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Clinical Cancer Research Vol. 12, 4491-4498, August 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Induction of Interleukin-6 by Hepatitis C Virus Core Protein in Hepatitis C–Associated Mixed Cryoglobulinemia and B-Cell Non–Hodgkin's Lymphoma

Georg Feldmann1, Hans Dieter Nischalke1, Jacob Nattermann1, Brigitte Banas1, Thomas Berg2, Christian Teschendorf3, Wolff Schmiegel3, Ulrich Dührsen4, Juliane Halangk2, Agathe Iwan1, Tilman Sauerbruch1, Wolfgang H. Caselmann1 and Ulrich Spengler1

Authors' Affiliations: 1 Department of Internal Medicine 1, University of Bonn, Bonn, Germany; 2 Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charite, Campus Virchow-Klinikum, Humboldt-Universität, Berlin, Germany; 3 Department of Internal Medicine, Ruhr-Universität Bochum, Knappschaftskrankenhaus, Bochum, Germany; and 4 Department of Haematology, University of Duisburg-Essen, Essen, Germany

Requests for reprints: Georg Feldmann, Department of GI Pathology, Johns Hopkins University School of Medicine, CRB2, Room 316, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-955-3511; Fax: 410-614-0671; E-mail: gfeldma4{at}jhmi.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Chronic hepatitis C carries the risk to develop mixed cryoglobulinemia (MC) and B-cell non–Hodgkin's lymphoma (B-NHL), possibly because viral antigens stimulate the host's inflammatory response via extracellular pattern recognition receptors (PRR). To clarify this issue, we studied whether recognition of hepatitis C virus (HCV) proteins by PRR is involved in the pathogenesis of HCV-associated MC or B-NHL.

Experimental Design: Peripheral blood mononuclear cells of patients with HCV-associated B-NHL (n = 12), MC (n = 14), uncomplicated hepatitis C (n = 12), and healthy volunteers (n = 12) were incubated with the recombinant HCV proteins E2, core, and NS3 to study induction of cytokine production, stimulation of B-cell proliferation, and immunoglobulin secretion. In addition, serum levels of interleukin-6 (IL-6) were measured by ELISA.

Results: HCV core was the only studied protein, which induced production of IL-6 and IL-8 in CD14+ cells. IL-6 induction was mediated via Toll-like receptor 2 (TLR2) and lead to increased B-cell proliferation in vitro. TLR2 expression on monocytes and IL-6 serum concentrations were increased in all groups of HCV-infected patients compared with healthy controls and were highest in MC (P < 0.05).

Conclusions: Increased secretion of IL-6 via stimulation of TLR2 by HCV core protein may play a role in the pathogenesis of hepatitis C–associated MC and B-NHL.

In addition to a high risk for developing liver cirrhosis and hepatocellular carcinoma, longstanding chronic infection with hepatitis C virus (HCV) has been shown to be associated with mixed cryoglobulinemia (MC; ref. 1). Cryoglobulins are cold-precipitable immune complexes (2), the deposition of which in small- and medium-sized blood vessels can lead to systemic vasculitis. MC is considered to be a benign B-cell proliferative disorder with clonal expansion of distinct B cells that can eventually turn into frank B-cell malignancy (3). Indeed, several groups have shown an increased incidence of B-cell non–Hodgkin's lymphoma (B-NHL) in patients with chronic HCV infection (4), and an increased prevalence of HCV infections in patients with B-NHL has been reported (5, 6). The hypothesis of an association between B-NHL and HCV infection is further supported by the observation that eradication of HCV by treatment with IFN-{alpha} and ribavirin could induce complete remission of splenic lymphomas with villous lymphocytes in seven of nine patients with chronic hepatitis C, whereas the same treatment had no effect in six patients with lymphomas who were HCV negative (7). These findings have prompted the hypothesis that HCV-associated B-NHL result from chronic immune stimulation by HCV antigens, which may make B-lymphocytes more vulnerable to acquire transforming mutations, such as bcl-2 translocations or mutations affecting the c-myc gene (8–10).

