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(Journal of Leukocyte Biology. 2004;76:1180-1186.)
© 2004 by Society for Leukocyte Biology

A hepatitis C virus-encoded, nonstructural protein (NS3) triggers dysfunction and apoptosis in lymphocytes: role of NADPH oxidase-derived oxygen radicals

Fredrik Thorén*,1, Ana Romero*, Magnus Lindh*, Claes Dahlgren{dagger} and Kristoffer Hellstrand*

* Departments of Clinical Virology and
{dagger} Rheumatology and Inflammation Research, Sahlgrenska Academy, Göteborg University, Sweden

1 Correspondence: Department of Clinical Virology, Göteborg University, Guldhedsgatan 10b, S-413 46 Göteborg, Sweden. E-mail: fredrik.thoren{at}microbio.gu.se


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ABSTRACT
 
The persistent infection caused by hepatitis C virus (HCV) is presumably explained by a deficient immune response to the infection, but the basis for the inefficiency of immune-mediated virus eradication is not known in detail. This study addresses mechanisms of relevance to dysfunction of cytotoxic lymphocytes in HCV infection, with a focus on the role of phagocyte-derived oxygen radicals. We show that NS3, a nonstructural, HCV-encoded protein, induces a prolonged release of oxygen radicals from mononuclear and polymorphnuclear phagocytes by activating a key enzyme in radical formation, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The NS3-activated phagocytes, in turn, induced dysfunction and/or apoptosis in three major subsets of lymphocytes of relevance to defense against HCV infection: CD3+/56– T cells, CD3–/56+ natural killer (NK) cells, and CD3+/56+ NKT cells. Two inhibitors of the NADPH oxidase, histamine and diphenylene iodonium, suppressed the NS3-induced oxygen radical production and efficiently protected lymphocytes against NS3-induced apoptosis and dysfunction. In conclusion, we propose that NS3, by triggering oxygen radical formation in phagocytes, may contribute to the dysfunction of antiviral lymphocytes in HCV-infected liver tissue and that strategies to circumvent oxidative stress may be useful in preventing HCV-associated carcinogenesis and facilitating lymphocyte-mediated clearance of infected cells.

Key Words: CTL • NK cells • oxidative burst • phagocytes


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INTRODUCTION
 
In the majority of subjects infected with hepatitis C virus (HCV), the acute infection is followed by chronic disease [1 ], with a continuous synthesis of virions in infected hepatocytes. Unfortunately, spontaneous recovery from chronic disease is rare, if not exceptional; instead, the infection is frequently complicated by liver cirrhosis, liver failure, and hepatocellular carcinoma [1 ].

HCV induces multiple immune responses, and several lines of evidence suggest that cytotoxic, antiviral lymphocytes play a role in eradicating the acute infection and in controlling the chronic infection [2 3 4 5 6 7 8 ]. Indeed, the HCV-associated liver pathology is characterized by massive lymphocyte infiltration in the infected organ, but HCV apparently evades or limits the effectiveness of this immune response [9 10 11 12 13 14 ]. In particular, the immune response to HCV has been found to be impaired in patients chronically infected with HCV, as suggested by reports of impaired cytokine production by peripheral blood lymphocytes [11 , 12 , 15 ], a higher propensity of apoptotic cell death in lymphocytes [16 , 17 ], and a pronounced depression of T cell responses to defined HCV antigens in these patients [15 , 18 , 19 ]. Understanding the mechanisms underlying the dysfunction of lymphocytes in HCV infection may help, not only to explain the persistence of the virus but also to design more efficacious, immunotherapeutic regimens.

In recent years, much attention has been directed to the ability of tumor-infiltrating mononuclear phagocytes to adversely affect lymphocyte function [20 ], with a distinct focus on the role of phagocyte-derived oxygen radicals [21 22 23 ]. These toxic compounds have been proposed to account for the dysfunction of tumor-killing lymphocytes, not only in malignant diseases, such as colorectal carcinoma and metastatic melanoma, but also in chronic viral and bacterial infections [24 ]. This mechanism of immunosuppression, lymphocyte inhibition by mononuclear phagocyte-derived oxygen radicals, may be of particular relevance in liver tissue: The liver contains 80% of the host’s mononuclear phagocytic system [25 ], and the production of oxygen radicals in HCV-infected liver tissue is reportedly enhanced by a factor of 100,000 [26 ].

The abundance of oxygen radical-producing phagocytes in liver tissue along with the recent findings that HCV proteins may stimulate the formation of oxygen radicals in infected tissue [27 28 29 30 ] led us to examine potential interactions among phagocytes, lymphocytes, and HCV-encoded proteins. We report that NS3, a nonstructural HCV protein, elicits dysfunction and apoptosis of three main subsets of cytotoxic lymphocytes, which are prevalently found in chronically HCV-infected liver tissue: conventional T lymphocytes (with CD3+/56– phenotype), CD3–/56+ natural killer (NK) cells, and CD3+/56+ NK/T cells. The dysfunction and apoptosis resulted from an NS3-induced release of reactive oxygen species (ROS) from phagocytes. We hypothesize that the immune dysfunction and chronicity associated with HCV infection may, in part, be related to oxygen radical induction by HCV-encoded proteins.


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MATERIALS AND METHODS
 
Separation of mononuclear cells
Peripheral venous blood was obtained as freshly prepared leukopacks from healthy blood donors at the Blood Centre, Sahlgren University Hospital (Göteborg, Sweden). The blood (65 ml per donor) was mixed with 92.5 ml Iscoves’ medium, 35 ml 6% dextran, and 7.5 ml acid citrate dextrose (ACD). After incubation for 15 min at room temperature, the supernatant was carefully layered on top of a Ficoll-Hypaque (Lymphoprep) density gradient. After centrifugation at 380 g for 15 min, mononuclear cells were collected at the interface. The cells were washed and resuspended in Iscoves’ medium supplemented with 10% AB+ serum. Cells were further separated into lymphocytes and monocytes using a counter-current centrifugal elutriation technique in which the sedimentation rate of cells in a spinning rotor is balanced by a counter-directed flow through the chamber. By slowly increasing the flow rate, fractions of cells of well-defined sizes can be collected. The mononuclear cells were resuspended in elutriation buffer, buffered NaCl supplemented with 0.5% bovine serum albumin (BSA) and 0.1% EDTA, and were fed into a Beckman J2-21 ultracentrifuge with a JE-6B rotor (Beckman Coulter, Fullerton, CA) at 2100 rpm. A fraction with >90% monocytes was obtained at a flow rate of ~19 ml/min. A lymphocyte fraction enriched for NK cells (CD3–/56+ phenotype) and T cells (CD3+/56– phenotype) was recovered at flow rates of 14–15 ml/min. Using flow cytometry, this latter fraction was shown to consist of CD3–/56+ NK cells (35–45%), CD3+/56– T cells (35–40%), CD3–/56– cells (5–10%), and CD3+/56+ cells (1–5%) with <3% contaminating monocytes [21 ]. Pure preparations of NK cells (CD3–/56+), T cells (CD3+/56–), and NKT cells (CD3+/56+) were obtained by cell sorting using FACSAria (Becton Dickinson, San José, CA).

Determination of ROS production
Mononuclear phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was measured with an isoluminol-dependent, peroxidase-enhanced chemiluminescence technique, described in detail by Dahlgren and Karlsson [31 ]. Briefly, mononuclear phagocytes were suspended in Krebs Ringer glucose buffer supplemented with isoluminol (10 µg/ml) and horseradish peroxidase (HRP; 4 U/ml) and incubated for 5 min at 37°C. Stimuli were added, and the release of ROS was measured in a six-channel Berthold Biolumat LB 9505 (Berthold Technologies, Wildbad, Germany).

NK cell cytotoxicity assay
51Cr-labeled cells from the NK cell-sensitive cell line K562, originally derived from patients suffering from chronic myelogenous leukemia in blast crisis, were used as target cells. For labeling, cells were incubated with 51Cr (150 µCi/ml) for 2 h at 37°C. The chromium-labeled cells were washed thoroughly and resuspended in culture. Ten thousand target cells were then incubated at 37°C with lymphocytes (100,000 cells/well) in 96-well microplates (Nunc, Roskilde, Denmark) in the presence or absence of monocytes (5000–60,000 cells/well). Compounds were added at the onset of incubation if not otherwise stated. After 16 h, the supernatant was collected using a tissue-collecting system (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and the radioactive content was determined in a {gamma}-counter. Results were expressed as percentage specific release: % Specific release = 100 x (experimental release–spontaneous release)/(maximum release–spontaneous release). Spontaneous release was measured by incubating labeled target cells with culture medium and maximum release, by incubating target cells with Triton X-100.

More than 90% of the lymphocyte cytotoxicity was depleted by the removal of CD56+ NK cells, using anti-CD56-coated beads, and removal of CD3+ T cells did not reduce cytotoxicity significantly.

Apoptosis in lymphocytes
Lymphocyte apoptosis was carefully monitored over time using different methods. One of the earliest events in the apoptotic process is depolarization of the mitochondrial transmembrane potential. The mitochondrial membrane sensor kit (BD Clontech, Palo Alto, CA) was used to identify cells with altered mitochondrial transmembrane potential according to the manufacturer’s protocol. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled Annexin V (BD PharMingen, San Diego, CA) was used to identify cells that had lost the asymmetrical distribution of membrane phospholipids and thus were exposing phosphatidyl serine on the extracellular side of the plasma membrane. Loss of structural integrity of the plasma membrane was monitored by adding the cationic dye, To-Pro-3 (2 µM; Molecular Probes, Junction City, OR), right before the flow cytometry analysis. Finally, the percentage of end-stage apoptotic cells was determined based on the altered scattering properties displayed by end-stage apoptotic cells, i.e., reduced forward-scatter and increased right angle-scatter.

Compounds
The following compounds were used: isoluminol, formylated Met-Leu-Phe (fMLF), and diphenylene iodonium (DPI; Sigma Chemical Co., St. Louis, MO); HRP, superoxide dismutase, and catalase (Boehringer-Mannheim, Mannheim, Germany); dextran (Kabi Pharmacia, Stockholm, Sweden); ACD (Baxter, Deerfield, IL); sodium chromate (Na251CrO4; Amersham Pharmacia Biotech, Uppsala, Sweden); BSA (ICN Biomedicals, Aurora, OH); EDTA and hydrogen peroxide (VWR, Göteborg, Sweden); Ficoll-Hypaque, Lymphoprep (Nycomed, Oslo, Norway); histamine dihydrochloride (Maxim Pharmaceuticals, San Diego, CA); ranitidine dihydrochloride (Glaxo Wellcome, Mölndal, Sweden). A recombinant, truncated HCV protein NS3 (genotype 1b, amino acids 1007–1534), produced in Escherichia coli and solubilized in 25 mM Tris, 190 mM glycine, and 0.1% sodium dodecyl sulfate (SDS), was obtained from Mikrogen Research (Munich, Germany). Pure FITC-, PE-, peridinin chlorophyll protein-, and allophycocyanin-conjugated monoclonal antibodies against various surface markers were purchased from Becton Dickinson (Stockholm, Sweden).


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RESULTS
 
Phagocyte ROS release triggered by NS3
We investigated the ability of the HCV protein NS3 to activate the oxygen radical-generating NADPH oxidase in mononuclear and polymorphnuclear phagocytes. NS3 was found to induce a rapid-onset production of ROS in both cell types, as manifested as an increase in release of ROS within seconds upon addition of NS3 (Fig. 1a and 1b ).



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  		Figure 1. NS3-induced oxygen radical production. Phagocytes were stimulated with 0.1 µM NS3 (NS3 lo), 0.3 µM NS3 (NS3 hi), fMLF (0.1 µM), or the corresponding buffer (Buff), and the extracellular release of oxygen radicals was measured by isoluminol-amplified chemiluminescence for 10 min. Chemiluminescence responses are given as Mcpm (106 cpm). (a) The kinetics of a representative experiment with mononuclear phagocytes (main graph) and polymorphnuclear phagocytes (inset). In accordance with earlier reports [32 ], the SDS-containing protein diluent was found to slightly but significantly activate the NADPH oxidase. However, NS3 at 0.1 and 0.3 µM significantly enhanced the release of ROS from mononuclear phagocytes over that induced by corresponding concentrations of SDS (b). Data are integral values expressed as percent of corresponding control (mean+SEM, n=5). (c) Mononuclear phagocytes were stimulated with fMLF or NS3 (0.1 µM) in the presence of DPI (0.3 µM), histamine (His; 100 µM), or histamine and ranitidine (Ran; 100 µM), and the release of oxygen radicals was measured for 15 min. The release of oxygen radicals from mononuclear phagocytes was significantly reduced when histamine (P<0.01) or DPI (P<0.05) was added to the NS3-stimulated cells (paired samples t-test). Data are integral values expressed as percent of NS3 response (mean+SEM, n=3–7).

To establish a role for the NADPH oxidase for the observed ROS production by NS3, we used two earlier-characterized NADPH oxidase inhibitors, DPI and histamine [33 , 34 ]. Both inhibitors reduced the NS3-triggered ROS formation (Fig. 1c) . The histamine effect was mediated by H2receptors, as illustrated by the finding that the H2 antagonist ranitidine prevented the histamine-induced reduction of ROS production (Fig. 1c) .

Inhibition of NK cell cytotoxicity by NS3
Previous studies have shown that the cytotoxicity of NK cells is impaired in HCV-infected individuals [9 ]. The suggestion that this impairment may be related to toxicity inflicted by ROS [35 ] along with our earlier findings, showing that NADPH oxidase-derived ROS are strongly inhibitory for NK cell function and viability [21 , 34 ], incited us to investigate the impact of phagocyte ROS release induced by NS3 on NK cell cytotoxic function.

In a series of experiments, mononuclear phagocytes were incubated with NK cell-enriched lymphocytes in the presence or absence of NS3, and the ability of NK cells to lyse K562 cells was monitored. NS3 was found to induce a mononuclear phagocyte-dependent suppression of NK cell function (Fig. 2a ). The suppression was clearly mediated by ROS, as the addition of catalase, a scavenger of hydrogen peroxide, restored NK cell cytotoxic activity. Cytotoxicity was also restored when the NADPH oxidase inhibitors, DPI or histamine, were added to the incubation mixture (Fig. 2b) .



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  		Figure 2. NS3-induced reduction of NK cell cytotoxic function; reversal by antioxidative treatment. NK cell-enriched lymphocytes (105 cells/well) were incubated with autologous monocytes at various mononuclear phagocyte (Mo)/lymphocyte (Ly) ratios for 16 h at 37°C and assayed for cytotoxicity against 51Cr-labeled K562 cells (104 cells/well). (a) Cells were treated with culture medium or NS3 (0.1 µM), added 15 min after the onset of incubation. NS3 did not significantly affect cytotoxicity in the absence of mononuclear phagocytes but enhanced the phagocyte-induced inhibition of cytotoxicity at all phagocyte/lymphocyte ratios investigated; P values ranging from 0.003 to 0.042 (paired samples t-test, n=5). Data are expressed as relative reduction of NK cytotoxicity induced by NS3 at various phagocyte/lymphocyte ratios. The inset shows a representative experiment (mean±SEM of quadruplicates). (b) Addition of 200 U/ml catalase (Cat; P=0.012), 0.3 µM DPI (P=0.029), or 100 µM histamine (His; P=0.024) reversed the NS3-induced inhibition of NK cell cytotoxicity (paired samples t-test). Data are mean ± SEM of four experiments.

NS3 triggers apoptosis in NK cells and T cells
To determine whether the NS3-elicited release of toxic oxygen metabolites also affected lymphocyte viability, lymphocytes were incubated with NS3-stimulated phagocytes and assayed for apoptosis. Within 2 h, lymphocytes displayed altered mitochondrial transmembrane potential, {Delta}{psi}m – an early marker for apoptosis (Fig. 3 ). The proportion of lymphocytes with altered {psi}m increased over time, and this process paralleled Annexin V staining (not shown). With time, apoptotic cells became increasingly stained with the cell-impermeant dye To-Pro-3 and changed their scatter characteristics. Mononuclear phagocytes alone induced some apoptosis, and polymorphnuclear phagocytes induced lower levels of apoptosis (Figs. 3 and 4a ). However, the addition of NS3 significantly enhanced the extent of phagocyte-induced lymphocyte apoptosis, and the addition of buffer did not (Fig. 4a) . Lymphocytes were fully protected from NS3-induced apoptosis by the addition of catalase, DPI, or histamine (Fig. 4b) , suggesting that apoptosis was mediated by NADPH oxidase-derived ROS.



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  		Figure 3. NS3 induces depolarization of the mitochondrial transmembrane potential. NK cell-enriched lymphocytes were incubated with phagocytes in the presence or absence of NS3. After 2 h, lymphocytes were stained with the mitosensor reagent and analyzed by flow cytometry. One early event in the apoptotic process is mitochondrial depolarization, which is detected as an increase in green fluorescence in mitosensor-stained cells. Stimulation of polymorphonuclear phagocytes (PMN; upper panels) with NS3 triggered mitochondrial depolarization in lymphocytes. In the presence of mononuclear phagocytes (Mo; lower panels), a larger fraction of lymphocytes displayed altered {psi}m, but stimulation with NS3 increased the fraction of lymphocytes with altered mitochondrial transmembrane potential. DPI fully protected lymphocytes from mitochondrial alterations induced by NS3-stimulated phagocytes. Similar results were obtained in three experiments with cells from different donors.



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  		Figure 4. NS3 triggers apoptosis in lymphocytes. NK cell-enriched lymphocytes were incubated overnight with autologous phagocytes at different ratios. After incubation, cells in a lymphocyte gate were assayed for morphological features of end-stage apoptosis (reduced forward- and increased right angle-scatter) by use of flow cytometry. Cells were treated with culture medium or NS3 (0.1 µM), added 15 min after the onset of incubation. (a) Results from representative experiments showing the effects of NS3 on lymphocyte apoptosis at different phagocyte/lymphocyte ratios (PMN/Ly Ratio, left panel; Mo/Ly Ratio, right panel). NS3 significantly enhanced the frequency of apoptotic lymphocytes at the highest PMN/Ly ratio (P=0.007, paired samples t-test, n=3) and at Mo/Ly ratios above 0.25 (P values ranging from 0.013 to 0.001, paired samples t-test, n=4–5), and the addition of buffer did not affect lymphocyte viability significantly. (b) NS3-stimulated phagocytes (PMN/Ly Ratio, left panel; Mo/Ly Ratio, right panel) were incubated with lymphocytes at a phagocyte/lymphocyte ratio of 0.6 in the presence of various compounds. Lymphocytes were fully protected by 200 U/ml catalase (Cat; P=0.02 and 0.003 for PMN and Mo, respectively), 0.3 µM DPI (P=0.02 and 0.003), or 100 µM histamine (His; P=0.01 and 0.002; paired samples t-test). The protective effect of histamine was abolished by addition of equimolar concentrations of the H2 antagonist, ranitidine (P=0.002, not shown). Data are mean + SEM of three (PMN) or four (Mo) experiments.

To clarify whether these NS3-elicited events applied to all the main lymphocyte subsets infiltrating the HCV-infectedliver, i.e., CD3+/56– T cells, CD3–/56+ NK cells, and CD3+/56+ NKT cells, lymphocytes were stained with antibodies to CD3 and CD56 before assessment of end-stage apoptosis. The results showed that NS3-stimulated mononuclear phagocytes induced apoptosis in CD3+/56– T cells and CD3–/56+ NK cells (Fig. 5a ). NK cells were highly sensitive to ROS-induced apoptosis, and CD3+/56– T cells appeared more resistant. However, the quantification of apoptotic cells of a specific lymphocyte subtype in a mixed lymphocyte preparation is flawed by the propensity of apoptotic NK cells to alter their expression of the CD56 antigen, as well as by the nonspecific binding of antibodies to the surface of apoptotic T cells. There is thus an inherent risk of underestimating NK and T cell apoptosis, and apoptosis of double-positive cells may be overestimated as a result of recruitment of inadequately stained, apoptotic T cells into the double-positive gate. To circumvent this problem, we determined ROS-induced apoptosis in preparations of pure CD3+/56– T cells, CD3–/56+ NK cells, and CD3+/56+ NKT cells. These experiments confirmed that apoptosis was induced in all liver-type lymphocyte subsets studied (Fig. 5b) and that NK cells were indeed more susceptible to ROS-induced apoptosis than were T cells or NKT cells.



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  		Figure 5. NK cell-enriched lymphocytes were incubated overnight with NS3-stimulated mononuclear phagocytes at indicated ratios and assayed for morphological features of end-stage apoptosis. Data are the frequency of apoptotic CD3+ cells or CD56+ cells (mean+SEM of five experiments; a). At the mononuclear phagocyte/lymphocyte (Mo/Ly) ratio 0.6, the frequency of apoptotic cells was significantly higher in CD56+ cells than in CD3+ cells (P value=0.003; paired samples t-test, n=5). (b) Pure preparations of CD3+/56– T cells, CD3–/56+ NK cells, and CD3+/56+ NKT cells were obtained by cell sorting using a BD FACSAria. These cells were incubated overnight with autologous mononuclear phagocytes and assayed for end-stage apoptosis. SSC, Side-scatter; FSC, forward-scatter.


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DISCUSSION
 
The liver tissue destruction and carcinogenesis associated with chronic HCV infection have been proposed to be mediated by an excessive formation of ROS in the infected tissue [26 ]. In this study, we investigated the stimulatory properties of the HCV-encoded protein NS3 on NADPH oxidase-dependent oxygen radical formation by human phagocytes, along with monitoring the effects of NS3-stimulated mononuclear phagocytes on the function and viability of lymphocyte subsets. Our data demonstrate that oxidative stress adversely affects lymphocytes, which are destined to eliminate transformed as well as HCV-infected cells.

Activators of oxygen radical production in phagocytes commonly trigger a specialized electron-transporting system, the NADPH oxidase, which ferries electrons to molecular oxygen. In this way, oxygen is reduced to toxic oxygen products. The NS3 protein was reported recently to weakly trigger oxygen radical production in monocytes [28 ]. However, the chemiluminescence technique used by these authors has limitations, which may lead to underestimation of the release of radicals. By using a modified and highly sensitive method for oxygen radical detection [31 ], we found that the NS3 protein induced a robust oxygen radical production with a magnitude comparable with that induced by other phagocyte activators [36 , 37 ].

NS3 is not the first HCV protein with purported ROS-generating capacity. HCV core and NS5a were shown recently to inflict oxidative damage in human cell lines and in murine hepatocytes [27 , 29 , 30 ]. These different HCV proteins might work in concert, each contributing to the emergence of oxidative damage to lymphocytes and other surrounding cells. Oxidative stress is a hallmark of HCV infection, and the reports of oxidative damage to hepatic as well as nonhepatic cells are numerous [38 39 40 41 42 43 ]. Although the oxidative stress is more severe in cirrhotic patients, it is clearly not only a late-stage phenomenon, as it reportedly occurs even before alanine aminotransferase levels are elevated [42 , 43 ].

Our finding that NS3-stimulated phagocytes induced dysfunction and apoptosis in antiviral lymphocytes may indicate that hepatic lymphocytes in HCV-infected patients become dysfunctional and undergo apoptosis as a result of the oxidative stress prevailing in the infected liver [26 ]. This view is supported by earlier reports showing that lymphocytes from HCV-infected individuals are frequently apoptotic and unusually susceptible to apoptosis-inducing stimuli [16 , 17 ]. Furthermore, NK cells, which are probably protective at early stages of HCV infection [44 ] before the development of specific immunity, are reportedly dysfunctional in HCV infection: Corado et al. [9 ] found NK cell cytotoxicity to be significantly reduced, and Barbaro et al. [35 ] reported that depletion of lymphocyte stores of reduced glutathione was associated with decreased peripheral blood mononuclear cell cytotoxicity in HCV-infected individuals. These findings imply that oxidative stress at an early stage of HCV infection may adversely affect the immune-mediated clearance of infected cells. A tentative hypothesis emerging from our study is that HCV can exploit the phagocyte NADPH oxidase to generate ROS, which in turn disable key antiviral lymphocytes; in this way, HCV may evade the immune system, enabling viral persistence and contributing to disease chronicity.

The consequences of uncontrolled formation of ROS might be especially problematic in HCV infection, as HCV is associated with elevated levels of iron [35 , 38 , 45 ]. Elevated levels of free iron ions further promote oxidative stress, as iron can catalyze the generation of highly reactive hydroxyl radicals via the Fenton reaction. In addition, iron can propagate the oxidative processes by catalyzing the decomposition of lipid peroxides into cytotoxic aldehydes [46 ]. Furthermore, the continuous release of mutagenic and potentially carcinogenic oxygen radicals on the one hand and dysfunctional immune cells relevant to the defense against malignancies on the other may have implications for the increased risk of hepatocellular carcinoma associated with chronic hepatitis C.

There are, thus, at least two reasons for including antioxidants in the treatment arsenal against HCV infection: By neutralizing ROS, antioxidants can alleviate the tissue damage of the liver and as suggested by the findings reported in this study, protect critical immune cells against oxygen radical-induced, functional inhibition and apoptosis, thereby increasing the likelihood of virus eradication. During the past decade, various antioxidants have indeed been tested as adjuncts to the standard treatment of chronic HCV infection, interferon-{alpha} (IFN-{alpha}), but results have been disappointing [47 , 48 ]. However, the antioxidants being tested have predominantly been scavengers, which react with already formed ROS with a limited spectrum of neutralizing activity. A more attractive strategy may be to prevent ROS from being produced by the phagocyte NADPH oxidase, and in this study, we show that two such inhibitors, histamine and DPI, maintain the cytotoxicity and viability of antiviral lymphocytes in the presence of NS3-stimulated mononuclear phagocytes in vitro. One of these compounds, histamine, is currently evaluated as a supplement to IFN therapy in chronic HCV infection [49 ]. A deeper understanding of the mechanism underlying the immunosuppression in chronic HCV infection will be helpful in the development of new measures to protect lymphocytes from the inhibitory signals in the HCV-infected liver.


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ACKNOWLEDGEMENTS
 
This study was supported by the Swedish Cancer Foundation (Cancerfonden), the Swedish Medical Research Council, and Maxim Pharmaceuticals Inc. We thank Marie-Louise Landelius for excellent technical assistance.

Received July 6, 2004; revised August 19, 2004; accepted August 20, 2004.


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REFERENCES
 
    1
  1. EASL International Consensus Conference on Hepatitis C. Paris, 26–28, February 1999, Consensus Statement. European Association for the Study of the Liver J. Hepatol. 1999;30,956-961[CrossRef][Medline]
    2. 2
  2. Diepolder, H. M., Zachoval, R., Hoffmann, R. M., Wierenga, E. A., Santantonio, T., Jung, M. C., Eichenlaub, D., Pape, G. R. (1995) Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection Lancet 346,1006-1007[CrossRef][Medline]
    3. 3
  3. Missale, G., Bertoni, R., Lamonaca, V., Valli, A., Massari, M., Mori, C., Rumi, M. G., Houghton, M., Fiaccadori, F., Ferrari, C. (1996) Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response J. Clin. Invest. 98,706-714[Medline]
    4. 4
  4. Freeman, A. J., Pan, Y., Harvey, C. E., Post, J. J., Law, M. G., White, P. A., Rawlinson, W. D., Lloyd, A. R., Marinos, G., Ffrench, R. A. (2003) The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection J. Hepatol. 38,349-356[CrossRef][Medline]
    5. 5
  5. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P., Salazar-Mather, T. P. (1999) Natural killer cells in antiviral defense: function and regulation by innate cytokines Annu. Rev. Immunol. 17,189-220[CrossRef][Medline]
    6. 6
  6. Lucas, M., Gadola, S., Meier, U., Young, N. T., Harcourt, G., Karadimitris, A., Coumi, N., Brown, D., Dusheiko, G., Cerundolo, V., Klenerman, P. (2003) Frequency and phenotype of circulating V{alpha}24/Vß11 double-positive natural killer T cells during hepatitis C virus infection J. Virol. 77,2251-2257[Abstract/Free Full Text]
    7. 7
  7. Kakimi, K., Guidotti, L. G., Koezuka, Y., Chisari, F. V. (2000) Natural killer T cell activation inhibits hepatitis B virus replication in vivo J. Exp. Med. 192,921-930[Abstract/Free Full Text]
    8. 8
  8. Einav, S., Koziel, M. J. (2002) Immunopathogenesis of hepatitis C virus in the immunosuppressed host Transpl. Infect. Dis. 4,85-92[CrossRef][Medline]
    9. 9
  9. Corado, J., Toro, F., Rivera, H., Bianco, N. E., Deibis, L., De Sanctis, J. B. (1997) Impairment of natural killer (NK) cytotoxic activity in hepatitis C virus (HCV) infection Clin. Exp. Immunol. 109,451-457[CrossRef][Medline]
    10. 10
  10. Par, G., Rukavina, D., Podack, E., Horanyi, M., Szekeres-Bartho, J., Hegedus, G., Paal, M., Szereday, L., Mozsik, G., Par, A. (2002) Decrease in CD3-negative-CD8dim(+) and V{delta}2/V{gamma}9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection J. Hepatol. 37,514-522[CrossRef][Medline]
    11. 11
  11. Gruener, N. H., Lechner, F., Jung, M. C., Diepolder, H., Gerlach, T., Lauer, G., Walker, B., Sullivan, J., Phillips, R., Pape, G. R., Klenerman, P. (2001) Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus J. Virol. 75,5550-5558[Abstract/Free Full Text]
    12. 12
  12. Wedemeyer, H., He, X. S., Nascimbeni, M., Davis, A. R., Greenberg, H. B., Hoofnagle, J. H., Liang, T. J., Alter, H., Rehermann, B. (2002) Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection J. Immunol. 169,3447-3458[Abstract/Free Full Text]
    13. 13
  13. Tseng, C. T., Klimpel, G. R. (2002) Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions J. Exp. Med. 195,43-49
    14. 14
  14. Crotta, S., Stilla, A., Wack, A., D’Andrea, A., Nuti, S., D’Oro, U., Mosca, M., Filliponi, F., Brunetto, R. M., Bonino, F., Abrignani, S., Valiante, N. M. (2002) Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein J. Exp. Med. 195,35-41
    15. 15
  15. Lechner, F., Wong, D. K., Dunbar, P. R., Chapman, R., Chung, R. T., Dohrenwend, P., Robbins, G., Phillips, R., Klenerman, P., Walker, B. D. (2000) Analysis of successful immune responses in persons infected with hepatitis C virus J. Exp. Med. 191,1499-1512[Abstract/Free Full Text]
    16. 16
  16. Nuti, S., Rosa, D., Valiante, N. M., Saletti, G., Caratozzolo, M., Dellabona, P., Barnaba, V., Abrignani, S. (1998) Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for V{alpha}24+ T cells and rapid elimination of effector cells by apoptosis Eur. J. Immunol. 28,3448-3455[CrossRef][Medline]
    17. 17
  17. Toubi, E., Kessel, A., Goldstein, L., Slobodin, G., Sabo, E., Shmuel, Z., Zuckerman, E. (2001) Enhanced peripheral T-cell apoptosis in chronic hepatitis C virus infection: association with liver disease severity J. Hepatol. 35,774-780[CrossRef][Medline]
    18. 18
  18. Chang, K. M., Thimme, R., Melpolder, J. J., Oldach, D., Pemberton, J., Moorhead-Loudis, J., McHutchison, J. G., Alter, H. J., Chisari, F. V. (2001) Differential CD4(+) and CD8(+) T-cell responsiveness in hepatitis C virus infection Hepatology 33,267-276[CrossRef][Medline]
    19. 19
  19. Thimme, R., Oldach, D., Chang, K. M., Steiger, C., Ray, S. C., Chisari, F. V. (2001) Determinants of viral clearance and persistence during acute hepatitis C virus infection J. Exp. Med. 194,1395-1406[Abstract/Free Full Text]
    20. 20
  20. Elgert, K. D., Alleva, D. G., Mullins, D. W. (1998) Tumor-induced immune dysfunction: the macrophage connection J. Leukoc. Biol. 64,275-290[Abstract]
    21. 21
  21. Hansson, M., Asea, A., Ersson, U., Hermodsson, S., Hellstrand, K. (1996) Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites J. Immunol. 156,42-47[Abstract]
    22. 22
  22. Kono, K., Salazar-Onfray, F., Petersson, M., Hansson, J., Masucci, G., Wasserman, K., Nakazawa, T., Anderson, P., Kiessling, R. (1996) Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing {zeta} molecules and inhibits tumor-specific T cell- and natural killer cell-mediated cytotoxicity Eur. J. Immunol. 26,1308-1313[Medline]
    23. 23
  23. Samlowski, W. E., Petersen, R., Cuzzocrea, S., Macarthur, H., Burton, D., McGregor, J. R., Salvemini, D. (2003) A nonpeptidyl mimic of superoxide dismutase, M40403, inhibits dose-limiting hypotension associated with interleukin-2 and increases its antitumor effects Nat. Med. 9,750-755[CrossRef][Medline]
    24. 24
  24. Kiessling, R., Wasserman, K., Horiguchi, S., Kono, K., Sjoberg, J., Pisa, P., Petersson, M. (1999) Tumor-induced immune dysfunction Cancer Immunol. Immunother. 48,353-362[CrossRef][Medline]
    25. 25
  25. Saba, T. M. (1970) Physiology and physiopathology of the reticuloendothelial system Arch. Intern. Med. 126,1031-1052[Abstract/Free Full Text]
    26. 26
  26. Valgimigli, M., Valgimigli, L., Trere, D., Gaiani, S., Pedulli, G. F., Gramantieri, L., Bolondi, L. (2002) Oxidative stress EPR measurement in human liver by radical-probe technique. Correlation with etiology, histology and cell proliferation Free Radic. Res. 36,939-948[CrossRef][Medline]
    27. 27
  27. Gong, G., Waris, G., Tanveer, R., Siddiqui, A. (2001) Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-{kappa} B Proc. Natl. Acad. Sci. USA 98,9599-9604[Abstract/Free Full Text]
    28. 28
  28. Bureau, C., Bernad, J., Chaouche, N., Orfila, C., Beraud, M., Gonindard, C., Alric, L., Vinel, J. P., Pipy, B. (2001) Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase J. Biol. Chem. 276,23077-23083[Abstract/Free Full Text]
    29. 29
  29. Moriya, K., Nakagawa, K., Santa, T., Shintani, Y., Fujie, H., Miyoshi, H., Tsutsumi, T., Miyazawa, T., Ishibashi, K., Horie, T., Imai, K., Todoroki, T., Kimura, S, Koike, K. (2001) Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis Cancer Res. 61,4365-4370[Abstract/Free Full Text]
    30. 30
  30. Okuda, M., Li, K., Beard, M. R., Showalter, L. A., Scholle, F., Lemon, S. M., Weinman, S. A. (2002) Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein Gastroenterology 122,366-375[CrossRef][Medline]
    31. 31
  31. Dahlgren, C., Karlsson, A. (1999) Respiratory burst in human neutrophils J. Immunol. Methods 232,3-14[CrossRef][Medline]
    32. 32
  32. Shpungin, S., Dotan, I., Abo, A., Pick, E. (1989) Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme J. Biol. Chem. 264,9195-9203[Abstract/Free Full Text]
    33. 33
  33. Robertson, A. K., Cross, A. R., Jones, O. T., Andrew, P. W. (1990) The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte-derived macrophages J. Immunol. Methods 133,175-179[CrossRef][Medline]
    34. 34
  34. Hellstrand, K., Asea, A., Dahlgren, C., Hermodsson, S. (1994) Histaminergic regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites J. Immunol. 153,4940-4947[Abstract]
    35. 35
  35. Barbaro, G., Di Lorenzo, G., Ribersani, M., Soldini, M., Giancaspro, G., Bellomo, G., Belloni, G., Grisorio, B., Barbarini, G. (1999) Serum ferritin and hepatic glutathione concentrations in chronic hepatitis C patients related to the hepatitis C virus genotype J. Hepatol. 30,774-782[CrossRef][Medline]
    36. 36
  36. Mellqvist, U. H., Hansson, M., Brune, M., Dahlgren, C., Hermodsson, S., Hellstrand, K. (2000) Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine Blood 96,1961-1968[Abstract/Free Full Text]
    37. 37
  37. Betten, A., Bylund, J., Cristophe, T., Boulay, F., Romero, A., Hellstrand, K., Dahlgren, C. (2001) A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis J. Clin. Invest. 108,1221-1228[CrossRef][Medline]
    38. 38
  38. Farinati, F., Cardin, R., De Maria, N., Della Libera, G., Marafin, C., Lecis, E., Burra, P., Floreani, A., Cecchetto, A., Naccarato, R. (1995) Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis J. Hepatol. 22,449-456[CrossRef][Medline]
    39. 39
  39. De Maria, N., Colantoni, A., Fagiuoli, S., Liu, G. J., Rogers, B. K., Farinati, F., Van Thiel, D. H., Floyd, R. A. (1996) Association between reactive oxygen species and disease activity in chronic hepatitis C Free Radic. Biol. Med. 21,291-295[CrossRef][Medline]
    40. 40
  40. Boya, P., de la Pena, A., Beloqui, O., Larrea, E., Conchillo, M., Castelruiz, Y., Civeira, M. P., Prieto, J. (1999) Antioxidant status and glutathione metabolism in peripheral blood mononuclear cells from patients with chronic hepatitis C J. Hepatol. 31,808-814[CrossRef][Medline]
    41. 41
  41. Farinati, F., Cardin, R., Degan, P., De Maria, N., Floyd, R. A., Van Thiel, D. H., Naccarato, R. (1999) Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection Free Radic. Biol. Med. 27,1284-1291[CrossRef][Medline]
    42. 42
  42. Cardin, R., Saccoccio, G., Masutti, F., Bellentani, S., Farinati, F., Tiribelli, C. (2001) DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver disease: validation in an open population study J. Hepatol. 34,587-592[CrossRef][Medline]
    43. 43
  43. Jain, S. K., Pemberton, P. W., Smith, A., McMahon, R. F., Burrows, P. C., Aboutwerat, A., Warnes, T. W. (2002) Oxidative stress in chronic hepatitis C: not just a feature of late stage disease J. Hepatol. 36,805-811[CrossRef][Medline]
    44. 44
  44. Biron, C. A., Brossay, L. (2001) NK cells and NKT cells in innate defense against viral infections Curr. Opin. Immunol. 13,458-464[CrossRef][Medline]
    45. 45
  45. Di Bisceglie, A. M., Axiotis, C. A., Hoofnagle, J. H., Bacon, B. R. (1992) Measurements of iron status in patients with chronic hepatitis Gastroenterology 102,2108-2113[Medline]
    46. 46
  46. Halliwell, B. (1987) Oxidants and human disease: some new concepts FASEB J. 1,358-364[Abstract]
    47. 47
  47. Ideo, G., Bellobuono, A., Tempini, S., Mondazzi, L., Airoldi, A., Benetti, G., Bissoli, F., Cestari, C., Colombo, E., Del Poggio, P., Fracassetti, O., Lazzaroni, S., Marelli, A., Paris, B., Prada, A., Rainer, E., Roffi, L. (1999) Antioxidant drugs combined with {alpha}-interferon in chronic hepatitis C not responsive to {alpha}-interferon alone: a randomized, multicentre study Eur. J. Gastroenterol. Hepatol. 11,1203-1207[Medline]
    48. 48
  48. Look, M. P., Gerard, A., Rao, G. S., Sudhop, T., Fischer, H. P., Sauerbruch, T., Spengler, U. (1999) Interferon/antioxidant combination therapy for chronic hepatitis C—a controlled pilot trial Antiviral Res. 43,113-122[CrossRef][Medline]
    49. 49
  49. Lurie, Y., Nevens, F., Aprosina, Z. G., Fedorova, T. A., Kalinin, A. V., Klimova, E. A., Ilan, Y., Maevskaya, M. V., Warnes, T. W., Yuschuk, N. D., Hellstrand, K., Gehlsen, K. R. (2002) A multicentre, randomized study to evaluate the safety and efficacy of histamine dihydrochloride and interferon-{alpha}-2b for the treatment of chronic hepatitis C J. Viral Hepat. 9,346-353[CrossRef][Medline]



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