Rainer P. Woitas, Uwe Petersen, Dirk Moshage, Tilman Sauerbruch, Ulrich Spengle, Department of Internal Medicine I, University of Bonn, 53105 Bonn, Germany
Hans H. Brackmann, Institute of Experimental Hematology, University of Bonn, 53105 Bonn, Germany
Bertfried Matz, Institute of Medical Microbiology and Immunology, University of Bonn, 53105 Bonn, Germany
Correspondence to: Dr. Rainer P. Woitas, Medizinische Kliniku. Poliklinik I, -Allgemeine Innere Medizin-, Universitt Bonn, Sigmund-Freud-Strae 25, D-53105 Bonn, Germany. woitas@uni-bonn.de
Received 2002-03-12 Accepted 2002-04-25

Abstract
AIM: Cytokine release by macrophages critically determines the type of immune response to an antigen. Therefore, we studied hepatitis C virus (HCV)-specific induction of interleukins-1β, -10, -12 (IL-1β, IL-10, IL-12), and tumor necrosis factor-α (TNF-α) in monocytes.

METHODS: Intracellular cytokine expression was studied by flow cytometry in 23 patients with chronic hepatitis C, 14 anti-HCV seropositives without viremia and 11 controls after stimulation of peripheral blood mononuclear cells with recombinant core, NS3, NS4, NS5a and NS5b proteins.

RESULTS: Patients with HCV viremia revealed greater spontaneous expression of IL-1β, TNF-α, and IL-10. Furthermore, greater than twofold higher IL-10 expression was induced by the HCV antigens in chronic hepatitis C than in the other two groups (P<0.05). In contrast, neither IL-12 nor TNF-α was induced preferentially.

CONCLUSION: In chronic hepatitis C antigen-specific cytokine induction in monocytes is apparently shifted towards predominant IL-10 induction - not counterbalanced by antiviral type 1 cytokines. This may contribute to persistent viral replication.

Woitas RP, Petersen U, Moshage D, Brackmann HH, Matz B, Sauerbruch T, Spengler U. HCV-specific cytokine induction in monocytes of patients with different outcomes of hepatitis C.World J Gastroenterol 2002;8(3):562-566

INTRODUCTION
Resistance or susceptibility to viral infections is critically linked to cytokine release, which can be polarized towards a type 1 (IFN-γ, TNF-α, IL-2) or type 2 (IL-4, IL-10, IL-13) pattern in helper as well as cytotoxic T lymphocytes^[1]. Type 1 and type 2 T lymphocytes are not derived from different lineages but develop from the same precursors, and their differentiation is influenced by the environment during priming. The most important signals are cytokines themselves: IL-12 produced by activated macrophages is the principal cytokine inducing type 1 responses, whereas the development of type 2 T lymphocytes is induced by IL-4 and IL-10.
      Hepatitis C virus (HCV) infection frequently leads to persistent viral replication, which may be facilitated by selective alterations in the host's immune response^[2]. In this context, studies of T cell functions in patients with chronic hepatitis C have indicated an inappropriately low production of antiviral type 1 cytokines in response to HCV antigens^[3-7] possibly facilitating persistent infection. However, it is not clear, whether the imbalance in the cytokine pattern is due to direct alterations of T cell function or a consequence of altered T cell priming. Because of their critical role for the type of immune reaction triggered in response to an antigen, we used flow cytometric detection of intracytoplasmic cytokines to study at the single cell level the HCV-specific induction of IL-1β, IL-10, IL-12 and TNF-α in peripheral blood monocytes.

MATERIALS AND METHODS
Patients
Three groups of patients were included into this study: Group 1 consisted of 23patients with chronic hepatitis C (male, n=20; female, n=3; median age 33, range 21-61), elevated liver enzymes and detectable HCV-RNA in the serum. The mean virus load was 6.3×10⁶ copies/mL (SD 1.0×10⁶ copies/mL) (Quantiplex^TM HCV RNA 2.0 assay, Chiron, Emeryville, CA). Group 2 consisted of 14 carefully selected patients with previous HCV infection (male, n=12; female,n=2; median age 28.5, range 18-63), who had consistently normal aminotransferases without detectable viral RNA on repeated examination over at least 2 years. Finally, 11 anti-HCV negative volunteers (male, n=5; female, n=6; median age 30, range 24-66) served as a control group (group 3).
      There were no significant differences between the two anti-HCV groups with respect to total immunoglobulin levels, and all were free from cryoglobulins or autoantibodies. None of the individuals in this study had hepatitis B virus or human immunodeficiency virus co-infection. The study was approved by the local ethical committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Diagnosis of HCV Infection
HCV antibodies were detected with a microparticle enzyme immunoassay (MEIA) (Axsym, Abbott, Wiesbaden, Germany) according to the instructions of the manufacturer. Positive results were confirmed by dot immunoassay (Matrix, Abbott, Wiesbaden, Germany). HCV RNA was detected with a nucleic acid purification kit (Viral Kit, Qiagen, Hilden, Germany) followed by reverse transcription and nested polymerase chain reaction as described elsewhere^[8]. Quantitative determination of HCV RNA copies was done via branched DNA technology (Chiron, Emeryville, CA). In group 1, genotypes of the infecuing HCV strains were determined by the INNO-LiPA HCV II test (Innogenetics, Zwijndrecht, Belgium) except one patient, who could not be genotyped. This group revealed the following isolates: 1a (n=8), 1b (n=9), 2b (n=1), 3a (n=1), and mixed (n=3; genotypes 1b/2b; 2a/2c; 4c/4d).
      Patients of group 2 were characterized by genotype-specific antibodies to NS4 (Murex, Abbott Wiesbaden, Germany). Serotypes 1 and 4 were found in 10 and 1 patients, respectively. Three patients had indeterminate serotypes.