However, the mechanisms leading to cryoglobulinemia in chronic HCV infection and malignant B-cell lymphoma are poorly understood. It has been proposed that direct infection of CD34-positive stem cells (11) may play a role for developing B-cell proliferative disorders (10).

Alternatively, cross-linking of the ubiquitously expressed tetraspanin CD81 by the HCV envelope protein E2 may modulate natural killer cell function (12–14) or induce cytokine secretion (15), both of which can contribute to the development of HCV-associated B-cell proliferative diseases.

Furthermore, there is now accumulating evidence that viral components, i.e., single-stranded or double-stranded RNA motifs as well as viral proteins, may trigger pattern recognition receptors (PRR; ref. 16). For instance, Toll-like receptor 3 (TLR3) recognizes double-stranded RNA derived from influenza A virus (17) and cytomegalovirus (18). TLR7 is triggered by single-stranded RNA molecules derived from influenza virus (19) and HIV-1 (20). TLR9 recognizes cytomegalovirus, herpes simplex virus-1, or herpes simplex virus-2 genomic DNA (18, 21–23).

In addition, Toll-like receptors on the host cell surface may directly be activated by viral proteins. For example, TLR2 can recognize cytomegalovirus (24) and herpes simplex virus-1 virions (25) as well as measles virus hemagglutinin protein (26). Moreover, TLR4 is triggered by respiratory syncytial virus (27). As a result of TLR stimulation, proinflammatory cytokines are released that activate the host immune system.

In the present study, we show that HCV core protein triggers production of the proinflammatory cytokine IL-6, which stimulates B-cells and thus probably contributes to the development of HCV-associated cryoglobulinemia and B-NHL.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Subjects. The study population comprised 50 patients who were stratified into the following four groups: (a) healthy volunteers (n = 12), (b) patients with uncomplicated chronic HCV infection (n = 12), (c) patients with HCV-associated MC (n = 14), and (d) patients with HCV-associated B-cell non–Hodgkin's lymphoma (B-NHL; n = 12). Patient characteristics are given in Table 1 .


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  		Table 1. Patient characteristics

 
Informed consent was obtained from each patient. The study protocol conformed to the guidelines of the declaration of Helsinki as approved by our ethical committee.

Diagnosis of HCV infection. Diagnosis of chronic hepatitis C was based on a >6-month history of liver disease together with a positive HCV antibody test (second-generation enzyme immunoassay) and detectable HCV RNA by reverse transcription PCR (Amplicor HCV, Roche Diagnostics, Mannheim, Germany). Viral load was measured using a branched DNA assay (Versant HCV RNA 3.0 bDNA), and HCV genotypes were determined by Versant INNO-LiPA HCV II line probe assay (both assays from Bayer Healthcare Diagnostics, Fernwald, Germany).

Diagnosis of cryoglobulinemia. Cryoglobulinemia was diagnosed following established guidelines (2). Briefly, 9 mL blood were drawn in S-Monovettes (Sarstedt, Nümbrecht, Germany) and immediately incubated at 37°C for 60 minutes. After centrifugation, 3 mL serum were transferred into Nissel tubes (Assistent GmbH, Sondheim, Germany) and incubated at 4°C for 3 days in an upright position. Cryoprecipitates were reported as cryocrit and were checked for resolution upon rewarming. Immunoelectrophoresis was done to classify cryoglobulinemia into MC type II (monoclonal IgM) or type III (polyclonal IgM fraction) according to Brouet et al. (28).

HCV antigens. Chinese hamster ovary–expressed full-length HCV E2 protein with a physiologic glycosylation pattern was a kind gift of M. Houghton, Chiron Corporation (Emeryville, CA), who also donated Chinese hamster ovary–expressed full-length HCV core protein. Truncated recombinant HCV core protein [amino acids (aa) 1-115] and NS3 protein (aa 1,007-1,534) were purchased from Mikrogen (Martinsried, Germany). All HCV proteins were derived from the HCV-1 prototype sequence (29).

Isolation of peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC) were separated from EDTA-blood by centrifugation on a Ficoll Hypaque density gradient (Lymphozytenseparationsmedium, PAA Laboratories GmbH, Linz, Austria) at 400 x g for 20 minutes. Cells were collected and washed twice with PBS. PBMCs were cryoconserved in a medium containing 90% FCS and 10% DMSO and stored in liquid nitrogen until use.

Stimulation of PBMC with recombinant HCV proteins. PBMCs were resuspended in RPMI 1640 containing 0.1 mg/mL penicillin/streptomycin and 2 mmol/L glutamine (PAA Laboratories GmbH) supplemented with 10% FCS (Biochrom KG, Berlin, Germany). Stimulations were carried out with 500,000 PBMC in 1 mL medium on 24-well plates (Greiner, Frickenhausen, Germany). Cells were incubated overnight in a humidified atmosphere at 37°C with 5% CO2. Then, recombinant HCV proteins (E2, core, and NS3) were added. Stimulation with 1 µg/mL lipopolysaccharide (LPS; Sigma, Deisenhofen, Germany) and 10 µg/mL phytohemagglutinin (Biochrom) served as positive controls. Cell culture supernatants were collected to measure IL-6 concentrations. Cells in the pellet were used for RNA extraction and determination of cytokine mRNA levels.

For denaturation, HCV protein solution was heated to 90°C for 30 minutes. To further exclude endotoxin contamination, stimulations were also done in the presence of polymyxin B (Sigma).

To study whether IL-6 induction was related to TLR stimulation, 100,000 PBMCs from HCV-naïve as well as from HCV-positive patients were incubated on 96-well plates (200 µL/well) with azide-free monoclonal mouse IgG2a anti-human TLR2 or TLR4 antibodies (clones TL2.1 and HTA125, BioCarta, Hamburg, Germany) together with recombinant HCV core protein (1.5 µg/mL). TLR stimulation was further confirmed in TLR2 and TLR4/MD-2–transfected HEK293 cells kindly provided by G. Szabo (University of Massachusetts, Worcester, MA; ref. 30). Stimulation with peptidoglycan and LPS, natural ligands for TLR2 and TLR4, respectively, were used as positive controls.

Determination of cytokine mRNA levels by real-time reverse transcription-PCR. RNA extraction was carried out using the QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany) with on-column DNase 1 digestion.

Fifteen microliters of RNA were reverse transcribed into cDNA in a total volume of 30 µL at 37°C for 60 minutes using the Omniscript Reverse Transcriptase kit (Qiagen) with oligo-dT-primers (N420-01, Invitrogen, Karlsruhe, Germany).

Real-time PCR was carried out on a LightCycler using the LightCycler-FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics). Two microliters of cDNA were amplified over 35 cycles in a total volume of 10 µL using sequence-specific primers (0.5 µmol/L) in the presence of 3.5 mmol/L Mg2+. The starting amount of template RNA was calculated using LightCycler Software Version 3 (Roche Molecular Biochemicals, Mannheim, Germany) and gauged relative to the amount of ß-actin–specific mRNA. Specificity was checked by melting curve analysis and agarose gel electrophoresis. The denaturation step was 95°C for 1 second in all reactions. Primer sequences and PCR conditions are given in Supplementary Table S1.

Determination of IL-6. IL-6 concentrations in cell culture supernatants and in patient sera were assessed by QuantiGlo IL-6 chemiluminescence immunoassay (R&D Systems, Wiesbaden, Germany).

To exclude that differences in IL-6 serum concentrations between the study groups were due to differences in the distribution of IL-6 high- or low-producer genotypes (31, 32), the –174 C/G IL-6 promoter polymorphism was determined using the One Lambda cytokine genotyping kit (BmT, Meerbusch, Germany).

Identification of IL-6-producing cells by intracellular cytokine staining. One milliliter of heparinized blood was incubated with recombinant full-length HCV core protein (1.5 µg/mL), buffer alone, or 1 µg LPS (Sigma) in the presence of 10 µL brefeldin A (Sigma-Aldrich Chemie, Steinheim, Germany). After incubation at 37°C for 6 hours, aliquots of 100 µL were stained with 10 µL anti-CD3-peridinin-chlorophyll-protein (PerCP), anti-CD19-FITC, anti-CD14-FITC or anti-CD3-PerCP, and anti-CD56-FITC for 15 minutes at room temperature in the dark to label PBMC subpopulations. Then, erythrocytes were lysed and mononuclear cells were permeabilized to enable intracellular IL-6 staining with the FastImmune Anti-Human IL-6 kit (BD Biosciences, Heidelberg, Germany). Fluorescence-activated cell sorting (FACS) analysis was done using a FACSCalibur cytometer and Cellquest Pro software package (BD Biosciences).

Analysis of TLR2 expression on monocytes. One million PBMCs were stained with phycoerythrin-conjugated monoclonal TLR2 antibody (clone TL2.1, eBioscience, Boston, MA) and counterstained with anti-CD14-PerCP. Phycoerythrin-conjugated mouse IgG2a isotype control antibody (eBioscience) served as a reference. Results are expressed as normalized mean fluorescence intensity (nMFI): nMFI = [mean fluorescence intensity (TLR2 stained sample) – mean fluorescence intensity (isotype control)] / mean fluorescence intensity (isotype control).

B-cell proliferation assays. PBMC (106/mL) isolated from HCV-positive and from HCV-naïve donors were seeded into 24-well plates and incubated for 24 hours with recombinant full-length HCV core protein (Chiron Corporation), using incubation with buffer alone or 1 µg/mL LPS as negative or positive controls, respectively. Then, cell culture supernatants were collected and stored at –20°C until further use. Autologous B cells were isolated by negative selection using the Macs B-cell Isolation kit II (Miltenyi, Bergisch Gladbach, Germany). Purity of B cells was checked by FACS analysis after staining of cells with monoclonal CD3-FITC and CD19-phycoerythrin antibodies (BD Biosciences).

B-lymphocytes (5 x 104 per well) were then incubated in 96-well plates with 100 µL supernatant obtained from stimulation experiments with HCV-core, HCV-NS3, buffer, or LPS. Proliferation was determined by [3H]thymidine incorporation during the last 4 hours of 48-hour cultures. Cells were harvested onto glass filters with a filtermate 196 cell harvester (Perkin-Elmer, Shelton, CT) and counted using a TopCount NXT microplate scintillation counter (Perkin-Elmer).

For blocking experiments, B-cell proliferation was also analyzed flow cytometrically by carboxyfluorescein succinimidy ester labeling (Invitrogen), as this method allows determination of B-cell proliferation, given as percentage of proliferating B-cells, after incubation of whole PBMC with the recombinant viral proteins without the necessity to physically separate B cells. Briefly, 107 PBMCs were incubated over 15 minutes in 5 mL PBS at 37°C in the presence of 1 µmol/L carboxyfluorescein succinimidy ester. Then, labeling was stopped by adding 5 mL RPMI 1640 supplemented with 10% FCS. B cells were identified with an APC-labeled CD19 antibody (BD Biosciences) and analyzed on a FACSCalibur flow cytometer. Cell viability was measured by 7-amino-actinomycin D (Sigma) incorporation.

To induce B-cell proliferation, PBMCs were stimulated over 72 hours with 1.5 µg/mL HCV core protein and immobilized IgM (Irvine Scientific, Santa Ana CA). In blocking experiments, B-cell proliferation was measured in the presence of 10 µg/mL neutralizing IL-6 antibody AF-206-NA or the irrelevant isotype control antibody AB-108-C (both from R&D Systems), respectively.

Statistical analysis. Mann-Whitney U test and Kruskal-Wallis analysis were done using SPSS release 11.0.1 for Microsoft Windows. P < 0.05 was regarded as statistically significant. Two-tailed t test was done using GraphPad Prism for Windows version 4.00. To check whether IL-6 was correlated to patient group allocation or confounding variables, we studied viral loads, alanine aminotransferase, {gamma}-glutamyltransferase, 174 C/G IL-6 promoter polymorphism, Brouet subtype of cryoglobulinemia, presence of cirrhosis, and age as potential confounding variables in a univariate linear correlation analysis and also calculated a multivariate forward conditional regression model (including variables with P < 0.1 in the univariate analysis into the calculation and excluding all variables with P > 0.05 from the final model).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patients with HCV-associated cryoglobulinemia (median age 62 years) and with HCV-associated B-NHL (median age 68 years) were significantly older than patients with uncomplicated HCV infection (median age 45 years; P = 0.0168 and P = 0.0064, respectively), possibly reflecting the fact that MC and B-NHL require longstanding HCV infection (see Table 1). Histologic subtypes of B-NHL are given in Table 2 . In the MC group, four patients had MC type II (28.6%) and 10 had MC type III (71.4%). Cryocrit levels had a median of 17% (range 1-83.3%). There were no differences in the distribution of the IL-6 –174 C/G genotypes between the patient groups (see Supplementary Table S2). All patients with B-NHL, in whom the HCV genotype was determined, had infection with genotype 1 strains.


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  		Table 2. Histologic subtypes of HCV-associated B-NHLs

 
In vitro induction of IL-6 by HCV core protein. As illustrated in Fig. 1 , recombinant HCV core protein induced significant up-regulation of IL-6 in each patient group after 24 hours of stimulation (P < 0.05), but differences in stimulation indices between the patient groups were not statistically significant. HCV core protein induced IL-6 production at the protein level in a dose-dependent manner (Fig. 2A ). Induction of IL-6 was confirmed when a truncated recombinant HCV core protein (aa 1-115) was used. FACS analysis revealed that IL-6 production was induced exclusively in CD14-positive, CD3-, CD19-, and CD56-negative cells (Fig. 2B). Although absolute monocyte counts showed considerable interindividual variation (range 120-970/µL), we did not find any statistically significant differences in monocyte counts between the study groups nor were absolute monocyte counts correlated to IL-6 induction indices. After in vitro stimulation, IL-6-positive monocytes accounted for 35% to 60% of the monocytes without any significant differences between the four patient groups (data not shown).


Figure 1
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  		Fig. 1. Induction of IL-6 mRNA by HCV core protein. Induction of IL-6 mRNA production in PBMC from healthy control persons (group A), patients with uncomplicated HCV infection (group B), HCV-associated cryoglobulinemia (group C), and HCV-associated B-NHL (group D) after incubation with recombinant HCV core protein. Columns, mean IL-6/ß-actin mRNA ratios; bars, SD. *, statistical significance versus baseline. Intergroup differences did not yield statistical significance due to considerable individual variability of stimulation indices.

 

Figure 2
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  		Fig. 2. Specificity of IL-6 induction by HCV core protein. A, dose-response relationship of IL-6 induction by HCV core. Recombinant HCV core protein induces IL-6 synthesis in PBMC in a dose-dependent manner. Representative example of the dose-response relationship in a healthy donor. PBMCs (100,000) were incubated with different concentrations (3.0, 1.5, 0.75, 0.38, and 0.0 µg/mL) of recombinant full-length HCV core protein for 24 hours. LPS (1 µg/mL) served as a positive control. IL-6 concentrations in the supernatant were determined by ELISA, and measurements were done in triplicates. Columns, mean IL-6 concentrations; bars, SD. B, representative stimulation experiment with 1.5 µg/mL recombinant HCV core protein in PBMC from a healthy individual. Intracellular IL-6 was detected by flow cytometry after blocking cytokine secretion with brefeldin A and permeabilization of the cells. The experiment illustrates that CD14-positive cells were the source of IL-6 production after stimulation with HCV core protein. CD14-positive cells stimulated with LPS (1.0 µg/mL) served as positive controls; cells incubated with PBS alone served as negative controls. The numbers give the percentages of cells within each quadrant. C, PBMC from HCV-negative donors were incubated over 6 hours with buffer alone, HCV core protein (5 µg/mL), denatured core protein (5 µg/mL), and core protein (5 µg/mL) plus polymyxin B (10 µg/mL), respectively. IL-6-producing monocytes determined by FACS analysis as described in (B). The graph illustrates that induction of IL-6 synthesis was abrogated by heat denaturation of the HCV core protein but not by addition of polymyxin B.

 
HCV core also induced IL-8 mRNA and protein, but none of the other cytokine mRNA species [MIP-1{alpha}, MIP-1ß, RANTES, MIP-3ß, MIP-3{alpha}, MIG, I-TAC, interleukin (IL)-2, and IFN-{gamma}] were induced by any of the studied HCV proteins. Although we were unable to show significant IL-6 mRNA induction, small amounts of IL-6 protein became detectable after stimulation with our truncated NS3 protein.

Denaturing HCV core protein completely abolished IL-6 induction, whereas addition of polymyxin B did not reduce IL-6 synthesis, thus excluding effects due to inadvertent endotoxin contamination (Fig. 2C). Induction of IL-6 secretion by HCV core was blocked by the monoclonal anti-TLR2 antibody TL2.1 in a dose-dependent manner, but not by the anti-TLR4 antibody HTA125 (Fig. 3A ). Activation of TLR2, but not TLR4, by HCV core protein was confirmed by the observation that HCV core induced IL-8 synthesis exclusively in TLR2-transfected HEK293 cells (Fig. 3B). Of note, TLR2 expression on monocytes was increased in all patients with HCV infection compared with the healthy controls (P = 0.032) and was highest in the subgroup of patients with hepatitis C and MC (Fig. 4A ).


Figure 3
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  		Fig. 3. HCV core selectively triggers TLR2. A, IL-6 production induced by incubation of PBMC with HCV core protein is blocked by anti-TLR2 monoclonal antibody (mAb) in a dose-dependent manner. White column on the left, buffer control; gray columns, experiments with HCV core protein (1.5 µg/mL) to which varied concentrations of TLR2-specific antibody TL2.1 had been added; dark column, control stimulation experiment done with HCV core (1.5 µg/mL) in the presence of an isotype control (25 µg/mL); shaded column on the right, HCV core stimulation in the presence of TLR4 antibody HTA125 (25 µg/mL). The graph illustrates that HCV core–triggered IL-6 production was inhibited by the TLR2 antibody but not by the TLR4 antibody. B, HEK293 cells stably transfected with human TLR2 (HEK/TLR2), TLR4 together with its coreceptor MD-2 (HEK/TLR4), and untransfected HEK293 cells (HEK/–) were incubated with recombinant HCV core protein. Stimulation with peptidoglycan (PGN, a natural ligand for TLR2) and LPS (a natural ligand for TLR4) were used as positive controls, respectively. IL-8 secretion was used as a readout system for Toll receptor stimulation in these experiments. The graph shows that recombinant core protein triggers TLR2-transfected HEK cells but not TLR4-transfected nor untransfected HEK cells.

 

Figure 4
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  		Fig. 4. TLR2 expression on monocytes and IL-6 serum levels in different patient groups. A, TLR2 expression on monocytes in healthy control persons (group A), patients with uncomplicated HCV infection (group B), HCV-associated MC (group C), and HCV-associated B-NHL (group D). Results are expressed as normalized mean fluorescence intensity (nMFI), which was calculated as nMFI = [mean fluorescence intensity (TLR2-stained sample) – mean fluorescence intensity (isotype control)] / mean fluorescence intensity (isotype control). B, IL-6 serum concentrations of healthy control persons (group A), patients with uncomplicated HCV infection (group B), HCV-associated MC (group C), and HCV-associated B-NHL (group D) were determined by ELISA. Box plots: lower and the upper hinges of the boxes, 25th and 75th percentiles, respectively. Line across the respective box, median; whiskers, ranges. P values were calculated using Mann-Whitney U test after performing Kruskal-Wallis analysis.

