Structural proteins of Hepatitis C virus induce interleukin 8 production and apoptosis in human endothelial cells

Anuradha Balasubramanian1, Neru Munshi1, Margaret J. Koziel2, Zongyi Hu3, T. Jake Liang3, Jerome E. Groopman1,{dagger} and Ramesh K. Ganju1,{dagger}

1 Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 4 Blackfan Circle, 3rd Floor, Boston, MA 02115, USA
2 Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Harvard Medical School, 4 Blackfan Circle, 3rd Floor, Boston, MA 02115, USA
3 Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence
Ramesh K. Ganju
rganju{at}bidmc.harvard.edu
Jerome E. Groopman
jgroopma{at}bidmc.harvard.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) infection is associated with inflammation of liver endothelium, which contributes to the pathogenesis of chronic hepatitis. The mechanism of this endothelitis is not understood, since the virus does not appear to infect endothelial cells productively. Here, an ‘innocent bystander’ mechanism related to HCV proteins was hypothesized and it was investigated whether the binding of HCV particles to human endothelium induced functional changes in the cells. Exposure of human umbilical vein endothelial cells (HUVECs) to HCV-like particles (HCV-LPs) resulted in increased interleukin 8 (IL8) production and induction of apoptosis. The IL8 supernatants collected after stimulation of HUVECs with HCV-LPs, BV-GUS (control baculovirus containing {beta}-glucuronidase) and appropriate controls were used to assay the transendothelial migration of neutrophils. This assay confirmed that HCV-LP-induced IL8 was functionally active. Using specific NF-{kappa}B inhibitors, it was also shown that HCV-LP-induced NF-{kappa}B activity mediated IL8 production in HUVECs. Apoptosis appeared to be mediated by the Fas/Fas-L pathway, as neutralizing antibodies for Fas and Fas-L significantly protected HUVECs against HCV-LP-induced apoptosis. Treatment of HUVECs with HCV-LPs also enhanced cellular Fas-L expression and augmented caspase-3 activation. This was confirmed by using a specific caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone. As shown by blocking of specific chemokine receptors for IL8 on HUVECs, the induction of IL8 did not appear to contribute to HCV-LP-induced apoptosis. These results suggest that HCV proteins can trigger the release of inflammatory chemokines such as IL8 and cause endothelial apoptosis, thereby facilitating endothelitis.

{dagger}These authors contributed equally to this work.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) is a major cause of end-stage liver disease (Hoofnagle, 1997). Persistent infection can lead to chronic hepatitis complicated by cirrhosis and/or carcinoma (Saito et al., 1990; Tong et al., 1995; Okuda, 1997; Trepo et al., 1998; Ohsawa et al., 1999). HCV belongs to the family Flaviviridae (Choo et al., 1989; Kato et al., 1990) and its RNA genome encodes a long polyprotein. Proteolytic processing of this polyprotein produces 10 different proteins. These include three HCV structural proteins, the core protein and the two envelope glycoproteins, E1 and E2 (Miyamura & Matsuura, 1993). The core protein has been reported to modulate immune functions. It can repress the transcriptional activity of various genes including the p53 promoter in vitro (Kim et al., 1994; Ray et al., 1995, 1997) and has been implicated in Fas-mediated apoptosis both in vitro and in vivo (Ruggieri et al., 1997).

HCV has the ability to persist, despite a strong immune response. Chronic infection may result in the development of cirrhosis. A more vigorous inflammatory response is associated with a more rapid progression to cirrhosis (Poynard et al., 1997). Characteristic pathological changes associated with HCV include lymphoid nodules, steatosis, subendothelial inflammation of portal and/or terminal hepatic veins and endothelial cell damage (Lory & Zimmermann, 1997). The cause of this endothelial pathology is not yet defined. One hypothesis suggests that there is a release of chemotactic cytokines that recruit specific lymphocytes to the liver and enhance inflammatory leukocyte–endothelial cell interactions (Mackay, 1996; Adams & Lloyd, 1997; Luster, 1998; Shields et al., 1999).

We examined the possibility that HCV structural proteins might be toxic to endothelial cells independent of direct viral infection, a so-called ‘innocent bystander’ effect. Such a ‘bystander effect’ is believed to be important in the pathophysiology of human immunodeficiency virus (HIV) infection (Meyaard et al., 1992; Groux et al., 1992; Ameisen et al., 1995; Oyaizu et al., 1997). HIV and its envelope protein gp120 can induce the apoptosis of bystander CD4+ cells via binding to specific chemokine co-receptors (Banda et al., 1992). Recently, gp120 has also been shown to interact with endothelium via the CXCR4 receptor and cause endothelial cell dysfunction (Huang et al., 1999). To investigate a possible ‘innocent bystander’ mechanism of HCV-related endothelitis, we exposed endothelial cells to HCV particles containing structural proteins expressed in a baculovirus system. The particles contained the nucleocapsid or core protein, and the two envelope glycoproteins, E1 and E2 (Baumert et al., 1998, 1999). Exposure of human umbilical vein endothelial cells (HUVECs) to these HCV-like particles (HCV-LPs) caused the release of the inflammatory chemokine interleukin 8 (IL8) via NF-{kappa}B and triggered Fas-mediated apoptosis. Further studies revealed that these particles induced the upregulation of the Fas ligand (Fas-L) and activated caspase-3.

Our results indicate that HCV proteins can interact with endothelium and cause the release of inflammatory mediators, as well as induce programmed cell death. Such events may contribute to the pathological finding of endothelitis seen in HCV hepatitis.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and cell culture.
HUVECs were grown at 37 °C in 5 % CO2 in endothelial cell growth medium (Clonetics) containing 2 % fetal bovine serum (FBS), 12 µg bovine brain extract ml–1, 10 ng human recombinant epidermal growth factor ml–1, 1 µg hydrocortisone ml–1 and GA-1000 (gentamicin and amphotericin B, 1 µg ml–1), according to the recommendation of the supplier (Clonetics).

