Nonstructural 3 Protein of Hepatitis C Virus Triggers an Oxidative Burst in Human Monocytes via Activation of NADPH Oxidase*

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47 PHOX phosphorylation and translocation showed that NADPH twice with phosphate-buffered saline, and incubated with a fluorescein 5-isothiocyanate-labeled sheep anti-mouse IgG (Jackson Immuno Research (Montluc¸on, 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 6 S.E. of three separate experiments. For each experiment, the data were subjected to one-way analysis of variance followed by the means multiple compari-son method of Tukey (24). p , 0.05 was considered as the level of statistical significance.

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 pro-teases, giving rise to at least 10 mature proteins, which are encoded on the viral RNA in the following order: NH 2 -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)(13)(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)(18)(19)(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 (LaCl 3 ), U73122, calphostin C, antibody specific for p38 and N G -methyl-L-arginine (LNMMA) were purchased from Sigma. Antibodies specific for p47 PHOX and p67 PHOX were obtained from Becton Dickinson (Le Pont de Claix, France). Antibodies specific for mitogen-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. activated protein kinases and herbimycin A were purchased from BIOMOL Research Laboratories, and Sepharose beads were purchased from Calbiochem. [ 32 P]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% CO 2 . Nonadherent cells were removed by washing with Hepes-buffered saline solution (Biomedia, Boussens, France), and the remaining adherent cells (Ͼ85% monocytes) were incubated in serum-free medium.
Assay for ROS Production-Mononuclear cells 1.5 ϫ 10 5 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 Victor 2 , Finland). The generation of chemiluminescence was monitored continuously for 30 min after incubation of the cells with luminol (66 M) in basal conditions and in the presence of either NS3 (10 Ϫ8 , 10 Ϫ9 , or 10 Ϫ10 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, H 2 O 2 ), and LNMMA (inhibitor of nitrogen monoxide production). Statistical analysis was performed using the area under the curve expressed in counts ϫs. 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 Ca 2ϩ ). Then adherent monocytes (6 ϫ 10 5 ) were loaded with 11.5 ϫ 10 Ϫ6 M Fluo 3-AM for 10 min at 37°C. The time course of the intracytosolic Ca 2ϩ level was recorded every 0.5 s for a total period of 3 min after the addition of NS3 (10 Ϫ8 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).
Immunoprecipitation Electrophoresis and Immunoblotting Experiments-The cleared lysate was incubated with p47 PHOX antibody for 2 h. Then 50 l 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-p47 PHOX monoclonal antibody 1/1000). Immuno complexes were revealed by an anti-mouse Ig peroxidaseconjugated antibody (1/1000) and then visualized using the Amersham Pharmacia Biotech ECL system. 32 P-Labeled p47 PHOX 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-p47 PHOX or anti-p67 PHOX (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 Ϯ 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 Ͻ 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 Ϫ8 , 10 Ϫ9 , 10 Ϫ10 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 Ϫ8 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 (1-115), NS4 (1616 -1862), NS5A, and NS5B (and did not observe any ROS production (data not shown).
NS3 Suppresses Oxidative Burst Induced by PMA-Human monocytes were incubated with luminol and stimulated by several doses of NS3 (10 Ϫ8 , 10 Ϫ9 , 10 Ϫ10 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.
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 . , H 2 O 2 , 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).
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.
NS3 Induces p47 PHOX 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 p47 PHOX . Indeed phosphorylation of p47 PHOX 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 32 P and stimulated with NS3 (10 -8 M) or PMA or medium for 2 min. Then p47 PHOX was immunoprecipitated with a specific antibody. Fig. 5 presents the PhosphorImager radioscan (middle) and the corresponding Western blot analysis of p47 PHOX (bottom). Histograms represent the ratio of radioactivity on the corresponding protein level. Phosphorylation of p47 PHOX 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.
NS3 Induces a p67 PHOX and p47 PHOX Translocation-To confirm the activation of NADPH oxidase induced by NS3, we used immunofluorescence staining with confocal analysis to see whether p67 PHOX and p47 PHOX were translocated to the membrane. Immunofluorescence confocal microscopy (Fig. 6) shows that, in resting conditions, p67 PHOX and p47 PHOX were located in the cytoplasm. Stimulation with NS3 resulted in condensation of p67 PHOX and p47 PHOX 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.

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 (LaCl 3 ), an inhibitor of extracellular calcium influx, BAPTA, an intracellular

NS3 and Oxidative Stress in HCV
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. 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.
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 signalregulated 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. Calcium Inflow, Tyrosine Kinases, and p38 Are Involved in p47 PHOX Phosphorylation-To assess the role of calcium inflow, tyrosine kinases, and p38 in the activation of NADPH oxidase, p47 PHOX phosphorylation was investigated in the absence or presence of corresponding specific inhibitors. Fig. 5 shows that phosphorylation of p47 PHOX was diminished by SB203580, lanthanum chloride, and herbimycin A. These results are in accordance with those obtained with measurement of ROS pro- duction 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 NFB 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 pro-duction 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 p47 PHOX was found to be increased. Furthermore, immunofluorescence staining with confocal analysis evidenced the translocation of p47 PHOX and p67 PHOX 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 FIG. 6. Effects of NS3 stimulaton on p47 PHOX and p67 PHOX translocation. Monocytes were stimulated with NS3 (10 Ϫ8 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-p47 PHOX or anti-p67 PHOX (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 p67 PHOX and p47 PHOX in particular spots.

FIG. 7. Effect of different inhibitors of calcium signal on NS3triggered 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 LaCl 3 , an inhibitor of calcium influx, BAPTA, an intracellular calcium trap, and with U73122, an inhibitor of phospholipase C. Data are the means Ϯ S.E. of three separate experiments, each performed in triplicate. *, p Ͻ 0.05. 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 p47 PHOX , demonstrating that both p38, tyrosine kinases, and calcium signal are involved in the phosphorylation of p47 PHOX , 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 stressactivated 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 NFB activation and inhibits the proliferation of immune cells (42)(43)(44). Tai et al. (45) show that NFB was activated in HCV-infected liver. Whether these phenomena play a role in the natural history of HCV infection remains to be assessed. 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 ϫ 10 Ϫ6 M Fluo 3-AM for 10 min at 37°C. A, the time course of the intracytosolic Ca 2ϩ 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 LaCl 3 , 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 Ϯ S.E. of three separate experiments, each performed in triplicate 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 Ϯ S.E. of three separate experiments, each performed in triplicate. *, p Ͻ 0,05