From the Departments of Pediatrics, ¶ Internal
Medicine,
Sealy Center for Molecular Sciences,
Microbiology and Immunology, University of
Texas Medical Branch, Galveston, Texas 77555-0366
Received for publication, February 19, 2001
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ABSTRACT |
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Respiratory syncytial virus (RSV) produces
intense pulmonary inflammation, in part, through its ability to induce
chemokine synthesis in infected airway epithelial cells. RANTES
(regulated upon activation, normal T-cells expressed and secreted) is a
CC chemokine which recruits and activates monocytes, lymphocytes, and
eosinophils, all cell types present in the lung inflammatory infiltrate
induced by RSV infection. In this study we investigated the role of
reactive oxygen species in the induction of RANTES gene
expression in human type II alveolar epithelial cells (A549), following
RSV infection. Our results indicate that RSV infection of airway
epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of
2',7'-dichlorofluorescein. Pretreatment of airway epithelial cells with
the antioxidant butylated hydroxyanisol (BHA), as well a panel of
chemically unrelated antioxidants, blocks RSV-induced RANTES gene
expression and protein secretion. This effect is mediated through the
ability of BHA to inhibit RSV-induced interferon regulatory factor
binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator
factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3
nuclear translocation. Together, these data indicates that a
redox-sensitive pathway is involved in RSV-induced IRF activation, an
event necessary for RANTES gene expression.
Respiratory syncytial virus
(RSV)1 is an enveloped,
negative-sense single-stranded RNA virus (1). Since its isolation, RSV has been identified as a leading cause of epidemic respiratory tract
illness in children in the United States and worldwide. In fact, RSV is
so ubiquitous that it will infect 100% of children before the age of
3. It is estimated that 40-50% of children hospitalized with
bronchiolitis and 25% of children with pneumonia are infected with
RSV, resulting in 100,000 hospital admissions annually in the United
States alone (1). In addition to acute morbidity, there are long-term
consequences of RSV infection in infancy: RSV has been shown to
predispose to the development of hyperreactive airway disease (2) and
recurrent episodes of wheezing in asthmatic children are often
precipitated by RSV infection. The mechanisms of RSV-induced airway
disease and its long-term consequences are largely unknown, but the
delicate balance between immunopathology and immunoprotection in the
airway mucosa may be altered by an exuberant and unwanted local
inflammatory response. Airway infiltration of monocytes and lymphocytes
is typical of RSV infection (1), and activation of eosinophil and
basophil leukocytes has been shown to correlate with the severity of
acute RSV disease (3, 4).
The composition of the cellular response at sites of tissue
inflammation is controlled by gradients of chemokines, a family of
small chemotactic cytokines, which direct leukocyte transendothelial migration and movement through the extracellular matrix. RANTES is a CC
chemokine highly chemoattractant for T lymphocytes, monocytes, eosinophils, and basophils (5), all cell types which are present or
activated in the inflammatory infiltrate that follows RSV infection of
the lung. Recent in vivo studies have shown elevated RANTES concentrations in nasal washes and bronchoalveolar lavages of children
infected with RSV (6, 7) and we have recently shown that RANTES is
strongly expressed in RSV-infected respiratory epithelial cells (8, 9),
which are the primary target for viral infection. Therefore, it is
likely that RANTES produced by infected epithelial cells plays an
important role in the pathogenesis of RSV-induced airway inflammation.
Reactive oxygen species (ROS) are ubiquitous, highly diffusable and
reactive molecules produced as a result of reduction of molecular
oxygen, including species such as hydrogen peroxide, superoxide anion,
and hydroxyl radical, and they have been implicated in damaging
cellular components like lipids, proteins, and DNA. In the past few
years, there has been increased recognition of their role as redox
regulators of cellular signaling (reviewed in Refs. 10 and 11).
Inducible ROS generation has been shown following stimulation with a
variety of molecules, like cytokines and growth factors, and infection
with certain viruses, like HIV, hepatitis B, and influenza (reviewed in
Ref. 12). Changes in the level of ROS, generated in response to some of
these stimuli, have been shown to modulate the expression of several
genes (10). Among the different members of the chemokine family,
interleukin (IL)-8 is the only one for which redox-sensitive signaling
pathways have been identified (13, 14). The contribution of ROS in RANTES gene expression, as well as in other CC chemokine induction, has
not been defined yet. Therefore, the purpose of this study was to
investigate the effect of RSV infection on ROS generation in human
airway epithelial cells and the role of ROS in RSV-induced RANTES
production. Our results indicate that RSV infection of airway
epithelial cells induces ROS production, as measured by intracellular
oxidation of 2',7'-dichlorofluorescein, and that treatment of airway
epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as
well as a panel of chemically unrelated antioxidants, blocks
RSV-induced RANTES protein secretion and gene expression. This effect
is mediated through the inhibition of RSV-induced interferon regulatory
factor (IRF) binding to the RANTES interferon-stimulated responsive
element (ISRE), an event that is absolutely required for RSV-stimulated
RANTES gene transcription. In infected A549 cells, ISRE binds IRF-1,
-3, and -7. IRF-1 and -7 are inducible upon RSV infection of alveolar
epithelial cells and treatment with BHA inhibits their gene expression
and protein synthesis. In contrast, IRF-3 is constitutively expressed
and antioxidant treatment blocks its nuclear translocation. These data
strongly indicate that a redox-sensitive pathway is involved in
RSV-induced IRF induction and RANTES gene expression. This study
provides novel insights on the role of ROS in viral-induced RANTES
secretion and IRF protein activation. Identification of the molecular
mechanisms involved in RANTES gene expression is fundamental for
developing strategies to modulate the inflammatory response associated
with RSV infection of the lung.
RSV Preparation--
The human Long strain of RSV (A2) was grown
in Hep-2 cells and purified by centrifugation on discontinuous
sucrose gradients as described elsewhere (15). The virus titer of the
purified RSV pools was 8-9 log10 plaque forming units/ml using
a methylcellulose plaque assay. No contaminating cytokines were found
in these sucrose-purified viral preparations (16).
Lipopolysaccharide, assayed using the limulus hemocyanin
agglutination assay, was not detected. Virus pools were aliquoted,
quick-frozen on dry ice/alcohol, and stored at Cell Culture and Infection of Epithelial Cells with
RSV--
A549, human alveolar type II-like epithelial cells (ATCC,
Manassas, VA), were maintained in F12K medium containing 10% (v/v) fetal bovine serum, 10 mM glutamine, 100 IU/ml penicillin,
and 100 µg/ml streptomycin. Small alveolar epithelial cells (SAE) were obtained from Clonetics, San Diego, CA, and grown according to the
manufacturer's instructions. Cell monolayers were infected with RSV at
a multiplicity of infection (m.o.i.) of 1 (unless otherwise stated), as
described (17). An equivalent amount of a 20% sucrose solution was
added to uninfected A549 cells, as a control. For the antioxidant
experiments, cells were pretreated with the antioxidants for 1 h
and then infected in the presence of the antioxidants. Since BHA was
diluted in ethanol, equal amounts of ethanol were added to untreated
cells, as a control. Total number of cells, cell viability, and viral
replication, following antioxidant treatment, were measured by trypan
blue exclusion and by plaque assay, respectively.
RANTES Enzyme-linked Immunosorbent Assay--
Immunoreactive
RANTES was quantitated by a double antibody enzyme-linked immunosorbent
assay kit (DuoSet, R&D Systems, Minneapolis, MN) following the
manufacturer's protocol.
Northern Blot--
Total RNA was extracted from control and
infected A549 cells by the acid guanidium thiocyanate-phenol chloroform
method (18). Twenty micrograms of RNA were fractionated on a 1.2%
agarose-formaldehyde gel, transferred to nylon membranes, and
hybridized to a radiolabeled RANTES, IRF-1, -3, and -7 cDNAs
(RANTES cDNA plasmid was a generous gift of Dr A. Krensky,
Stanford, CA; IRF-1, -3, and -7 cDNAs were a generous gift of Dr.
Lin, Lady Davis Institute for Medical Research, Montreal,
Quebec, Canada), as previously described (19). Hybridization temperature for all probes was 55 °C. After washing, membranes were
exposed for autoradiography using Kodak X-AR film at Assesment of Intracellular ROS Generation--
A549 cells were
grown in 96-well tissue culture plates and infected with RSV at 0.1, 0.5 and 1 multiplicity of infection (m.o.i.). At different times
post-infection, cells were washed with Hank's balanced salt solution
and loaded with 10 µM 2,7-dichlorofluorescein diacetate
(DCF-DA) in Hank's balanced salt solution medium containing 25 mM HEPES, pH 7.4, for 30 min at 37 °C. The cells were
then washed twice and fluorescence intensity was determined at 485 nm
excitation and 590 nm emission, using an automated fluorescence reader
(Flurocount, Hewlett-Packard Instruments, IL). For the experiments in
which H2O2 was used as a stimulus for ROS
production, cells were preloaded with 10 µM DCF-DA for 30 min, washed, and then fluorescence intensity was measured at different
times following addition of H2O2. Measurements
were performed in triplicates and results expressed as fluorescence
mean ± S.D. of n = 3 independent experiments.
Plasmid Construction and Cell Transfection--
A fragment of
the human RANTES promoter spanning from
Logarithmically growing A549 cells were transfected in triplicate in
60-mm Petri dishes by DEAE-dextran, as previously described (20). Cells
were incubated in 2 ml of HEPES-buffered Dulbecco's modified Eagle's
medium (10 mM Hepes, pH 7.4) containing 20 µl of 60 mg/ml
DEAE-dextran (Amersham Pharmacia Biotech) premixed with 6 µg of
RANTES-pGL2 plasmids and 1 µg of cytomegalovirus- Electrophoretic Mobility Shift Assay--
Nuclear extracts of
uninfected and infected A549 cells were prepared using
hypotonic/nonionic detergent lysis, as previously described (20).