Baculovirus constructs.
Recombinant HCV structural proteins (HCV-LPs) and the control baculovirus preparation were produced as described (Baumert et al., 1998). Briefly, cDNA for the HCV structural proteins (from HCV-J strain, genotype 1b) was used to generate the recombinant baculovirus HCV particles (HCV-LPs). pFastBacHCV (which contains the coding sequences for the core, E1 and E2 proteins) was generated by PCR with the following primers: 5'-GAGACAGACGTGCCTGCTACTTAGCAACACGCG-3' (sense, nt 1918–1951) and 5'-TCGAAAGCTTAGGCCTCAGCCTGGGCTATCAGC-3' (antisense, nt 2567–2543). A stop codon and the HindIII site (shown in bold) were introduced at the 3' end of the p7 protein-coding region. The NotI/HindIII digestion product of the PCR fragment was subcloned into the NotI/HindIII site (multiple cloning site) of pFastBacHCV.S (which contains the coding sequences for the core, E1, E2 and p7 proteins as well as 21 aa of the NS2 protein), as described previously (Baumert et al., 1998). pFastBacGUS containing the coding sequence of {beta}-glucuronidase (GUS) was used to generate the control baculovirus preparation (BV-GUS).

Reagents.
Protease inhibitors aprotinin, leupeptin and pepstatin, trypsin inhibitor and lipopolysaccharide (LPS) (Escherichia coli 0111 : B4) were obtained from Sigma. Anti-Fas and anti-Fas-L rabbit polyclonal immunoglobulin G (IgG) antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad. Caspase-3 and Fas-L-neutralizing antibodies were obtained from PharMingen. Fas receptor-neutralizing antibody, ZB4, was obtained from Medical & Biological Laboratories. Monoclonal antibody to human IL8 receptor A (IL8RA or CXCR-1) was from PharMingen and monoclonal anti-IL8RB (anti-CXCR-2) was a gift from LeukoSite. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and the specific caspase-3 inhibitor Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) were purchased from Enzyme System Products. Bay 11-7082, NF-{kappa}B SN50 cell-permeable inhibitory peptide and NF-{kappa}B SN50M cell-permeable control peptide were obtained from BIOMOL Research Laboratories. Electrophoresis reagents and nitrocellulose membrane were obtained from Bio-Rad.

Inflammatory studies
IL8 measurement.
IL8 production was measured by ELISA (Endogen). Briefly, cells were plated in a 24-well plate and grown to 90 % confluence. Cells were then treated with 1000 ng HCV-LP or the control BV-GUS ml–1. Supernatants were collected at the indicated time periods and assayed for the production of IL8 using an ELISA kit according to the manufacturer's instructions (Endogen). Similarly, specific ELISAs were used to measure tumour necrosis factor {alpha} (TNF-{alpha}), interferon {gamma} (IFN-{gamma}) and monocyte chemoattractant protein-1 (MCP-1) (Endogen) in HCV-treated and control supernatants, according to the manufacturer's protocols.

Isolation of neutrophils from human blood.
Neutrophils were isolated by discontinuous Ficoll-Hypaque gradient centrifugation of heparinized blood obtained from disease-free volunteers (Boyum et al., 1991).

Transendothelial migration of neutrophils.
Transendothelial migration was performed as previously described (Smith et al., 1991) with slight modifications. A confluent monolayer of HUVECs was grown in the Transwell membrane of the 24-well culture tray. Neutrophils were added at a concentration of 5x105 cells per well to the top chamber with 2·5 % BSA and the supernatants that were collected from the cells stimulated with HCV-LPs along with appropriate controls were added to the lower chamber with 2·5 % BSA. The neutrophils were incubated at 37 °C for 20 h, after which the migrated neutrophils in the lower chamber were collected and counted. Transmigration was expressed as a percentage of the neutrophils added.

Transient transfection and NF-{kappa}B–luciferase reporter assay.
The NF-{kappa}B reporter assay was performed as described earlier (Balasubramanian et al., 2003) in HUVECs after transfection with 1 µg pNF{kappa}B-Luc vector and pTAL-Luc vector (negative control) (BD Biosciences Clontech) per well using Lipofectamine Plus reagent according to the manufacturer's protocol (Invitrogen) for 48 h. To assess transfection efficiency, cells were co-transfected with pSV-{beta}-galactosidase control vector (Promega). Cells were stimulated with 1000 ng HCV-LPs, control BV-GUS or LPS ml–1 for 1 h. The chemiluminescence (BD Biosciences Clontech) of the luciferase activity was detected using a microplate luminescence counter. {beta}-Galactosidase activity was determined using a Promega kit to verify the reproducibility between the quadruplicate transfections in all experiments.

Apoptosis detection assays
Sandwich ELISA for histone-associated DNA fragments.
HUVEC apoptosis was assessed by a photometric enzyme immunoassay using a cell death detection kit (Boehringer Mannheim). Briefly, 1x104 cells were plated in a 96-well plate and grown to 90 % confluence. The cells were placed in low-serum medium (0·5 % FBS) for 2 h and subsequently left untreated or treated with varying concentrations of HCV-LPs or the control BV-GUS for 24 h at 37 °C. The cells were then lysed and centrifuged at 200 g to separate the cytoplasmic and nuclear fractions. Twenty microlitres of supernatant was added to a streptavidin-coated microtitre plate. Biotin-labelled anti-histone antibody was then added, followed by horseradish peroxidase-conjugated anti-DNA antibody. The increase in nucleosome degradation was calculated by comparing the values from the HCV-LP or BV-GUS cultures with that of cells in 0·5 % FBS. Statistical analysis was done using Student's t-test.

TdT-mediated dUTP nick-end labelling (TUNEL).
The level of chromatin cleavage due to apoptosis was quantified using the Fluorescein In situ Cell Death Detection kit (Boehringer Mannheim). Briefly, cells were plated in chamber slides (Nalge Nunc International) and then cultured in low-serum medium with or without varying concentrations of HCV-LPs or BV-GUS for 24 h. After 24 h, the medium was aspirated off and the cells washed with 1x PBS. The air-dried cells were fixed with a 4 % paraformaldehyde solution for 1 h at room temperature. Cells were then washed with PBS and incubated in permeabilization solution (0·1 % Triton X-100, 0·1 % sodium citrate) for 2 min on ice. Slides were rinsed twice with PBS and 50 µl of TUNEL reaction mixture was added to each sample. Samples were then incubated at 37 °C in the dark for 60 min. After washing the slides again with PBS, the samples were directly analysed under a fluorescence microscope.

