Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M407640200 on July 30, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43479-43486, October 15, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/42/43479    most recent
M407640200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eisen-Vandervelde, A. L.
Right arrow Articles by Hahn, Y. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eisen-Vandervelde, A. L.
Right arrow Articles by Hahn, Y. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Hepatitis C Virus Core Selectively Suppresses Interleukin-12 Synthesis in Human Macrophages by Interfering with AP-1 Activation*

Audrey L. Eisen-Vandervelde{ddagger}§, Stephen N. Waggoner{ddagger}§, Zhi Qiang Yao{ddagger}, Evan M. Cale{ddagger}, Chang S. Hahn||, and Young S. Hahn{ddagger}§**

From the {ddagger}Beirne Carter Center for Immunology Research and the Departments of §Microbiology and Pathology, University of Virginia, Charlottesville, Virginia 22908 and ||Immunology Platform, Aventis Pharmaceuticals, Bridgewater, NJ 08807

Received for publication, July 7, 2004 , and in revised form, July 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection, suggesting that it has evolved one or more strategies aimed at evading the host immune response. T cell responses, including interferon-{gamma} production, are severely suppressed in chronic HCV patients. The HCV core protein has been previously shown to circulate in the bloodstream of HCV-infected patients and inhibit host immunity through an interaction with gC1qR. To determine the role of the HCV core-gC1qR interaction in modulation of inflammatory cytokine production, we examined interleukin (IL)-12 production, which is critical for the induction of interferon-{gamma} synthesis, in lipopolysaccharide-stimulated human monocyte/macrophages. We found that core protein binds the gC1qR displayed on the cell surface of monocyte/macrophages and inhibits the production of IL-12p70 upon lipopolysaccharide stimulation. This inhibition was found to be selective in that HCV core failed to affect the production of IL-6, IL-8, IL-1{beta}, and tumor necrosis factor {alpha}. In addition, suppression of IL-12 production by core protein occurred at the transcriptional level by inhibition of IL-12p40 mRNA synthesis. Importantly, core-induced inhibition of IL-12p40 mRNA synthesis resulted from impaired activation of AP-1 rather than enhanced IL-10 production. These results suggest that the HCV core-gC1qR interaction may play a pivotal role in establishing persistent infection by dampening TH1 responses.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCV,1 a positive-stranded RNA virus of ~9.6 kb, is a serious and growing health threat, having already infected roughly 170 million people worldwide (1, 2). Transmission of HCV has been linked to a blood-borne route (i.e. blood transfusions, organ transplants, and IV drug use) (3). Following a 10–20-year subclinical phase, greater than 80% of patients progress to persistent HCV infection, a leading cause of chronic liver disease associated with the development of cirrhosis, hepatocellular carcinoma, and autoimmune disorders (3–9). The propensity of HCV to persist in the vast majority of infected individuals suggests that the virus has evolved one or more mechanism(s) of evading host immunity. Unfortunately, the current anti-HCV therapy, IFN-{alpha} treatment in conjunction with the nucleoside analog ribavirin, has proven ineffectual, and attempts to generate a vaccine against viral proteins have been unsuccessful (10, 11).

The activation of virus-specific cytotoxic T lymphocytes (CTL) is critical for the elimination of virus-infected cells. Virus-specific CTL, both in the liver and periphery, are significantly diminished in chronic HCV patients as compared with those observed for other persistent viruses, such as hepatitis B virus and human immunodeficiency virus (12, 13). This apparent suppression of CTL responses suggests that HCV may interfere with CD4+ helper T cell activation and differentiation, thus allowing for the establishment of chronic infection. IL-12, which is known to regulate proinflammatory T helper (TH) 1 responses by promoting IFN-{gamma} production, provides an important link between innate and adaptive immunity. Patients chronically infected with HCV demonstrate a diminished capacity to up-regulate IL-12 and generate TH1-type immunity (14–16). In addition to HCV, several other infectious organisms, including measles virus, human immunodeficiency virus, and Legionella pneumophila, prevent the establishment of a TH1 environment by inhibiting IL-12 production (17–19). This suppression of TH1 polarization by infectious organisms may represent a novel mechanism of host immune evasion. Although both lymphocytes and macrophages are targeted for infection by HCV, the rate of infectivity is relatively low (20, 21). Thus, it is unlikely that infection of these cells alone accounts for the immune dysregulation observed in chronic HCV patients, suggesting that the virus may utilize an indirect mechanism, such as the production of an immunomodulatory factor, to persist.

HCV core (21 kDa), which is the first protein to be produced upon viral infection, demonstrates multiple functions, affecting numerous operations within both the host and virus. In addition to forming the viral nucleocapsid, HCV core has been observed to modulate host cellular responses, including apoptosis and immunity (22–25). Studies on mice infected with a panel of recombinant vaccinia viruses expressing the various HCV proteins demonstrated the HCV core protein to be sufficient for the increased morbidity and mortality because of failure of vaccinia viruses clearance (26). This core-induced immune suppression was found to result from inhibition of IFN-{gamma}-mediated CTL activation. Interestingly, activated murine macrophages stably transfected with a vector containing HCV core exhibit severely diminished IL-12 and nitric oxide production (27). This result corresponds well with the clinical observation that HCV-infected individuals with a self-limited course of disease demonstrate a TH1 dominance, whereas those who progress to chronic infection tend toward a TH2 phenotype (15, 16, 28, 29).

In addition to cytoplasmic and nuclear localization, HCV core protein is secreted from stably transfected cell lines. Indeed, circulating core protein is detectable in the bloodstream of infected patients, where it may provide the virus with an indirect, infection-independent mechanism of immune dysregulation (30, 31). Recently, we demonstrated that exogenous addition of HCV core protein to mixed lymphocyte culture resulted in suppression of IFN-{gamma} production and T cell proliferation (3). Importantly, this core-induced inhibition of T cell responsiveness was dependent upon its interaction with a complement receptor, gC1qR, on the surface of immune cells (3, 32). The natural ligand of gC1qR, C1q, functions to elicit diverse responses within cells of the immune system by modulating both innate and adaptive inflammatory responses. Although C1q affects innate immunity by enhancing phagocytosis in macrophages and mediating C1q-dependent cytotoxicity, C1q also influences adaptive immunity by suppressing cytokine expression and cellular proliferation (33–36). Notably, HCV core has been shown to elicit similar inhibitory effects on T cell responsiveness through its interaction with gC1qR, suggesting that HCV might usurp gC1qR signaling to down-regulate host immune responses (3). Indeed, gC1qR has been shown to interact with several pathogen-derived proteins in addition to HCV core, including HIV-1 Rev, EBV EBNA-1, and Listeria monocytogenes internalin B, potentially providing these organisms with a novel mechanism of immune subversion (37–39).

