 |
INTRODUCTION |
Hepatitis C virus (HCV)1
is a member of the Flaviviridae (1), and chronic infection with HCV is
a major cause of liver disease and liver cancer worldwide. The
viral genome is a 9.5-kilobase, positive-sense single-stranded RNA
molecule that contains a single open reading frame encoding a
polyprotein of 3,010-3,030 amino acids (2). The HCV polyprotein
undergoes proteolytic processing by both host signal peptidases and
viral proteases, giving rise to at least 10 mature proteins, which are
encoded on the viral RNA in the following order:
NH2-Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. Core,
E1, E2, and p7 are viral-structural proteins. The remaining viral
proteins (NS2 to NS5B) are believed to be nonstructural proteins,
components of the viral replication machinery. The catalytic domain of
the NS3 protease has been mapped to the N-terminal 180-amino acid
region of NS3, which contains a characteristic serine protease catalytic triad (3, 4). In addition to the N-terminal protease domain,
the C-terminal two-thirds domain of the NS3 protein contains conserved
sequence motifs, which are the hallmark of RNA helicases (5). Recent
experiments indicate that NS3 could suppress apoptosis and be involved
in persistent infection (6, 7). Borowski et al. (9) show
that NS3 also modulates signal transduction mediated by protein kinase
C (PKC)/PKA (8). Furthermore, these authors reported that NS3 could
inhibit reactive oxygen species (ROS) production triggered by phorbol
ester, a PKC agonist (10).
On the other hand, it has been suggested that liver injury and
mitochondrial dysfunction in hepatitis C could be partly caused by
HCV-mediated oxidative stress (11). Chronic infection was also
associated with ROS production in the liver and peripheral blood
mononuclear cells (12-14). ROS, especially superoxide anion (O
2), are essential contributors to host defense
against invading microorganisms. They are able to activate
transcription factors and, thus, modulate immune and inflammatory
responses and control cell survival (15, 16). During immune response,
release of ROS from sequestered phagocytes and activated resident
macrophages represent the predominant component of oxidative stress in
the liver. Monocytes could be target cells for HCV (17-20). In these cells, ROS production requires the activation of NADPH oxidase, which
catalyzes reduction of molecular oxygen to superoxide in conjunction
with oxidation of NADPH (21). They are key cells in antigen
presentation and in the inflammatory response and could thus be
involved in the natural history of HCV infection. However, little is
known about the ability of the monocyte to produce ROS in response to
HCV proteins. Consequently, the purpose of this study was to
investigate the ability of several proteins of HCV to activate ROS
production in human monocytes and to clarify the signaling pathway involved.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Phorbol 12-myristate 13-acetate (PMA), leupeptin,
pepstatin, aprotinin, sodium orthovanadate, catalase, superoxide
dismutase, cytochrome c, rotenone, thenoyltrifluoroacetone
(TTFA), myxothiazol, antimycin A, diphenyleneionodium chloride (DPI),
lanthanum chloride (LaCl3), U73122, calphostin C, antibody
specific for p38 and NG-methyl-L-arginine (LNMMA) were
purchased from Sigma. Antibodies specific for p47PHOX and
p67PHOX were obtained from Becton Dickinson (Le Pont
de Claix, France). Antibodies specific for mitogen-activated protein
kinases and herbimycin A were purchased from BIOMOL Research
Laboratories, and Sepharose beads were purchased from
Calbiochem. [32P]Orthophosphoric acid was obtained from
Amersham Pharmacia Biotech. Recombinant NS3 protein (1007-1534
amino acids) was produced in Escherichia coli by Mikrogen
Research (Germany) and solubilized in 25 mM Tris, 190 mM glycine, and 0.1% SDS. The final concentration of SDS
in cell culture was always less than 0.001%. Core, NS4, NS5A, and NS5B
were also produced in E. coli and purchased by Mikrogen
Research (Germany). Core protein (1-115 amino acid) does not include
E1, E2, and p7. NS4 protein (1616-1862) includes NS4A and NS4B.
Control medium was the buffer solution in which NS3 was solubilized.
Cell Preparation--
Mononuclear cells were obtained from buffy
coats from healthy blood donors by a standard Ficoll-Hypaque gradient
method. Human monocytes were isolated from mononuclear cells by
adherence to plastic for 2 h in special macrophage serum-free
medium (Life Technologies, Inc.) with L-glutamine at
37 °C in a humidified atmosphere containing 5% CO2.
Nonadherent cells were removed by washing with Hepes-buffered saline
solution (Biomedia, Boussens, France), and the remaining adherent cells
(>85% monocytes) were incubated in serum-free medium.
Assay for ROS Production--
Mononuclear cells 1.5 × 105 were placed in a 96-well microplate. ROS production was
measured by chemiluminescence in the presence of
5-amino-2,3-dihydro-1,4-phthalazinedione (luminol, Sigma) using a
thermostatically (37 °C) controlled luminometer (Wallac 1420 Victor2, Finland). The generation of
chemiluminescence was monitored continuously for 30 min after
incubation of the cells with luminol (66 µM) in basal
conditions and in the presence of either NS3 (10
8,
10
9, or 10
10 M) or 100 nM PMA. In some experiments, cells were incubated for 10 min before adding NS3 in the presence of different inhibitors. None of
the inhibitors used affected cell viability at the concentration used.
To assess superoxide anion production, chemiluminescence was measured
in the presence of superoxide dismutase (scavenger for O
2),
catalase (scavenger for hydrogen peroxide,
H2O2), and LNMMA (inhibitor of nitrogen
monoxide production). Statistical analysis was performed using the area
under the curve expressed in counts ×s.
Determination of Intracellular Calcium
Concentration--
Intracellular calcium concentration was measured in
single cells by a video digital microscopy technique using the
fluorescent probe Fluo 3-AM (Molecular Probe) as previously described
(22). Briefly, peripheral blood mononuclear cells were plated into
35-mm diameter plastic culture dishes, and nonadherent cells were
washed twice with Hepes-buffered saline solution (with and without
Ca2+). Then adherent monocytes (6 × 105)
were loaded with 11.5 × 10
6 M Fluo 3-AM
for 10 min at 37 °C. The time course of the intracytosolic Ca2+ level was recorded every 0.5 s for a total period
of 3 min after the addition of NS3 (10
8 M).
In parallel assays, cells were preincubated with several inhibitors for
10 min before the addition of NS3. Cells were visualized with an
inverted microscope (Nikon Diaphot 300). The light source was a xenon
lamp XBO 100 W (Orsam, Munich, Germany). Excitation (488 nm) and
emission (525 nm) were selected by a XF23 filter block (Nikon). These
wavelengths were acquired by an intensified camera LHESA, LH
5038-STD (Cergy-pontoise, France). Images were digitalized, and
fluorescence was analyzed using the IMSTAR starwise/fluo software system (Paris, France). Fluorescence calibration was performed
using ionomycin and a heavy metal as described previously (23).
[32]P Labeling and p47PHOX
Phosphorylation--
Monocytes (1.2 × 107 cells/ml)
were isolated from mononuclear cells by adherence to 60-mm plastic
dishes for 2 h in serum-free medium. After washing nonadherent
cells, the remaining monocytes were incubated in phosphate-free medium
(Dulbecco's modified Eagle's medium, Life Technologies, Inc.)
containing 500 µCi of 32P/107 cells/ml for
1 h at 37 °C. The cells were washed twice with Hepes-buffered saline solution. NS3 (10
8 M), PMA (100 nM), or medium was then added for 2 min in phosphate-free medium with or without inhibitors. Then 32P-labeled
monocytes were scraped off into ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium
deoxycholate, 1 mM EGTA, 1% Triton X-100 with aprotinin,
leupeptin, phenylmethylsulfonyl fluoride, orthovanadate, calpain
inhibitor) and centrifuged.