Western blotting.
Whole-cell lysates from the HCV-LP- or BV-GUS-treated cells were prepared by lysing the cells in RIPA buffer (50 mM Tris/HCl pH 7·4, 1 % NP-40, 0·25 % sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 10 µg each aprotinin, leupeptin and pepstatin ml–1, 10 mM sodium vanadate, 10 mM sodium fluoride and 10 mM sodium pyrophosphate). Proteins were separated by 10 % SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5 % non-fat milk for 2–3 h and then incubated with the respective primary and secondary antibodies for 2–3 h. Membranes were washed three to four times and developed by chemiluminescence (ECL system; Amersham Pharmacia Biotech).

Inhibition studies.
For studies with NF-{kappa}B inhibitors, various concentrations of Bay 11-7082 and/or SN50 were added to the culture medium 1 h prior to stimulation with 1000 ng HCV-LP, control BV-GUS or LPS ml–1. After 24 h of stimulation, the supernatants were used for estimation of IL8 production as described above.

Cells were treated with the Fas antagonistic ZB4 antibody or Fas-L antagonistic NOK-1 antibody or their respective isotype-matched control antibodies. Antibodies were applied 2 h before the HCV-LP treatment and cell death was evaluated by sandwich ELISA.

Z-VAD-FMK, a cell-permeable general caspase inhibitor, or Z-DEVD-FMK, a specific caspase-3 inhibitor, was added to the cells at a 20 µM concentration, 2 h prior to treatment with HCV-LPs. Z-Phe-Ala-fluoromethyl ketone (Z-FA-FMK) at the same concentration was used as an inhibitor control. Cells were incubated with virus particles and the caspase inhibitors for 24 h. Apoptosis was measured by ELISA.

HUVECs were placed in low-serum medium alone or containing varying concentrations of either CXCR-1 (IL8RA)- or CXCR-2 (IL8RB)-neutralizing antibodies for 4 h. Cells were then washed and stimulated with 1000 ng HCV-LP or control BV-GUS ml–1 for 24 h. After 24 h, cells were lysed as described elsewhere and subjected to Western blot analysis to check for the expression of Fas-L.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HCV-LP treatment of HUVECs leads to increased IL8 production
When HUVECs were exposed to HCV-LPs, increased production of the inflammatory chemokine IL8 was observed. As shown in Fig. 1(a), the IL8 level was about 2·5-fold higher in the HCV-LP-treated sample at 24 h (P<0·023) than with either the medium control or the BV-GUS-treated HUVECs. HCV particles did not induce the production of other inflammatory cytokines such as TNF-{alpha}, IFN-{gamma} or MCP-1 (data not shown). To determine whether IL8 induced by HCV-LPs was functionally active, the transendothelial migration of neutrophils was assessed (Fig. 1b). IL8 supernatants collected from HUVECs after stimulation with BV-GUS, HCV-LP, LPS, heat-inactivated BV-GUS or heat-inactivated HCV-LPs, along with the unstimulated control, were used for the assay. A 2·5-fold increase in the transmigration of neutrophils was observed in the presence of the HCV-LP-induced IL8 supernatant when compared with supernatant from the unstimulated control.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Stimulation of HUVECs with HCV-LPs leads to the production of functionally active IL8. (a) HUVECs were plated in a 24-well plate, grown to 90 % confluence and then left untreated or treated with 1000 ng HCV-LP or the control BV-GUS ml–1 in low-serum medium (0·5 % FBS). Supernatants were collected at the indicated times and assayed for the production of IL8. *, P<0·023. Data are the means of two independent experiments performed in duplicate. (b) A monolayer of HUVECs was grown on each membrane of the Transwell plate. Simultaneously, the serum-free supernatants, collected from HUVECs that were untreated or treated with 1000 ng HCV-LPs, control BV-GUS, heat-inactivated HCV-LPs ({Delta}HCV-LPs), heat-inactivated BV-GUS ({Delta}BV-GUS) or LPS ml–1 were used in the lower chamber along with 2·5 % BSA. Neutrophils (5x105 cells per well) prepared from healthy human blood were placed in the upper chamber with 2·5 % BSA and incubated for 20 h at 37 °C. The migration of neutrophils was determined by counting the number of cells in the lower chamber and the percentage migration was then calculated. *, P<0·0005. The data are from one of two independent experiments performed in duplicate.

 
HCV-LPs induces NF-{kappa}B activity
NF-{kappa}B has been shown to activate various inflammatory genes. It is also widely known that IL8 gene expression is regulated by NF-{kappa}B and/or AP-1 transcription factors. HUVECs that were stimulated with HCV-LPs induced NF-{kappa}B activity. As shown in Fig. 2, when HUVECs transfected with the pNF-{kappa}B-Luc vector were stimulated with HCV-LPs, we found a 6·4-fold increase in NF-{kappa}B activity compared with the unstimulated control. The reporter assay further showed that HUVECs transfected with the pTAL-Luc vector (control vector) did not induce NF-{kappa}B activity.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. HCV-LPs induce NF-{kappa}B activity as detected by the NF-{kappa}B–luciferase reporter assay. HUVECs were transiently transfected with the pTAL-Luc (control) vector or pNF-{kappa}B-Luc vector. After 48 h, transfection efficiency was determined by measuring {beta}-galactosidase activity. HUVEC transfectants were stimulated with 1000 ng HCV-LP, BV-GUS or LPS ml–1 for 1 h. Cells were lysed and luciferase activity was detected by chemiluminescence using a microplate luminescence counter. Luminescence was measured as counts s–1. *, P<0·05. Data presented are from one of two independent experiments performed in duplicate.