Here we sought to determine whether the interaction between HCV core and gC1qR on the surface of monocyte/macrophages (M/M{phi}) might suppress IL-12 production, a key cytokine for induction of IFN-{gamma} and TH1 type CD4+ T cell responses. To address this possibility, we examined the effect of HCV core on the production of IL-12 by human M/M{phi} stimulated with the toll-like receptor 4 (TLR4) ligand LPS, which is known to induce production of inflammatory cytokines, including IL-12. We demonstrated that HCV core is able to bind the surface of M/M{phi} and that this binding involves an interaction with gC1qR. Further, we found that exogenous addition of HCV core to human PBMC stimulated with either LPS or SAC/anti-IL10 antibody results in suppression of both IL-12p70 and IFN-{gamma} production. We subsequently demonstrated that M/M{phi} are targeted for IL-12p70 inhibition by HCV core within the bulk PBMC population and that this suppression involves a diminished capacity to up-regulate IL-12p40 mRNA by interfering with AP-1 activation. Importantly, HCV core-mediated inhibition of IL-12p40 mRNA synthesis depends upon its interaction with gC1qR. These results indicate that the HCV core-gC1qR interaction may be significant in the establishment of chronic HCV infection through the suppression of TH1 responses, and, for the first time, report the role of gC1qR in immunomodulation of antigen presenting cell function. Therapies aimed at disrupting this interaction may prove invaluable in the prevention of HCV persistence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures—PBMC were isolated from whole blood or buffy coats of donors (Virginia Blood Services) by Ficoll-Hypaque gradient (Amersham Biosciences). The cells were washed twice and cultured in RPMI 1640 (Invitrogen) containing 10% (v/v) fetal bovine serum (HyClone Laboratories, Logan, UT), penicillin/streptomycin (100 µg/ml; Invitrogen), L-glutamine (2 mM), and 2-mercaptoethanol (55 µM; Invitrogen) at 37 °C with 5% CO2 in a humidified atmosphere. THP-1 cells (American Type Culture Collection, Manassas, VA), a human monocytic leukemia cell line, were cultured in RPMI 1640 containing 2 mM L-glutamine adjusted to contain 0.15% sodium bicarbonate, 0.45% glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 10% fetal bovine serum (Invitrogen) at 37 °C with 5% CO2 in a humidified atmosphere.

Quantification of Cytokine Production by ELISA—PBMC (2 x 105cells/well) were stimulated to produce IL-12p70 and/or IFN-{gamma} by incubation in complete medium containing 0.005% (v/v) Staphylococcus aureus Cowen strain (SAC; Sigma) and 10 µg/ml anti-IL-10 antibody (BD Pharmingen, San Diego, CA) in a round-bottomed, 96-well tissue culture plate for 24 h. Primary human M/M{phi} (5 x 105 cells/well) were isolated from the bulk PBMC by attachment to a 48-well, flat-bottomed polystyrene tissue culture plate for 20 min at 37 °C. Macrophage maturation of THP-1 cells was driven by incubation of 6 x 105 cells in complete medium containing 1.2% Me2SO for 24 h at 37 °C. These isolated M/M{phi} and matured THP-1 cells were presensitized with 100 ng/ml recombinant human (rh)-IFN-{gamma} (BD Pharmingen) for 16 h and were stimulated with 5 µg/ml LPS (Sigma) in the presence of varying doses of either {beta}-gal-fused HCV core (Virogen, Watertown, MA) or {beta}-gal control protein for 24 h at 37 °C. The supernatants were obtained to quantify the production of IL-12p70 or IFN-{gamma} by ELISA (BD Pharmingen). For quantification of TNF-{alpha}, IL-1{beta}, IL-6, IL-10, and IL-8, primary M/M{phi} were activated as above, and cytokine levels were determined using a BDTM cytometric bead array multiplex assay (BD Biosciences), as per the manufacturer's instructions.

Flow Cytometric Analysis—To determine core binding on the cell surface of M/M{phi}, 1 x 106 PBMC were incubated for 1 h at 37 °C in the presence or absence of His6-tagged HCV core (80 nM). His6-tagged HCV core was expressed as six histidine residues located at the N terminus of the full-length HCV core protein (amino acids 2–191) and was purified under native conditions as described previously (32). The cells were washed three times in fluorescence-activated cell sorter media (RPMI 1640 supplemented with 10% fetal bovine serum, 0.1% NaN3). The cells were resuspended in 50 µl of fluorescence-activated cell sorter media and incubated with anti-gC1qR (University of Virginia Hybridoma Center) or anti-core (Affinity Bioreagents, Inc., Golden, CO) monoclonal antibodies on ice for 1 h. After washing three times, the cells were incubated with fluorescein isothiocyanate anti-mouse IgG (BD Pharmingen) on ice for 1 h in the dark. The cells were washed three times at 4 °C and fixed with 1% paraformaldehyde in phosphate-buffered saline and analyzed by flow cytometry (BD Biosciences, San Jose, CA). Unstained cells and primary isotype controls were used to determine the levels of background fluorescence. The viable cell gates were used to collect 30,000 events. To address the role of gC1qR in core binding to macrophages, anti-gC1qR polyclonal antibody, prebleed control sera (1:10 (v/v) dilution; QED Bioscience, San Diego, CA), or soluble His6-gC1qR or His6-DHFR (prepared in our laboratory) were co-incubated with His6 core and PBMC. Core binding was assessed by flow cytometry as described above.

Determination of Cytokine mRNA Levels—RT-PCR was employed to determine the levels of IL-12p40, IL-10, and {beta}-actin mRNA. RNA was isolated by TRIzol (Invitrogen) according to the manufacturer's instructions. 1 µg of RNA was treated with DNase to digest genomic DNA and 0.27 µg of RNA was then reverse transcribed using murine leukemia virus reverse transcriptase under conditions of 10 min at room temperature, 20 min at 42 °C, 5 min at 99 °C, and 5 min at 5 °C. Various volumes of cDNA generated in the RT reaction were added to the PCR. PCR was carried out using the following primer pairs: IL-12p40 sense 5'-CAGCAGTTGGTCATCTCTTG-3', antisense 5'-CCAGCAGGTGAAACGTCCAG-3'; IL-10 sense 5'-ATGCCCCAAGCTGAGAACCA-3', antisense 5'-TCTCAAGGGGCTGGGTCAGC-3'; and {beta}-actin sense 5'-CGAGCGGGAAATCGTGCGTGACAT-3', antisense 5'-CGTCATACTCCTGCTTGCTGATCCACATCT-3' for 35 cycles of 95 °C for 40 s, 60 °C for 20 s, 72 °C for 40 s, followed by 10 min of incubation at 72 °C.

