Role of oxidants in influenza virus-induced gene expression

Katharine Knobil1, Augustine M. K. Choi1, Gordon W. Weigand2, and David B. Jacoby1

1 Division of Pulmonary and Critical Care Medicine and 2 Division of Clinical and Molecular Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Influenza virus-induced epithelial damage may be mediated, in part, by reactive oxygen intermediates (ROIs). In this study, we investigated the role of ROIs in the influenza virus-induced gene expression of antioxidant enzymes and in the activation of nuclear factor-kappa B (NF-kappa B), an oxidant-sensitive transcriptional factor. Influenza virus infection increased production of intracellular ROIs in A549 pulmonary epithelial cells. Induction of manganese superoxide dismutase (MnSOD) mRNA correlated with increased MnSOD protein and enzyme activity. Influenza virus infection also activated NF-kappa B binding as determined by an electrophoretic mobility shift assay. Pretreatment of A549 cells with N-acetyl-L-cysteine attenuated virus-induced NF-kappa B activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. In contrast, pyrrolidine dithiocarbamate blocked activation of NF-kappa B and induction of MnSOD and IL-8 mRNAs. Treatment with pyrrolidine dithiocarbamate also markedly decreased virus-induced cell death. Thus oxidants are involved in influenza virus-induced activation of NF-kappa B, in the expression of IL-8 and MnSOD, and in virus-induced cell death.

manganese superoxide dismutase; interleukin-8; N-acetyl-L-cysteine; pyrrolidine dithiocarbamate; nuclear factor-kappa B

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

INFLUENZA VIRUS INFECTION causes pathological changes predominantly in the epithelial cell layer of the respiratory tract. A role for reactive oxygen intermediates (ROIs) as mediators of virus-induced epithelial damage is supported by studies in which antioxidant treatment decreases lung damage and mortality in influenza-infected mice (1). The source of these oxidants may be leukocytes, which are activated and primed by influenza virus infection (1), or xanthine oxidase, which is increased in influenza-infected lungs (1). Alternatively, the source of the ROIs may be the epithelial cells of the lung themselves. Airway epithelial cells produce hydrogen peroxide (H2O2) during oxidant stress (15). Jacoby and Choi (14) previously showed that influenza virus infection causes an oxidant stress response in cultured airway epithelial cells.

There are several major cellular enzymes that defend against oxidant stress. Superoxide (O<SUP>−</SUP><SUB>2</SUB>⋅) can be metabolized via the dismutation reaction (O<SUP>−</SUP><SUB>2</SUB>⋅ O<SUP>−</SUP><SUB>2</SUB>⋅ + 2H+ right-arrow O2 + H2O2), which is catalyzed by copper-zinc superoxide dismutase (Cu,ZnSOD) or manganese superoxide dismutase (MnSOD). Cu,ZnSOD is a cytoplasmic enzyme that is constitutively expressed, whereas MnSOD is a mitochondrial enzyme that is induced in response to oxidant stress (24). The H2O2 produced in the dismutase reaction is converted to H2O by catalase in peroxisomes and by glutathione peroxidase (GP) in the cytoplasm (24). Superoxide can also be metabolized by indoleamine 2,3-dioxygenase (IDO), which uses O<SUP>−</SUP><SUB>2</SUB>⋅ in the oxidation of tryptophan. This reaction does not produce H2O2 (32). Expression of both MnSOD and IDO is induced by influenza virus infection in mouse lungs (7, 32) and in primary cultures of human airway epithelial cells (14).

Although oxidant production may be directly involved in tissue damage, endogenous oxidants may also be involved in signal transduction pathways that activate transcriptional factors and induction of gene expression in influenza-infected cells. Choi and Jacoby (6) showed that influenza virus infection of human airway epithelial cells in culture induces expression of the strongly chemotactic cytokine interleukin-8 (IL-8). Expression of the IL-8 gene is influenced by the oxidant-sensitive transcription factor nuclear factor-kappa B (NF-kappa B) (16), which is activated in the lungs of influenza-infected mice (14). We postulate that production of oxidants by influenza-infected lung epithelial cells plays an important role in intracellular signaling, in activating oxidant-sensitive transcription factors, and in inducing expression of both proinflammatory cytokines and antioxidant genes.