HCV antigens
The purified recombinant proteins [r-core (truncated): aa 1-115, r-NS3: aa 1007-1534, r-NS4: aa 1616-1862, r-NS5: aa 2007-2268] derived from the HCV-1 prototype sequence^[9] were purchased from Mikrogen, Munich, Germany. The bacterial lipopolysaccharide content of the proteins was between 4.0-20 pg/μg recombinant protein as determined by the Limulus assay. Lipopolysaccharide from E. coli OH101 (Sigma, Munich, Germany) was used in the control experiments.

Antibodies
Fluoresceinisothiocyanate (FITC)- and phycoerythrin (PE)-labelled antibodies were purchased from the following companies: FITC- and PE-labelled -anti-CD14 (mouse IgG2b, clone MΦP9) from Becton Dickinson (Heidelberg, Germany); FITC- and PE-labelled anti-IL-1β (mouse IgG1, clone H9.5) was purchased by Holtzel Diagnostica (Cologne, Germany). Anti-IL-10/PE (rat IgG2a, clone JES3-19F1), anti-IL-12/PE (mouse IgG1, clone C11.5.14), anti-TNF-α/PE (mouse IgG1, clone MAb11) as well as appropriate isotype controls from Pharmingen (Hamburg, Germany). Unlabelled mAbs for blocking experiments were purchased from Pharmingen (Hamburg, Germany) except for IL-1β that was a gift of Holtzel Diagnostica (Cologne, Germany).

Cells and cell culture
PBMC (1.1×10⁶/ml) isolated from fresh EDTA blood by Ficoll density-gradient centrifugation (Biochrom, Berlin, Germany) were resuspended in low endotoxin level culture medium (RPMI 1640, Biochrom, Berlin, Germany) containing 10% autologous human serum, 100 units/ml penicillin, 100 units/ml streptomycin and incubated at 37℃ with 5% CO₂ in 96 well microtiter plates (Sarstedt, Berlin, Germany) in the presence of recombinant HCV proteins (1μg/ml) or LPS (10-200ng/ml). Kinetic experiments showed that the antigen-specific cytokine induction indicated a maximum after 12 hours of stimulation. Further experiments revealed 1.0μM monensin (Sigma, Munich Germany) for 12hours to be optimal to enhance the signal/noise ratio as well as to exclude relevant toxicity^[10-12].

Dual-colour flow cytometry for immunophenotyping and intracytoplasmic staining of cytokines
CD14 and iotracytoplasmic cytokines were detected by direct immunofluorescence using a paraformaldehyde (PFA)-saponin procedure with 4% PFA as fixative and 0.2% saponin for permeabilisation. In brief, cultured cells were washed twice in Hank's balanced salt solution (HBSS) (Gibco, Eggenstein, Germany) and stained for the surface markers (20 minutes incubation at 4℃ in the dark). After one further wash, the cells were fixed in ice cold HBSS containing 4% PFA for 5 minutes and washed again. Cells were resuspended in HBSS containing 0.2% saponin (saponin buffer). Then cytokine specific antibodies diluted in saponin buffer were added at a concentration of 0.5-3.0μg/ml and incubated for 30 minutes at room temperature in the dark. Cells were washed in saponin buffer and analyzed by dual-colour flow cytometry on a FACSort flowcytometer (Becton Dickinson, Heidelberg, Germany). Forward and side scatter as well as gating for CD14⁺, cells were used to identify monocytes. The data were analyzed with the CellQuest( software (Becton Dickinson) after counting 5000 CD14⁺ cells. All experiments were performed in triplicate.
      To ensure specificity of the cytokine staining procedures, the binding of each mAb was blocked with an excess of unlabelled mAb.

Statistics
Results are given as median and range. Differences between the groups were analyzed by the Kruskal-Wallis test, Mann-Whitney U test and Wilcoxon signed rank test, as appropriate. All calculations were performed on a personal computer with Statview 4.5 software (Abacus Concepts inc., Berkeley CA, USA). P values <0.05 were regarded as significant.