Nuclear Extracts and Electrophoretic Mobility Shift Assays—5 x 106 THP-1 cells/well were matured and primed with rh-IFN-{gamma} as before, followed by activation with 5 µg/ml LPS for various time points. The nuclei were isolated by suspending cells in extraction buffer (200 mM HEPES, pH 7.9, 0.5 M EDTA, 200 mM dithiothreitol, 0.1 M phenylmethylsulfonyl fluoride, complete protease inhibitor mixture (Roche Applied Science), 2.5 µl of Nonidet P-40/ml) for 15 min on ice. The cells were centrifuged for 10 min at 800 x g and 4 °C. Supernatant was removed and stored as cytoplasmic fraction. The nuclear extracts were prepared by resuspending nuclei in extraction buffer containing 0.1 M NaCl, incubating on ice for 15 min, and centrifuging for 15 min at 16,000 x g and 4 °C. 20% glycerol was added to stabilize isolates, and extracts were mixed with 32P-labeled probes for either NF-{kappa}B, 5'-ACTTCTTGAAATTCCCCCAGAAGG-3', AP-1, consensus binding motif 5'-TGACTCA-3' (Santa Cruz Biotechnology), or NF-IL-6, 5'-TCGATCAGTTGCAAATCGT-3'. The mixtures were run on a 4% acrylamide gel, which was prerun for 20 min at 200V. Supershift was conducted using antibodies specific for the p65 or p50 subunits of NF-{kappa}B (Santa Cruz Biologicals). To control for nonspecific binding of the probe, binding was also carried out in the presence of a 10x unlabeled competitor. The gel was dried and exposed to Kodak X-Omat AR film at –80 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HCV Core Binds the Surface of Human Monocytes/Macrophages in a gC1qR-dependent Manner—We previously demonstrated that HCV core interaction with gC1qR results in suppression of IFN-{gamma} production and T cell proliferation in a mixed lymphocyte reaction (3, 32). To determine whether the HCV core-gC1qR interaction alters the production of inflammatory cytokines (i.e. IL-12) and inhibits IFN-{gamma} production, we first examined the ability of HCV core to bind gC1qR displayed on the cell surface of primary human monocytes/macrophages (M/M{phi}). As shown in Fig. 1a, 94.3% of primary M/M{phi} were positive for the expression of cell surface gC1qR by flow cytometric analysis. Additionally, when cells were incubated with recombinant His6 core, 64.0% of primary M/M{phi} displayed core protein bound on their surface (Fig. 1b). To assess the role of gC1qR in the association of core with M/M{phi}, we determined the ability of {alpha}-gC1qR antibody to prevent core binding on the cell surface of M/M{phi}. In the presence of specific {alpha}-gC1qR antibody, but not prebleed control sera, the percentage of core-bound M/M{phi} was reduced by 54%, whereas the mean intensity of staining decreased 68% (Fig. 1c). Likewise, we examined the ability of soluble recombinant gC1qR to antagonize core association with M/M{phi}. Excess His6-gC1qR (molar ratio of 6x core) reduced the frequency as well as intensity of core staining in M/M{phi} by 42 and 59%, respectively (Fig. 1d). In contrast, an equivalent excess amount of His6-DHFR control protein did not affect core binding to M/M{phi}. These results suggest that gC1qR is as a receptor for binding HCV core on the cell surface of M/M{phi}.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1.
HCV core binds the cell surface of human macrophages through an interaction with gC1qR. a, expression of cell surface gC1qR on human macrophages was determined by incubating 1 x 106 PBMC with anti-gC1qR monoclonal Ab followed by staining with fluorescein isothiocyanate-conjugated anti-mouse Ig (solid line) or isotype control Ab (shaded histogram). After the cells were fixed in 1% paraformaldehyde/phosphate-buffered saline, gC1qR expression was determined by flow cytometry analysis. The M/M{phi} population was gated based on forward and side scatter patterns. Histogram counts are normalized to total cell counts and denoted as percentages of the maximum. b, core binding to the cell surface gC1qR on macrophages was assessed by incubating 1 x 106 PBMC with 80 nM His6 core for 1h at 37 °C. Core binding was visualized by staining with anti-core monoclonal antibody followed by fluorescein isothiocyanate anti-mouse Ig (solid line) or isotype control Ab (shaded histogram). M/M{phi} were gated as above. c, to determine the role of gC1qR in core association with M/M{phi}, PBMC were incubated with 1:10 (v/v) anti-gC1qR polyclonal Ab or prebleed control sera in addition to 80 nM His6 core. Core binding on PBMC was determined as described above. Shown in histogram are isotype control (shaded), core (solid thin line), core plus {alpha}-gC1qR antibody (solid heavy line), core plus control antibody (dashed line). d, core binding (solid thin line) was also assessed as above in the presence of molar excess (6x) soluble His6-gC1qR (solid heavy line) or the control protein His6-DHFR (dashed line).

 
HCV Core Inhibits IL-12p70 and IFN-{gamma} Production by Activated PBMC—IL-12p70, the biologically functional form of IL-12, is responsible for inducing IFN-{gamma} production and T cell proliferation (40). To examine the effect of HCV core on modulation of IL-12p70 production by activated PBMC, we determined IL-12p70 production in PBMC stimulated with SAC/{alpha}-IL-10 antibody in the presence or absence of {beta}-gal core by ELISA. As shown in Fig. 2a, IL-12p70 production in SAC/{alpha}-IL-10-stimulated PBMC is significantly inhibited by HCV core in a concentration-dependent manner as compared with that in {beta}-gal control protein-treated cells. In addition, His6 core prepared in our laboratory is able to inhibit IL-12p70 production similar to that seen with the commercially available {beta}-gal core (data not shown). To ensure that the inhibition of IL-12p70 production is not as a result of cell death induced by core protein, LDH release as well as trypan blue exclusion analyses were performed and revealed little cell death following the 24-h incubation period under any of the treatment conditions (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 2.
HCV core inhibits IL-12p70 and IFN-{gamma} production by activated PBMC. PBMC (5 x 105 cells/well) were plated in 96-well tissue culture plates and stimulated by 0.005% (v/v) SAC/anti-IL-10 antibody (10 µg/ml) for 24 h at 37 °C in the presence of various dose (25, 50, and 100 nM) of either {beta}-gal core (open circles) or {beta}-gal (closed circles). The supernatants were harvested, and the IL-12p70 (a) and IFN-{gamma} (b) concentrations were determined by ELISA. *, values below the level of detection limit are defined as zero. These results were found to be reproducible in three independent experiments.

 
IL-12 secreted by antigen presenting cells is able to induce IFN-{gamma} production in T and NK cells (40). In addition, chronic HCV patients exhibit depressed peripheral IFN-{gamma} levels (15, 41). Based on the apparent suppression of IL-12 observed above, we next sought to determine the effect of HCV core on IFN-{gamma} production by activated PBMC. As shown in Fig. 2b, HCV core demonstrates a significant, dose-dependent inhibition of IFN-{gamma} production by SAC/{alpha}-IL-10-stimulated PBMC as compared with those treated with control protein {beta}-gal. These data indicate that HCV core can suppress IL-12 as well as IFN-{gamma} production from human PBMC. It implicates that the inhibition of IFN-{gamma} production by HCV core might be responsible for preventing TH1 type CD4+ T cell responses as observed in chronic HCV patients.

HCV Core Selectively Inhibits IL-12 Production by Activated Macrophages—Macrophages and dendritic cells are the major sources of IL-12 produced during an inflammatory response (42). Thus, the observed suppression of IL-12p70 by HCV core might be due to a direct interference with macrophage/dendritic cells activation by HCV core. To examine whether HCV core affects macrophage-derived IL-12p70 production, primary M/M{phi} were isolated from the bulk PBMC by adherence to polystyrene. Because LPS is a potent inducer of inflammatory cytokine production including IL-12, isolated M/M{phi} were activated by LPS in the presence of varying doses of either {beta}-gal core or {beta}-gal. As shown in Fig. 3a, HCV core inhibits IL-12 production by activated M/M{phi} in a dose-dependent manner. A 4-fold decrease in IL-12p70 was detected in cells treated with 100 nM core. In contrast, control protein {beta}-gal fails to elicit the inhibitory effect on IL-12p70 production. The HCV core-mediated inhibition of IL-12 production was reproducible in primary M/M{phi} derived from 10 different blood donors (data not shown). These results suggest that HCV core interferes with macrophage activation and is able to inhibit IL-12 production. In addition, HCV core also suppressed IL-12p70 production in LPS- or CD40L-stimulated human dendritic cells (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3.
HCV core suppresses IL-12p70 production by activated M/M{phi}. a, purified human M/M{phi} (5 x 105 cells/well) were presensitized with 100 ng/ml rh-IFN-{gamma} for 16h at 37 °C. LPS (5 µg/ml) was added for 24hat37 °C in the presence of various doses (12.5, 25, 50, and 100 nM) of {beta}-gal core (open circles) or a {beta}-gal control protein (closed circles). The supernatants were harvested, and IL-12p70 production was determined by ELISA. b, THP-1 (6 x 105 cells/well) were plated on a 96-well tissue culture plate for 24 h at 37 °C in medium containing 1.2% Me2SO. Me2SO-containing medium was aspirated, and medium containing 100 ng/ml rh-IFN-{gamma} was added for 16 h at 37 °C. LPS (5 µg/ml) was added, and IL-12p70 concentration was determined as above. These results were reproducible in three independent experiments.

 
To confirm the ability of HCV core to inhibit IL-12 production by primary M/M{phi}, we examined the ability of HCV core to affect IL-12p70 production by the human monocytic leukemia cell line, THP-1. As shown in Fig. 3b, HCV core elicited a similar dose-dependent inhibition of IL-12p70 production by LPS-stimulated THP-1 (10-fold decrease in IL-12p70 at 100 nM core) as observed in primary M/M{phi}. It is notable that the amount of IL-12p70 produced by activated THP-1 in this experiment was ~10-fold greater than that observed for primary M/M{phi}. In contrast, the {beta}-gal control protein did not affect IL-12p70 production by these cells.

In addition to IL-12, macrophages also produce other inflammatory cytokines including TNF-{alpha}, IL-6, IL-1{beta}, and IL-8 followed by the binding of LPS on TLR4 (43). To determine whether HCV core-gC1qR engagement affects the production of other cytokines, we examined the effect of HCV core on secretion of TNF-{alpha}, IL-6, IL-1{beta}, and IL-8 by LPS-stimulated primary M/M{phi} using a cytometric bead array. As shown in Fig. 4, HCV core elicited no significant effect on production of TNF-{alpha}, IL-6, IL-1{beta}, or IL-8 in LPS-stimulated primary M/M{phi}. This suggests that the ligation of gC1qR by HCV core selectively inhibits IL-12p70 production in macrophages in response to LPS stimulation.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4.
HCV core does not affect production of TNF-{alpha}, IL-6, IL-1{beta}, and IL-8. Purified human M/M{phi} (5 x 105 cells/well) were stimulated with LPS (5 µg/ml) for 24 h at 37 °C in the presence or absence of various concentrations of {beta}-gal core after IFN-{gamma} presensitization. The supernatants were obtained, and cytokine production was determined using a cytometric bead array as per the manufacturer's instructions. These results were reproducible in two independent experiments.

 
HCV Core-induced Inhibition of IL-12 Production Is Not Mediated by Overproduction of IL-10—IL-10 is responsible for the negative regulation of IL-12 (40). Thus, IL-10 is a crucial factor for maintenance of a balance between effective resistance against pathogens and detrimental systemic inflammation. To determine whether enhanced IL-10 production is involved in HCV core-mediated inhibition of IL-12, we examined the effect of core on the expression of IL-10 by primary M/M{phi}.

As shown in Fig. 5a, secretion of IL-10 from LPS-activated primary M/M{phi} in the presence of {beta}-gal core is not enhanced, and in fact, expression levels appear to be slightly decreased by high core concentrations (100 nM). Similarly, treatment of THP-1 cells with {beta}-gal core did not enhance but rather antagonized LPS-induced IL-10 mRNA synthesis (Fig. 5b). These results indicate that suppression of IL-12 by HCV core is not mediated by enhanced expression of IL-10. This is also consistent with results demonstrating that core-mediated suppression of IL-12p70 production occurs in IL-10-depleted culture by treatment of anti-IL-10 antibody as shown in Fig. 2.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5.
HCV core does not up-regulate IL-10. a, purified human M/M{phi} (5 x 105 cells/well) were presensitized with IFN-{gamma} and activated with LPS (5 µg/ml) for 24 h at 37 °C in the presence or absence of various concentrations (0, 25, 50, and 100 nM) of {beta}-gal core. The supernatants were obtained, and IL-10 production was determined using a cytometric bead array as per the manufacturer's instructions. These results were reproducible in three independent experiments. b, 1 x 106 THP-1 were presensitized with rh-IFN-{gamma} and were stimulated with LPS (5 µg/ml) in the presence or absence of {beta}-gal core (100 nM). RT-PCR was performed by using 1, 5, and 10 µl of RT products to amplify IL-10 and {beta}-actin genes. These results were reproducible in two independent experiments.