 
NF-{kappa}B mediates HCV-LP-induced IL8
To confirm that the NF-{kappa}B pathway was involved in the HCV-LP-induced production of IL8, HUVECs were stimulated with HCV-LPs in the presence of different inhibitors of NF-{kappa}B along with appropriate controls, and the supernatants were then used for IL8 estimation. HCV-LP-induced IL8 was reduced significantly (1·6-fold) in the presence of 10 µM Bay 11-7082, an inhibitor of cytokine-induced I{kappa}B{alpha} phosphorylation (Fig. 3a). A gradual decrease in the HCV-LP-induced production of IL8 was observed when increasing concentrations of the inhibitor were used. Similarly, in the presence of 10 µg SN50 ml–1, a cell-permeable inhibitory peptide that blocks nuclear translocation of the NF-{kappa}B active complex, a 1·5-fold decrease in IL8 expression induced by HCV-LPs was noticed (Fig. 3b). A gradual reduction in the amount of IL8 induced by HCV-LPs was observed with increasing concentrations of SN50. In parallel, there was no obvious change in the amount of HCV-LP-induced IL8 in the presence of SN50M, a cell-permeable control peptide. These results clearly suggest that the NF-{kappa}B pathway might be involved in the induction of IL8 by HCV-LPs.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. HCV-LPs induce NF-{kappa}B-mediated IL8 production. (a) HUVECs were untreated (–) or pretreated with Bay 11-7082 (1–10 µM) for 1 h. Cells were then washed and stimulated with 1000 ng HCV-LP or BV-GUS ml–1 in low-serum medium (0·5 % FBS). Heat-inactivated BV-GUS and heat-inactivated HCV-LPs (1000 ng ml–1) were used as negative controls and LPS was used as the positive control. Supernatants were collected after 24 h and assayed for the production of IL8. *, P<0·0005. (b) HUVECs were untreated or pretreated with SN50 or SN50M (control peptide) (0·5–10 µg ml–1) for 1 h. Subsequently, the cells were washed and untreated (–) or treated (+) with 1000 ng HCV-LP or BV-GUS ml–1 in low-serum medium (0·5 % FBS). Supernatants were collected after 24 h and assayed for the production of IL8. *, P<0·05. Data presented are from one of two independent experiments performed in duplicate.

 
HCV-LPs induce apoptosis in HUVECs
HCV-LP treatment of HUVECs resulted in an approximate 2·5-fold (P<0·0005) increase in apoptosis over BV-GUS-treated or untreated HUVECs as measured by the histone ELISA method. As shown in Fig. 4(a), the degree of apoptosis at 24 h was found to be dose-dependent with maximal effect at a concentration of 1000 ng HCV-LPs ml–1. The degree of apoptosis after treatment of HUVECs with the control baculovirus BV-GUS was not significantly different from the untreated HUVEC cultures over the same time periods. The induction of apoptosis measured by ELISA was confirmed using the TUNEL method. The positively stained cells, as shown in Fig. 4(b), were counted to quantitate the degree of apoptosis. About 68 % of the cells were found to be TUNEL positive after HCV-LP treatment (1000 ng ml–1) (Fig. 4b, panel v), while 32 % of the cells were TUNEL positive after treatment with a similar concentration of BV-GUS (Fig. 4b, panel iv). Untreated HUVECs showed ~28 % TUNEL-positive cells (Fig. 4b, panel i, control). A similar result was observed at a lower concentration (100 ng ml–1) of HCV-LPs (Fig. 4b, panel iii) (35 % TUNEL positive). HUVECs treated with 100 ng of the control baculovirus ml–1 showed ~28 % TUNEL-positive staining (Fig. 4b, panel ii). These data indicate that HCV-LP treatment of HUVECs results in the induction of apoptosis.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. HCV-LPs induce endothelial apoptosis in a concentration-dependent manner. (a) HUVECs were grown in a 96-well plate and then left untreated or treated with HCV-LPs or BV-GUS (control preparation) at different concentrations in low-serum medium (0·5 % FBS). Apoptosis was measured by sandwich ELISA for histone-associated DNA fragments. The fold increase in nucleosome degradation was calculated by comparing the optical density values of the HCV-LP-treated HUVECs with that of the untreated cells. *, P<0·0005. Data are the means of two independent experiments performed in quadruplicate. (b) HUVECs were grown in chamber slides and treated with different concentrations of HCV-LPs or the control vector in low-serum medium for 24 h. Samples were analysed for apoptosis using the TUNEL method. Green fluorescent cells represent apoptotic cells. (i) Untreated HUVECs; (ii) 100 ng BV-GUS ml–1; (iii) 100 ng HCV-LPs ml–1; (iv) 1000 ng BV-GUS ml–1; and (v) 1000 ng HCV-LPs ml–1.

 
HCV particles induce Fas-mediated apoptosis in HUVECs
To explore further the mechanism of apoptosis in HUVECs, we considered the possible involvement of known apoptotic pathways such as Fas. To this end, HUVECs were treated with 1000 ng HCV-LPs or the control BV-GUS ml–1 in low-serum medium. Immunoblot analysis (Fig. 5a) showed that HCV-LP treatment of cells caused an increase in the production of Fas-L. However, there was no change in Fas receptor expression over the same period of time under similar conditions (Fig. 5b). These data indicate that HCV-LPs may increase the susceptibility of HUVECs to Fas-mediated apoptosis by upregulating the expression of Fas-L. To establish further the involvement of the Fas/Fas-L pathway in HCV-LP-mediated endothelial apoptosis, we used the Fas-antagonistic antibody ZB4 and the Fas-L-antagonistic antibody NOK-1. HUVECs were treated with varying concentrations of either antibody or the isotype-matched control antibody for 2 h. Subsequently, HCV-LPs (1000 ng ml–1) were added to the cells along with the respective antibodies for 24 h. Cell death was assessed by ELISA. A dose-response study showed that the maximum inhibitory effect of these antibodies occurred at 1000 ng ml–1 (data not shown). Fig. 5(c) shows that HCV-LPs were unable to induce apoptosis in HUVECs in the presence of the ZB4 or NOK-1 antibody, suggesting that the Fas/Fas-L pathway may play a key role in HCV-induced endothelial cell death.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. HCV-LPs increase the expression of Fas-L and induce Fas-mediated apoptosis in HUVECs. (a, b) HUVECs were untreated or treated with HCV-LPs or control BV-GUS in low-serum medium. Cells were lysed at various time points. Lysates were analysed by Western blotting with either anti-Fas-L (a) or anti-Fas antibodies (b). (c) HUVECs were untreated or treated with HCV-LPs and apoptosis was assessed by ELISA. In some cultures, Fas-neutralizing antibody (ZB4) or Fas-L-neutralizing antibody (NOK-1) was added. Isotype-matched IgG served as a control. *, P<0·0005. Data are the means of two independent experiments performed in either triplicate or quadruplicate.