RESULTS
Using flowcytometric detection of intracytoplasmic cytokines in combination with CD14 staining, we were able to specifically measure spontaneous as well as HCV antigen-induced production of IL-1β, IL-10, IL-12 and TNF-α in peripheral blood monocytes (Figure 1). Following stimulation with LPS, intracytoplasmic expression of the cytokines did not show statistically significant differences between the study groups for the cytokines with the exception of IL-1β, which was higher in patients with chronic hepatitis C when compared to the controls (Table 1). Cytokine production after 12 hours of HCV-specific stimulation together with the corresponding spontaneous and LPS-induced production is summarized in Table 1.
      With respect to spontaneous production of IL-1β, TNF-α, IL-10, and IL-12, there was considerable individual variability which was most pronounced in the group with chronic hepatitis C. The numbers of monocytes with detectable spontaneous production of IL-12 were low in general (0.1-5.1%) and on average not significantly different between the three study groups. In contrast, average numbers with detectable spontaneous production of IL-1β, TNF-α, and IL-10 were higher in patients with chronic hepatitis C than in aviremic anti-HCV seropositives. However, due to considerable inter-individual variations, these differences between the groups reached statistical significance only for IL-1β and IL-10 (P<0.05).
      Antigen-specific stimulation of peripheral blood mononuclear cells resulted in increased numbers of monocytes with production of IL-1β, TNF-α, IL-10, and IL-12 in each study group for most of the tested HCV proteins (Table 1). However, after stimulation with the HCV proteins core, NS3, NS4, NS5a, and NS5b, the numbers of IL-1β and IL-10 producing monocytes were at least twofold higher in patients with chronic hepatitis C than in aviremic anti-HCV seropositives and the controls (P<0.05 for each HCV protein). In contrast, statistically significant differences (P<0.05) in the numbers of TOF-α and IL-12 producing monocytes between patients with chronic hepatitis C and the other groups were only seen after stimulation with HCV core and NS5b (Table 1). The marked difference in the balance between IL-1β and IL-10 producing monocytes,and TNF-α and IL-12 producing ones among the patients with chronic hepatitis C is illustrated in Figure 2, which shows the stimulation experiments with recombinant NS4 as a representative example. Since stimulation of monocytes was performed in the presence of T cells, we undertook control experiments with untouched isolated monocytes. When these purified monocytes were stimulated with HCV antigens or LPS, the cells showed a similar behaviour although the number of cytokine producing macrophages was strongly reduced compared to the PBMC assays (data not shown).

Figure 1(PDF)Contour plots of cytokine expression in CD14+ cells. These contour plots show representative experiments for the detection of spontaneous expression (buffer control, left column) and HCV NS5b-induced expression (1μg/mL, right column) of IL-1 (a), IL-10 (b), IL-12 (c) and TNF-( (d) in peripheral blood CD14+ monocytes of patient # 23 with chronic hepatitis C at 12 hours of incubation.
Figure2(PDF)Induction of cytokines by the HCV NS4 protein. The graph displays the fractions of IL-1 (a), IL-10 (b), IL-12 (c) and TNF-( (d) producing monocytes 12 hours after stimulation with recombinant NS4 protein (1μg/mL) for patients with chronic hepatitis C (filled squares), aviremic anti-HCV seropositives (upward triangles), and non-HCV related controls (downward triangles). Each dot represents the mean percentage of a single patient obtained from triplicate experiments. The horizontal bar gives the mean of each group.

Table 1 CD14⁺ monocytes with detectable intracellular cytokine expression

    § Significances:
    a=P<0.05 Chronic HCV infection vs. aviremic anti-HCV seropositives (Kruskal-Wallis test and Mann-Whitney U test)
    b=P<0.05 Chronic HCV infection vs. Controls (Kruskal-Wallis test and Mann-Whitney U test)
    c=P<0.05 Stimulated vs. spontaneous cytokine expression (Wilcoxon signed rank test)
    Data are given as median+ range.

DISCUSSION
Impaired function of macrophages has been described repeatedly in infection with HCV and also the closely related Dengue virus^[13-17]. Our study adds to these observation that in chronic hepatitis C altered macrophage function may also comprise the pattern of cytokines produced in response to HCV antigens. Of note, the various cytokines appeared to be involved differentially. Despite considerable individual variability, we found significantly greater average numbers of monocytes with spontaneous IL-1β and IL-10 production in patients with chronic hepatitis than that in aviremic anti HCV seropositives or the HCV-naive controls. This finding appears to be compatible with in vivo pre-activation of the monocytes from patients with chronic hepatitis C. However, the numbers of monocytes with spontaneous IL-12 production did not reveal any conspicuous differences between our study groups. Thus far, studies on the cytokine production in hepatitis C have produced conflicting results for macrophages, mainly because monocytes were assessed only indirectly by measuring cytokines in the supernatants of peripheral blood mononuclear cells after stimulation with mitogen or LPS. This flowcytometric study is the first inv