 
HCV Core-mediated Inhibition of IL-12 Production Occurs at the Level of IL-12p40 Gene Transcription in a gC1qR-dependent Manner—The biologically functional IL-12p70 molecule is a heterodimer comprised of a p40 and p35 subunit, respectively. IL-12p40 has been shown to be the regulatory subunit of the p70 heterodimer within macrophage populations (40). Thus, the observed inhibition of IL-12p70 by HCV core might be due to suppression of IL-12p40 mRNA synthesis. To test this possibility, we examined the effect of HCV core on IL-12p40 mRNA synthesis in LPS-stimulated primary M/M{phi} by RT-PCR. As shown in Fig. 6a, HCV core significantly inhibits LPS-induced synthesis of IL-12p40 mRNA but not the expression of {beta}-actin. These results suggest that HCV core-induced suppression of IL-12p70 production occurs at the level of inhibiting IL-12p40 mRNA synthesis. Also it suggests that HCV core-gC1qR engagement may inhibit one or more transcription factors critical for the induction of IL-12p40 mRNA synthesis.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 6.
HCV core selectively inhibits IL-12p40 mRNA synthesis through its interaction with gC1qR. a, purified M/M{phi} (1 x 106 cells/well) were presensitized with rh-IFN-{gamma} and then stimulated with LPS (5 µg/ml) for 6 h in the presence or absence of {beta}-gal core (100 nM). To compare IL-12p40 mRNA levels from core-treated and untreated cells, increasing volumes of cDNA (1, 5, and 10 µl) from the RT reaction were added for each reaction. b, IFN-{gamma} presensitized THP-1 (1 x 106 cells/treatment) were stimulated with LPS (5 µg/ml) in the presence or absence of {beta}-gal core (100 nM) and/or anti-gC1qR polyclonal Ab. Prebleed sera obtained from the same animal was employed as a control for the anti-gC1qR antibody. These results were found to be reproducible in two independent experiments.

 
We next sought to determine whether the observed core-induced suppression of IL-12p40 mRNA synthesis in activated M/M{phi} is gC1qR-dependent. As shown in Fig. 6b, LPS stimulation of THP-1 resulted in strong induction of IL-12p40 mRNA, which was significantly diminished upon the addition of HCV core. Importantly, co-incubation of these core-treated cells with {alpha}-gC1qR antibody was able to reverse the core-induced suppression of IL-12p40 mRNA synthesis. In contrast, treatment of prebleed control sera was unable to reverse HCV core-mediated inhibition of IL-12p40 mRNA synthesis. These results suggest that HCV core might usurp gC1qR signaling to influence the LPS-induced inflammatory response. In the absence of LPS, neither prebleed nor {alpha}-gC1qR antibody treatment possesses agonistic effect to induce IL-12p40 mRNA synthesis (data not shown).

HCV Core Blocks AP-1 Activation and Delays the Early Activation of NF-{kappa}B—The promoter region of IL-12p40 has been well characterized, with several known mechanisms of transcriptional regulation resulting from TLR4 signaling (42, 44–47). AP-1 is known to play a critical role in regulating IL-12p40 gene transcription. In addition to AP-1, NF-{kappa}B is involved in transcriptional regulation of IL-12p40, whereas the absolute necessity for NF-{kappa}B in IL-12p40 expression remains to be elucidated (44–47). Recent studies indicate two temporally separable NF-{kappa}B activation steps in TLR4 signaling: MyD88/TIR domain-containing adaptor protein (TIRAP)-mediated NF-{kappa}B activation occurs early after stimulation, whereas TIR domain-containing adaptor inducing IFN-{beta} (TRIF) induces later NF-{kappa}B activation (48–50).

To examine whether HCV core may affect the activation of AP-1 and NF-{kappa}B and inhibit IL-12p40 mRNA synthesis, we performed electrophoretic mobility shift assay analysis in core-treated THP-1 at various time points (0, 30, 60, 180, and 300 min) after LPS stimulation. As shown in Fig. 7a, HCV core significantly suppresses activation of AP-1 at all time points, when compared with those cells treated with LPS alone. We also examined the effect of HCV core on NF-{kappa}B activation using nuclear extracts of LPS-stimulated THP-1 cells by electrophoretic mobility shift assay. As shown in Fig. 7b, NF-{kappa}B rapidly accumulated in the nuclei of LPS-stimulated cells by 30 min, dissipating by 180 min. The addition of HCV core to these cells, however, diminished the early activation of NF-{kappa}B at 30 min after LPS stimulation, suggesting that core may disrupt the arm of TLR4 signaling responsible for early activation of NF-{kappa}B. However, HCV core did not affect the activation of the transcription factor NF-IL-6 (data not shown). Taken together, these results suggest that HCV core disrupts one or more TLR signaling pathways, thus preventing the activation of key transcription factors AP-1 and the delayed activation of NF-{kappa}B.



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 7.
HCV core suppresses AP-1 activation. a, THP-1 (5 x 106 cells/well) were presensitized with rh-IFN-{gamma} as before and were stimulated with LPS (5 µg/ml) for various time points (0, 30, 60, 180, and 300 min). Nuclear extracts were prepared, and 4 x 105 cell equivalents were incubated with 32P-labeled AP-1-specific probe. As a control for specificity of the AP-1 band, nuclear extracts were also incubated with cold competitor present at 10x that of the radiolabeled probe. These results were reproducible in three independent experiments. b, cells were treated as above for the indicated time points (0, 30, 60, and 180 min) and nuclear extracts were incubated with a 32P-labeled probe specific for NF-{kappa}B, in the presence or absence of a 10x cold competitor. Supershifts were performed using antibodies for the p50 and p65 subunits of NF-{kappa}B. Supershifts were not visible following 12h exposure (left panel), but were apparent after 24 h (right panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we demonstrate that HCV core selectively suppressed IL-12p70 production by activated human PBMC and macrophages following LPS stimulation, whereas levels of TNF-{alpha}, IL-1{beta}, IL-6, and IL-8 remained unaffected. Inhibition of IL-12p70 by HCV core led to the suppression of IFN-{gamma} secretion in SAC-stimulated PBMC. In addition, we found this suppression to occur at the transcriptional level through inhibition of IL-12p40 mRNA synthesis. The HCV core-induced inhibition of IL-12p40 mRNA synthesis resulted from the suppression of transcription factor AP-1 rather than the enhanced production of IL-10. Importantly, the HCV core-mediated inhibition of IL-12p40 mRNA synthesis could be reversed by anti-gC1qR antibody treatment, suggesting that an interaction between HCV core and gC1qR is crucial for suppressing IL-12 production. These results suggest that the HCV core-gC1qR-mediated inhibition of IL-12 production may play a pivotal role in establishing and maintaining a TH2 microenvironment in infected individuals.

IL-10 is well established as an important negative regulator of IL-12 production (40). However, we found the observed core-induced suppression of IL-12 synthesis in LPS-stimulated M/M{phi} to occur independently of IL-10 induction. Instead, the inhibition of IL-12 by HCV core occurs at the transcriptional level through suppression of IL-12p40 expression. This suggests that the ligation of gC1qR by HCV core might interfere with LPS-induced TLR4 signaling. It is also notable that the suppression of IL-12p40 expression has broader implications for inflammatory cytokine production because IL-12p40 associates with a p19 chain to form a covalently linked heterodimeric cytokine, IL-23 (51). IL-23 is produced by similar cell types as IL-12 and induces IFN-{gamma} production (51). Importantly, IL-23 was shown to induce stronger, more sustained CTL and TH1 immune responses in an HCV vaccination model (52). Given the inhibitory effect of HCV core on IL-12p40 expression, HCV core is also likely to suppress production of IL-23. We are currently investigating the alteration of IL-23 by HCV core.

