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
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ABSTRACT |
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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-B (NF-
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-
B
binding as determined by an electrophoretic mobility shift assay.
Pretreatment of A549 cells with
N-acetyl-L-cysteine
attenuated virus-induced NF-
B activation and interleukin (IL)-8 mRNA
induction but did not block induction of MnSOD mRNA. In contrast,
pyrrolidine dithiocarbamate blocked activation of NF-
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-
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-B
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INTRODUCTION |
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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 () can be metabolized via the dismutation reaction
(
+
+ 2H+
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
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-B
(NF-
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-B during influenza
virus infection. We further investigate the role of ROIs in regulating
MnSOD and IL-8 gene expression in this system.
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METHODS |
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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 × 106 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- (TNF-
) 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-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-
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-
B subunits binding to the oligonucleotide probe were determined by incubating with antibodies to the p50, p65, and
c-rel subunits of NF-
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.
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RESULTS |
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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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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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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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NF-B activation. After influenza infection,
NF-
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-
B was added to each reaction. Figure
4B shows that the intensity of the
band representing NF-
B was decreased by the addition of 1×
cold NF-
B and completely disappeared with 10× cold NF-
B. A
competition assay with a nonspecific transcription factor (SP-1) did
not displace the labeled NF-
B at any concentration (data not shown).
Supershift assays were performed with antibodies to the p50 and p65
subunits of NF-
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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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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Activation of NF-B was attenuated in cells treated with NAC before
and after infection. However, NF-
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-
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-
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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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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DISCUSSION |
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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-B is activated. Treatment with NAC and PDTC blocked NF-
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-B play critical roles in the early events controlling the
molecular response to ROIs (23). NF-
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 (I
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-
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-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-
B detected. When assaying for activated NF-
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-
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-
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-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-
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-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-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-
B activation after treatment with NAC. The
reason for the failure of NAC treatment to activate NF-
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-
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-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-
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-
B and induces antioxidant genes in mice, but
there has been no evidence that NF-
B has a direct effect on MnSOD
gene expression. In this study, we have demonstrated that NF-
B may
not be involved in MnSOD gene regulation during viral infection because
NAC blocks NF-
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- (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-
than are control cells (30). Although TNF-
is
known to be an important inducer of MnSOD, we found no detectable
TNF-
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-
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-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-
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-
B in IL-8 expression, suggests that oxidant-mediated
activation of NF-
B is responsible for IL-8 expression. In contrast,
although NAC blocked NF-
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-
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.
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ACKNOWLEDGEMENTS |
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We thank Bethany Yost for technical assistance.
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FOOTNOTES |
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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.
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