In the present study, we characterize the induction of antioxidant genes and IL-8 as well as the activation of NF-kappa B during influenza virus infection. We further investigate the role of ROIs in regulating MnSOD and IL-8 gene expression in this system.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture conditions. A549 cells (a human lung epithelial cell line derived from a malignant tumor) were obtained from the American Type Culture Collection (ATCC; Rockville, MD). A second epithelial cell line, 16 HBE 14o- cells, was a gift from Dieter Gruenert (University of California, San Francisco). This cell line was derived from human bronchial epithelial cells transformed with origin-deficient SV40. The cells were maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum, 4 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B. Cultures were maintained at 34°C in a humidified atmosphere of 5% CO2-95% air.

Influenza virus. Human influenza virus A/Port Chalmers/72 (H3N2) (ATCC) was grown in embryonated chicken eggs. Fluid collected from the eggs was stored at -70°C. Viral content of this virus stock was determined by a hemadsorption assay as described in Hemadsorption.

Viral inactivation. Influenza virus was inactivated by incubation at 37°C for 2 h after the addition of 0.25% propionolactone. Lack of infectivity was determined by hemadsorption assay as described in Hemadsorption. Control cell cultures were treated with inactivated virus as described for active virus.

Hemadsorption. Viral content was determined by exposing rhesus monkey kidney (RMK) cell monolayers to serial 10-fold dilutions of virus stock or supernatant from virus-infected cells. After 1 wk, the monolayers were incubated with guinea pig erythrocytes for 1 h at 4°C. The monolayers were washed with Hanks' buffered salt solution (HBSS) three times to remove nonadherant erythrocytes. Viral infection was demonstrated by adsorption of erythrocytes to virus-infected cells as seen under phase-contrast microscopy. The dilution that infected 50% of the monolayers (TCID50) was calculated.

Infection of epithelial cells. Because serum inhibits influenza replication, serum-containing medium was removed, and the cells were washed with HBSS. The cells were then exposed to 105 TCID50 influenza virus per milliliter in LHC-8e medium for 1 h at 34°C and were then washed with HBSS. The HBSS was replaced with LHC-8e medium, and RNA and proteins were collected at 1, 3, 6, 24, 48, and 72 h (see RNA isolation and Northern blot analysis and Cellular nuclear protein extraction). To demonstrate viral infection and replication, fresh RMK cell monolayers were exposed to serial 10-fold dilutions of the supernatants from infected epithelial cell monolayers. Infection of RMK cells was demonstrated by hemadsorption as described in Hemadsorption.

Measurement of oxidant production. Cells were grown in P 100 plates. One hour before infection, the medium was removed, the monolayers were washed, and serum-free medium either alone or containing pyrrolidine dithiocarbamate (PDTC; 100 µM) or N-acetyl-L-cysteine (NAC; 20 mM) was replaced in the cultures. One hour later, either growth medium or virus was added. After incubation for 72 h, cells were scraped, suspended at 1 × 10-6 cells/ml in HBSS, and incubated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate for 15 min at 37°C. In the presence of oxidants, the indicator is converted to the fluorescent dichlorofluorescein, which was detected with a FACStra+ flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) (4).

RNA isolation and Northern blot analysis. Total RNA was isolated by the RNAstat-60 method, with direct lysis of cells in RNAzol lysis buffer, followed by chloroform extraction (Tel-Test B, Friendswood, TX). Northern blot analysis was performed as previously described (5). Autoradiogram signals were quantified by densitometric scanning (Molecular Dynamics, Sunnyvale, CA). To control for variation in the amount of RNA, blots were hybridized with an oligonucleotide probe corresponding to the 18S rRNA after the original probe was stripped in wash buffer [1% sodium dodecyl sulfate (SDS), 40 mM phosphate buffer, pH 8.0, and 1 mM EDTA] at 95°C. All mRNA densitometric values obtained were normalized to values for 18S rRNA on the same blot. Quantitation of steady-state mRNA of treated cells is expressed in densitometric absorbance units and normalized to control samples.

cDNA and oligonucleotide probes. Rat MnSOD (pJL4) and Cu,ZnSOD (pCu/ZnSOD) cDNAs were provided by Dr. H. Nick (University of Florida, Gainesville). Human MnSOD cDNA was provided by Dr. Ye Shi Ho (Wayne State University, Detroit, MI). Human IL-8 cDNA was purchased from R&D Systems (Minneapolis, MN). Rat liver catalase cDNA (PMJ1010) was provided by Dr. S. Furuta (Shinshu University School of Medicine, Nagano, Japan). Rat liver GP cDNA (pGPX1211) was provided by Dr. S. Shetty (Pennsylvania State University, University Park). Mouse IDO cDNA was provided by Dr. R. Yoshida (Osaka Bioscience Institute, Japan). Human tumor necrosis factor-alpha (TNF-alpha ) cDNA was provided by Dr. Paul Noble (Johns Hopkins University, Baltimore, MD). A 24-base pair oligonucleotide (5'-ACG GTA TCT GAT CGT CTT CGA ACC-3') complementary to 18S RNA was synthesized with a DNA synthesizer (Applied Biosystems, Foster, CA). cDNA probes were labeled with [32P]CTP with a random-primer kit (Boehringer Mannheim, Mannheim, Germany). Oligonucleotides were 32P labeled at the 3'-end with terminal deoxynucleotidyltransferase (Bethesda Research Laboratories, Gaithersburg, MD).

Western blot analysis. Cells were lysed in buffer containing Nonidet P-40 (NP-40). An equal volume of double-strength SDS-sample buffer [0.125 M tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.4, 4% SDS, and 20% glycerol] was added, and the samples were boiled for 5 min. Samples were subjected to electrophoresis in a 12% SDS-polyacrylamide gel (Novex, San Diego, CA) for 2 h at 20 mA. The gel was transferred electophoretically (Bio-Rad) onto a 0.45-µm polyvinylidine fluoride membrane (Immobilon-P, Millipore, Bedford, MA) and incubated for 2 h in 5% nonfat powdered milk in 1× Tween 20-Tris-buffered saline solution. The membranes were then incubated for 2 h with rabbit polyclonal antibody against rat MnSOD (1:400 dilution; Larry Oberley, University of Iowa). After three 5-min washes in Tween 20-Tris-buffered saline solution, the membranes were incubated with peroxidase-labeled goat anti-rabbit immunoglobulin G antibody (Amersham, Arlington Heights, IL) for 2 h. The membranes were then washed three times for 5 min each, and binding of the antibody was detected with an enhanced chemiluminescence system (ECL System, Amersham).

MnSOD activity. A549 cells were infected with influenza as outlined in Infection of epithelial cells. At 72 h, the cells were harvested in 1 ml of cold phosphate-buffered saline, centrifuged at 14,000 g at 4°C for 5 min, and then resuspended in 0.25 M Tris. MnSOD activity was measured with the xanthine oxidase-cytochrome c method (9). One unit of SOD is the amount that halves the rate of reduction of cytochrome c.

Cellular nuclear protein extraction. Cells were scraped in cold phosphate-buffered saline and centrifuged at 14,000 g at 4°C for 10 min. The supernatant was discarded, and the cell pellet was lysed in lysis buffer containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol (DTT), 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The nuclei were isolated by centrifugation at 1,500 g, washed in lysis buffer without NP-40, and centrifuged again at 1,500 g for 5 min. The pellet was resuspended in nuclear resuspension buffer containing 25 mM Tris, pH 7.8, 60 mM KCl, 1 mM DTT, and 1 mM PMSF. The nuclei were frozen and thawed three times and then centrifuged at 14,000 g for 15 min. The nuclear protein was kept in nuclear resuspension buffer and stored at -80°C. Protein concentrations of the suspensions used for electrophoretic mobility shift assay (EMSA) were determined by the Bradford assay (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA).

EMSA. Mobility shift assays were performed as described by Barberis et al. (3) with minor modifications. DNA binding activity was determined after incubating 3 µg of nuclear protein extracts with 10 fmol (20,000 counts/min) of the 22-mer 32P-labeled oligonucleotide encompassing the NF-kappa B site (5'-GATCGAGGGGACTTTCCCTAGC-3'; Stratagene, La Jolla, CA) in reaction buffer containing 10 mM HEPES (pH 7.9), 1 mM DTT, 1 mM EDTA, 80 mM KCl, 1 µg polydeoxyinosinic-deoxycytidylic acid [poly(dIdC) · (dIdC)] and 4% Ficoll. After a 20-min incubation, the reaction mixture was electrophoresed on a 6% polyacrylamide gel. The gel was transferred to DE81 ion-exchange chromatography paper (Whatman, Maidstone, UK) and dried before exposure to autoradiographic film. Self-competitions were carried out under the same conditions with 1-, 10-, and 100-fold molar excess of the unlabeled NF-kappa B oligonucleotide probes. Nonspecific competitions were performed with an unlabeled oligonucleotide probe encompassing an SP-1 transcription factor binding site (5'-GATCGATCGGGGCGGGGCGATC-3'; Stratagene). The NF-kappa B subunits binding to the oligonucleotide probe were determined by incubating with antibodies to the p50, p65, and c-rel subunits of NF-kappa B (Santa Cruz Biochemical, Santa Cruz, CA). Binding of the antibodies to the activated subunits was detected as a further increase in the molecular mass of the complex (a "supershift").

Treatment with antioxidants. Cells that were to be infected with virus were treated with antioxidant either both pre- and postinfection, preinfection only, or postinfection only. Cells pretreated with antioxidant were incubated with 20 mM NAC or 100 µM PDTC for 1 h. The cells were then infected with influenza virus as outlined in Infection of epithelial cells. Cells treated after viral infection had the same concentration of antioxidant added back to the medium after the virus was washed away. Negative controls included cells treated with LHC-8e medium or with inactivated virus. All treated cells were harvested at 24 h.

Cytotoxicity studies. To determine whether virus-induced cell death was mediated by the production of oxidants by infected epithelial cells, we tested the effect of PDTC on cell viability 72 h after infection with influenza virus. Cells were pretreated with PDTC (100 µM) for 1 h, after which they were infected as described in Infection of epithelial cells. The medium contained PDTC for the entire 72-h period after infection. The cells were then trypsinized, stained with trypan blue (0.04%), counted in a hemocytometer, and divided into those that excluded trypan blue (live cells) and those that stained with trypan blue (dead cells).

Statistical analysis. Data are expressed as means ± SE. Differences in measured variables between the experimental and control groups were assessed with Student's t-test. Statistical calculations were performed on a Macintosh personal computer with the Statview II statistical package (Abacus Concepts, Berkeley, CA). Statistical difference was accepted at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Viral infections. Influenza virus infection of epithelial cells was confirmed in each experiment by recovering the virus from the culture medium into RMK cell monolayers and performing hemadsorption assays. There was no inhibiton of viral infection or replication by NAC or PDTC (data not shown).

Fluorescent measurement of oxidant production. Oxidant production, measured as the fluorescence of dichlorofluorescein, was increased by viral infection (Fig. 1A). This effect was blocked by treatment with PDTC. In contrast, treatment with NAC in itself increased oxidant production, whereas the combination of NAC and viral infection led to a level of oxidant production that was attenuated compared with either NAC or viral infection alone. Quantitation of oxidant production as measured by fluorescence of dichlorofluorescein is shown in Fig. 1B.


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Fig. 1.   Flow cytometry analysis of oxidant production by A549 cells. Cells were harvested 72 h after treatment and/or infection and treated with 10 µM 2',7'-dichlorodihydrofluorescein diacetate. A (top to bottom): control, virus-infected, pyrrolidine dithiocarbamate (PDTC)-treated, PDTC-treated and virus-infected, N-acetyl-L-cysteine (NAC)-treated, and NAC-treated and virus-infected cells. B: quantitation of oxidant production by fluorescence of dichlorofluorescein (%gated cells). Data are means ± SE from 3-5 independent experiments. Significantly different from control cells: * P < 0.0004; @ P < 0.05. # Significantly different from virus-infected cells, P < 0.0001. ** Significantly different from NAC-treated cells, P < 0.001.

Northern blot analysis. With the use of the rat MnSOD probe, increased expression of MnSOD mRNA peaked 72 h after infection (Fig. 2). Medium control cells and cells treated with inactivated virus showed no increase in MnSOD mRNA. Hybridization with human MnSOD cDNA produced the identical result (data not shown). IL-8 mRNA expression was also induced by influenza virus infection (Fig. 2). The kinetics of IL-8 induction were similar to those of MnSOD in that induction was first seen at 24 h and increased through 72 h, but the level of induction was 20-fold higher than baseline. In 16-HBE 14o- cells, induction of MnSOD mRNA after viral infection was also increased (data not shown).


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Fig. 2.   A: kinetics of manganese superoxide dismutase (MnSOD), interleukin-8 (IL-8), and indoleamine 2,3-dioxygenase (IDO) mRNA expression in A549 cells after influenza virus infection. Total RNA was extracted 1, 3, 6, 24, and 72 h after influenza virus infection and analyzed for MnSOD, IL-8, and IDO mRNAs by Northern blot analysis. B: quantitation of Northern blots. 18S rRNA hybridization was used as a normalization control. V, virus; C, medium control; I, inactivated virus. Subscript nos., time in h.

Expression of IDO, catalase, Cu,ZnSOD, and GP was also analyzed by Northern blot analysis. IDO mRNA was increased fourfold after influenza virus infection (Fig. 2), but catalase, Cu,ZnSOD, and GP mRNAs were not induced after influenza virus infection (data not shown).

Western blotting and activity. Figure 3A shows the Western blot for MnSOD 72 h after viral infection. There is a threefold increase in the amount of MnSOD protein in infected cells compared with control cells. MnSOD enzyme activity increased nearly fivefold 72 h after influenza virus infection (Fig. 3B).


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Fig. 3.   A: Western blot of MnSOD protein showing a 3-fold increase in protein 72 h after viral infection. B: MnSOD enzyme activity 72 h after viral infection. Values are means ± SE. * P < 0.04.

NF-kappa B activation. After influenza infection, NF-kappa B was activated and was first detected at ~12 h (Fig. 4A). The level of activation continued to increase at 72 h (data not shown). To assess the specificity of the assay, a 1- to 100-fold molar excess of unlabeled NF-kappa B was added to each reaction. Figure 4B shows that the intensity of the band representing NF-kappa B was decreased by the addition of 1× cold NF-kappa B and completely disappeared with 10× cold NF-kappa B. A competition assay with a nonspecific transcription factor (SP-1) did not displace the labeled NF-kappa B at any concentration (data not shown). Supershift assays were performed with antibodies to the p50 and p65 subunits of NF-kappa B and with antibodies to the NH2- and COOH-terminal ends of c-rel. Migration of the complex was retarded with the addition of antibodies to p50 (Fig. 4C) but not with p65 or c-rel antibodies (data not shown).


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Fig. 4.   Electrophoretic mobility shift assay of cellular protein extracts for nuclear factor-kappa B (NF-kappa B) binding activity after influenza virus infection. A: nuclear protein extracts were obtained from A549 cells 6, 12, 24, and 48 h after influenza virus infection and analyzed for NF-kappa B binding with a 22-mer double-stranded oligonucleotide probe that contained an NF-kappa B consensus sequence. B: competition of NF-kappa B binding in cells 24 h after influenza virus infection with unlabeled NF-kappa B. Competitions were carried out with 1-, 10-, and 100-fold excess of unlabeled NF-kappa B. C: supershift assay of cellular protein obtained 24 h after influenza virus infection with antibodies to p50 and p65.

Effects of antioxidants. Treatment of A549 cells with NAC (20 mM) did not attenuate the induction of MnSOD mRNA during influenza virus infection (Fig. 5). However, IL-8 mRNA induction was diminished with this treatment. PDTC (100 µM) markedly decreased both MnSOD and IL-8 mRNA induction (Fig. 6). For both genes, the effect of the antioxidant was more pronounced for cells treated pre- and postinfection or postviral infection than for pretreated cells.


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Fig. 5.   Effect of NAC on MnSOD mRNA expression after influenza virus infection. Northern blot analysis of total RNA was performed 24 h after influenza virus infection. PRE/POST, cells treated both before and after infection; PRE, cells treated only before infection.


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Fig. 6.   Effect of PDTC on MnSOD mRNA expression after influenza virus infection. Northern blot analysis of total RNA was performed 24 h after influenza virus infection. POST, cells treated only after infection.

Activation of NF-kappa B was attenuated in cells treated with NAC before and after infection. However, NF-kappa B activation was not affected in cells treated before viral infection only (Fig. 7). The same was true for PDTC, in that there was complete obliteration of the band corresponding to activated NF-kappa B but only if the cells were treated with PDTC after viral infection (Fig. 8). There was no difference between the pre- and posttreated cells and the posttreated cells. Viral infection of PDTC-treated cells activated a protein of lower molecular mass that bound to the NF-kappa B probe, and this band was easily competed off with excess unlabeled probe (data not shown). Addition of antibodies to p50, p65, or c-rel did not retard the migration of this complex (data not shown).


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Fig. 7.   Effect of NAC on activation of NF-kappa B after influenza virus infection. Nuclear protein was extracted from A549 cells 24 h after influenza virus infection and analyzed for NF-kappa B activation.


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Fig. 8.   Effect of PDTC on activation of NF-kappa B after influenza virus infection. Nuclear protein was extracted from A549 cells 24 h after influenza virus infection and analyzed for NF-kappa B activation.

Cytotoxicity. Viral infection caused substantial cytotoxicity, with only 30% of cells excluding trypan blue after 72 h of infection (Fig. 9). This effect was markedly attenuated by treatment with PDTC.


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Fig. 9.   Effects of viral infection and treatment with PDTC on cell viability expressed as percent survival (ability to exclude trypan blue).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

These data show that influenza A virus infection leads to the production of oxidants by airway epithelial cells. This has consequences for both intracellular signaling and cell damage. Expression of the genes encoding MnSOD, IDO, and IL-8 is induced, and NF-kappa B is activated. Treatment with NAC and PDTC blocked NF-kappa B activation and IL-8 expression. Treatment with PDTC, but not with NAC, blocked virus-induced MnSOD gene expression.

Recent evidence suggests that transcriptional regulatory proteins such as NF-kappa B play critical roles in the early events controlling the molecular response to ROIs (23). NF-kappa B has been found in many cell types and has been implicated in the activation of genes in inflammatory and immune responses (2). It remains in the cytoplasm complexed with its inhibitor (Ikappa B) until activated by a stimulus such as oxidant stress. It then enters the nucleus where it mediates gene transcription. It has been shown that activation of NF-kappa B by a wide variety of compounds can be blocked by treatment with NAC, suggesting that the production of reactive oxygen intermediates may act as a common pathway for a diverse range of stimuli (23).

NF-kappa B, as assayed by EMSA, represents a family of protein dimers, including, among others, various combinations of 50- and 65-kDa subunits (termed p50 and p65) and c-rel. Treatment of nuclear proteins from virus-infected cells with an antibody to p50, but not to p65 or c-rel, resulted in the formation of a complex of higher molecular mass, indicating that p50, but not p65 or c-rel, is part of the activated NF-kappa B detected. When assaying for activated NF-kappa B by EMSA, treatment with PDTC after viral infection caused the appearance of a band of lower molecular mass than that seen with viral infection alone. The band represents a protein that binds to the labeled NF-kappa B probe, but it does not bind to antibodies to p50, p65, or c-rel. This protein may be a different member of the NF-kappa B family of proteins, or it may be a transcriptional mediator that has yet to be identified. It is unlikely that it is a degradation product caused by PDTC because PDTC alone does not cause the appearance of this band.

The production of oxidants and the ability of antioxidants to block NF-kappa B activation and IL-8 gene expression in epithelial cells suggest that there is oxidant-mediated intracellular signaling after influenza virus infection. Expression of influenza hemagglutinin (the protein that mediates binding and fusion of the virus to cells) alone may be sufficient for activation of NF-kappa B. This has been demonstrated in HeLa cells transfected with the hemagglutinin gene (19). Hemagglutinin was expressed in our A549 cells as demonstrated by the fact that they exhibited hemadsorption (data not shown).

Production of oxidants by virus-infected epithelial cells also provides a potential mechanism by which viruses cause cell damage and death. In vivo data in mice with influenza infections demonstrate that antioxidants can ameliorate lung damage and decrease mortality (1). However, because in vivo viral infections cause an influx of activated leukocytes into the lung, the source of the oxidants responsible for the damage is unclear. Our in vitro studies demonstrate that the epithelial cells themselves are capable of producing oxidant. The ability of PDTC to decrease cell damage suggests that epithelial oxidants are involved in virus-induced cytotoxicity.

IL-8 is one of a family of chemotactic cytokines that contributes to virus-induced inflammation. Increased IL-8 gene expression has been demonstrated under conditions of oxidant stress (11), and its regulation involves NF-kappa B activation (16). It has been previously demonstrated that the addition of antioxidants to cells undergoing oxidant stress inhibits IL-8 production (11). It has also been shown that epithelial cells infected with respiratory syncytial virus produce IL-8 (17) and that this effect is blocked by the addition of antioxidants (17). These findings support the hypothesis that production of ROIs after influenza virus infection may be involved in the regulation of genes during the inflammatory response to viral infection.

PDTC, but not NAC, was effective in preventing the induction of MnSOD mRNA after influenza virus infection. The difference between the two antioxidants may have to do with the relative antioxidant properties of each. It has been shown that NAC is a relatively weak antioxidant and, in cell culture, requires high concentrations to be effective (18). PDTC is more effective as an antioxidant, although its full mechanism of action is not known. PDTC and NAC are thiol compounds and may exert their antioxidant effects via glutathione. Dithiocarbamates have also been used as metal chelators (25) and may prevent ROI production via the Fenton reaction (22).

The ability of treatment with NAC and PDTC to suppress activation of NF-kappa B and induction of IL-8 gene expression is likely to involve the antioxidant effects of these substances. However, our studies using flow cytometry with cells labeled with the oxidant-senstive dye dichlorofluorescein suggest that assumptions regarding the effects of NAC on cellular oxidants should be made with caution. Although the increase in intracellular glutathione that results from NAC treatment is likely to ameliorate the effects of subsequent oxidant production, our data show that treatment with NAC can itself increase oxidant production. This has been previously demonstrated by Das et al. (10), who also demonstrated NF-kappa B activation after treatment with NAC. The reason for the failure of NAC treatment to activate NF-kappa B or to induce expression of antioxidants or IL-8 in our hands may relate to the culture conditions in our study, in that infection of cells with influenza requires serum-free conditions, whereas the previous study (10) that demonstrated NAC-induced NF-kappa B activation included serum in the medium.

Our study also demonstrated the unexpected finding that although either viral infection or treatment with NAC increased epithelial cell oxidant production, the viral infection of NAC-treated cells did not. The reason for this consistent finding is not clear. It is conceivable that the oxidants produced during viral infection are different from those produced by NAC treatment. Because both ROIs and nitric oxide are detected by dichlorofluorescein (12), whereas, under some circumstances, ROI production can be inhibited by nitric oxide (20), the production of one class of oxidants by NAC treatment and the other by viral infection might yield these results.

The induction of the MnSOD gene in virus-infected cells and the inhibition of its induction by PDTC also suggest a response to epithelial oxidant production. The role of NF-kappa B in the regulation of the MnSOD gene is not yet known. The sequence of the human MnSOD gene has been determined, and it is known that there is one NF-kappa B consensus sequence, as well as multiple SP-1 and activator protein-2 sites (28). Choi et al. (7) previously showed that influenza virus infection activates NF-kappa B and induces antioxidant genes in mice, but there has been no evidence that NF-kappa B has a direct effect on MnSOD gene expression. In this study, we have demonstrated that NF-kappa B may not be involved in MnSOD gene regulation during viral infection because NAC blocks NF-kappa B activation but not induction of MnSOD mRNA.

Induction of MnSOD has been shown to be important in other models of oxidant stress. MnSOD is induced by asbestos exposure (13), endotoxin (21), and TNF-alpha (31) and, when instilled intratracheally, protects against the effects of hyperoxia (27). Cells that overexpress MnSOD, either by induction with endotoxin or by transfection of the MnSOD gene, are protected against further oxidant stress if exposed to hyperoxia (8), ozone exposure (21), or paraquat (29). More importantly, cells that express antisense MnSOD RNA are much more sensitive to cell killing by TNF-alpha than are control cells (30). Although TNF-alpha is known to be an important inducer of MnSOD, we found no detectable TNF-alpha mRNA by Northern blot analysis (data not shown). These findings suggest that expression of the MnSOD gene is important for cell viability during oxidant stress; however, TNF-alpha is not required for its induction.

In the face of oxidant stress, not all antioxidant genes are induced. In the present study, there was no increase in the induction of GP, Cu,ZnSOD, and catalase genes after influenza virus infection. Other studies have shown that oxidant stress induces only MnSOD gene expression, whereas the expression of most other antioxidant genes remains unchanged or decreases (14, 21, 24). The exception is IDO, which has also been shown to increase after influenza virus infection (7, 32), and this finding is confirmed by the present study. Little is known about the antioxidant properties of IDO, and its effectiveness may be hindered by the depletion of intracellular tryptophan required by IDO as a substrate (26).

Thus viral infection of cultured epithelial cells causes significant oxidant stress. This can be demonstrated directly with dichlorofluorescein and can also be seen in the response of the cell, which is characterized by NF-kappa B activation and induction of MnSOD expression. Cell damage and death, which are substantial after viral infections, are likely to involve oxidant stress because treatment with PDTC markedly decreases virus-induced cytotoxicity. NF-kappa B activation and IL-8 induction in virus-infected cells can be blocked by both NAC and PDTC. This finding, along with previous studies demonstrating the role of NF-kappa B in IL-8 expression, suggests that oxidant-mediated activation of NF-kappa B is responsible for IL-8 expression. In contrast, although NAC blocked NF-kappa B activation, it did not block virus-induced MnSOD expression. Because PDTC blocks MnSOD induction, this virus-induced expression is likely to be oxidant mediated, but it does not involve the activation of NF-kappa B. The finding that oxidants both participate in virus-induced cytotoxicity and mediate the induction of the proinflammatory cytokine IL-8 (which would, in vivo, recruit and activate inflammatory cells, thereby amplifying the oxidant stress) points toward a central role for epithelial oxidant production in influenza pathogenesis.

    ACKNOWLEDGEMENTS

We thank Bethany Yost for technical assistance.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-47126 and HL-54659 (to D. B. Jacoby); National Institute on Aging Grant AG-00516 (to A. M. K. Choi); Interdisciplinary Training Program in Lung Diseases, NHLBI Grant HL-07534 (to K. Knobil); a grant from the American Heart Association (to D. B. Jacoby); and a Research Grant from the American Lung Association (to A. M. K. Choi).

Address for reprint requests: D. B. Jacoby, Division of Pulmonary and Critical Care, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

Received 15 April 1996; accepted in final form 15 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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