It is also crucial to point out that core treatment inhibited TLR4-induced activation of the transcription factor AP-1, a key regulator of IL-12p40 mRNA synthesis. However, activation of NF-{kappa}B appeared to be delayed rather than abolished. This might explain the selective inhibition of IL-12 production by HCV core while most other cytokines remain unaffected. Recently, two distinct, temporally separable pathways mediate TLR4 signaling: MyD88/TIRAP-dependent and TRIF-dependent pathways. Interestingly, similar to results observed in HCV core-treated macrophages, the early activation of NF-{kappa}B was only suppressed, and late activation of NF-{kappa}B was sustained in macrophages isolated from TIRAP- and MyD88-deficient mice (49, 50). This suggests that the TIRAP/MyD88-mediated signaling pathway might be responsible for early activation of NF-{kappa}B, whereas TRIF-mediated signaling controls later, more sustained NF-{kappa}B activity. In addition, TIRAP/MyD88 deficiency completely abolished IL-12p40 production (50, 53, 54). Thus, the binding of HCV core to gC1qR might selectively impair the TIRAP/MyD88-dependent pathway of TLR4 signaling. These results suggest cross-talk between gC1qR ligation and TLR4 signaling.

However, the signaling intermediates of gC1qR have yet to be identified. The ligation of gC1qR by HCV core may lead to induction of negative regulatory molecules, such as the Src homology 2-containing tyrosine phosphatase (SHP-1), which inhibit a distinct TLR4 signaling pathway and thus modulate IL-12 production (55–57). SHP-1 is a protein-tyrosine phosphatase expressed primarily in cells of the immune system. In addition to its functions in hematopoiesis, in vivo studies have revealed that SHP-1 plays an integral role in the negative regulation of immune cell activity (57). SHP-1 was also implicated in the CD46-mediated suppression of IL-12 by measles virus (55). Ongoing research in our lab is focused on determining whether HCV core-gC1qR engagement might induce a negative regulatory molecule, such as SHP-1, to inhibit the proximal events of TLR4 signaling.

IL-12 is known to play a pivotal role in the generation of TH1-type immune responses and provides a critical link between innate and adaptive immunity (42). The establishment of a TH1 environment in the periphery of HCV-infected individuals is thought to be a key determinant of disease progression. Specifically, those who demonstrate TH2 dominance tend toward chronic infection, whereas those with a TH1 phenotype tend to clear the virus (14–16). Despite this apparent immune dysregulation, HCV exhibits a relatively low infectivity rate, suggesting that the virus utilizes an indirect mechanism of immune dysregulation to persist. Because the hepatocyte is the likely primary cell type to support HCV replication and viral persistence, core protein secreted from HCV-infected hepatocyte will bind to gC1qR displayed on macrophages (Kupffer cells) in the local environment (liver) and suppress the inflammatory response mediated by macrophages. These results raise the possibility that HCV core-mediated inhibition of inflammatory response and local innate immune response may play a pivotal role in the establishment of persistent infection. Furthermore, it indicates that the immunosuppressive action of core protein may allow the virus an avenue of escape by down-modulating TH1 responses in favor of switching to TH2 responses.

This immunomodulatory behavior of HCV core may be genotype-specific, because HCV core from certain genotypes have not been found to be immunosuppressive or may represent a generalized mechanism of immune evasion utilized by the virus (58). It is likely that discrepancies in the abilities of these core proteins to affect immune responses might result from a disparity in the magnitude of core secreted during the acute phase of infection, as well as the sequence variability in the gC1qR-binding site within core protein, thus influencing the relative affinity of these core proteins for the gC1qR. In addition, gC1qR polymorphism may play a role in influencing HCV core-gC1qR-meidated immune dysregulation in HCV-infected individuals.

HCV continues to spread and persist among people worldwide, making it a leading indicator for liver transplantation. The ability of HCV core to usurp the complement cascade to afford the virus an indirect mechanism of immune evasion presents a potential therapeutic target. The impaired immune responses by HCV core-gC1qR interaction could occur at two levels. First, the interaction between HCV core and gC1qR on the surface of T cells directly suppresses T cell function (3, 32). Second, the binding of HCV core on gC1qR displayed on the cell surface of antigen presenting cells (i.e. macrophages and dendritic cells) interferes with cell-mediated immune responses by modulating inflammatory cytokine production. The design of molecules aimed at antagonizing the HCV core-gC1qR interaction may provide us with a novel treatment for HCV, allowing the host immune system to effectively clear the virus.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK66754. 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. Back

** To whom correspondence should be addressed: Dept. of Microbiology, Pathology, and Beirne Carter Center, University of Virginia HSC, Box 801386, Charlottesville, VA 22903. Tel.: 434-924-1155; Fax: 434-924-1221; E-mail: ysh5e{at}virginia.edu.

1 The abbreviations used are: HCV, hepatitis C virus; IFN, interferon; CTL, cytotoxic T lymphocyte(s); HIV, human immunodeficiency virus; TLR, toll-like receptor; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor; PBMC, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; SAC, S. aureus Cowen strain; {beta}-gal, {beta}-galactosidase; RT, reverse transcriptase; rh, recombinant human; TIRAP, TIR domain-containing adaptor protein; TRIF, TIR domain-containing adaptor inducing IFN-{beta}; SHP-1, Src homology 2-containing tyrosine phosphatase; Ab, antibody. Back


