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Originally published online as doi:10.1189/jlb.0204113 on September 10, 2004

Published online before print September 10, 2004
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(Journal of Leukocyte Biology. 2004;76:1207-1213.)
© 2004 by Society for Leukocyte Biology

Activation of cytotoxic lymphocytes by interferon-{alpha}: role of oxygen radical-producing mononuclear phagocytes

Markus Hansson*, Ana Romero{dagger}, Fredrik Thorén{dagger}, Svante Hermodsson{dagger} and Kristoffer Hellstrand{dagger},1

* Department of Hematology, University of Lund, Sweden; and
{dagger} Department of Virology, University of Göteborg, Sweden

1 Correspondence: Department of Virology, University of Göteborg, S-413 46 Göteborg, Sweden. E-mail: kristoffer.hellstrand{at}microbio.gu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A significant part of the therapeutic benefit of interferon-{alpha} (IFN-{alpha}) therapy in malignant diseases and in chronic viral infections is assumed to result from activation of lymphocytes with natural killer (NK) and T cell phenotype. In tumor tissue and in chronically infected tissue, the function and viability of these lymphocytes are frequently impaired. Mononuclear phagocyte (MP)-derived reactive oxygen species (ROS) have been proposed to contribute to the lymphocyte suppression in these tissues. Here, we report that three types of human cytotoxic lymphocytes of relevance to immunoactivation by IFN-{alpha}, CD3{varepsilon}+/8+/56– T cells, CD3{varepsilon}–/56+ NK cells, and CD3{varepsilon}+/56+ NK/T cells became anergic to IFN-{alpha} induction of the cell-surface activation marker CD69 after exposure to autologous MPs in vitro. In addition to their incapacity to express CD69, cytotoxic lymphocytes acquired features characteristic of apoptosis after incubation with MPs. The lymphocyte apoptosis and nonresponsiveness to IFN-{alpha} were prevented by two inhibitors of reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of ROS in MPs, histamine dihydrochloride and diphenylene ionodonium, as well as by catalase, a scavenger of ROS. We conclude that MP-derived ROS may negatively affect IFN-{alpha}-induced immunostimulation and propose that ROS inhibitors or scavengers may be useful to improve lymphocyte activation during treatment with IFN-{alpha}.

Key Words: T cell • NK cell • NK/T cell • histamine • ROS • CD69


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The leukocyte-derived cytokine interferon-{alpha} (IFN-{alpha}) is currently used as a therapeutic in hematology (chronic myelogenous and hairy cell leukemia), oncology (lymphoma, adjuvant treatment of resected melanoma), and infectious disease (chronic hepatitis B and C) [1 2 3 ]. The precise mechanism underlying the benefit of IFN-{alpha} in these diseases is not known, but it seems well established that immunostimulatory properties of IFN-{alpha} contribute to its antineoplastic and antiviral efficiency [3 4 5 6 ]. These activating properties comprise inter-alia triggering of tumoricidal and antiviral lymphocytes such as natural killer (NK) cells and cytotoxic T cells. Early studies demonstrated that IFN-{alpha} induces cytolytic activity in NK cells, expands their target cell spectrum, and induces transcription of cytokine genes and the de novo appearance of activation markers in T cells as well as NK cells. The lymphocyte activation is assumed to result from interaction between IFN-{alpha} and defined IFN receptors on lymphocytes [7 , 8 ]. More recent studies suggest that IFN-{alpha} also up-regulates stimulatory molecules on antigen-presenting dendritic cells to indirectly activate cytotoxic lymphocytes [9 ].

Phagocytes, in particular, mononuclear phagocytes (MPs), form a major part of the cellular inflammatory infiltrate within human malignant tumors and in chronically infected tissues [10 , 11 ]. These phagocytic cells produce reactive oxygen sepcies (ROS) by assembling and activating a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex at the cell membrane. A first step in the NADPH oxidase-dependent formation of ROS is the reduction of oxygen to superoxide anions, which are rapidly metabolized into hydrogen peroxide and other oxygen species. Earlier studies have revealed that phagocyte-derived ROS induce dysfunction and apoptosis of NK cells and T cells [12 13 14 15 16 ], and lymphocyte inhibition by phagocyte-derived ROS has been proposed as an important immune escape mechanism in tumors and in chronically infected tissue (reviewed in refs. [17 18 19 ]).

Understanding the impact of MPs and in particular, their production of ROS, on the inducibility of cytotoxic lymphocytes by IFN-{alpha} may therefore have therapeutic implications. CD69 (Leu-23 or MLR-3), a transmembrane glycoprotein with signal-transducing properties, is induced on the surface of T cells or NK cells by IFN-{alpha}, and the CD69 induction correlates to cytotoxicity and proliferation in cytotoxic lymphocytes [20 21 22 ]. In this study, we used the surface expression of CD69 as a marker for IFN-{alpha}-induced activation of cytotoxic lymphocytes and studied the impact of ROS-producing phagocytes on these immunostimulatory events. We report that ROS, constitutively produced by autologous MPs, suppress or abolish IFN-{alpha} stimulation in NK cells, CD8+ T cells, and NK/T cells, a hybrid of NK and T cells with a CD3{varepsilon}+/56+ phenotype [23 ]. In addition to becoming nonresponsive to IFN-{alpha}, a sizable fraction of these cytotoxic lymphocytes acquired features of apoptosis after exposure to ROS-producing phagocytes. Three strategies to circumvent oxidative stress—the NADPH-oxidase inhibitors histamine dihydrochloride and diphenylene iodonium (DPI) and the hydrogen peroxide scavenger catalase—all rescued NK, NK/T, and T cells from MP-induced apoptosis and restored their responsiveness to IFN-{alpha}. We conclude that MP-derived ROS limit the IFN-{alpha}-induced activation of cytotoxic lymphocytes and propose that adjunctive, antioxidative therapy may be useful in enhancing the immunostimulatory efficiency of IFN-{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Separation of mononuclear cells (MNC)
Peripheral venous blood was obtained as freshly prepared leukopacks from healthy blood donors at the Blood Center, Sahlgren’s Hospital (Göteborg, Sweden). The blood (65 ml) was mixed with 92.5 ml Iscove’s medium, 35 ml 6% Dextran (Kabi Pharmacia, Stockholm, Sweden), and 7.5 ml acid citrate dextrose (Baxter, Deerfield, IL). After incubation for 15 min at room temperature, the supernatant was carefully layered onto Ficoll-Hypaque (Lymphoprep, Nyegaard, Norway). The MNC were collected at the interface after centrifugation at 380 g for 15 min and washed twice in phosphate-buffered saline (PBS) and resuspended in Iscove’s medium supplemented with 10% human AB+ serum. During all further separation of cells, the cell suspensions were kept in siliconized test tubes (Vacuette, Greiner, Stockholm).

The cells were further separated into lymphocytes and MPs using the counter-current centrifugal elutriation technique, as described in detail elsewhere [12 ]. Briefly, the MNC were resuspended in elutriation buffer containing 0.05% bovine serum albumin and 0.015% EDTA in buffered NaCl and fed into a Beckman J2-21 ultracentrifuge with a JE-6B rotor at 2100 rpm. A fraction with >90% MPs was obtained at a flow rate of 18 ml/min. A lymphocyte fraction enriched for NK cells (CD3{varepsilon}–/56+ phenotype) and T cells (CD3{varepsilon}+/56–) was recovered at flow rates of 14–15 ml/min. This fraction contained <3% MPs and consisted of CD3{varepsilon}–/56+ NK cells (15–20%), CD3{varepsilon}+/56– T cells (60–65%), CD3{varepsilon}–/56– cells (1–5%), and CD3{varepsilon}+/56+ cells (15–20%), as judged by flow cytometry. In some experiments, dynabeads (Dynal A/S, Oslo, Norway) coated with anti-CD56 were used to obtain purified lymphocyte preparations of T cells, as described in detail elsewhere [24 ]. In some experiments, the lymphocyte fraction was further separated by the FACSAria cell-sorting system obtaining CD3{varepsilon}+/CD56+ NK/T cells. The mixture of NK and T cells or sorted NK/T cells was exposed to autologous, elutriated MPs in microplates. The lymphocytes (150,000 cells/well in 200 µl) were incubated with or without MPs (150,000 cells/well) for 16 h at 37°C.

Assays of apoptosis
Apoptosis was monitored by use of flow cytometry. Nonapoptotic T cells or NK cells were gated after exposure to MPs, as described elsewhere [25 ]. The gate was set to comprise lymphocytes with reduced forward-scatter and increased right angle-scatter characteristics of apoptosis [16 ]. The level of apoptosis determined in lymphocytes using this assay was earlier shown to correlate closely with Annexin V staining [15 ], analysis of DNA strand breaks by terminal deoxynucleotidyl transferase-mediated bromolated deoxyuridine triphosphate nick end-labeling of DNA fragments [26 ], and analysis of reduced intracellular glutathione [16 ].

Detection of surface antigens
One million cells were incubated with appropriate fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, and peridinin chlorophyll protein (PerCP)-conjugated monoclonal antibodies (mAb; Becton Dickinson, Stockholm, Sweden; 1 µl/106 cells) on ice for 30 min. The cells were washed twice in PBS and resuspended in 500 µl sterile-filtrated PBS and analyzed by use of flow cytometry on a FACSort with a Lysys II software program (Becton Dickinson) or FACSAria cell-sorting system with a CellQuest software program (Becton Dickinson). Lymphocytes were gated on the basis of forward- and right angle-scatter. The flow rate was adjusted to <200 cells x s–1, and at least 104 cells were analyzed for each sample, if not otherwise stated.

Compounds
Human recombinant IFN-{alpha}-2b (IntronA, Schering-Plough, Stockholm, Sweden), histamine dihydrochloride (Maxim Pharmaceuticals, San Diego, CA; Sigma Chemcials, Stockholm, Sweden), ranitidine hydrochloride (Glaxo, Mölndal, Sweden), AH202399AA (Glaxo), DPI (Sigma Chemical Co., St. Louis, MO), catalase (Boehringer Mannheim, Germany), and L-nitro-arginine-methyl-ester (L-NAME; Sigma Chemical Co.) compounds were readily dissolved in culture medium. FITC-, PE-, and PerCP-conjugated mAb against CD3{varepsilon}, CD4, CD8, CD56, and CD69 were purchased from Becton Dickinson.

Statistics
For statistical analysis, Mann-Whitney U-test or Wilcoxons sign-rank test was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD69 activation antigen induction in T, NK/T, and NK cells
We determined the expression of CD69 on T, NK/T, and NK cells, incubated in the presence or absence of autologous MPs. The following lymphocyte subsets were studied: CD3{varepsilon}+/4+ (T helper cells), CD3{varepsilon}+/8+/56– (T suppressor/cytotoxic cells), CD3{varepsilon}–/8–/56+ (NK cells), and CD3{varepsilon}+/56+ (NK/T cells). In concurrence with earlier studies [27 ], T cells with CD3{varepsilon}, CD4, or CD8 phenotype acquired CD69 when treated with IFN-{alpha} (100 U/ml) in the absence of MPs. CD69 appeared at 2–4 h after the onset of treatment with IFN-{alpha} with an expression maximum at 16–24 h. The induction was dose-dependent at final IFN-{alpha} concentrations of 1–100 U/ml (data not shown).

The IFN-{alpha}-induced expression of CD69 in CD8+ T cells was significantly reduced by the addition of MPs. In contrast, CD4+ T cells significantly increased their expression of CD69 after incubation with MPs (Table 1 ). Histamine (50 µM) did not significantly alter the baseline expression of CD69 in either subset of unstimulated T cells incubated without MPs. In IFN-{alpha}-stimulated CD4+ T cells, histamine significantly increased the CD69 expression of cells incubated without MPs. This effect of histamine was not observed in CD8+ T cells; however, histamine counteracted the MP-induced inhibition of IFN-{alpha}-induced CD69 expression in CD8+ T cells. Furthermore, histamine treatment yielded a fivefold increase of IFN-{alpha}-induced expression of CD69 in CD8+ T cells incubated with MPs (Table 1) .


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  		Table 1. Expression of the CD69 Activation Antigen in T Cells and NK Cells

 
The constitutive expression of CD69 on CD56+ NK cells and CD56+/CD3{varepsilon}+ NK/T cells was significantly higher than that observed in CD4+ or in CD8+ T cells, in concordance with earlier studies [21 ]. NK cells and NK/T cells incubated with IFN-{alpha} expressed CD69 equally strongly, with a time- and dose-dependence similar to that observed for CD8+ T cells. However, in the presence of MPs, IFN-{alpha} only weakly induced CD69 in NK cells or NK/T cells. Histamine restored the IFN-{alpha}-induced expression of CD69 in NK cells to the level observed in the absence of MPs (Table 1) . In NK/T cells, histamine significantly restored the MP-induced inhibition of CD69 (Table 2 ).


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  		Table 2. Expression of the CD69 Activation Antigen in NK/T Cells

 
The effects of histamine on IFN-{alpha}-induced expression of CD69 in cytotoxic lymphocytes were dose-dependent at final histamine concentrations of 0.1–50 µM, with an ED50 of ~2 µM (see Fig. 2 ), i.e. similar to that previously reported for histamine H2 receptor (H2R)-mediated effects on leukocyte functions [28 ] (data not shown). Further, histamine-induced reversal of the T cell, NK cell, and NK/T cell anergy to IFN-{alpha} was completely antagonized by ranitidine, an antagonist at H2-type histamine receptors used at concentrations equimolar or tenfold lower than histamine (see Fig. 1 and not shown). To exclude nonspecific effects of ranitidine, we used a ranitidine analog AH20399AA (C13H22O4), in which the thioether group of ranitidine has been replaced by an ether, thereby reducing its affinity for H2R > 50-fold [12 ]. AH20399AA did not block the histamine effect (data not shown).



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  		Figure 2. Protection of NK/T cells from oxidative stress inflicted by MPs. NK/T cells (150,000 cells/well in a total volume of 200 µl) were activated with IFN-{alpha} (100 U/ml), cultured with or without MP (150,000 cells/well), and concomitantly treated with catalase (200 U/ml), DPI (10 µM), and L-NAME (100 µM), as indicated. Data shown are percent of viable and activated CD3{varepsilon}+/CD56+ NK/T cells, measured as nonapoptotic and CD69+ cells by flow cytometry. The presented data are from one representative experiment of seven similar. The inset table shows the mean % ± SEM of morphologic apoptosis among flow cytometry-sorted, IFN-{alpha}-stimulated NK/T cells, subjected to two concentrations of MPs in the presence or absence of histamine as indicated in six experiments. Histamine significantly protected NK/T cells from MP-induced apoptosis; **P < 0.01 (Wilcoxons’ signed rank test). LY, Lymphocytes.

 


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  		Figure 1. (A–D) Inhibition of NADPH oxidase and ROS scavenging protected different populations of lymphocytes against MP-induced down-regulation of CD69. Lymphocytes and MPs were recovered from PBMC by centrifugal elutriation [12 ]. The lymphocytes (150,000 cells/well in a total volume of 200 µl) were incubated with or without MPs (150,000 cells/well) and were concomitantly treated with IFN-{alpha} (100 U/ml), histamine (hist; 50 µM), catalase (cat; 100 U/ml), DPI (10 µM), ranitidine (ran; 50 µM) culture medium (med), or combinations as indicated for 16 h at 37°C. CD69 expression was monitored in indicated subsets of lymphocytes by use of flow cytometry in gates comprising all viable lymphocytes. Data show the percent of respective cells carrying CD69. The data show the mean percentage of CD69+ cells with respective phenotype in one representative experiment of seven similar.

 
Role of ROS
Earlier studies have shown that MP-derived ROS induce dysfunction of NK cells and other cytotoxic lymphocytes and that histamine protects lymphocytes by suppressing or inhibiting ROS formation [12 , 16 , 28 , 29 ]. To investigate whether the observed MP-induced inhibition of lymphocyte responsiveness to IFN was related to ROS production, we incubated lymphocytes and MPs with ROS production inhibitors and scavengers. DPI, an inhibitor of NADPH-oxidase activity in MPs [30 ], was found to reverse the MP-induced inhibition of IFN-{alpha} activation in CD4+ and CD8+ T cells (Fig. 1A 1B 1C ) and NK cells (Fig. 1D) . In addition, DPI is an inhibitor of nitric oxide synthetase (NOS) [30 ], and MPs are known to produce reactive nitrogen intermediates of which NO and hydroxylnitrate are the ultimate effector-molecules. To clarify whether NO induction in MPs contributed to the observed T and NK cell anergy to IFN-{alpha}, we used a specific NOS inhibitor, L-NAME. This compound, used at concentrations sufficient to inhibit NOS in MPs [24 ], did not affect the MP-induced suppression of T cells, NK cells (data not shown), or NK/T cells (Fig. 2 ).

Catalase, a scavenger of hydrogen peroxide [13 ], significantly reversed the MP-induced inhibition of IFN-{alpha}-induced CD69 expression in T cells and NK cells at concentrations exceeding 50 U/ml (Fig. 1A 1B 1C 1D) , whereas superoxide dismutase, a scavenger of superoxide anion [13 ], was ineffective at concentrations sufficient to scavenge >90% of superoxide anion (200 U/ml; data not shown).

Apoptotic cell death in T cells and NK cells
In concordance with the results of an earlier report [16 ], a large fraction of T and NK cells acquired a feature characteristic of apoptosis with a reduced forward-scatter and an increased right angle-scatter after overnight incubation with MPs (Table 3 ). Analyses of DNA fragmentation, Annexin V, and depleted intracellular glutathione [16 ] confirmed apoptosis in these cells. The three subsets of cytotoxic lymphocytes studied differed in their propensity of acquiring apoptotic features after incubation with MPs. Thus, NK/T cells were found to be intermediately sensitive and acquired characteristics of apoptosis to a lesser degree than NK cells but higher than T cells (Fig. 3 ). The apoptosis in the NK/T cell population was studied in a presorted population, as some NK cells aquired unspecific binding of anti-CD3{varepsilon} while undergoing apoptosis (data not shown), thereby confounding the analysis of apoptosis in NK/T cells. The sorting experiments also confirmed that NK cells were significantly more susceptible to apoptosis induction by MPs than CD3+/56– T cells (Fig. 3) .


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  		Table 3. Apoptosis in NK and T Cells after Coculture with MPs and IFN-{alpha} Stimulation

 


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  		Figure 3. MP induced apoptosis in NK/T cells compared with NK cells and T cells. Lymphocytes and MPs were recovered from PBMC by centrifugal elutriation. The lymphocytes were then stained and sorted with a flow cytometer for CD3{varepsilon}+, CD56+, or CD3{varepsilon}+/CD56+ cells. The sorted lymphocytes (150,000 cells/well in a total volume of 200 µl) were incubated with MPs (150,000 cells/well 50% MP and 75,000 cells/well 25% MP; control) for 16 h at 37°C. Apopotosis was measured in indicated subsets of lymphocytes by use of flow cytometry scatter plots [16 ]. The data show the mean percentage of apoptosis ± SEM in lymphocytes from the respective phenotype recovered from five blood donors. NK cells were significantly more sensitive to both ratios of monocytes tested than T cells, P < 0.01; or NK/T cells, P < 0.05. T cells, conversely, were significantly less sensitive to the highest monocyte ratio than NK/T cells, P < 0.05 (Mann-Whitney U-test).

 
IFN-{alpha} did not significantly alter the MP-induced apoptosis in T, NK, or NK/T cells. In accordance with earlier studies [29 ], histamine, DPI, and catalase protected T, NK, and NK/T cells (Fig. 2 , inset table) from MP-induced apoptosis. The same pattern was observed in IFN-{alpha}-stimulated T, NK, and NK/T cells (Fig. 4 and data not shown). To illustrate the impact of ROS for the IFN inducibility of cytotoxic lymphocytes, we calculated the number of viable, CD69-expressing lymphocytes with or without MPs and with or without histamine. This calculation revealed that IFN-{alpha} alone augmented the number of viable and activated CD4+ T cells 9.2-fold in the presence of MPs (as compared with untreated CD4+ T cells incubated with MPs), whereas the combination of histamine and IFN-{alpha} increased the number of activated and viable cells 30.6-fold (P<0.001 vs. IFN-{alpha}, Mann-Whitney U-test). When CD8+ T cells were admixed with MPs, IFN-{alpha} increased the number of activated CD8+ T cells 2.3-fold over control, whereas the combination of histamine and IFN-{alpha} increased the number of activated viable cells 17.5-fold (P<0.001 vs. IFN-{alpha}, Mann-Whitney U-test). The corresponding figures for NK cells and NK/T cells were a 1.3-fold and 1.7-fold increase, respectively, of viable and activated cells by IFN-{alpha} compared with control, whereas the increase by the combination of histamine + IFN-{alpha} was 11.0-fold and 11.4-fold, respectively (P<0.001 vs. IFN-{alpha} for NK cells and NK/T cells, Mann-Whitney U-test).



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  		Figure 4. Protection of a different lymphocyte population from MP-induced apoptosis by scavenging of ROS. Lymphocytes and MPs were recovered from PBMC by centrifugal elutriation [12 ]. The lymphocytes (150,000 cells/well in a total volume of 200 µl) were incubated with MPs (150,000 cells/well) and were concomitantly treated with IFN-{alpha} (100 U/ml), histamine (hist; 50 µM), ranitidine (ran; 50 µM), catalase (cat; 100 U/ml), DPI (10 µM), culture medium (med), or combinations as indicated for 16 h at 37°C. Apoptosis was monitored in indicated subsets of lymphocytes by use of flow cytometry. Data show the percent of respective cells with apoptotic morphology in one representative experiment of seven similar.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The object of this study was to determine the role of MP-derived ROS for the activation of human T cells, NK cells, and NK/T cells by IFN-{alpha}. Therefore, we exposed NK cells, NK/T cells, and subsets of T cells to oxidative stress, inflicted by autologous MPs, and monitored activation by use of the CD69 (Leu-23) antigen. In short, our data demonstrate that CD8+ T cells, CD56+ NK cells, and CD56+/CD3{varepsilon}+ NK/T cells only weakly acquire cell-surface CD69 in response to IFN-{alpha} when autologous MPs are present. In CD4+ T cells, by contrast, CD69 expression was significantly induced only in the presence of MPs, suggesting the IFN-{alpha}-induced triggering of CD69 in CD4+ T cells depends on an as-yet undefined interaction with MPs. Incubation with MPs was followed by apoptosis in all subsets of lymphocytes studied: CD4+, CD8+ T cells, in CD56+ NK cells, and CD56+/CD3{varepsilon}+ NK/T cells. The lymphocyte anergy and apoptosis were apparently inflicted by ROS, as suggested by the findings that two inhibitors of ROS synthesis, histamine and DPI, and a scavenger of hydrogen peroxide, catalase, prevented the MP-induced nonresponsiveness and apoptosis.

These data suggest that ROS production by MPs limits the activation of cytotoxic cells by IFN-{alpha}. Earlier studies show that cytokine-induced activation of NK cell-mediated cytotoxicity and proliferation is inhibited by MP-derived ROS, and MPs have been reported to inhibit IL-2-induced CD69 expression in several phenotype lymphocytes [12 , 16 , 31 ]. Our results confirm and extend these findings by demonstrating that an additional triggering pathway, IFN-{alpha} activation, is inhibited by MP-derived ROS.

The populations of lymphocytes studied showed different sensitivity against MP-induced, ROS-induced anergy and apoptosis. Earlier studies have revealed that NK cells are more sensitive to this suppressive mechanism than CD3{varepsilon}+ T cells, as reflected by the finding that a higher frequency of NK cells became apoptotic after contact with ROS-producing MPs [16 , 28 ] or after exposure to exogenously supplied hydrogen peroxide [13 , 15 , 28 ]. Our data extend this finding by demonstrating the following order of sensitivity to the ROS-mediated, apoptosis-inducing signal: NK cells > NK/T cells = CD8+ T cells > CD4+ T cells. The mechanisms responsible for the differential sensitivity to MP-induced apoptosis in these cell populations remain to be defined.

In recent years, much attention has been directed toward the ability of MPs to adversely affect lymphocyte function [32 ], and ROS generated by inflammatory MPs have been proposed to account for a significant part of the dysfunction of tumor-killing lymphocytes in malignant diseases and in chronic viral infection [13 , 19 , 33 , 34 ]. This mechanism of immunosuppression may be of particular relevance in liver tissue, which contains 80% of the host’s MPs [35 ]. Furthermore, the production of ROS in hepatitis C virus (HCV)-infected liver tissue is reportedly enhanced by a factor of 50,000 [36 ], possibly as the result of triggering of ROS formation by HCV-encoded proteins [37 ]. Therefore, our finding that NADPH-oxidase inhibitors and a scavenger of hydrogen peroxide protected IFN-{alpha}-activated T cells, NK cells, and NK/T cells, which are assumed to participate in the eradication of HCV-infected cells [38 39 40 41 ], from MP-induced apoptosis and IFN-{alpha} nonresponsiveness may be applied therapeutically. One of these compounds, histamine dihydrochloride, is currently evaluated as an adjunct to IFN-{alpha} therapy in chronic hepatitis C [42 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, and Maxim Pharmaceuticals (San Diego, CA). We are indebted to Marie-Louise Landelius for expert technical assistance.

Received February 26, 2004; revised June 17, 2004; accepted July 26, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wai, C. T., Lok, A. S. (2002) Treatment of hepatitis B J. Gastroenterol. 37,771-778[CrossRef][Medline]
  2. Chander, G., Sulkowski, M. S., Jenckes, M. W., Torbenson, M. S., Herlong, H. F., Bass, E. B., Gebo, K. A. (2002) Treatment of chronic hepatitis C: a systematic review Hepatology 36,S135-S144[CrossRef][Medline]
  3. Kirkwood, J. (2002) Cancer immunotherapy: the interferon-{alpha} experience Semin. Oncol. 29,18-26
  4. Herberman, R.B. (1997) Effect of {alpha}-interferons on immune function Semin. Oncol. 24,S9–78-S9–80
  5. Burchert, A., Wolfl, S., Schmidt, M., Brendel, C., Denecke, B., Cai, D., Odyvanova, L., Lahaye, T., Muller, M. C., Berg, T., Gschaidmeier, H., Wittig, B., Hehlmann, R., Hochhaus, A., Neubauer, A. (2003) Interferon-{alpha}, but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia Blood 101,259-264[Abstract/Free Full Text]
  6. Morishima, C., Musey, L., Elizaga, M., Gaba, K., Allison, M., Carithers, R. L., Gretch, D. R., McElrath, M. J. (2003) Hepatitis C virus-specific cytolytic T cell responses after antiviral therapy Clin. Immunol. 108,211-220[CrossRef][Medline]
  7. Edwards, B. S., Hawkins, M. J., Borden, E. C. (1984) Comparative in vivo and in vitro activation of human natural killer cells by two recombinant {alpha}-interferons differing in antiviral activity Cancer Res. 44,3135-3139[Abstract/Free Full Text]
  8. Dumont, F. J. (1986) Treatment of resting T lymphocytes with interferon-{alpha}/ß augments their proliferative response to activation signals delivered through their surface Ly-6 antigen Cell. Immunol. 101,625-632[CrossRef][Medline]
  9. Jinushi, M., Takehara, T., Kanto, T., Tatsumi, T., Groh, V., Spies, T., Miyagi, T., Suzuki, T., Sasaki, Y., Hayashi, N. (2003) Critical role of MHC class I-related chain A and B expression on IFN-{alpha}-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection J. Immunol. 170,1249-1256[Abstract/Free Full Text]
  10. Vose, B. M., Moore, M. (1985) Human tumor-infiltrating lymphocytes: a marker of host response Semin. Hematol. 22,27-40[Medline]
  11. Rugge, M., Vanstapel, M. J., Ninfo, V., Realdi, G., Tremolada, F., Montanari, P. G., van Damme, B., Fevery, J., de Groote, J., Desmet, V. (1983) Comparative histology of acute hepatitis B and non-A, non-B in Leuven and Padova Virchows Arch. A Pathol. Anat. Histopathol. 401,275-288[CrossRef][Medline]
  12. 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]
  13. 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]
  14. 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]
  15. Betten, A., Dahlgren, C., Hermodsson, S., Hellstrand, K. (2001) Serotonin protects NK cells against oxidatively induced functional inhibition and apoptosis J. Leukoc. Biol. 70,65-72[Abstract/Free Full Text]
  16. Hansson, M., Hermodsson, S., Brune, M., Mellqvist, U. H., Naredi, P., Betten, A., Gehlsen, K. R., Hellstrand, K. (1999) Histamine protects T cells and natural killer cells against oxidative stress J. Interferon Cytokine Res. 19,1135-1144[CrossRef][Medline]
  17. Hellstrand, K. (2003) Melanoma immunotherapy: a battle against radicals? Trends Immunol. 24,232-233[CrossRef][Medline]
  18. Whiteside, T. L. (1999) Signaling defects in T lymphocytes of patients with malignancy Cancer Immunol. Immunother. 48,346-352[CrossRef][Medline]
  19. 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]
  20. Lanier, L. L., Buck, D. W., Rhodes, L., Ding, A., Evans, E., Barney, C., Phillips, J. H. (1988) Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen J. Exp. Med. 167,1572-1585[Abstract/Free Full Text]
  21. Gerosa, F., Scardoni, M., Tommasi, M., Benati, C., Snelli, L., Gandini, G., Libonati, M., Tridente, G., Carra, G. (1991) Interferon {alpha} induces expression of the CD69 activation antigen in human resting NK cells, while interferon {gamma} and tumor necrosis factor {alpha} are ineffective Int. J. Cancer 48,473-475[Medline]
  22. Rabinowich, H., Herberman, R. B., Whiteside, T. L. (1993) Differential effects of IL12 and IL2 on expression and function of cellular adhesion molecules on purified human natural killer cells Cell. Immunol. 152,481-498[CrossRef][Medline]
  23. Wilson, M. T., Singh, A. K., Van Kaer, L. (2002) Immunotherapy with ligands of natural killer T cells Trends Mol. Med. 8,225-231[CrossRef][Medline]
  24. Asea, A., Hansson, M., Czerkinsky, C., Houze, T., Hermodsson, S., Strannegard, O., Hellstrand, K. (1996) Histaminergic regulation of interferon-{gamma} (IFN-{gamma}) production by human natural killer (NK) cells Clin. Exp. Immunol. 105,376-382[CrossRef][Medline]
  25. 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]
  26. Allen, R. T., Hunter, W. J., III, Agrawal, D. K. (1997) Morphological and biochemical characterization and analysis of apoptosis J. Pharmacol. Toxicol. Methods 37,215-228[CrossRef][Medline]
  27. Sun, S., Zhang, X., Tough, D., Sprent, J. (2000) Multiple effects of immunostimulatory DNA on T cells and the role of type I interferons Springer Semin. Immunopathol. 22,77-84[CrossRef][Medline]
  28. Hansson, M., Asea, A., Hermodsson, S., Hellstrand, K. (1996) Histaminergic regulation of NK-cells: protection against monocyte-induced apoptosis Scand. J. Immunol. 44,193-196[CrossRef][Medline]
  29. Hellstrand, K., Asea, A., Hermodsson, S. (1990) Role of histamine in natural killer cell-mediated resistance against tumor cells. Monocyte-mediated suppression of IL-2-induced NK-cell activation. Regulation by 5-HT1A-type serotonin receptors J. Immunol. 145,4365-4370[Abstract]
  30. Miesel, R., Kurpisz, M., Kroger, H. (1996) Suppression of inflammatory arthritis by simultaneous inhibition of nitric oxide synthase and NADPH oxidase Free Radic. Biol. Med. 20,75-81[CrossRef][Medline]
  31. Hellstrand, K., Asea, A., Hermodsson, S. (1994) Histaminergic regulation of antibody-dependent cellular cytotoxicity of granulocytes, monocytes, and natural killer cells J. Leukoc. Biol. 55,392-397[Abstract]
  32. Alleva, D. G., Walker, T. M., Elgert, K. D. (1995) Induction of macrophage suppressor activity by fibrosarcoma-derived transforming growth factor-ß 1: contrasting effects on resting and activated macrophages J. Leukoc. Biol. 57,919-928[Abstract]
  33. Kono, H., Rusyn, I., Yin, M., Gabele, E., Yamashina, S., Dikalova, A., Kadiiska, M. B., Connor, H. D., Mason, R. P., Segal, B. H., Bradford, B. U., Holland, S. M., Thurman, R. G. (2000) NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease J. Clin. Invest. 106,867-872[Medline]
  34. 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]
  35. Saba, T. M. (1970) Physiology and physiopathology of the reticuloendothelial system Arch. Intern. Med. 126,1031-1052[CrossRef][Medline]
  36. 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]
  37. 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]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Trinchieri, G. (1989) Biology of natural killer cells Adv. Immunol. 47,187-376[Medline]



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