 
The effect of HCV-LPs on Fas-mediated apoptosis may be due to augmentation of caspase-3 activation
To assess which downstream mediators might be operative in this HCV-LP-induced apoptosis, we examined the activation of caspase-3, a primary activator of several key apoptosis target proteins. As shown in Fig. 6(a), at 24 h the expression of the cleaved active caspase-3 fragment (17 kDa) was higher in the HCV-LP-treated cells compared with that of either the untreated or control BV-GUS-treated cells. This clearly indicates that HCV-LP treatment caused the cleavage of procaspase-3. This enhanced enzyme activation suggests that HCV-LPs may induce caspase-3 as part of the Fas downstream signalling pathway. In order to assess further the role of caspases in this apoptotic process, we used the cell-permeable inhibitors Z-VAD-FMK (a general caspase inhibitor) or Z-DEVD-FMK (a specific caspase-3 inhibitor), or DMSO as a diluent control. HUVECs were treated with HCV-LPs (1000 ng ml–1) alone or in combination with the various inhibitors for 24 h. Apoptosis was measured based on the degree of nucleosome degradation by ELISA (Fig. 6b). We found a twofold reduction (P<0·0005) in apoptosis with either the general caspase inhibitor or the specific caspase-3 inhibitor compared with cells treated only with HCV-LPs. However, no inhibition was observed when cells were treated with the inhibitor control Z-FA-FMK in the presence of HCV-LPs under similar conditions.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6. HCV particles augment caspase-3 activation and inhibit apoptosis in HUVECs in the presence of caspase inhibitors. (a) HUVECs were untreated or treated with 1000 ng HCV-LPs or BV-GUS ml–1 in low-serum medium for 24 h. Cells were lysed and caspase-3 activation was detected by blotting with caspase-3 antibody. Caspase-3, in an inactive form as procaspase-3 (32 kDa), is cleaved to an active form (17 kDa). (b) HUVECs were untreated or treated for 24 h with 1000 ng HCV-LPs ml–1 alone or in the presence of 20 µM of the general caspase pathway inhibitor Z-VAD-FMK, the specific caspase-3 inhibitor Z-DEVD-FMK or the control inhibitor Z-FA-FMK. Cell death was measured by sandwich ELISA. *, P<0·0005. Data are the means of two independent experiments performed in either triplicate or quadruplicate.

 
IL8 production does not mediate HCV-LP-induced apoptosis in HUVECs
We next sought to determine whether IL8 participated in apoptotic induction in HUVECs. To this end, we pretreated HUVECs with neutralizing antibodies to the IL8-specific receptors CXCR-1 (IL8RA) and CXCR-2 (IL8RB). Fig. 7(a) and (b) show that at all three concentrations (10, 100 and 1000 ng ml–1) of these antibodies, HCV-LPs were still able to induce the expression of Fas-L, suggesting that IL8 production was not responsible for the induction of HUVEC apoptosis.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. HCV-LP-induced expression of Fas-L in the presence of CXCR-1- and CXCR-2-neutralizing antibodies. HUVECs were untreated or pretreated with varying concentrations (10–1000 ng ml–1) of either IL8RA (CXCR-1) (a) or IL8RB (CXCR-2) (b) for 4 h. Subsequently, cells were washed and untreated or treated with 1000 ng HCV-LP or BV-GUS ml–1. The expression of Fas-L was analysed by Western blotting.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C infection often results in damage in different cell types within the liver. Hepatocytes are susceptible to HCV infection. Peripheral blood mononuclear cells are also thought to be productively infected by HCV (Zignego et al., 1992; Lerat et al., 1996). However, endothelial cells do not appear to be directly infected with the virus. It has previously been suggested that chemokines regulate the adherence of lymphocytes to endothelium before extravasation from blood into tissue (Campbell et al., 2003) by activating leukocytes through changes in integrin affinity/avidity (Baltus et al., 2003). This results in firm adhesion to the vessel wall and eventual transmigration of the lymphocyte through the endothelium and into the underlying tissue to sites of infection (Johnston & Butcher, 2002). Previous studies by others suggest that in HCV infection specific chemokine–chemokine receptor interactions are involved in the recruitment of lymphocytes into the liver via either portal or sinusoidal endothelium (Shields et al., 1999; Grant et al., 2002), but little is known about the signals that position and retain lymphocytes at epithelial surfaces within the liver (Heydtmann et al., 2001). The inflammatory reaction against HCV that is believed to contribute to hepatic pathology includes polyclonal B- and T-cell responses, some of which are HCV specific (Koziel et al., 1995). Non-specific recruitment of T cells into the inflamed liver may also occur (Unutmaz et al., 1994; Nuti et al., 1998).

Since endothelial cells do not appear to be susceptible to HCV infection, we hypothesized that the binding of the HCV particles rather than infection per se might contribute to endothelial damage. Using recombinant HCV particles obtained from the cDNA of the HCV structural proteins, we analysed the effects of structural proteins on HUVECs. In support of our theory that HCV-LPs bind to endothelial cells, earlier studies have indeed reported that HCV-LPs bind to liver sinusoidal endothelial cells (LSECs), a specialized endothelial cell type with antigen-presenting cell function (Ludwig et al., 2004). Ludwig et al. (2004) further reported that this interaction of HCV-LPs with the LSECs might be through the L-SIGN antigen receptor. L-SIGN is known to be expressed in LSECs (Bashirova et al., 2001; Pohlmann et al., 2001) and it has recently been demonstrated that the C-type lectins DC-SIGN and L-SIGN/DC-SIGNR may be involved in HCV binding through their interaction with the HCV envelope glycoprotein E2 (Gardner et al., 2003; Lozach et al., 2003; Pohlmann et al., 2003). It has been suggested that L-SIGN expressed in LSECs may capture HCV from the blood and mediate the infection of adjacent hepatocytes, the main target cells of HCV.

In our study, HCV-LPs induced IL8 production but not the production of other inflammatory cytokines such as MCP-1, TNF-{alpha} or IFN-{gamma}. In prior immunohistochemical studies of HCV infection, IL8 was shown to be expressed in infiltrating cells in the portal tract and fibrotic septa and within hepatic lobules in patients (Napoli et al., 1994; Koziel et al., 1995). Moreover, it has been reported that in HCV patients there was a correlation between the serum IL8 levels and liver fibrosis (Kaplanski et al., 1997), and that intrahepatic mRNA IL8 expression was associated with hepatic inflammation and fibrosis (Fukuda et al., 1996; Shimoda et al., 1998; Masumoto et al., 1998; Mahmood et al., 2002). We have also observed enhanced IL8 production upon HCV E2 and HIV gp120 treatment in hepatocytes (Balasubramanian et al., 2003). Transmigration of neutrophils across HUVECs in the presence of the HCV-LP-induced IL8 supernatants further confirmed the functional activity of IL8. While our results also showed that IL8 expression induced by HCV-LPs was mediated by the NF-{kappa}B pathway, we do not yet know the HCV-LP-induced mechanism of NF-{kappa}B activation.

Although in chronic liver inflammation due to HCV it has been reported that E-selectin, ICAM-1 and V-CAM-1 are strongly expressed on endothelial cells from sinusoidal vessels and may play an important role in leukocyte extravasation (Volpes et al., 1992), we did not find any significant change in the basal expression of the inflammatory adhesion molecules ICAM-1, VCAM-1, E-selectin and P-selectin when HUVECs were treated with HCV-LPs (data not shown). In addition, previous studies have shown that chemokines with chemotactic activity on leukocytes may also be important in the pathogenesis of chronic hepatitis (Baggiolini et al., 1994), which is in correlation with our study showing induction of the chemokine IL8 by HCV-LPs.

Our studies also revealed that HCV-LPs can induce programmed cell death in endothelial cells. Previously, the apoptosis of hepatocytes has been histologically demonstrated in patients with chronic hepatitis C (Roberts et al., 1993). To address the mechanism of this cell death, we focused on the involvement of a known apoptotic mediator, Fas. In chronic HCV infection, the immunohistochemical detection of Fas antigen in liver tissue has been demonstrated, but whether this upregulation of Fas in hepatocytes and Fas ligand in T lymphocytes is due to HCV infection or a secondary phenomenon is unknown (Hiramatsu et al., 1994). Here, we observed that treatment of HUVECs with HCV-LPs caused the upregulation of Fas-L. We further demonstrated the functional involvement of the Fas pathway by abrogating apoptosis with neutralizing antibodies for Fas and Fas-L. In addition, we recently showed that HCV E2 in conjunction with HIV gp120 induces apoptosis in hepatocytes via the Fas-mediated pathway (Munshi et al., 2003).

The binding of Fas-L to the Fas receptor is known to lead to the activation of a series of death-associated molecules including FADD (Chinnaiyan et al., 1995), an adaptor protein with a death domain. FADD in turn binds and activates procaspase-8, eventually triggering activation of other caspases such as caspase-7, -3 and -6 (Muzio et al., 1996). To characterize Fas-mediated apoptosis induced by HCV particles in more detail, we examined the effects on the activation of the caspase cascade by assessing activation of caspase-3. Caspase-3 is a downstream caspase that cleaves anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-XL. This cleavage by caspase-3 destroys their anti-apoptotic functions and causes the release of their C-terminal fragments, which are pro-apoptotic (Wolf & Green, 1999). Treatment of HUVECs with HCV-LPs resulted in increased activation of caspase-3. This was confirmed using a specific caspase-3 inhibitor, Z-VAD-FMK.

With these observations in hand, we asked whether IL8 release might participate in apoptosis in HUVECs. IL8 belongs to the CXC subfamily of pro-inflammatory chemokines and is known for the induction of several cellular functions such as chemotaxis, cell adhesion and growth-modulating effects (Detmers et al., 1991; Loetscher et al., 1994; Jones et al., 1997; Rainger et al., 1998; Wolf & Green, 1999). IL8 has been shown to mediate these effects by binding to the G protein-coupled CXCR-1 and CXCR-2 receptors (Murphy, 1997). Neutralizing antibodies against CXCR-1 and CXCR-2 had no effect on HCV-LP-induced apoptosis (data not shown), nor did these antibodies inhibit the production of Fas-L. These results indicated that IL8 production did not contribute to the induction of apoptosis.

It appears that HCV, like HIV and other viruses, may encode proteins that bind specifically to cells such as endothelium that are not susceptible to direct infection and cause damage via an ‘innocent bystander’ effect. Therapeutic interventions targeted against the interaction of endothelium and HCV structural proteins may be beneficial in limiting the pathology of HCV infection.


   ACKNOWLEDGEMENTS
 
We thank Janet Delahanty for editing the manuscript and Daniel Kelley for preparation of the figures. This work was supported in part by National Institutes of Health grants DA15008 (J. E. G.) and AI49140 (R. K. G.).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adams, D. H. & Lloyd, A. R. (1997). Chemokines: leucocyte recruitment and activation cytokines. Lancet 349, 490–495.[CrossRef][Medline]

Ameisen, J. C., Estaquier, J., Idziorek, T. & De Bels, F. (1995). Programmed cell death and AIDS pathogenesis: significance and potential mechanisms. Curr Top Microbiol Immunol 200, 195–211.[Medline]

Baggiolini, M., Dewald, B. & Moser, B. (1994). Interleukin-8 and related chemotactic cytokines – CXC and CC chemokines. Adv Immunol 55, 97–179.[Medline]

Balasubramanian, A., Ganju, R. K. & Groopman, J. E. (2003). Hepatitis C virus and HIV envelope proteins collaboratively mediate IL-8 secretion through activation of p38 MAP kinase and SHP2 in hepatocytes. J Biol Chem 278, 35755–35766.[Abstract/Free Full Text]

Baltus, T., Weber, K. S., Johnson, Z., Proudfoot, A. E. & Weber, C. (2003). Oligomerization of RANTES is required for CCR1-mediated arrest but not CCR5-mediated transmigration of leukocytes on inflamed endothelium. Blood 102, 1985–1988.[Abstract/Free Full Text]

Banda, N. K., Bernier, J., Kurahara, D. K., Kurrle, R., Haigwood, N., Sekaly, R. P. & Finkel, T. H. (1992). Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med 176, 1099–1106.[Abstract/Free Full Text]

Bashirova, A. A., Geijtenbeek, T. B., van Duijnhoven, G. C. & 10 other authors (2001). A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 193, 671–678.[Abstract/Free Full Text]

Baumert, T. F., Ito, S., Wong, D. T. & Liang, T. J. (1998). Hepatitis C virus structural proteins assemble into virus-like particles in insect cells. J Virol 72, 3827–3836.[Abstract/Free Full Text]