   ACKNOWLEDGMENTS
 
We thank our colleagues for invaluable suggestions and criticisms. We also thank Travis Lillard and Sue Landes for excellent technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alter, M. J. (1997) Hepatology 26, 62S–65S[CrossRef][Medline] [Order article via Infotrieve]
  2. Cerny, A., and Chisari, F. V. (1999) Hepatology 30, 595–601[CrossRef][Medline] [Order article via Infotrieve]
  3. Kittlesen, D. J., Chianese-Bullock, K. A., Yao, Z. Q., Braciale, T. J., and Hahn, Y. S. (2000) J. Clin. Invest. 106, 1239–1249[Medline] [Order article via Infotrieve]
  4. Houghton, M., Weiner, A., Han, J., Kuo, G., and Choo, Q. L. (1991) Hepatology 14, 381–388[CrossRef][Medline] [Order article via Infotrieve]
  5. Polakos, N. K., Drane, D., Cox, J., Ng, P., Selby, M. J., Chien, D., O'Hagan, D. T., Houghton, M., and Paliard, X. (2001) J. Immunol. 166, 3589–3598[Abstract/Free Full Text]
  6. Duenas-Carrera, S., Alvarez-Lajonchere, L., Alvarez, J. C., Ramos, T., Pichardo, D., and Morales, J. (2001) Biotechnol. Appl. Biochem. 33, 47–51[CrossRef][Medline] [Order article via Infotrieve]
  7. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q. L., Houghton, M., and Kuo, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6547–6549[Abstract/Free Full Text]
  8. Ferri, C., Greco, F., Longombardo, G., Palla, P., Moretti, A., Marzo, E., Mazzoni, A., Pasero, G., Bombardieri, S., and Highfield, P. (1991) Clin. Exp. Rheumatol. 9, 621–624[Medline] [Order article via Infotrieve]
  9. Johnson, R. J., Gretch, D. R., Yamabe, H., Hart, J., Bacchi, C. E., Hartwell, P., Couser, W. G., Corey, L., Wener, M. H., Alpers, C. E., and Willson, R. (1993) N. Engl. J. Med. 328, 465–470[Abstract/Free Full Text]
  10. Hino, K., Yamaguchi, Y., Fujiwara, D., Katoh, Y., Korenaga, M., Okazaki, M., Okuda, M., and Okita, K. (2000) J. Viral Hepat. 7, 36–42[CrossRef][Medline] [Order article via Infotrieve]
  11. Gerotto, M., Sullivan, D.G., Polyak, S. J., Chemello, L., Cavalletto, L., Pontisso, P., Alberti, A., and Gretch, D. R. (1999) J. Virol. 73, 7241–7247[Abstract/Free Full Text]
  12. Rehermann, B., Chang, K. M., McHutchinson, J., Kokka, R., Houghton, M., Rice, C. M., and Chisari, F. V. (1996) J. Virol. 70, 7092–7102[Abstract/Free Full Text]
  13. Nelson, D. R., Marousis, C. G., Davis, G. L., Rice, C. M., Wong, J., Houghton, M., and Lau, J. Y. (1997) J. Immunol. 158, 1473–1481[Abstract]
  14. Amaraa, R., Mareckova, H., Urbanek, P., and Fucikova, T. (2002) Immunol. Lett. 83, 209–214[CrossRef][Medline] [Order article via Infotrieve]
  15. Sarih, M., Bouchrit, N., and Benslimane, A. (2000) Immunol. Lett. 74, 117–126[CrossRef][Medline] [Order article via Infotrieve]
  16. Kanto, T., Hayashi, N., Takehara, T., Tatsumi, T., Kuzushita, N., Ito, A., Sasaki, Y., Kasahara, A., and Hori, M. (1999) J. Immunol. 162, 5584–5591[Abstract/Free Full Text]
  17. Karp, C. L., Wysocka, M., Wahl, L. M., Ahearn, J. M., Cuomo, P. J., Sherry, B., Trinchieri, G., and Griffin, D. E. (1996) Science 273, 228–231[Abstract]
  18. Chehimi, J., Starr, S. E., Frank, I., D'Andrea, A., Ma, X., MacGregor, R. R., Sennelier, J., and Trinchieri, G. (1994) J. Exp. Med. 179, 1361–1366[Abstract/Free Full Text]
  19. Matsunaga, K., Klein, T. W., Newton, C., Friedman, H., and Yamamoto, Y. (2001) Infect. Immun. 69, 1929–1933[Abstract/Free Full Text]
  20. Gunji, T., Kato, N., Hijikata, M., Hayashi, K., Saitoh, S., and Shimotohno, K. (1994) Arch. Virol. 134, 293–302[CrossRef][Medline] [Order article via Infotrieve]
  21. Lerat, H., Berby, F., Trabaud, M. A., Vidalin, O., Major, M., Trepo, C., and Inchauspe, G. (1996) J. Clin. Invest. 97, 845–851[Medline] [Order article via Infotrieve]
  22. McLauchlan, J. (2000) J. Viral Hepat. 7, 2–14[CrossRef][Medline] [Order article via Infotrieve]
  23. Dolganiuc, A., Kodys, K., Kopasz, A., Marshall, C., Do, T., Romics, L., Jr., Mandrekar, P., Zapp, M., and Szabo, G. (2003) J. Immunol. 170, 5615–5624[Abstract/Free Full Text]
  24. Yao, Z. Q., Eisen-Vandervelde, A., Ray, S., and Hahn, Y. S. (2003) Virology 314, 271–282[CrossRef][Medline] [Order article via Infotrieve]
  25. Large, M. K., Kittlesen, D. J., and Hahn, Y. S. (1999) J. Immunol. 162, 931–938[Abstract/Free Full Text]
  26. Accapezzato, D. Francavilla, V., Rawson, P., Cerino, A., Cividini, A., Mondelli, M. U., and Barnaba, V. (2004) Eur. J. Immunol. 34, 438–446[CrossRef][Medline] [Order article via Infotrieve]
  27. Lee, C. H., Choi, Y. H., Yang, S. H., Lee, C. W., Ha, S. J., and Sung, Y. C. (2001) Virology 279, 271–279[CrossRef][Medline] [Order article via Infotrieve]
  28. Piazzolla, G., Tortorella, C., Schiraldi, O., and Antonaci, S. (2000) J. Clin. Immunol. 20, 54–61[CrossRef][Medline] [Order article via Infotrieve]
  29. Tsai, S. L., Liaw, Y. F., Chen, M. H., Huang, C. Y., and Kuo, G. C. (1997) Hepatology 25, 449–458[Medline] [Order article via Infotrieve]
  30. Kanto, T., Hayashi, N., Takehara, T., Hagiwara, H., Mita, E., Naito, M., Kasahara, A, Fusamoto, H., and Kamada, T. (1995) J. Hepatol. 22, 440–448[CrossRef][Medline] [Order article via Infotrieve]
  31. Sabile, A., Perlemuter, G., Bono, F., Kohara, K., Demaugre, F., Kohara, M., Matsuura, Y., Miyamura, T., Brechot, C., and Barba, G. (1999) Hepatology. 30, 1064–1076[CrossRef][Medline] [Order article via Infotrieve]
  32. Yao, Z. Q., Nguyen, D. T., Hiotellis, A. I., and Hahn, Y. S. (2001) J. Immunol. 167, 5264–5272[Abstract/Free Full Text]
  33. Kishore, U., and Reid, K. B. (2000) Immunopharmacology 49, 159–170[CrossRef][Medline] [Order article via Infotrieve]
  34. Ghebrehiwet, B., Habicht, G. S., and Beck, G. (1990) Clin. Immunol. Immunopathol. 54, 148–160[CrossRef][Medline] [Order article via Infotrieve]
  35. Chen, A., Gaddipati, S., Hong, Y., Volkman, D. J., Peerschke, E. I., and Ghebrehiwet, B. (1994) J. Immunol. 153, 1430–1440[Abstract]