Proteins were normalized by protein assay (Protein Reagent, Bio-Rad,
Hercules, CA) and used to bind to a duplex oligonucleotide corresponding to the RANTES ISRE, whose sequences is shown:
5'-GATCCATATTTCAGTTTTCTTTTCCGT-3', 3'-TATAAAGTCAAAAGAAAAGGCATCTAG-5'.
DNA-binding reactions contained 10-15 µg of nuclear proteins, 5%
glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol, 5 mM Mg2Cl, 0.5 mM EDTA, 1 µg of poly(dI-dC), and 40,000 cpm of
32P-labeled double-stranded oligonucleotide in a total
volume of 20 µl. The nuclear proteins were incubated with the probe
for 15 min at room temperature and then fractionated by 6%
nondenaturing polyacrylamide gels (PAGE) in TBE buffer (22 mM Tris-HCl, 22 mM boric acid, 0.25 mM EDTA, pH 8). After electrophoretic separation, gels were
dried and exposed for autoradiography using Kodak X-AR film at
Microaffinity Isolation Assay--
Microaffinity purification of
proteins binding to the RANTES ISRE was performed using a two-step
biotinylated DNA-streptavidin capture assay (20). In this assay, duplex
oligonucleotides are chemically synthesized containing 5'-biotin on a
flexible linker (Genosys, The Woodlands, TX). Four hundred micrograms
of 12-h infected A549 cells nuclear extracts were incubated at 4 °C
for 30 min with 50 pmol of biotin ISRE, in the absence or presence of
10-fold molar excess of non-biotinylated ISRE wild type (WT) or mutated
(MUT). The binding buffer contained 8 µg of poly(dI-dC) (as
nonspecific competitor) and 5% (v/v) glycerol, 12 mM
HEPES, 80 mM NaCl, 5 mM dithiothreitol, 5 mM Mg2Cl, 0.5 mM EDTA. One hundred
microliters of a 50% slurry of pre-washed streptavidin-agarose beads
was then added to the sample, and incubated at 4 °C for an
additional 20 min with gentle rocking. Pellets were washed twice with
500 µl of binding buffer, and the washed pellets were resuspended in
100 µl of 1 × SDS-PAGE buffer, boiled, and fractionated on a
10% SDS-polyacrylamide gel. After electrophoresis separation, proteins
were transferred to polyvinylidene difluoride membrane for Western blot analysis.
Western Immunoblot--
Total cell lysates and cytoplasmic and
nuclear proteins were prepared as previously described, fractionated by
SDS-PAGE, and transferred to polyvinylidene difluoride membrane (20).
Membranes were blocked with 5% albumin in TBS-Tween and incubated
overnight with a rabbit polyclonal antibody to IRF-1, -3, and -7 (Santa Cruz Biotechnology, Santa Cruz, CA). For secondary detection, we used a
horseradish-coupled anti-rabbit or anti-mouse antibody in the enhanced
chemiluminescence assay (Amersham Pharmacia Biotech, Arlington Heights,
IL).
Statistical Analysis--
Data from experiments involving
multiple samples subject to each treatment were analyzed by the Student
Newman Keuls t test for multiple pairwise comparisons.
Results were considered significantly different at a p value
<0.05.
RSV Induces Reactive Oxygen Species Formation--
To determine
whether RSV infection induced ROS production, A549 cells were grown to
~90% of confluency and infected with RSV. At different time points
after infection, cells were loaded with the membrane permeable compound
2',7'-DCF-DA, which is trapped intracellularly following cleavage by
cellular esterases. DCF oxidation was measured by changes in mean
fluorescence intensity in control versus infected cells (21,
22). When cells were infected with m.o.i. of 1, the production of ROS
was detectable as early as 2 h post-infection, reaching a plateau
around 4 h and declining thereafter, although the level of
cellular ROS in infected cells was still higher than in control cells
at 24 h post-infection (Fig. 1).
When cells were infected with a lower multiplicity of infection, such
as 0.1 and 0.5, the kinetic of ROS production was delayed of a few
hours, reaching a plateau between 6 and 8 h post-infection,
reflecting the lower number of cells infected at the earliest time
points.
Antioxidants Block RSV-induced RANTES Secretion--
We have
recently demonstrated that RSV is a potent stimulus for RANTES
production in cultured human nasal, bronchial, and alveolar epithelial
cells (8, 9). In all epithelial cell types, synthesis of RANTES
required replicating virus and was dose- and
time-dependent, with increased steady state levels of RANTES mRNA observed between 6 and 12 h after infection (8, 9). To determine the contribution of RSV-induced ROS generation in
RANTES secretion, A549 cells were infected with RSV in the absence or
presence of the chemically unrelated antioxidants dimethyl sulfoxide
(Me2SO), N-acetyl·cystein (NAC), tetramethyl
thiourea (TMTU), and butylated hydroxyanisol (BHA). In preliminary
studies different concentrations of antioxidants were used to identify the most effective ones in inhibiting RANTES secretion (data not shown). We found that 2% (v/v) Me2SO, 20 mM
NAC, 20 mM TMTU, and 400 µM BHA were
sufficient to significantly block RSV-induced RANTES production, with
BHA being the most effective (Fig. 2, panel A). To confirm these results in a normal cell type,
similar experiments were performed in SAE cells infected with RSV. SAE cells are derived from the small bronchioli of the lung and they show a
similar pattern of RANTES induction, following RSV infection, compared
with A549 cells (9). As in A549 cells, the antioxidants TMTU, NAC, and
BHA significantly reduced RSV-induced RANTES production (Fig. 2,
panel B), suggesting that indeed inducible RANTES secretion is regulated in a redox-sensitive manner. Antioxidant treatment did not
significantly affect cell viability or viral replication (data not
shown).
Since BHA was the most effective compound in reducing RSV-induced
RANTES secretion in both A549 and SAE cells, we selected this
antioxidant to perform all the subsequent experiments. To directly
confirm the ability of BHA to inhibit ROS, A549 cells were stimulated
with 200 µM H2O2 in the absence
or presence of 400 µM BHA and the amount of cellular ROS
was monitored by oxidation of 2'7'-DCF. As shown in Fig.
3, H2O2 was able
to induce a high level of ROS production, which was almost completely
inhibited by treatment with BHA. These data indicate that BHA function
as a potent antioxidant in airway epithelial cells.
BHA Inhibits RSV-induced RANTES Gene Expression--
To determine
if the reduction in RSV-induced RANTES secretion by BHA was paralled by
changes in the steady-state level of RANTES mRNA, A549 cells were
infected with RSV for various lenghts of time, in the absence or
presence of the antioxidant, and total RNA was extracted from control
and infected cells for Northern blot analysis. A small increase in
RANTES mRNA expression was first detected at 6 h
post-infection, with maximal induction between 12 and 24 h (Fig.
4). There was no further increase in
mRNA levels at later time points (data not shown). Treatment with
400 µM BHA completely inhibited RSV-induced RANTES
mRNA induction at 6 and 12 h post-infection and greatly
reduced it at 24 h (Fig. 4). This dramatic change was not due to a
nonspecific effect since total cell number and viability in the group
treated with antioxidant were unchanged (data not shown) and levels of
the housekeeping gene
Inducible RANTES gene expression is controlled at both transcriptional
and post-transcriptional levels (23-25). We have previously shown that
in A549 cells RSV-induced RANTES promoter activation mirrors the
induction of the endogenous RANTES gene mRNA, suggesting that in
alveolar epithelial cells RANTES expression, following RSV infection,
is controlled mainly at the level of
transcription.2 To determine
whether the antioxidant effect of BHA influenced RANTES gene
transcription, A549 cells were transiently transfected with a construct
containing the first 220 nucleotides of the human RANTES promoter
linked to the luciferase reporter gene, defined as pGL2-220. This
fragment of the promoter contains all the necessary regulatory elements
that drive regulated luciferase expression in A549 cells following RSV
infection.2 The day after, cells were infected with RSV for
24 h in the absence or presence of BHA. Similar to what we have
observed for mRNA levels, treatment with BHA almost completely
abolished RSV-induced luciferase activity (Fig.
5), indicating that the antioxidant effect occurs mainly by interfering with RANTES gene
transcription.
Effects of BHA Treatment on RSV-induced Transcription Factor
Activation--
We have recently investigated the promoter
cis-regulatory elements and nuclear factors involved in the
regulation of RANTES gene transcription following RSV infection of
human airway epithelial cells. The results of that study have indicated
that RSV-induced RANTES transcription requires cooperation of multiple
response elements, including the ISRE.2 The ISRE is
absolutely required for RSV-induced promoter activation, since its
mutation completely blocks RSV-induced luciferase activity (Fig.
6). To determine if BHA-induced
inhibition of RANTES transcription was due to changes in the abundance
of DNA-binding proteins recognizing the RANTES ISRE, we performed
electrophoretic mobility shift assays using nuclear extracts prepared
from A549 cells control or infected with RSV for 12 h, in the
absence or presence of BHA. As shown in Fig.
7, RSV infection induced a dramatic
increase in ISRE binding, which was completely abolished by treatment
with BHA.
The major components of the RSV-induced ISRE binding complex are IRF-1,
-3, and -7.2 It has been previously shown that IRF-7 gene
expression and protein synthesis is viral inducible, while IRF-3 is
constitutively expressed and translocates to the nucleus when
phosphorylated in response to a viral infection (26). We have
previously shown that RSV infection of A549 cells induces de
novo synthesis of IRF-1 (20). To determine if RSV infection of
A549 cells induced IRF-7 synthesis and IRF-3 activation, we performed
Western blot analysis of cytoplasmic and nuclear proteins extracted
from A549 cells uninfected or infected for various lengths of time. As
shown in Fig. 8, RSV infection induced
de novo synthesis of IRF-7 and its nuclear translocation starting around 12 h post-infection. By contrast, IRF-3 was
constitutively expressed and RSV infection induced its nuclear
translocation starting around 6 h post-infection (Fig. 8). The
cytoplamic abundance of IRF-3 was lower at 12 h post-infection,
compared with control cells, likely due to the combined effect of
nuclear translocation and cytoplasmic proteosome-mediated degradation
of the activated form (27). Antioxidant treatment of RSV-infected
alveolar epithelial cells greatly reduced the nuclear abundance of
IRF-1 and -7 and one of the two detectable nuclear forms of IRF-3,
as shown in Fig. 9.
Since the other RSV-inducible nuclear form of IRF-3 was not affected by
BHA treatment, we questioned if this form was able to bind to the
RANTES ISRE. For this purpose, we used a two-step microaffinity
isolation/Western blot assay. In this assay, biotinylated ISRE was used
to bind nuclear extracts of control and 12-h infected A549 cells (20).
ISRE-binding proteins were captured by the addition of
streptavidin-agarose beads, washed, and the presence of bound IRF-3 was
detected by Western blot. As shown in Fig. 10, there was little detectable binding
of IRF-3 in control nuclear extracts, but its abundance was
greatly increased after RSV infection. BHA treatment almost completely
abolished IRF-3 binding, indicating that the nuclear form of IRF-3 not
inhibitable by antioxidant treatment is not able to bind to and
therefore to transactivate the RANTES ISRE.
To determine if the reduction in nuclear abundance of IRF-1, -3, and -7 following antioxidant treatment was due to inhibition of IRF gene
expression, protein synthesis, or nuclear translocation, we performed
Western blot analysis of whole cell extracts prepared from control and
RSV-infected A549 cells, in the absence or presence of BHA. RSV
infection induced a strong increase of IRF-1 and -7 protein expression
and it caused a shift in the electrophoretic mobility of one of the
three detectable forms of IRF-3, an event likely due to changes in
IRF-3 phosphorylation, as it has been previously described in Sendai
virus-infected cells (28). BHA treatment almost completely abolished
RSV-induced IRF-1 and -7 protein synthesis and IRF-3 mobility shift
(Fig. 11).
Since the amount of IRF-1 and -7 protein present in the cell is
dependent on their gene expression, we performed Northern blot analysis
of IRF-1 and -7 mRNA following RSV infection in the absence or
presence of BHA. Both IRF-1 and -7 mRNA accumulation was almost
completely blocked by antioxidant treatment at 6 and 12 h
post-infection and significantly reduced at 24 h (Fig.
12). On the other hand, IRF-3 mRNA
level was not increased following RSV infection and was unaffected by
the antioxidant treatment (data not shown). In summary, these
results strongly suggest that a redox-sensitive signaling pathway is
involved in IRF activation and RANTES gene expression following RSV
infection of alveolar epithelial cells.
Under normal conditions, airway epithelial cells represent an
important interface between the external environment and the host. Upon
infection or injury they play an important role in initiating the
mucosal immune response by producing soluble factors, like chemokines,
a family of small chemotactic cytokines, which are able to recruit and
activate leukocytes in a cell-type specific manner (29). The
immunomodulatory activity of the airway epithelium is of particular
relevance to RSV infection, since the inflammatory response triggered
by RSV infection appears to be an essential pathogenic component of
RSV-induced lung damage (30). RANTES is a CC chemokine highly
chemoattractant for T lymphocytes, monocytes, eosinophils, and
basophils, all cell types which are present or activated in the
inflammatory infiltrate that follows RSV infection of the lung. RANTES
concentrations are elevated in nasal washes and bronchoalveolar lavages
of children infected with RSV (6, 7) and RANTES gene is strongly
expressed in RSV-infected respiratory epithelial cells (8, 9),
suggesting that its production by infected epithelial cells may indeed
play an important role in the pathogenesis of RSV-induced airway inflammation.
Free radicals and reactive oxygen species have recently been shown to
function as second messengers influencing a variety of molecular and
biochemical processes, including expression of a number of genes
(reviewed in Ref. 10). The results of this study demonstrate that RSV
infection of alveolar epithelial cells induces ROS formation and
activates a redox-sensitive signaling pathway leading to de
novo synthesis of the transcription factors IRF-1 and -7 and
nuclear translocation of IRF-3 events that play a fundamental role in
viral-induced RANTES gene expression2 (31). This is the
first report of increased ROS production in airway epithelial cells
following RSV infection. Our data show that the kinetic of ROS
generation in infected cells is quite fast and precedes RSV-induced
transcription factor activation and increase in RANTES mRNA.
Several studies have pointed to the ability of certain viruses,
including influenza, Sendai, hepatitis B, and HIV, to induce the
formation of ROS (reviewed in Ref. 12). In most cases, the ROS
generation was a consequence of the activation of professional
phagocytes like monocytes and polymorphonuclear cell, similar to what
we have shown for eosinophils, in which RSV infection can induce
superoxide production (32). The relationship between viral-induced ROS
production and molecular and biochemical processes occurring in
infected cells has been more carefully investigated only for HIV.
HIV-induced ROS generation has been linked to gene expression and
apoptosis (12), although a role for ROS has also recently been claimed
in influenza-induced transcription factor activation and gene
expression (33). That RSV infection of epithelial cells could induce
ROS production was suggested by a previous study by Mastronarde
et al. (34), who showed that antioxidant treatment of
infected cells was able to block protein synthesis and mRNA
induction of interleukin-8, a pro-inflammatory chemokine that has been
extensively investigated in the past few years, whose activation
involves ROS-sensitive signaling pathways (13, 14). However, that study
did not show directly RSV-induced ROS generation.
Since the first evidence that reactive oxygen species can serve as
subcellular messengers in signal transduction pathways leading to
modification of gene transcription, there has been an explosion in the
number of genes whose expression has been reported to be influenced by
cellular redox changes. To date, there was only one report indicating a
possible role of ROS in RANTES gene expression, where antioxidant
treatment of mesangial cells stimulated with aggregated
immunoglobulins, which can enhance ROS formation, was able to inhibit
RANTES mRNA induction (35). However, the mechanism of RANTES
inhibition by the antioxidant treatment was not investigated. In this
study we were able to show that pretreatment of RSV-infected airway
epithelial cells with a panel of chemically unrelated antioxidants can
effectively inhibit RANTES secretion, mRNA induction, and
transcription, confirming the involvement of ROS in RANTES gene
expression. Although in this study we did not identify which species of
ROS are specifically induced in alveolar epithelial cells infected with
RSV, they probably do not include nitric oxide. Indeed, we were unable
to measure changes in RSV-induced RANTES secretion in cells treated
with the nitric-oxide synthase inhibitor Nitro-L-arginine
methyl ester (L-NAME) (data not shown). Furthermore, a similar
result was reported by Mastronarde et al. (34) for
RSV-induced IL-8 secretion.
IRF transcription factors have been shown to play a fundamental role in
the induction of several genes involved in the immune/inflammatory response to viral infections, including interferon Inhibition of phosphorylation and subsequent nuclear translocation is
likely the mechanism by which BHA inhibits RSV-induced IRF-3
activation. IRF-3 gene is constitutively expressed and IRF-3 protein is
present in cells in multiple forms due to different levels and sites of
its phosphorylation, as recently demonstrated by Servant et
al. (28). They have recently shown that IRF-3 exists as two forms
in unstimulated cells: form I represents nonphosphorylated IRF-3 and
form II a basally phosphorylated form. Infection with Sendai virus, as
well as Newcastle disease and measle viruses, all paramyxoviruses like
RSV, induces the appearance of form III and IV, which represent
C-terminal phosphorylation of IRF-3. The latter two forms are able to
translocate to the nucleus to induce gene expression. In the case of
A549 cells, we were able to detect three forms of IRF-3 in unstimulated
cells. Following RSV infection, we could detect a fourth band, which is
likely to represent a hyperphosphorylated form II of IRF-3, whose
formation was inhibited by antioxidant treatment. This
hyperphosphorylated form would migrate to the nucleus to bind RANTES
ISRE and would correspond to the nuclear form of IRF-3 which disappears
following BHA treatment and therefore is no longer present in the
RSV-induced ISRE binding complex, as shown by microaffinity isolation
assay. The signaling pathway leading to IRF-3 activation is currently
unknown. Faure et al. (40) have recently shown that IRF-1
was necessary for the NOS-2 in retinal epithelial cells stimulated with
lipopolysaccharide/interferon- In summary, our study indicates that the signaling pathway leading to
IRF-1 and -7 protein expression and IRF-3 activation involves one or
more redox-sensitive molecules that could be different depending on the
stimulus applied. Current studies are in progress to identify those
signaling molecules activated by RSV infection and leading to IRF
protein induction and activation and RANTES production. Identification
of the molecular mechanisms involved in RSV-induced gene expression is
fundamental for developing strategies to modulate the inflammatory
response associated with RSV infection of the lung.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until used.
70 °C, using
intensifying screens. After exposure, membranes were stripped and
rehybridized with a
-actin probe.
220 to +55 nucleotides (nt),
relative to the mRNA start site designated +1 was cloned into the
luciferase reporter gene vector pGL2 (Promega, Madison, WI) and defined
as pGL2-220. Site mutations of the RANTES ISRE in the context of
pGL2-220 plasmid were introduced by polymerase chain reaction using the
following upstream and downstream mutagenic primers (mutations in
lowercases): 5'-CATATTTCAGTaaaCTaaaCCGT-3' and
3'-TATAAAGTCAtttGAtttGGCAT-5'.
-galactosidase internal control plasmid. After 3 h, media was removed and 0.5 ml
of 10% (v/v) Me2SO in phosphate-buffered saline was added
to the cells for 2 min. Cells were washed with phosphate-buffered saline and cultured overnight in 10% fetal bovine serum/Dulbecco's modified Eagle's medium. The next morning, cells were infected with
RSV and at 24 h post-infection cells were lysed to measure independently luciferase and
-galactosidase reporter activity, as
previously described (20). Luciferase was normalized to the internal
control
-galactosidase activity. All experiments were performed in
duplicate or triplicate.
70 °C using intensifying screens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of ROS production in A549 cells
infected with RSV. A549 cells were infected with RSV at three
different multiplicity of infections. At various time points after
infection, cells were loaded with DCF-DA and fluorescence was measured
in control and infected cells. Mean fluorescence intensity is plotted
as a function of time. The error bars represent S.D. from
three independent experiments.
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Fig. 2.
Effect of antioxidants on RSV-induced RANTES
secretion. A549 cells (panel A) or SAE cells
(panel B) were infected with RSV in the absence or presence
of 2% (v/v) Me2SO, 20 mM NAC, 20 mM TMTU, and 400 µM BHA. Culture
supernatants, from control and infected cells were assayed 24 h
later for RANTES production by enzyme-linked immunosorbent assay. Data
are expressed as mean ± S.D. of three independent experiments
performed in triplicates. *, p < 0.01 relative to
RSV-infected cells not treated with antioxidants.
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Fig. 3.
BHA neutralization of
H2O2-induced ROS production. DCF-DA-loaded
A549 cells were left untreated or stimulated with 200 µM
H2O2 in the absence or presence of 400 µM BHA. Mean fluorescence intensity is plotted as a
function of time. Data are representative of one of three independent
experiments with similar results. *, p < 0.01 relative
to H2O2 only treated cells.
-actin were not systematically reduced
compared with untreated cells (Fig. 4).
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Fig. 4.
Northern blot of RANTES mRNA in
RSV-infected A549. A549 cells were infected with RSV for various
lengths of time in the absence or presence of 400 µM BHA.
Total RNA was extracted from control and infected cells and 20 µg of
RNA were fractionated on a 1.2% agarose-formaldehyde gel, transferred
to nylon membrane, and hybridized to a radiolabeled RANTES cDNA
probe. Membrane was stripped and hybridized with a radiolabeled
-actin probe, to show equal loading of the samples.
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Fig. 5.
Effect of BHA on RANTES promoter activation
following RSV infection. A549 cells were transiently transfected
with pGL2-220 plasmid and infected with RSV in the absence or presence
of 400 µM BHA. At 24 h post-infection, cells were
harvested to measure luciferase activity. Uninfected plates served as
controls. For each plate luciferase was normalized to the
-galactosidase reporter activity. Data are expressed as mean ± S.D, of normalized luciferase activity. *, p < 0.01 relative to RSV-infected plates not treated with BHA.
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Fig. 6.
Effect of site mutations of RANTES promoter
ISRE on RSV-inducible luciferase activity. A549 cells were
transiently transfected with pGL2-220 RANTES promoter either wild type
(WT) or mutated (MUT) in the ISRE site and
infected with RSV for 24 h. Uninfected plates served as controls.
For each plate luciferase was normalized to the -galactosidase
reporter activity. Data are expressed as mean ± S.D. of
normalized luciferase activity.
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Fig. 7.
Electrophoretic mobility shift assay of
RANTES ISRE binding complexes in response to antioxidant
treatment. Nuclear extracts were prepared from control and cells
infected with RSV for 12 h, in the absence or presence of 400 µM BHA, and used for binding to the RANTES ISRE in
electrophoretic mobility shift assay. Shown is the nucleoprotein
complex formed on the RANTES ISRE in response to RSV infection.
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Fig. 8.
Western blot of IRF-3 and -7 in RSV-infected
A549 cells. Cytoplasmic and nuclear proteins were prepared from
control and A549 cells infected for various length of time,
fractionated on a 10% SDS-PAGE, transferred to polyvinylidene
difluoride membranes, and probed with the appropriate antibody.
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Fig. 9.
Effect of BHA on IRF nuclear abundance in
A549 cells infected with RSV. A549 cells were infected with RSV
for 3, 6, 12, and 24 h, in the absence or presence of 400 µM BHA. Cells were harvested to prepare nuclear extracts
and equal amounts of protein from control and infected cells were
assayed for IRF-1, -3, and -7 by Western blot. Arrows
indicate the two nuclear forms of IRF-3 detectable after RSV
infection.
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Fig. 10.
Microaffinity isolation/Western blot for
IRF-3. Nuclear extracts, prepared from A549 cells control and
infected with RSV for 12 h, in the absence or presence of BHA,
were affinity purified using biotinylated ISRE oligonucleotide. After
capture with streptavidin-agarose beads, complexes were eluted and
assayed for IRF-3 by Western blot. Asterisk (*) indicates a
nonspecific band.
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Fig. 11.
Effect of BHA on IRF protein expression in
A549 cells infected with RSV. A549 cells were infected with RSV
for 12 h, in the absence or presence of 400 µM BHA.
Cells were harvested to prepare total cell lysates and equal amounts of
protein from control and infected cells were assayed for IRF-1, -3, and
-7 by Western blot. I, II, III, and IV indicates
the four different bands detected by the anti-IRF-3 antibody.
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Fig. 12.
Northern blot of IRF mRNA in
RSV-infected A549. A549 cells were infected with RSV for various
lengths of time in the absence or presence of 400 µM BHA.
Total RNA was extracted from control and infected cells and 20 µg of
RNA were fractionated on a 1.2% agarose-formaldehyde gel, transferred
to nylon membrane, and hybridized to a radiolabeled IRF-1 or -7 cDNA probe. Membrane was stripped and hybridized with a
radiolabeled -actin probe, to show equal loading of the
samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
,
cytokines like IL-15, adhesion molecules, MHC I molecules, and
inducible NOS (reviewed in Ref. 36). RSV has been shown to predispose to the development of asthma (2) and recurrent episodes of wheezing in
asthmatic children are often precipitated by RSV infection. Increased
IRF-1 expression has been recently found in airway epithelial cells of
patients with asthma, but not in normal individuals or patients with
chronic bronchitis (37). IRF protein binding to the ISRE of RANTES
promoter is necessary for viral induction of RANTES transcription and
gene expression2 (31). In alveolar epithelial cells IRF-1,
-3, and -7 are present in the DNA-nucleoprotein complex formed on the
ISRE following RSV infection and all three are involved in RSV-induced
RANTES promoter activation.2 In this study, we show for the
first time that the antioxidant treatment interferes with RSV-induced
RANTES transcription by inhibition of IRF binding to the ISRE, due its
multiple effects including blocking of IRF gene expression, protein
synthesis, or nuclear translocation. IRF-1 and -7 mRNA and protein
levels are clearly increased in A549 cells following RSV infection and greatly decreased by the treatment with BHA. The decrease of IRF-1 and
-7 protein synthesis is likely the major mechanism for their reduced
nuclear abundance in RSV-infected cells treated with BHA. However, it
is possible that BHA also affects IRF-1 and -7 phosphorylation, which
is important for their nuclear translocation and DNA binding (36).
IRF-1 gene expression is induced by interferon-
and cytokines through the activation of STAT and NF-
B transcription factors (38).
Similarly, interferon-
activates IRF-7 gene transcription through an
ISRE site that binds members of the STAT family (39). BHA treatment of
A549 cells did not affect RSV-induced NF-kB nuclear translocation and
DNA binding (data not shown). Therefore, it is possible that BHA
treatment affects RSV-induced STAT activation, leading to inhibition of
both IRF-1 and -7 gene expression.
and that the antioxidant pyrrolidine
dithiocarbamate was able to inhibit NOS-2 induction by interfering with
lipopolysaccharide/interferon-
-induced IRF-1 activation. In the case
of NOS-2, ERK and p38 MAP kinases are potential candidates as
redox-sensitive signaling molecules involved in IRF-1 activation, since
both kinases are important for its gene expression (40), both can be
activated by H2O2 stimulation and both are
inhibited by antioxidant treatment (reviewed in Ref. 41). However, this
may not be the case for RSV-induced IRF activation. In fact, even if we
have evidence that ERK is also involved in RSV-induced RANTES
secretion, inhibitors of ERK activation do not affect RSV induced
binding to the RANTES ISRE.3
This result is in agreement with the recently published observation by
Servant et al. (28) showing that pharmacological inhibitors of both ERK and p38 MAP kinases did not affect IRF-3 activation by
Sendai virus infection.
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ACKNOWLEDGEMENTS |
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We thank Todd Elliott for excellent technical assistance and Drs. A. Kamijo and R. M. Goldblum for help with the DCF-DA oxidation assay.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI 40218 and AI 15939 (NIAID) and P30ES0 6676 (NIEHS).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.
§ Child Health Research Center Young Investigator supported by Child Health and Human Development Grant HD 27841. To whom correspondence should be addressed: Dept. of Pediatrics, Div. of Child Health Research Center 301 University Blvd., Galveston, TX 77555-0366. Tel.: 409-747-0581; Fax: 409-772-1761; E-mail: ancasola@utmb.edu.
** Established Investigator of the American Heart Association.
Published, JBC Papers in Press, March 20, 2001, DOI 101074/jbc.M101526200
2 A. Casola, R. P. Garofalo, H. Haeberle, T. F. Elliott, M. Jamaluddin and A. R. Brasier, J. Virol. (in press).
3 A. Casola, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: RSV, respiratory syncytial virus; RANTES, regulated upon activation, normal T-cells expressed and secreted; ROS, reactive oxygen species; NAC, N-acetylcystein; TMTU, tetramethyl thiourea; BHA, butylated hydroxyanisol; ISRE, interferon-stimulated responsive element; IRF, interferon regulatory factor; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HIV, human immunodeficiency virus; IL, interleukin; SAE, small alveolar epithelial cells; m.o.i., multiplicity of infection; DCF-DA, 2,7-dichlorofluorescein diacetate; PAGE, polyacrylamide gel electrophoresis; STAT, signal transducers and activators of transcription.
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