 
When supernatants of HCV core–stimulated PBMCs were transferred to purified B-cell cultures, we observed increased proliferation rates compared with supernatants from unstimulated PBMC (Fig. 5A ). This increase in B-cell proliferation was confirmed with a complementary assay, when whole PBMC isolates were incubated with core protein and B-cell proliferation was determined by FACS analysis. HCV core–induced B-cell proliferation was significantly blocked (P = 0.029) by adding a neutralizing IL-6 antibody during stimulation, whereas an isotype control antibody had no effect (Fig. 5B). Supernatants from cells stimulated with the truncated HCV-NS3 protein did not induce significant B-cell proliferation.


Figure 5
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  		Fig. 5. Proliferation in HCV core–stimulated B cells. A, increased proliferation of B cells in response to supernatants from HCV core–stimulated cells. To study proliferation PBMCs were incubated for 24 hours with HCV core protein (1.5 µg/mL), PBS alone (negative control), and LPS (1.0 µg/mL, positive control), respectively. Next, separated B cells of the same individual were incubated with the supernatants and proliferation was quantified by [3H]thymidine incorporation during the last 4 hours of the 48-hour incubation. All experiments were done in quadruplicates. One representative experiment out of four. B, increase in B-cell proliferation was confirmed when determined by FACS analysis after incubation of PBMC with HCV core and staining of the cells with carboxyfluorescein diacetate succinimidyl ester. PBMC of a HCV-negative donor were incubated with recombinant HCV core protein (1.5 µg/mL) or buffer alone. Results of four experiments illustrating that HCV-core stimulation induced increase in B-cell proliferation, which was blocked by 10 µg/mL neutralizing IL-6 antibody but not by the isotype control antibody.

 
Elevated IL-6 serum concentrations in HCV patients. Although IL-8 serum levels did not differ between the different groups of patients (data not shown), IL-6 serum levels were significantly higher in all groups of HCV-infected patients compared with healthy donors (all P < 0.039). Furthermore, IL-6 serum concentrations were significantly higher in the group with HCV-associated cryoglobulinemia than in patients with uncomplicated HCV infection (P = 0.014) or with HCV-associated B-NHL (P = 0.028; Fig. 4B). IL-6 serum concentrations were also correlated to the presence of cirrhosis (P = 0.032) and age (P = 0.011) but not to viral loads, alanine aminotransferase, {gamma}-glutamyltransferase, or cryocrit values (data not shown). However, when the effects of potentially confounding variables were compared by multivariate analysis, only study group allocation was identified as an independent determinant of IL-6 serum levels (P < 0.001).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Despite marked regional differences (33), most epidemiologic studies report an association between long-lasting chronic HCV infection and malignant B-NHL (4–6, 34–37).

MC is a B-cell proliferative disorder, which shows strong correlation to HCV infection and is considered to be a potential precursor of HCV-associated B-NHL (2, 34).

Here, we report that recombinant HCV core proteins induce production of IL-6 and IL-8 in CD14+ cells. We further showed that HCV core protein triggers IL-6 and IL-8 production via stimulation of TLR2. These findings are in line with a recently published study by Dolganiuc et al. (30), who also found induction of IL-6, IL-8, and tumor necrosis factor-{alpha} in peripheral blood monocytes isolated from healthy donors or HCV-infected individuals. Based on experiments with TLR2- and TLR4-transfected human embryonal kidney cells, they concluded that TLR2 was involved in HCV core protein binding and induction of IL-6, IL-8, and tumor necrosis factor-{alpha}. In our experiments, IL-6 was also induced by a truncated core protein, locating the putative TLR2 binding site on HCV core between aa 1 and 115 in line with the report of Dolganiuc et al. Unlike the data reported by Dolganiuc et al., we found induction of only small amounts of IL-6 at the protein level and none at the mRNA level with our recombinant HCV NS3 protein. This difference can probably be explained by the fact that Dolganiuc et al. identified a region between aa 1,450 and 1,643 as the major site for HCV NS3-TLR2 interactions by using two different truncated forms of the NS3 protein. This region, however, was partially missing on our truncated NS3 protein (aa 1,007-1,534).

Here, we present the novel finding that IL-6 serum levels were elevated in patients chronically infected with HCV compared with healthy controls, suggesting that TLR2-mediated induction of IL-6 may have in vivo relevance. Of note, Grungreiff et al. (38) showed that IL-6 serum concentrations normalized when HCV was eradicated by IFN-{alpha} treatment, supporting the idea that elevated IL-6 serum concentrations may reflect immune stimulation by HCV. In contrast, induction of IL-8 protein was too weak as to cause detectable increases in the serum levels of our patient groups.

It is a novel finding in our study that IL-6 serum concentrations were particularly high in patients with HCV-associated cryoglobulinemia compared with the other HCV-infected groups. This finding may have significant implications for the pathogenesis of HCV-associated B-cell proliferative diseases, because IL-6 is a potent B-cell growth and maturation factor (39). This concept is corroborated by our finding of increased B-cell growth when supernatants of HCV core–stimulated PBMCs were transferred to B-cell cultures as well as when PBMCs were directly coincubated with recombinant HCV core protein. Of note, HCV core–stimulated B-cell proliferation could be significantly blocked, when a neutralizing IL-6 antibody was added during stimulation. It has been proposed for a long time that IL-6 secretion may contribute to the pathogenesis of multiple myeloma (40). IL-6 has antiapoptotic effects (41) and enhances proliferation of myeloma cells in vitro (42, 43). Moreover, in a mouse model, i.p. injection of J3V1 retrovirus induced B-cell neoplasia in 30% to 50% of mice carrying the wild-type allele of the IL-6 gene, whereas B-cell neoplasia could not be induced in IL-6-deficient mice (44). Furthermore, Ishikawa et al. (45) found that transfection with HCV core induced malignant follicular center cell type B-cell lymphomas in 80% of susceptible mice at ages over 20 months.

There are obviously striking parallels between the proposed concept concerning the pathogenesis of HCV-associated, B-cell proliferative disorders and the putative pathogenesis of MALT lymphomas seen in chronic Helicobacter pylori infection, which are presumably triggered by a chronic immune response to H. pylori antigens, including recognition of H. pylori–derived LPS by TLR2 (46). Up-regulated TLR2 expression has been reported in chronic hepatitis C (47, 48). Furthermore, a role of TLR2 triggering by HCV antigens in the pathogenesis of HCV-associated lymphoproliferative disease is also substantiated in our study by finding increased TLR2 surface expression on monocytes particularly in HCV-infected patients with MC, possibly indicating in vivo sensitized cells.

Other cofactors may also contribute to the pathogenesis of HCV-associated MC and B-NHL. Liver cirrhosis was quite prevalent in our patients with MC and B-NHL, and both decreased clearance of IL-6 and increased endotoxinemia associated with cirrhosis (49) may enhance the effects of TLR2 stimulation, thus increasing the risk of HCV-induced B-cell proliferative disorders in patients with long duration of HCV infection. In line with this reasoning, presence of cirrhosis and old age were also identified as contributing factors in a univariate correlation analysis. In addition, B cells can also be activated by HCV antigens by other signaling pathways, e.g., stimulation via CD81 after binding of HCV E2 (50). Thus, further studies are necessary to elucidate the relative contribution of IL-6 triggering to the pathogenesis of HCV-associated lymphoproliferative disorders. In particular, it is worthwhile finding out whether IL-6 blocking will offer new therapeutic perspectives to prevent or treat HCV-associated lymphoproliferative disorders in patients with longstanding HCV infection.


   Acknowledgments
 
We thank Monika Schulz for excellent technical assistance, U. Kullig for help in the collection of sample material, and G. Szabo for the human embryonal kidney (HEK293) cells stably transfected with human TLR2 and TLR4 together with its coreceptor MD-2 (30), given to us with the permission of D. Golenbock.


   Footnotes
 
Grant support: German Network of Competence for Hepatitis (Hep-Net) grant 01KI0416.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

G. Feldmann and H.D. Nischalke contributed equally.

Hep-Net was also used to identify patients with Hepatitis C–associated B-cell Non–Hodgkin's Lymphoma.

Received 1/23/06; revised 5/ 3/06; accepted 5/18/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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