  36. Yamada, M., Oritani, K., Kaisho, T., Ishikawa, J., Yoshida, H., Takahashi, I., Kawamoto, S., Ishida, N., Ujiie, H., Masaie, H., Botto, M., Tomiyama, Y., and Matsuzawa, Y. (2004) Eur. J. Immunol. 34, 221–230[CrossRef][Medline] [Order article via Infotrieve]
  37. Luo, Y., Yu, H., and Peterlin, B. M. (1994) J. Virol. 68, 3850–3856[Abstract/Free Full Text]
  38. Matthews, D. A., and Russell, W. C. (1998) J. Gen. Virol. 79, 1677–1685[Abstract]
  39. Braun, L., Ghebrehiwet, B., and Cossart, P. (2000) EMBO J. 19, 1458–1466[CrossRef][Medline] [Order article via Infotrieve]
  40. Trinchieri, G. (2003) Nat. Rev. Immunol. 3, 133–146[CrossRef][Medline] [Order article via Infotrieve]
  41. Ulsenheimer, A., Gerlach, J. T., Gruener, N. H., Jung, M. C., Schirren, C. A., Schraut, W., Zachoval, R., Pape, G. R., and Diepolder, H. M. (2003) Hepatology 37, 1189–1198[CrossRef][Medline] [Order article via Infotrieve]
  42. Trinchieri, G. (1998) Adv. Immunol. 70, 83–243[Medline] [Order article via Infotrieve]
  43. Magor, B. G., and Magor, K. E. (2001) Dev. Comp. Immunol. 25, 651–682[CrossRef][Medline] [Order article via Infotrieve]
  44. Becker, C., Wirtz, S., Ma, X., Blessing, M., Galle, P. R., and Neurath, M. F. (2001) J. Immunol. 167, 2608–2618[Abstract/Free Full Text]
  45. Sanjabi, S., Hoffmann, A., Liou, H. C., Baltimore, D., and Smale, S. T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12705–12710[Abstract/Free Full Text]
  46. Zhu, C., Gagnidze, K., Gemberling, J. H., and Plevy, S. E. (2001) J. Biol. Chem. 276, 18519–18528[Abstract/Free Full Text]
  47. Weinmann, A. S., Mitchell, D. M., Sanjabi, S., Bradley, M. N., Hoffmann, A., Liou, H. C., and Smale, S. T. (2001) Nat. Immunol. 2, 51–57[CrossRef][Medline] [Order article via Infotrieve]
  48. Takeda, K., and Akira, S. (2004) Semin. Immunol. 16, 3–9[CrossRef][Medline] [Order article via Infotrieve]
  49. Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002) Nature 420, 329–333[CrossRef][Medline] [Order article via Infotrieve]
  50. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324–329[CrossRef][Medline] [Order article via Infotrieve]
  51. Trinchieri, G., Pflanz, S., and Kastelein, R. A. (2003) Immunity. 19, 641–644[CrossRef][Medline] [Order article via Infotrieve]
  52. Ha, S. J., Kim, D. J., Baek, K. H., Yun, Y. D., and Sung, Y. C. (2004) J. Immunol. 172, 525–531[Abstract/Free Full Text]
  53. Takeuchi, O., Takeda, K., Hoshino, K., Adachi, O., Ogawa, T., and Akira, S. (2000) Int. Immunol. 12, 113–117[Abstract/Free Full Text]
  54. Takeuchi, O., Hoshino, K., and Akira, S. (2000) J. Immunol. 165, 5392–5392[Abstract/Free Full Text]
  55. Kurita-Taniguchi, M., Fukui, A., Hazeki, K., Hirano, A., Tsuji, S., Matsumoto, M., Watanabe, M., Ueda, S., and Seya, T. (2000) J. Immunol. 165, 5143–5152[Abstract/Free Full Text]
  56. Knutson, K. L., Hmama, Z., Herrera-Velit, P., Rochford, R., and Reiner, N. E. (1998) J. Biol. Chem. 273, 645–652[Abstract/Free Full Text]
  57. Zhang, J., Somani, A. K., and Siminovitch, K. A. (2000) Semin. Immunol. 12, 361–378[CrossRef][Medline] [Order article via Infotrieve]
  58. Liu, Z. X., Nishida, H., He, J. W., Lai, M. M., Feng, N., and Dennert, G. (2002) J. Virol. 76, 990–997[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Xu, N. Xiao, F. Liu, H. Ren, and J. Gu
Inhibition of RIG-I and MDA5-dependent antiviral response by gC1qR at mitochondria
PNAS, February 3, 2009; 106(5): 1530 - 1535.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
D. L. Montgomery, A. Van Olphen, H. Van Campen, and T. R. Hansen
The Fetal Brain in Bovine Viral Diarrhea Virus-infected Calves: Lesions, Distribution, and Cellular Heterogeneity of Viral Antigen at 190 Days Gestation
Vet. Pathol., May 1, 2008; 45(3): 288 - 296.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. R. Lukens, M. W. Cruise, M. G. Lassen, and Y. S. Hahn
Blockade of PD-1/B7-H1 Interaction Restores Effector CD8+ T Cell Responses in a Hepatitis C Virus Core Murine Model
J. Immunol., April 1, 2008; 180(7): 4875 - 4884.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. N. Waggoner, C. H. T. Hall, and Y. S. Hahn
HCV core protein interaction with gC1q receptor inhibits Th1 differentiation of CD4+ T cells via suppression of dendritic cell IL-12 production
J. Leukoc. Biol., December 1, 2007; 82(6): 1407 - 1419.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Chang, A. Dolganiuc, and G. Szabo
Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins
J. Leukoc. Biol., September 1, 2007; 82(3): 479 - 487.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. A. Byrnes, D.-Y. Li, K. Park, D. Thompson, C. Mocilnikar, P. Mohan, J. P. Molleston, M. Narkewicz, H. Zhou, S. F. Wolf, et al.
Modulation of the IL-12/IFN-{gamma} axis by IFN-{alpha} therapy for hepatitis C
J. Leukoc. Biol., March 1, 2007; 81(3): 825 - 834.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
A. R. Ciccaglione, E. Stellacci, C. Marcantonio, V. Muto, M. Equestre, G. Marsili, M. Rapicetta, and A. Battistini
Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes
J. Virol., January 1, 2007; 81(1): 202 - 214.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. N. Waggoner, M. W. Cruise, R. Kassel, and Y. S. Hahn
gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway
J. Immunol., October 1, 2005; 175(7): 4706 - 4714.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
A. Matsumori
Hepatitis C Virus Infection and Cardiomyopathies
Circ. Res., February 4, 2005; 96(2): 144 - 147.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/42/43479    most recent
M407640200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eisen-Vandervelde, A. L.
Right arrow Articles by Hahn, Y. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eisen-Vandervelde, A. L.
Right arrow Articles by Hahn, Y. S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement