for GPx activity. The data are expressed as nanomoles of NADPH oxidized/min/milligram of protein and are the means ± S.E. of values from five independent experiments. *, p < 0.0001 versus control (Student's t test). B, sensitized mice were subjected to ovalbumin (OVA) challenge either with or without selenite pretreatment. The lung tissue was removed 48 h after challenge, and the cytosolic extracts were prepared from lung tissue and then analyzed as in A. Lung tissue from naive control mice was similarly processed. The data are the means ± S.E. of values from five animals/group. *, p < 0.0001; dagger , p < 0.05 versus respective control (Student's t test). Insets, extracts were also subjected to immunoblot analysis with rabbit polyclonal antibodies to GPx.

Inhibition by Selenite of the Intracellular Generation of H2O2 in A549 Cells-- The intracellular generation of H2O2 in response to a variety of exogenous stimuli is thought to contribute to the activation of NF-kappa B (42). The overexpression of H2O2-scavenging enzymes such as GPx and catalase thus prevents activation of the NF-kappa B signaling pathway (43, 44). We measured the intracellular generation of H2O2 by flow cytometry in A549 cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin diacetate; 2',7'-dichlorofluorescin diacetate is converted inside cells to 2',7'-dichlorofluorescin, which is oxidized by H2O2 to the fluorescent 2',7'-dichlorofluorescein. The intracellular concentration of H2O2 was increased in a time-dependent manner by exposure of control A549 cells to TNF-alpha , reaching a maximum at 12 min (Fig. 7). In contrast, the generation of H2O2 in response to TNF-alpha was not detected in A549 cells that had been cultured in the presence of 100 nM selenite. Furthermore, the basal concentration of H2O2 in the selenite-supplemented cells was reduced compared with that in the control cells.


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  		Fig. 7.   Effect of selenite on the intracellular generation of H2O2 in A549 cells. A, the cells were cultured in medium containing 10% dialyzed fetal bovine serum for 7 days and incubated in the absence (open circles) or presence (closed circles) of 100 nM selenite (Se) for 3 days. They were then loaded with 20 µM 2',7'-dichlorofluorescin diacetate for 30 min, washed with PBS, detached from the culture dish by exposure to trypsin, isolated by centrifugation, resuspended in medium containing 10% dialyzed fetal bovine serum, and subjected to flow cytometry (FACSCalibur, Becton Dickinson). 2',7'-Dichlorofluorescein fluorescence was excited at 488 nm and measured at 530 nm. Base-line fluorescence was monitored for 2 min, after which the cells were exposed (arrows) to TNF-alpha (40 ng/ml). The data are presented as 2',7'-dichlorofluorescein fluorescence index and are from a representative experiment. B, 2',7'-dichlorofluorescein fluorescence index at 12 min after the addition of TNF-alpha was quantitated for cells pretreated or not with selenite and was then expressed as a percentage of the basal value for cells not exposed to selenite. The data are the means ± S.E. of values from three independent experiments. *, p < 0.01 versus the basal value for cells not exposed to selenite (Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although selenium has been implicated as a determinant of asthma severity (13, 14), the mechanism for such an association has been unclear. We have now investigated the anti-inflammatory effect of selenium on asthma by intraperitoneal injection of selenite in allergen-sensitized mice. Such treatment with selenite greatly reduced the extent of NF-kappa B activation, NF-kappa B-dependent inflammatory protein expression, and eosinophil infiltration in the lung induced by allergen challenge. Selenite treatment also increased GPx activity in the lung. Furthermore, supplementation of A549 airway epithelial cells with selenite also resulted in an increase in GPx activity as well as in removal of H2O2 and in inhibition of NF-kappa B activation induced by TNF-alpha . Selenite also reversed in vitro the activation of NF-kappa B induced by TNF-alpha in A549 cells. Together, our results thus indicate that selenite regulates NF-kappa B activity by increasing the activity of GPx and possibly also by direct oxidation of essential sulfhydryl groups of this transcription factor, thereby greatly ameliorating the allergen-induced asthmatic response (Fig. 8).


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  		Fig. 8.   Proposed mechanism for the inhibitory effect of selenite on allergen-induced asthma.

Selenium is an essential trace element in mammals. It is incorporated into selenoproteins as selenocysteine, which is encoded by a UGA codon that normally functions as a signal for the termination of protein synthesis (15). Selenoproteins include the antioxidants GPx, selenoprotein P, and thioredoxin reductase as well as thyroxine 5'-deiodinase (45). The activity of these proteins thus depends on the concentration of selenium available for their biosynthesis (19-21).

In a mouse model of asthma, we have now shown that ovalbumin-induced asthmatic symptoms, such as lung obstruction and eosinophil recruitment to lung airways, were greatly reduced in animals pretreated with selenite. In investigating the mechanism of this anti-inflammatory effect, we showed by EMSA analysis that the increase in the DNA binding activity of NF-kappa B in lung tissue induced by ovalbumin challenge was markedly inhibited by pretreatment of mice with selenite. Selenite also inhibited the DNA binding activity of NF-kappa B in nuclear extracts prepared from A549 cells treated with TNF-alpha . This inhibition was reversed by the subsequent addition of 2 mM DTT to the nuclear extracts. Furthermore, the addition of selenite to A549 cell cultures resulted in a time-dependent inhibition of the activation of NF-kappa B by TNF-alpha .

NF-kappa B plays a critical role in the transcription of genes for various inflammatory mediators, including cytokines, eotaxin, and cell adhesion molecules (6-8). The expression of cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin on endothelial cells during inflammation is thus regulated through NF-kappa B activation (6, 8, 10). We have now shown that the expression of ICAM-1, VCAM-1, and E-selectin is increased in the lung tissue of ovalbumin-challenged mice and that this increased expression is abolished by pretreatment with selenite. These data thus suggest that the transcription of the genes for these cell adhesion molecules in lung endothelium is controlled by NF-kappa B and that inhibition of NF-kappa B activation by selenite blocks their expression as well as the consequent recruitment of eosinophils to the lung airway.

Overexpression of GPx inhibits NF-kappa B-dependent signaling (46). We have now shown that the activity of selenium-dependent GPx was increased in the lungs of selenite-treated mice as well as in extracts of A549 cells cultured in the presence of selenite. These selenite-induced increases in GPx activity were accompanied by increases in the abundance of GPx protein. Moreover, the concentration of total glutathione in the blood of mice treated with selenite was twice that in mice not treated with selenite (data not shown). The rate of glutathione depletion in cells is increased by selenite supplementation, with the oxidized form of glutathione being released from the cells, and this increased rate of glutathione depletion is indicative of an increased GPx activity (21). The selenite-induced increase in GPx activity in the lungs of ovalbumin-challenged mice thus likely results in the rapid removal of ROI (such as H2O2) that contribute to the activation of NF-kappa B.

ROI generated in cells are thought to function as second messengers in the NF-kappa B signaling pathway (43, 44, 46-52). Our present data support this notion: (i) TNF-alpha induced the intracellular accumulation of H2O2 in A549 cells; (ii) preincubation of the cells with selenite prevented this effect of TNF-alpha ; and (iii) the TNF-alpha -induced activation of NF-kappa B in A549 cells was blocked by pretreatment with buthionine sulfoximine, an inhibitor of glutathione synthesis that increases the basal concentration of peroxide (data not shown).

The NF-kappa B signaling pathway has been proposed as a promising target for therapeutic intervention in inflammatory diseases (53-56). Our results are consistent with this proposal. In summary, we have shown that GPx activity is increased in the lung tissue of selenite-treated mice and in selenite-supplemented A549 cells. Furthermore, selenite blocks the TNF-alpha -induced generation of H2O2 and activation of NF-kappa B in A549 cells as well as the allergen-induced activation of NF-kappa B, inflammatory mediator gene expression, and eosinophil infiltration in the lungs of mice. Together, our data indicate that selenite ameliorates asthmatic symptoms and that ROI (such as H2O2) play a pivotal role in allergen-induced inflammatory signaling.

    ACKNOWLEDGEMENT

We thank Dr. Ho Zoon Chae (Chonnam National University, Kwangju, Korea) for kindly providing the antibodies to GPx.

    FOOTNOTES

* This work was supported by Grant KOSEF 2000-2-20900-008-5 from the Korea Science and Engineering Foundation (to I. Y. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this study.

|| To whom correspondence should be addressed: Laboratory of Cellular and Molecular Biochemistry, Graduate School of Biotechnology, Korea University, 1 5-Ka, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Korea. Tel.: 82-2-3290-3449; Fax: 82-2-3290-3449; E-mail: ickkim@korea.ac.kr.

Published, JBC Papers in Press, March 15, 2002, DOI 10.1074/jbc.M200808200

    ABBREVIATIONS

The abbreviations used are: ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; GPx, glutathione peroxidase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; ROI, reactive oxygen intermediate(s); PBS, phosphate-buffered saline; BAL, bronchoalveolar lavage; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; NAC, N-acetylcysteine; TNF-alpha , tumor necrosis factor alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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