Baumert, T. F., Vergalla, J., Satoi, J., Thomson, M., Lechmann, M., Herion, D., Greenberg, H. B., Ito, S. & Liang, T. J. (1999). Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate. Gastroenterology 117, 1397–1407.[Medline]

Boyum, A., Lovhaug, D., Tresland, L. & Nordlie, E. M. (1991). Separation of leukocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand J Immunol 34, 697–712.[Medline]

Campbell, D. J., Kim, C. H. & Butcher, E. C. (2003). Chemokines in the systemic organization of immunity. Immunol Rev 195, 58–71.[CrossRef][Medline]

Chinnaiyan, A. M., O'Rourke, K., Tewari, M. & Dixit, V. M. (1995). FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505–512.[CrossRef][Medline]

Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362.[Medline]

Detmers, P. A., Powell, D. E., Walz, A., Clark-Lewis, I., Baggiolini, M. & Cohn, Z. A. (1991). Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis. J Immunol 147, 4211–4217.[Abstract/Free Full Text]

Fukuda, R., Ishimura, N., Ishihara, S. & 11 other authors (1996). Intrahepatic expression of pro-inflammatory cytokine mRNAs and interferon efficacy in chronic hepatitis C. Liver 16, 390–399.[Medline]

Gardner, J. P., Durso, R. J., Arrigale, R. R., Donovan, G. P., Maddon, P. J., Dragic, T. & Olson, W. C. (2003). L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus. Proc Natl Acad Sci U S A 100, 4498–4503.[Abstract/Free Full Text]

Grant, A. J., Goddard, S., Ahmed-Choudhury, J., Reynolds, G., Jackson, D. G., Briskin, M., Wu, L., Hubscher, S. G. & Adams, D. H. (2002). Hepatic expression of secondary lymphoid chemokine (CCL21) promotes the development of portal-associated lymphoid tissue in chronic inflammatory liver disease. Am J Pathol 160, 1445–1455.[Abstract/Free Full Text]

Groux, H., Torpier, G., Monte, D., Mouton, Y., Capron, A. & Ameisen, J. C. (1992). Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J Exp Med 175, 331–340.[Abstract/Free Full Text]

Heydtmann, M., Shields, P., McCaughan, G. & Adams, D. (2001). Cytokines and chemokines in the immune response to hepatitis C infection. Curr Opin Infect Dis 14, 279–287.[Medline]

Hiramatsu, N., Hayashi, N., Katayama, K., Mochizuki, K., Kawanishi, Y., Kasahara, A., Fusamoto, H. & Kamada, T. (1994). Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 19, 1354–1359.[CrossRef][Medline]

Hoofnagle, J. H. (1997). Hepatitis C: the clinical spectrum of disease. Hepatology 26, 15S–20S.[CrossRef][Medline]

Huang, M. B., Hunter, M. & Bond, V. C. (1999). Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures. AIDS Res Hum Retroviruses 15, 1265–1277.[CrossRef][Medline]

Johnston, B. & Butcher, E. C. (2002). Chemokines in rapid leukocyte adhesion triggering and migration. Semin Immunol 14, 83–92.[CrossRef][Medline]

Jones, S. A., Dewald, B., Clark-Lewis, I. & Baggiolini, M. (1997). Chemokine antagonists that discriminate between interleukin-8 receptors. Selective blockers of CXCR2. J Biol Chem 272, 16166–16169.[Abstract/Free Full Text]

Kaplanski, G., Farnarier, C., Payan, M. J., Bongrand, P. & Durand, J. M. (1997). Increased levels of soluble adhesion molecules in the serum of patients with hepatitis C. Correlation with cytokine concentrations and liver inflammation and fibrosis. Dig Dis Sci 42, 2277–2284.[CrossRef][Medline]

Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Sugimura, T. & Shimotohno, K. (1990). Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A 87, 9524–9528.[Abstract/Free Full Text]

Kim, D. W., Suzuki, R., Harada, T., Saito, I. & Miyamura, T. (1994). Trans-suppression of gene expression by hepatitis C viral core protein. Jpn J Med Sci Biol 47, 211–220.[Medline]

Koziel, M. J., Dudley, D., Afdhal, N., Grakoui, A., Rice, C. M., Choo, Q. L., Houghton, M. & Walker, B. D. (1995). HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J Clin Invest 96, 2311–2321.[Medline]

Lerat, H., Berby, F., Trabaud, M. A., Vidalin, O., Major, M., Trepo, C. & Inchauspe, G. (1996). Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells. J Clin Invest 97, 845–851.[Abstract/Free Full Text]

Loetscher, P., Seitz, M., Clark-Lewis, I., Baggiolini, M. & Moser, B. (1994). Both interleukin-8 receptors independently mediate chemotaxis. Jurkat cells transfected with IL-8R1 or IL-8R2 migrate in response to IL-8, GRO alpha and NAP-2. FEBS Lett 341, 187–192.[CrossRef][Medline]

Lory, J. & Zimmermann, A. (1997). Endotheliitis-like changes in chronic hepatitis C. Histol Histopathol 12, 359–366.[Medline]

Lozach, P. Y., Lortat-Jacob, H., de Lacroix de Lavalette, A. & 9 other authors (2003). DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 278, 20358–20366.[Abstract/Free Full Text]

Ludwig, I. S., Lekkerkerker, A. N., Depla, E., Bosman, F., Musters, R. J. P., Depraetere, S., van Kooyk, Y. & Geijtenbeek, T. B. H. (2004). Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation. J Virol 78, 8322–8332.[Abstract/Free Full Text]

Luster, A. D. (1998). Chemokines – chemotactic cytokines that mediate inflammation. N Engl J Med 338, 436–445.[Free Full Text]

Mackay, C. R. (1996). Chemokine receptors and T cell chemotaxis. J Exp Med 184, 799–802.[CrossRef][Medline]

Mahmood, S., Sho, M., Yasuhara, Y. & 7 other authors (2002). Clinical significance of intrahepatic interleukin-8 in chronic hepatitis C patients. Hepatol Res 24, 413–419.[CrossRef][Medline]

Masumoto, T., Ohkubo, K., Yamamoto, K., Ninomiya, T., Abe, M., Akbar, S. M., Michitaka, K., Horiike, N. & Onji, M. (1998). Serum IL-8 levels and localization of IL-8 in liver from patients with chronic viral hepatitis. Hepatogastroenterology 45, 1630–1634.[Medline]