Immunoprecipitation Electrophoresis and Immunoblotting
Experiments--
The cleared lysate was incubated with p47PHOX
antibody for 2 h. Then 50 µl of Sepharose beads were added and
incubated overnight at 4 °C with gentle mixing. The beads were
washed extensively with lysis buffer. The immunoprecipitated proteins
were eluted by boiling in electrophoresis sample buffer. The beads were
then pelleted by brief centrifugation, and the proteins in the
supernatant were separated by SDS-polyacrylamide gel electrophoresis
(10%) according to Laemmli (46) for 80 min on a 4 Mini Trans
Blot electrophoretic transfer cell (Bio-Rad). Proteins were then
transferred to nitrocellulose membranes first blocked with
Tris-buffered saline containing 5% nonfat dried milk and 0.1%
Tween. Membranes were then incubated overnight with a specific primary
antibody (anti-p47PHOX monoclonal antibody 1/1000). Immuno
complexes were revealed by an anti-mouse Ig peroxidase-conjugated
antibody (1/1000) and then visualized using the Amersham Pharmacia
Biotech ECL system. 32P-Labeled p47PHOX were
detected by a PhosphorImager (Molecular Dynamics).
Immunofluorescence Confocal Microscopy--
Monocytic cells were
fixed in 2% paraformaldehyde in phosphate-buffered saline for 20 min
at room temperature and permeabilized with 0.1% Triton X-100 for 15 min. The cells were incubated with the primary antibody (mouse
anti-p47PHOX or anti-p67PHOX (1/200)) for 1 h at
room temperature, washed twice with phosphate-buffered saline, and
incubated with a fluorescein 5-isothiocyanate-labeled sheep anti-mouse
IgG (Jackson Immuno Research (Montluçon, France)). The cells were
visualized in a confocal microscope system (Leica TCS 4D) using
a laser-scanning head fitted to a Leitz DMIRB microscope with
double-label sequential detection.
Statistical Analysis--
The data are expressed as mean ± S.E. of three separate experiments. For each experiment, the data were
subjected to one-way analysis of variance followed by the means
multiple comparison method of Tukey (24). p < 0.05 was
considered as the level of statistical significance.
 |
RESULTS |
NS3 Early Activates ROS Production--
Human monocytes were
incubated with luminol and stimulated by several doses of NS3
(10
8, 10
9, 10
10
M), medium, or PMA (100 nM). ROS production was
measured by chemiluminescence. As shown in Fig.
1, NS3 induced a rapid, transient, and
significant increase in luminescence as compared with control. This
effect was dose-dependent and the peak observed with NS3
10
8 M occurred at 10 min. Chemiluminescence
returned to the basal level about 15 min after stimulation. We
performed the same experiments with the viral proteins, namely core
(1), NS4 (1616-1862), NS5A, and NS5B (and did not observe any ROS
production (data not shown).

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Fig. 1.
Time course of oxidative activity in response
to several doses of NS3 in human monocytes. The generation of
chemiluminescence was monitored continuously for 30 min after
incubation of the monocytes with luminol (66 µM) in the
absence of NS3 (Buffer), in the presence of NS3 at
10 8, 10 9, 10 10 M.
ROS production is expressed in counts/s. The curves are representative
of three separate experiments, each performed in triplicate.
|
|
NS3 Suppresses Oxidative Burst Induced by PMA--
Human monocytes
were incubated with luminol and stimulated by several doses of NS3
(10
8, 10
9, 10
10
M) or medium for 60 min. Monocytes were then stimulated
with 100 nM PMA. ROS production was inhibited when
monocytes were preincubated with NS3 (Fig.
2). Inhibition was
dose-dependent, and NS3 10-8 M
elicited a 80% decrease in ROS production in response to PMA.

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Fig. 2.
Effect of NS3 on the oxidative burst induced
by PMA. Monocytes were incubated with NS3 (10 8,
10 9, 10-10 M) or with buffer for
1 h. The generation of chemiluminescence was monitored
continuously for 40 min after incubation of the monocytes with luminol
(66 µM) and stimulation with 100 nM PMA. ROS
production is expressed in counts/s. The curves are representative of
three separate experiments, each performed in triplicate.
|
|
NS3 Triggers Superoxide Anion Production--
To investigate the
nature of the ROS produced by human monocytes after NS3 treatment, we
tested the effect of superoxide dismutase, catalase, and LNMMA
(respectively, scavengers for O
2,
H2O2, and NO) on chemiluminescence production.
The chemiluminescence was completely abolished when the cells were
preincubated with superoxide dismutase (Fig.
3, left panel). Catalase
and LNMMA had no effect (Fig. 3, right panel). These
results suggest that O
2 is the major oxygen radical produced
in response to NS3 stimulation. Furthermore, ROS produced in the
presence of NS3 were shown to reduce cytochrome c (data not
shown), a widely used method to assess O
2 production (25).

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Fig. 3.
Effect of inhibitors of different
oxygen reactive metabolites on NS3-triggered chemiluminescence
production in human monocytes. The generation of chemiluminescence
was monitored continuously for 30 min after stimulation with NS3 or
buffer. Right panel, monocytes were pretreated for 10 min with superoxide dismutase (SOD, a scavenger for
O 2) before stimulation with NS3. Left panel,
monocytes were pretreated for 10 min with catalase (scavenger for
H2O2) or LNMMA (an inhibitor of NO production)
before stimulation with NS3. ROS production is expressed in count/s.
The curves are representative of three separate experiments, each
performed in triplicate.
|
|
NADPH Oxidase Is Involved in the Production of ROS--
In
monocytes, the primary source of oxygen metabolites is NADPH oxidase.
However in conditions of cellular stress, the mitochondria have been
shown to generate ROS. Therefore several inhibitors of mitochondrial
complex I (rotenone (26)), II (TTFA (27)), and III (antimycin A and
myxothiazol (28)) and DPI, an inhibitor of the NADPH oxidase (29), were
used to assess whether mitochondria or NADPH oxidase was involved in
ROS production. We compared the effects of these different inhibitors
on the oxidative burst induced by NS3 or PMA. Rotenone and TTFA failed
to suppress chemiluminescence induced by either NS3 (Fig.
4) or PMA (data not shown). Myxothiazol or antimycin suppressed the effect of NS3 on monocytic cells (Fig. 4).
However, inhibition was similar to that observed with these agents on
ROS production after stimulation by PMA (data not shown). Furthermore
DPI totally abolished the effects of NS3 and PMA.

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Fig. 4.
Effects of different mitochondrial inhibitors
and DPI on the production of reactive oxygen radicals induced by
NS3. Total chemiluminescence emission (area under the curve
expressed in counts/15 min) was observed continuously for 15 min after
NS3 stimulation in the absence or the presence of either rotenone,
TTFA, myxothiazol, antimycin, or DPI. When inhibitors were used, the
monocytes were preincubated with them 10 min before stimulation.
Effects of the different inhibitors were also assessed in the absence
of NS3 stimulation. Data are means ± S.D. of three separate
experiments, each performed in triplicate. *, p < 0.05.
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|
NS3 Induces p47PHOX Phosphorylation--
These
observations suggest that NS3 is able to induce ROS production by
activating the phagocyte NADPH oxidase and led us to investigate the
phosphorylation of p47PHOX. Indeed phosphorylation of
p47PHOX is one of the first steps required for the activation
of NADPH oxidase in monocytes (21). To establish the activation of
NADPH oxidase by NS3, monocytes were loaded with 32P and
stimulated with NS3 (10-8 M) or PMA or medium
for 2 min. Then p47PHOX was immunoprecipitated with a specific
antibody. Fig. 5 presents the
PhosphorImager radioscan (middle) and the corresponding
Western blot analysis of p47PHOX (bottom).
Histograms represent the ratio of radioactivity on the corresponding
protein level. Phosphorylation of p47PHOX increased on average
2-fold after stimulation with NS3 as compared with control. These
results support the earlier chemiluminescence data and suggest that NS3
activates NADPH oxidase.

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Fig. 5.
Effect of NS3 and PMA on
p47PHOX phosphorylation. 32P-Labeled monocytes
were incubated with NS3 (10 8 M), PMA (100 nM), or buffer for 2 min after preincubation with
inhibitors SB203580, LaCl3, and herbimycin A
(HA), when present. TPA,
12-O-tetradecanoylphorbol-13-acetate. P47PHOX
was immunoprecipitated with a specific antibody as described under
"Experimental Procedures." Shown is a PhosphorImager radioscan
(middle) and the corresponding Western blot analysis
(bottom); histograms represent the ratio between
radioactivity and protein level.
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|
NS3 Induces a p67PHOX and p47PHOX
Translocation--
To confirm the activation of NADPH oxidase induced
by NS3, we used immunofluorescence staining with confocal analysis to
see whether p67PHOX and p47PHOX were translocated to
the membrane. Immunofluorescence confocal microscopy (Fig.
6) shows that, in resting conditions,
p67PHOX and p47PHOX were located
in the cytoplasm. Stimulation with NS3 resulted in condensation of
p67PHOX and p47PHOX in
particular spots (30). Control antibodies yielded no staining (data not
shown). These results confirm that NADPH oxidase is activated by NS3 in
human monocytes.

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Fig. 6.
Effects of NS3 stimulaton on
p47PHOX and p67PHOX translocation. Monocytes were
stimulated with NS3 (10 8 M for 5 min, fixed
in 2% paraformaldehyde, and permeabilized with 0.1% Triton X-100. The
cells were incubated with the primary antibody (mouse
anti-p47PHOX or anti-p67PHOX (1/200)) for 1 h and
incubated with a fluoerescein 5-isothiocyanate-labeled sheep
anti-mouse. The cells were visualized in a confocal microscope system
(Leica TCS 4D) using a laser-scanning head fitted to a Leitz
DMIRB microscope with double-label sequential detection. Stimulation
with NS3 resulted in condensation of
p67PHOX and p47PHOX in
particular spots.
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|
NS3-induced ROS Production Requires a Calcium Signal--
The
intracellular calcium concentration could be involved in the activation
of NADPH oxidase. To investigate the role of calcium in the NS3-induced
ROS production, cells were preincubated for 10 min with lanthanum
chloride (LaCl3), an inhibitor of extracellular calcium
influx, BAPTA, an intracellular calcium scavenger, or U73122, an
inhibitor of phospholipase C. BAPTA suppressed ROS production in
NS3-stimulated monocytes, which suggests that an increase in
intracellular calcium concentration is required for ROS production
triggered by NS3. Likewise, lanthanum chloride totally abolished the
oxidative burst, suggesting that extracellular calcium inflow was
essential. In contrast U73122 had no effect (Fig.
7), which indicates that mobilization of
intracellular calcium pools is not implicated in ROS production in our
model.

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Fig. 7.
Effect of different inhibitors of calcium
signal on NS3-triggered chemiluminescence production in human
monocytes. Total chemiluminescence emission (area under the curve
expressed in counts/15 min) was measured continuously for 15 min after
NS3 stimulation with or without inhibitor. When inhibitors were used,
cells were preincubated for 10 min with LaCl3, an inhibitor
of calcium influx, BAPTA, an intracellular calcium trap, and with
U73122, an inhibitor of phospholipase C. Data are the means ± S.E. of three separate experiments, each performed in triplicate. *,
p < 0.05.
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|
To support these results, intracellular calcium concentration was
measured in cells after stimulation by NS3 using a video digital
microscopy technique. The results presented in Fig.
8A clearly demonstrate that
NS3 enhanced the intracellular calcium concentration. Indeed NS3
induced an early, transient, and significant increase in intracellular
calcium concentration as compared with control medium. This increase
was inhibited when calcium-free medium was used. Then monocytes were
incubated with lanthanum chloride and U73122 for 10 min before adding
NS3. Only lanthanum chloride suppressed the increase of intracellular
calcium concentration (Fig. 8B). These results confirm that
the activation of ROS production requires a calcium influx triggered by
NS3.

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Fig. 8.
Effect of NS3 on intracellular calcium
concentration. Intracellular calcium concentration was measured in
single cells by a video digital microscopy technique using the
fluorescent probe Fluo 3-AM as described in "Experimental
Procedures." Adherent monocytes were loaded with 11.5 × 10 6 M Fluo 3-AM for 10 min at 37 °C.
A, the time course of the intracytosolic Ca2+
level was recorded every 0.5 s for a total period of 3 min after
the addition of NS3 or buffer or NS3 in calcium-free medium.
B, in similar conditions, cells were preincubated with
LaCl3, an inhibitor of calcium influx, and with U73122, an
inhibitor of phospholipase C for 10 min before the addition of NS3.
Data are the means ± S.E. of three separate experiments, each
performed in triplicate
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Tyrosine Kinases and P38 Are Involved in the NS3-induced ROS
Production--
PKC, tyrosine kinases, and mitogen-activated protein
kinase are known to activate NADPH oxidase. To assess the possible
involvement of PKC, tyrosine kinases, and mitogen-activated protein
kinases, cells were preincubated for 10 min with several inhibitors of these different signaling pathways, and ROS production was measured after stimulation by NS3. Fig. 9 shows
that calphostin C (inhibitor of PKC), PD980592, and U0126 (two
inhibitors of extracellular signal-regulated kinases (ERK) 1 and 2)
failed to suppress the effect of NS3. Conversely, the NS3-induced
respiratory burst was inhibited by SB 203580, an inhibitor of
stress-activated protein kinase 2 p38 (SAPK2/p38) and by herbimycin A
and AG 126, two inhibitors of tyrosine kinases. Thus SAPK2/p38 and
tyrosine kinases participate in the signal transduction pathway leading
to ROS production induced by NS3.

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Fig. 9.
Effect of several inhibitors of tyrosine
kinases, mitogen-activated protein kinases, and PKC on NS3-triggered
chemiluminescence production in human monocytes. Total
chemiluminescence emission (area under the curve expressed in counts/15
min) was measured continuously for 15 min after NS3 stimulation with or
without inhibitor. When inhibitors were used, cells were preincubated
for 10 min with herbimycin A and AG 126; two inhibitors of tyrosine
kinases, PD98059 and U0126; two inhibitors of extracellular
signal-regulated kinase 1 and 2; SB203580, a specific inhibitor of
SAPK2/p38; and calphostin C, an inhibitor of PKC. Data are the
means ± S.E. of three separate experiments, each performed in
triplicate. *, p < 0,05
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Calcium Inflow, Tyrosine Kinases, and p38 Are Involved in
p47PHOX Phosphorylation--
To assess the role of calcium
inflow, tyrosine kinases, and p38 in the activation of NADPH oxidase,
p47PHOX phosphorylation was investigated in the absence or
presence of corresponding specific inhibitors. Fig. 5 shows that
phosphorylation of p47PHOX was diminished by SB203580,
lanthanum chloride, and herbimycin A. These results are in accordance
with those obtained with measurement of ROS production and confirm that
NS3 is able to trigger ROS production in monocytic cells by activating
the phagocytic NADPH oxidase via calcium influx, tyrosine kinases, and p38.
 |
DISCUSSION |
ROS are essential contributors to host defense against invading
microorganisms owing to their direct cytotoxicity (31). They are also
able to modulate signal transduction by activating tyrosine kinases
(32) and transcription factors (33, 34). It has been demonstrated that
they can activate the NF
B pathway, which may trigger tumor necrosis
factor
production (15, 16, 33). Oxidative stress can also trigger
membrane and DNA damages (35). During the immune response, the release
of reactive oxygen species from sequestered phagocytes and activated
resident macrophages represents the predominant component of oxidative
stress in the liver. Thus, monocytes could be involved in the
inflammatory and immune response during hepatitis C virus infection and
in the pathogenesis of liver lesions. However, little is known about the ability of monocytes to produce ROS in response to HCV proteins. In
the present study, we investigated the ability of the Core, NS3, NS4,
and NS5 proteins of HCV to trigger ROS production in human monocytic
cells from healthy subjects; among them, only NS3 induced a
biphasic response on ROS production, which was initially activated,
then inhibited. The major ROS produced was O
2. Signal transduction involved NADPH oxidase, calcium inflow, tyrosine kinases,
and p38. Lipopolysaccharide contamination could not be implicated in
the modulation of ROS production because other proteins were also
produced in E. coli and had no significant effect.
The most informative method we used to assess these effects was
chemiluminescence. However, this method is not specific enough to
determine whether or not O
2 was the main ROS produced. This is
why we also used the cytochrome c reduction method, a
standard assay for measuring O
2 production (25). Actually,
this demonstrates that the NO pathway is not involved in the
NS3-induced ROS production.
The source of ROS was also questioned since they can originate from the
NADPH oxidase pathway or from mitochondria. DPI completely abolished
oxidative burst induced by NS3. DPI is known to inhibit NADPH oxidase
(29) but could also affect mitochondrial complex I (36). However,
rotenone, which inhibits mitochondrial complex I (26) and TTFA
(mitochondrial complex II inhibitor (27)), had no effect. Myxothiazol
and antimycin A (complex III inhibitors (28)) were able to suppress the
effect of NS3 on human monocytic cells. However, these two inhibitors
of mitochondrial complex III also inhibited the oxidative burst in
monocytes stimulated with PMA (results not shown), which is well known
to increase ROS production through NADPH oxidase activation (10).
Therefore, we cannot totally exclude that mitochondria are partially
involved in the ROS generation. However, the most likely explanation
for these discrepant results would be that antimycin and myxothiazol are not perfectly specific and partially inhibit NADPH oxidase activity. In support of this hypothesis, the phosphorylation of p47PHOX was found to be increased. Furthermore,
immunofluorescence staining with confocal analysis evidenced the
translocation of p47PHOX and p67PHOX from the cytoplasm
to the membrane, confirming that NADPH oxidase activity was not only
primed (37) but actually increased. We therefore conclude that
among HCV proteins, NS3 is specifically able to trigger O
2
through NADPH oxidase activation.
In a second set of experiments, we investigated how NS3 activated NADPH
oxidase. Different separate signal transduction pathways could be
involved in the activation of NADPH oxidase, among them: calcium
signal, PKC, tyrosine kinases, and mitogen-activated protein kinases.
After stimulation by NS3, a rapid and transient calcium signal was
evidenced by digital microscopy. Both calcium signal and oxidative
burst were totally abolished by a calcium channel inhibitor (lanthanum
chloride), whereas a specific inhibitor of phospholipase C (U73122) had
no effect. This strongly suggests that calcium influx but not
intracellular calcium mobilization is involved in the NS3-induced
activation of NADPH oxidase. The absence of intracellular calcium
increase when monocytes were incubated in a calcium-free medium
corroborates this interpretation.
The use of specific inhibitors showed that NS3-induced ROS production
involves tyrosine kinases and p38 pathways but neither extracellular
signal-regulated kinase 1 or 2 nor PKC. Inhibitors of p38, tyrosine
kinases, and lanthanum chloride decreased the phosphorylation of
p47PHOX, demonstrating that both p38, tyrosine kinases, and
calcium signal are involved in the phosphorylation of p47PHOX,
which thereafter activates NADPH oxidase. This is in keeping with
others studies showing that p38 plays a role in the activation of NADPH
oxidase in neutrophils (38, 39). Interestingly, p38, a member of the
stress-activated protein kinases is known to activate apoptosis but
could also be involved in cell proliferation and survival (40). The
relevance of these effects in the pathogenesis of hepatitis C remains
to be investigated.
After the initially triggering an oxidative burst, NS3 was shown to
secondarily inhibit the PMA-induced ROS production. This is in
accordance with the results of Borowski et al. (9), who showed a similar inhibition of PMA effects by NS3 in neutrophils. These
authors provide evidence that NS3 could disrupt PKC-mediated signal
transduction (8, 9). However the neurophils were permeabilized before
NS3 was added. This could explain why these authors did not observe the
initial oxidative burst we evidenced. Actually, when NS3 was added in
the medium and after a 1-h incubation time, PMA stimulation of ROS
production was abolished. This requires a tight interaction between NS3
and PKC (8, 9). Accordingly, this suggests that NS3 initially could
promote ROS production through a membrane receptor and, thereafter, is
internalized and interacts with PKC to inhibit ROS production. This
hypothesis clearly needs to be assessed by further experiments.
Several studies have provided evidence that ROS production activates
the transcription factor NF
in monocytes and, thus, modulates
immune and inflammatory responses as well as apoptosis by modulating
the transcription of several genes (15, 16, 33) Furthermore oxidative
genomic damage might be relevant in viral induced-carcinogenesis (35).
In human immunodeficiency virus infection, many studies have shown that
viral proteins, especially Tat, trigger ROS production (41). This
oxidative stress facilitates viral replication through NF
B
activation and inhibits the proliferation of immune cells (42-44). Tai
et al. (45) show that NF
B was activated in HCV-infected
liver. Whether these phenomena play a role in the natural history of
HCV infection remains to be assessed.