Meyaard, L., Otto, S. A., Jonker, R. R., Mijnster, M. J., Keet, R. P. & Miedema, F. (1992). Programmed death of T cells in HIV-1 infection. Science 257, 217–219.[Medline]

Miyamura, T. & Matsuura, Y. (1993). Structural proteins of hepatitis C virus. Trends Microbiol 1, 229–231.[CrossRef][Medline]

Munshi, N., Balasubramanian, A., Koziel, M., Ganju, R. K. & Groopman, J. E. (2003). Hepatitis C and HIV envelope proteins co-operatively induce hepatocytic apoptosis via an innocent bystander mechanism. J Infect Dis 188, 1192–1204.[CrossRef][Medline]

Murphy, P. M. (1997). Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol 34, 311–318.[Medline]

Muzio, M., Chinnaiyan, A. M., Kischkel, F. C. & 11 other authors (1996). FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817–827.[CrossRef][Medline]

Napoli, J., Bishop, G. A. & McCaughan, G. W. (1994). Increased intrahepatic messenger RNA expression of interleukins 2, 6, and 8 in human cirrhosis. Gastroenterology 107, 789–798.[Medline]

Nuti, S., Rosa, D., Valiante, N. M., Saletti, G., Caratozzolo, M., Dellabona, P., Barnaba, V. & Abrignani, S. (1998). Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for V{alpha}24+ T cells and rapid elimination of effector cells by apoptosis. Eur J Immunol 28, 3448–3455.[CrossRef][Medline]

Ohsawa, M., Shingu, N., Miwa, H., Yoshihara, H., Kubo, M., Tsukuma, H., Teshima, H., Hashimoto, M. & Aozasa, K. (1999). Risk of non-Hodgkin's lymphoma in patients with hepatitis C virus infection. Int J Cancer 80, 237–239.[CrossRef][Medline]

Okuda, K. (1997). Hepatitis C and hepatocellular carcinoma. In Liver Cancer, pp. 38–50. Edited by K. Okuda & E. Tabor. London: Churchill Livingstone.

Oyaizu, N., Adachi, Y., Hashimoto, F., McCloskey, T. W., Hosaka, N., Kayagaki, N., Yagita, H. & Pahwa, S. (1997). Monocytes express Fas ligand upon CD4 cross-linking and induce CD4+ T cells apoptosis: a possible mechanism of bystander cell death in HIV infection. J Immunol 158, 2456–2463.[Abstract]

Pohlmann, S., Soilleux, E. J., Baribaud, F., Leslie, G. J., Morris, L. S., Trowsdale, J., Lee, B., Coleman, N. & Doms, R. W. (2001). DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci U S A 98, 2670–2675.[Abstract/Free Full Text]

Pohlmann, S., Zhang, J., Baribaud, F. & 7 other authors (2003). Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 77, 4070–4080.[Abstract/Free Full Text]

Poynard, T., Bedossa, P. & Opolon, P. (1997). Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 349, 825–832.[CrossRef][Medline]

Rainger, G. E., Rowley, A. F. & Nash, G. B. (1998). Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of different chemotactic agents. Blood 92, 4819–4827.[Abstract/Free Full Text]

Ray, R. B., Lagging, L. M., Meyer, K., Steele, R. & Ray, R. (1995). Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 37, 209–220.[CrossRef][Medline]

Ray, R. B., Steele, R., Meyer, K. & Ray, R. (1997). Transcriptional repression of p53 promoter by hepatitis C virus core protein. J Biol Chem 272, 10983–10986.[Abstract/Free Full Text]

Roberts, J. M., Searle, J. W. & Cooksley, W. G. (1993). Histological patterns of prolonged hepatitis C infection. Gastroenterol Jpn 28, (Suppl. 5) 37–41.[Medline]

Ruggieri, A., Harada, T., Matsuura, Y. & Miyamura, T. (1997). Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein. Virology 229, 68–76.[CrossRef][Medline]

Saito, I., Miyamura, T., Ohbayashi, A. & 8 other authors (1990). Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci U S A 87, 6547–6549.[Abstract/Free Full Text]

Shields, P. L., Morland, C. M., Salmon, M., Qin, S., Hubscher, S. G. & Adams, D. H. (1999). Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J Immunol 163, 6236–6243.[Abstract/Free Full Text]

Shimoda, K., Begum, N. A., Shibuta, K., Mori, M., Bonkovsky, H. L., Banner, B. F. & Barnard, G. F. (1998). Interleukin-8 and hIRH (SDF1-{alpha}/PBSF) mRNA expression and histological activity index in patients with chronic hepatitis C. Hepatology 28, 108–115.[CrossRef][Medline]

Smith, W. B., Gamble, J. R., Clark-Lewis, I. & Vadas, M. A. (1991). Interleukin-8 induces neutrophil transendothelial migration. Immunology 72, 65–72.[Medline]

Tong, M. J., el-Farra, N. S., Reikes, A. R. & Co, R. L. (1995). Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 332, 1463–1466.[Abstract/Free Full Text]

Trepo, C., Berthillon, P. & Vitvitski, L. (1998). HCV and lymphoproliferative diseases. Ann Oncol 9, 469–470.[CrossRef][Medline]

Unutmaz, D., Pileri, P. & Abrignani, S. (1994). Antigen-independent activation of naive and memory resting T cells by a cytokine combination. J Exp Med 180, 1159–1164.[Abstract/Free Full Text]

Volpes, R., Van den Oord, J. J. & Desmet, V. J. (1992). Vascular adhesion molecules in acute and chronic liver inflammation. Hepatology 15, 269–275.[Medline]

Wolf, B. B. & Green, D. R. (1999). Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274, 20049–20052.[Free Full Text]

Zignego, A. L., Macchia, D., Monti, M. & 7 other authors (1992). Infection of peripheral mononuclear blood cells by hepatitis C virus. J Hepatol 15, 382–386.[CrossRef][Medline]

Received 22 March 2005; accepted 31 August 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Balasubramanian, A.
Articles by Ganju, R. K.
PubMed
PubMed Citation
Articles by Balasubramanian, A.
Articles by Ganju, R. K.
Agricola
Articles by Balasubramanian, A.
Articles by Ganju, R. K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS